Update October 2019

Recent work has shown that meningeal lymphatic vessels (mLVs), mainly in the dorsal part of the skull, are involved in the clearance of cerebrospinal fluid (CSF), but the precise route of CSF drainage is still unknown. Here we reveal the importance of mLVs in the basal part of the skull for this process by visualizing their distinct anatomical location and characterizing their specialized morphological features, which facilitate the uptake and drainage of CSF. Unlike dorsal mLVs, basal mLVs have lymphatic valves and capillaries located adjacent to the subarachnoid space in mice. We also show that basal mLVs are hotspots for the clearance of CSF macromolecules and that both mLV integrity and CSF drainage are impaired with ageing. Our findings should increase the understanding of how mLVs contribute to the neuropathophysiological processes that are associated with ageing.

Portal hypertension is a common complication of liver disease, either acute or chronic. Consequently, in chronic liver disease, such as the hypertensive mesenteric venous pathology, the coexisting inflammatory response is classically characterized by the splanchnic blood circulation. However, a vascular lymphatic pathology is produced simultaneously with the splanchnic arterio-venous impairments. The pathological increase of the mesenteric venous pressure, by mechanotransduction of the venous endothelium hyperpressure, causes an inflammatory response involving the subendothelial mast cells and the lymphatic endothelium of the intestinal villi lacteal. In portal hypertension, the intestinal lymphatic inflammatory response through the development of mesenteric-systemic lymphatic collateral vessels favors the systemic diffusion of substances with a molecular pattern associated with damage and pathogens of intestinal origin. When the chronic hepatic insufficiency worsens the portal hypertensive inflammatory response, the splanchnic lymphatic system transports the hyperplasied intestinal mast cells to the mesenteric lymphatic complex. Then, an acquired immune response regulating a new hepato-intestinal metabolic scenario is activated. Therefore, reduction of the hepatic metabolism would reduce its key centralized functions, such as the metabolic, detoxifying and antioxidant functions which would try to be substituted by their peroxisome activity, among other functions of the mast cells.

OBJECTIVE: ALK1 (activin-receptor like kinase 1) is an endothelial cell-restricted receptor with high affinity for BMP (bone morphogenetic protein) 9 TGF-beta (transforming growth factor-beta) family member. Loss-of-function mutations in ALK1 cause a subtype of hereditary hemorrhagic telangiectasia-a rare disease characterized by vasculature malformations. Therapeutic strategies are aimed at reducing potential complications because of vascular malformations, but currently, there is no curative treatment for hereditary hemorrhagic telangiectasia. APPROACH AND RESULTS: In this work, we report that a reduction in ALK1 gene dosage (heterozygous ALK1(+/)(-) mice) results in enhanced retinal endothelial cell proliferation and vascular hyperplasia at the sprouting front. We found that BMP9/ALK1 represses VEGF (vascular endothelial growth factor)-mediated PI3K (phosphatidylinositol 3-kinase) by promoting the activity of the PTEN (phosphatase and tensin homolog). Consequently, loss of ALK1 function in endothelial cells results in increased activity of the PI3K pathway. These results were confirmed in cutaneous telangiectasia biopsies of patients with hereditary hemorrhagic telangiectasia 2, in which we also detected an increase in endothelial cell proliferation linked to an increase on the PI3K pathway. In mice, genetic and pharmacological inhibition of PI3K is sufficient to abolish the vascular hyperplasia of ALK1(+/)(-) retinas and in turn normalize the vasculature. CONCLUSIONS: Overall, our results indicate that the BMP9/ALK1 hub critically mediates vascular quiescence by limiting PI3K signaling and suggest that PI3K inhibitors could be used as novel therapeutic agents to treat hereditary hemorrhagic telangiectasia.

BACKGROUND: Renal inflammation and immune cell infiltration are characteristic of several forms of hypertension. Our lab has previously demonstrated that renal inflammation-associated lymphangiogenesis occurs in salt-sensitive and nitric oxide inhibition-induced hypertension. Moreover, enhancing renal lymphatic density prevented the development of these two forms of hypertension. Here, we investigated the effects of angiotensin II-induced hypertension on renal lymphatic vessel density in male and female mice. METHODS AND RESULTS: Male and female mice were infused with angiotensin II for two or three weeks. Compared to vehicle controls, angiotensin II-infused male and female mice had significantly increased renal lymphatic vessel density in association with pro-inflammatory immune cells in the kidneys of these mice. Direct treatment of lymphatic endothelial cells with angiotensin II had no effect since they lack angiotensin II receptors, however angiotensin II treatment of isolated splenocytes and peritoneal CD11b+ cells induced secretion of the lymphangiogenic growth factor VEGF-C in vitro. Utilizing our genetic mouse model of inducible renal lymphangiogenesis, we demonstrated that greatly augmenting renal lymphatic density prior to angiotensin II infusion prevented the development of hypertension in male and female mice and this was associated with a reduction in renal CD11c+F4/80- monocytes. CONCLUSION: Renal lymphatics play a significant role in renal immune cell trafficking and blood pressure regulation, and represent a novel avenue of therapy for hypertension.

Combined germline and somatic second hit inactivating mutations of the RASA1 gene, which encodes a negative regulator of the Ras signaling pathway, cause blood and lymphatic vascular lesions in the human autosomal dominant vascular disorder capillary malformation-arteriovenous malformation (CM-AVM). How RASA1 mutations in endothelial cells (EC) result in vascular lesions in CM-AVM is unknown. Here, using different murine models of RASA1-deficiency, we found that RASA1 was essential for the survival of EC during developmental angiogenesis in which primitive vascular plexuses are remodeled into hierarchical vascular networks. RASA1 was required for EC survival during developmental angiogenesis because it was necessary for export of collagen IV from EC and deposition in vascular basement membranes. In the absence of RASA1, dysregulated Ras mitogen-activated protein kinase (MAPK) signal transduction in EC resulted in impaired folding of collagen IV and its retention in the endoplasmic reticulum (ER) leading to EC death. Remarkably, the chemical chaperone, 4-phenylbutyric acid, and small molecule inhibitors of MAPK and 2-oxoglutarate dependent collagen IV modifying enzymes rescued ER retention of collagen IV and EC apoptosis and resulted in normal developmental angiogenesis. These findings have important implications with regards an understanding of the molecular pathogenesis of CM-AVM and possible means of treatment.

Recent evidence has indicated that the lymphatic vessel endothelial hyaluronan receptor (LYVE-1) is implicated in chronic inflammation and the lymphatic immune response. The soluble form of LYVE-1 (sLYVE-1) is produced by ectodomain shedding of LYVE-1 under pathological conditions including cancer and chronic inflammation. In this study, 1014 consecutive patients who underwent coronary angiography from May 2015 to September 2015 were included to investigate whether serum sLYVE-1 is associated with coronary artery disease (CAD) and its concomitant diseases includes chronic kidney disease (CKD). Results showed that there was no significant difference in sLYVE-1 levels between patients with CAD and without. However, a significantly higher level of sLYVE-1 was seen in patients with renal dysfunction compared to those with a normal eGFR. Results were validated in a separate cohort of 259 patients who were divided into four groups based on their kidney function assessed by estimated glomerular filtration rate (eGFR). Simple bivariate correlation analysis revealed that Lg[sLYVE-1] was negatively correlated with eGFR (r = -0.358, p < 0.001) and cystatin C (r = 0.303, p < 0.001). Multivariable logistic regression analysis revealed that the increase in Lg[sLYVE-1] was an independent determinant of renal dysfunction (odds ratio = 1.633, p = 0.007). Therefore, renal function should be considered when serum sLYVE-1 is used as a biomarker for the detection of pathological conditions such as chronic inflammation and cancer. Further study is required to elucidate the exact role of sLYVE-1 in renal function.

Germinal matrix hemorrhage (GMH) results from the rupture of the immature thin-walled blood vessels and consequent bleeding into the subependymal germinal matrix and possible lateral ventricles. The purpose of this study is to investigate how astrogliosis impacts the glymphatic-meningeal lymphatic system in cerebrospinal fluid (CSF) reabsorption after GMH and how the anti-scarring agent olomoucine attenuates post-hemorrhagic hydrocephalus. GMH was induced by stereotaxic collagenase infusion into P7 Sprague-Dawley rats of both sexes. Western blot and immunofluorescence were used to assess astrogliosis and how astrogliosis affects glymphatic function by measuring Aquaporin-4 expression. Intracisternal injection of fluorescence tracer was used to measure CSF diffusion throughout the brain, its dispersion in the paravascular area and CSF drainage into the deep cervical lymph nodes at 28days after GMH. Both short-term and long-term behavioral tests were used to assess the neurological outcomes. Nissl staining was used to assess the morphological changes at 28days after hemorrhage. GMH elicited astrogliotic scarring and reduced the exchange between CSF and interstitial fluid, as well as CSF reabsorption through the meningeal lymphatic vessels. This might be associated with redistribution of Aquaporin-4. Olomoucine ameliorated scar tissue formation and attenuated post-hemorrhagic hydrocephalus. These findings of this study suggested that the glymphatic system might play a role in CSF reabsorption in neonates following GMH. Scar tissue formation impairs this CSF clearance route, and therefore astrogliosis inhibition might be a potential therapeutic strategy for neonatal post-hemorrhagic hydrocephalus.

Alzheimer's disease (AD) is a complex neurodegenerative disorder causing progressive cognitive decline, memory loss, and death of neural tissue. Current research suggests a connection between bulk flow of interstitial fluid and cerebrospinal fluid across the blood-brain barrier and the recently confirmed meningeal lymphatic channels of the brain. The main symptom of interest in AD is the spontaneous aggregation of amyloid beta (Abeta) proteins resulting from increased production or lack of clearance from brain tissues. These protein aggregates manifest as plaques in the capillary and artery lumina and the neuronal and dural tissues of the brain, and are known to contribute to cerebral amyloid angiopathy and a host of other neuroinflammatory conditions. The meningeal lymphatics contain a substantial population of immune cells and also serve as a drain into the deep cervical lymph nodes. In this study we discuss the molecular mechanisms by which Abeta could gain access to meningeal lymphatic channels through the blood-brain interface, including ways in which it can be cleared to preclude aggregation and plaque deposition.

The systemic circulation depends upon a highly organized, hierarchal blood vascular network that requires the successful specification of arterial and venous endothelial cells during development. This process is driven by a cascade of signaling events (including Hedgehog, vascular endothelial growth factor (VEGF), Notch, connexin (Cx), transforming growth factor-beta (TGF-beta), and COUP transcription factor 2 (COUP-TFII)) to influence endothelial cell cycle status and expression of arterial or venous genes and is further regulated by hemodynamic flow. Failure of endothelial cells to properly undergo arteriovenous specification may contribute to vascular malformation and dysfunction, such as in hereditary hemorrhagic telangiectasia (HHT) and capillary malformation-arteriovenous malformation (CM-AVM) where abnormal vessel structures, such as large shunts lacking clear arteriovenous identity and function, thereby compromising peripheral blood flow. This review provides an overview of recent findings in the field of arteriovenous specification and highlights key regulators of this process.

BACKGROUND: Surgical excision and/or radiation targeting of regional lymph nodes are an essential component in the clinical management of cancer. Importantly, a more accurate understanding of lymphatic anatomy could enable refinement of present treatment strategies. Given the spatial resolution limitations of contemporary imaging methods, our group sought to utilize noncontrast-enhanced microcomputed tomography (muCT) imaging to clarify regional lymphatic anatomy. METHODS AND RESULTS: This study was conducted with embalmed en bloc lymphatic tissue packets from six donors (three females and three males: medianage of death = 78 years). All specimens were investigated with noncontrast-enhanced muCT imaging using a conebeam-CT imaging system. Adipose and lymphatic tissues were segmented by radiodensity based on sampling regions of interest. To confirm the observations from muCT, lymph nodes from each packet were exposed to hematoxylin and eosin staining and anti-D240 immunostaining. Following muCT imaging, mean peak radiodensities of -203.14 ± 19.35 Hounsfield units (HU) and 37.25 ± 31.95 HU were revealed for adipose and lymphatic tissues, respectively (p < 0.01). By analyzing histograms of the radiodensity distributions, we determined a threshold of -82.42 HU to differentiate adipose and lymphatic tissue, to generate three-dimensional renderings, and to calculate quantitative metrics. On average, adipose tissue comprised 9.62 ± 3.60 cm(3) (73.6%) of the total packet volume, whereas lymphatic tissue comprised 3.47 ± 2.71 cm(3) (26.4%). Moreover, each en bloc packet contained four small lymph nodes (1–5 mm) and three to four large lymph nodes (>5 mm). Histology corroborated the observations from muCT. CONCLUSIONS: Altogether, a precise understanding of regional lymphatic anatomy elucidated by the present imaging modality may help refine clinical cancer treatment strategies.

The purpose of this study was to test the hypothesis that infections are linked to chromosomal anomalies that cause neurodevelopmental disorders. In children with disorders in the development of their nervous systems, chromosome anomalies known to cause these disorders were compared with foreign DNAs, including known teratogens. Genes essential for neurons, lymphatic drainage, immunity, circulation, angiogenesis, cell barriers, structure, epigenetic and chromatin modifications were all found close together in polyfunctional clusters that were deleted or rearranged in neurodevelopmental disorders. In some patients, epigenetic driver mutations also changed access to large chromosome segments. These changes account for immune, circulatory, and structural deficits that accompany neurologic deficits. Specific and repetitive human DNA encompassing large deletions matched infections and passed rigorous artifact tests. Deletions of up to millions of bases accompanied infection-matching sequences and caused massive changes in human homologies to foreign DNAs. In data from 3 independent studies of private, familial, and recurrent chromosomal rearrangements, massive changes in homologous microbiomes were found and may drive rearrangements and encourage pathogens. At least 1 chromosomal anomaly was found to consist of human DNA fragments with a gap that corresponded to a piece of integrated foreign DNA. Microbial DNAs that match repetitive or specific human DNA segments are thus proposed to interfere with the epigenome and highly active recombination during meiosis, driven by massive changes in human DNA-foreign DNA homologies. Abnormal recombination in gametes produces zygotes containing rare chromosome anomalies that cause neurologic disorders and nonneurologic signs. Neurodevelopmental disorders may be examples of assault on the human genome by foreign DNAs at a critical stage. Some infections may be more likely tolerated because they resemble human DNA segments. Even rare developmental disorders can be screened for homology to infections within altered epigenomes and chromatin structures. Considering effects of foreign DNAs can assist prenatal and genetic counseling, diagnosis, prevention, and early intervention.

How developing vascular networks acquire the right balance of arteries, veins and lymphatic vessels to efficiently supply and drain tissues is poorly understood. In zebrafish embryos, the robust and regular 50:50 global balance of intersegmental veins and arteries that form along the trunk prompts the intriguing question of how does the organism keep ‘count’? Previous studies have suggested that the ultimate fate of an intersegmental vessel (ISV) is determined by the identity of the approaching secondary sprout emerging from the posterior cardinal vein. Here, we show that the formation of a balanced trunk vasculature involves an early heterogeneity in endothelial cell behaviour and Notch signalling activity in the seemingly identical primary ISVs that is independent of secondary sprouting and flow. We show that Notch signalling mediates the local patterning of ISVs, and an adaptive flow-mediated mechanism subsequently fine-tunes the global balance of arteries and veins along the trunk. We propose that this dual mechanism provides the adaptability required to establish a balanced network of arteries, veins and lymphatic vessels.

PURPOSE OF REVIEW: In this review, we describe novel findings related to intestinal lipid transport in lymphatic vessels. RECENT FINDINGS: Studies have shown that chylomicron entry to lacteals and lymph movement in intestinal lymphatic capillaries is an active process. Regulators of this intestinal chylomicron transport include among others the autonomous nervous system, transcription factors like PLAGL2, and molecular regulators, such as VEGF-A/Nrp1/VEGFR1, VEGF-C/VEGFR3, DLL4, CALCRL and GLP-2. Chylomicron transport in intestinal lymphatics is now emerging not only as an option for drug delivery but also as a new candidate for drug targeting in lipid-related disorders. SUMMARY: Dysfunctions of lymphatic lipid transport can result in conditions such as dyslipidaemia. Intestinal lymphatics also provide several potential therapeutic possibilities: molecular regulation of lacteal cell-to-cell junctioning and lymph flow could provide new ways of treating conditions like hyperlipidaemia and associated diseases, such as atherosclerosis and other cardiovascular diseases, obesity, diabetes and fatty-liver disease. The intestinal lymphatic system can also be employed to deliver lipid nanoparticles as drug carriers to the venous circulation for improved treatment outcome. These findings highlight the importance and need for research on the different players of intestinal lymphatics in dietary lipid handling and therapeutic applications.

The mammalian circulatory system comprises both the cardiovascular system and the lymphatic system. In contrast to the closed, high-pressure and circular blood vascular circulation, the lymphatic system forms an open, low-pressure and unidirectional transit network from the extracellular space to the venous system. It plays a key role in regulating tissue fluid homeostasis, absorption of gastrointestinal lipids, and immune surveillance throughout the body. Despite the critical physiological functions of the lymphatic system, a complete understanding of the lymphatic vessels lags far behind that of the blood vasculatures due to the challenge of their visualization. During the last 20years, discoveries of underlying genes responsible for lymphatic vessel biology, combined with state-of-the-art lymphatic function imaging and quantification techniques, have established the importance of the lymphatic vasculature in the pathogenesis of cardiovascular diseases including lymphedema, obesity and metabolic diseases, dyslipidemia, hypertension, inflammation, atherosclerosis and myocardial infraction. In this review, we highlight the most recent advances in the field of lymphatic vessel biology, with an emphasis on the new identification techniques of lymphatic system, pathophysiological mechanisms of atherosclerosis and myocardial infarction, and new therapeutic perspectives of lymphangiogenesis.

Methotrexate (MTX) is a folic acid antagonist used as an effective drug to treat various kinds of cancers. However, MTX has limited use in cancer chemotherapy due to its adverse effects such as poor bioavailability, low specificity, drug resistance, and dose-dependent side effects. To improve lymphatic delivery and reduce toxicity of MTX, MTX-loaded nanoparticles (NPs) were prepared in the present study. NPs were prepared with double emulsion solvent evaporation method using poly(lactide-co-glycolide) (PLGA). NPs were assessed for size, encapsulation efficiency, morphology, Fourier-transform infrared spectroscopy, X-ray diffraction, and thermal characterization. In vitro release profiles and cytotoxicity of these NPs were also evaluated. Prepared NPs and free MTX were administered orally or intravenously (5 mg/kg as MTX) to rats to evaluate their pharmacokinetic characteristics and lymphatic delivery effects. Mean particle size and encapsulation efficiency of NPs were 163.7 ± 10.25 nm and 93.3 ± 0.5%, respectively. Prepared NPs showed a sustained release profile of MTX in vitro and may be effective to cancer cells. Area under the blood concentration-time curve, total clearance, half-life, and lymphatic targeting efficiency were significantly different (p < 0.05) between prepared NPs and free MTX. These results demonstrate that MTX-loaded PLGA NPs are good candidates for targeted delivery of MTX to the lymphatic system.

Lymphatic microvessel density (LMVD) contributes to fibrosis in patients with myocardial infarction. However, the role of LMVD in the process of myocardial fibrosis in hypertrophic obstructive cardiomyopathy (HOCM) patients is unclear. We studied LMVD in ventricular septal (VS) samples from 52 individuals (42 was HOCM patients who underwent a transaortic extended septal myectomy, and 10 traffic accident victims), and examined the relationships between the LMVD stained immunohistochemically with lymphatic vessel endothelial hyaluronan receptor (LYVE-1) antibodies, collagen volume fraction (CVF), and clinical characteristics. Compared with traffic accident victims, LMVD was significantly increased in VS of HOCM patients (132.0 ± 49.0 VS 57.8 ± 48.8/mm(2), p = 0.000). HOCM patients with syncope had higher level of LMVD than without syncope [166.7 (131.0–201.1) VS 116.4 (80.7–152.1)/mm(2), p = 0.017], and LMVD were positively correlated with Log (CVF) (r = 0.431, p = 0.004). On multiple variables regression analysis, LMVD was independently associated with Log (CVF) (r = 0.379, p = 0.009) and syncope (r = 0.335, p = 0.020). In conclusions, the LYVE-1-positive lymphatics have close associations with VS fibrosis in HOCM patients.

The post-transcriptional mechanisms contributing to molecular regulation of developmental lymphangiogenesis and lymphatic network assembly are not well understood. MicroRNAs are important post-transcriptional regulators during development. Here, we use high throughput small RNA sequencing to identify miR-204, a highly conserved microRNA dramatically enriched in lymphatic vs. blood endothelial cells in human and zebrafish. Suppressing miR-204 leads to loss of lymphatic vessels while endothelial overproduction of miR-204 accelerates lymphatic vessel formation, suggesting a critical positive role for this microRNA during developmental lymphangiogenesis. We also identify the NFATC1 transcription factor as a key miR-204 target in human and zebrafish, and show that NFATC1 suppression leads to lymphatic hyperplasia. The loss of lymphatics caused by miR-204 deficiency can be largely rescued by either endothelial autonomous expression of miR-204 or by suppression of NFATC1. Together, our results highlight a miR-204/NFATC1 molecular regulatory axis required for proper lymphatic development.

Filariases are diseases caused by arthropod-borne filaria nematodes. The related pathologies depend on the location of the infective larvae when their migration, the asymptomatic and least studied phase of the disease, comes to an end. To determine factors assisting in filariae dissemination, we image Litomosoides sigmodontis infective larvae during their escape from the skin. Burrowing through the dermis filariae exclusively enter pre-collecting lymphatics by mechanical disruption of their wall. Once inside collectors, their rapid and unidirectional movement towards the lymph node is supported by the morphology of lymphatic valves. In a microfluidic maze mimicking lymphatic vessels, filariae follow the direction of the flow, the first biomechanical factor capable of helminth guidance within the host. Finally, non-infective nematodes that rely on universal morpho-physiological cues alone also migrate through the dermis, and break in lymphatics, indicating that the ability to spread by the lymphatic route is an ancestral trait rather than acquired parasitic adaptation.

Lymphatic absorption in the peritoneal cavity may contribute to ultrafiltration failure in peritoneal dialysis (PD). Lymphatic vessels develop during PD-related peritoneal fibrosis. Connective tissue growth factor (CTGF, also called CCN2) is an important determinant of fibrotic tissue remodeling, but little is known about its possible involvement in lymphangiogenesis. In this study, we investigated the relationship between CTGF and peritoneal lymphangiogenesis. A positive correlation was observed between vascular endothelial growth factor-C (VEGF-C), a major lymphangiogenic growth factor, and the CTGF concentration in human PD effluents. CTGF expression was positively correlated with expression of lymphatic markers and VEGF-C in human peritoneal biopsies. We found a positive correlation between the increase in CTGF and the increase in VEGF-C in cultured human peritoneal mesothelial cells (HPMCs) treated with transforming growth factor-beta1 (TGF-beta1). The diaphragm is a central player in peritoneal lymphatic absorption. CTGF expression was also correlated with expression of VEGF-C and lymphatics in a rat diaphragmatic fibrosis model induced by chlorhexidine gluconate (CG). Furthermore, CTGF gene deletion reduced VEGF-C expression and peritoneal lymphangiogenesis in the mouse CG model. Inhibition of CTGF also reduced VEGF-C upregulation in HPMCs treated with TGF-beta1. Our results suggest a close relationship between CTGF and PD-associated lymphangiogenesis.

PURPOSE: Post-operative radiation therapy (RT) delivered to lymphatics is associated with an increased risk of developing lymphedema. Reported effects of RT on lymphatic vessels have varied, however, possibly due to the use of different animal models with varying surgery and radiation schedules, as well as the inability to directly and longitudinally image lymphatics in vivo. Here we report, using non-invasive imaging, changes in lymphatic remodeling and function in response to surgery and RT in a mouse model. METHODS AND MATERIALS: Popliteal lymphadenectomy in mice preceded single-dose gamma irradiation of the lower extremity at a single dose of 0, 20, or 40Gy. The right hind limb of intact mice was also radiated with 4 fractions (4 × 5Gy). Near-infrared fluorescence lymphatic imaging (NIRFLI) with indocyanine green (ICG) was performed over 6 months to monitor lymphatic vessel remodeling. RESULTS: Post-operative mice treated with 20Gy showed transient changes in lymphatic drainage; exacerbated vessel remodeling including qualitative vessel dilation and abnormal ICG pooling from week 1 to 2, and initiation of restoration of lymphatic vessels, although dermal back flow was occasionally observed. Mice treated with 40Gy showed steadily increasing lymphatic impairment until week 3 and extravasation of dye and dermal backflow in weeks 4 to 25. The ankles of mice treated with 40Gy were significantly swollen from weeks 2 to 4, as compared to mice treated with 0Gy or 20Gy. Mice with fractionated RT showed that similar lymphatic vessel remodeling occurred as when a single 20Gy dose was given; however, dermal backflow did not resolve as it did in the case of a single 20Gy dose. CONCLUSION: The degree of non-reversing lymphatic damage seen in our mouse model was dependent upon RT dose. Our results suggest that NIRFLI detection of early lymphatic changes can be used to predict development of lymphedema in cancer patients.

Forkhead transcription factor C2 (FOXC2) is a transcription factor regulating vascular and lymphatic development, and its mutations are linked to lymphedema-distichiasis syndrome. FOXC2 is also a crucial regulator of the epithelial-mesenchymal transition processes essential for tumor metastasis. Here, we report the crystal structure of the FOXC2-DNA-binding domain in complex with its cognate DNA. The crystal structure provides the basis of DNA sequence recognition by FOXC2 for the T/CAAAC motif. Helix 3 makes the majority of the DNA-protein interactions and confers the DNA sequence specificity. The computational energy calculation results also validate the structural observations. The FOXC2 and DNA complex structure provides a detailed picture of protein and DNA interactions, which allows us to predict its DNA recognition specificity and impaired functions in mutants identified in human patients.

Mitoxantrone (MTO) is used to treat certain types of cancer, mostly metastatic cancer. While the drug has poor aqueous solubility and high side effects. Self-assembly nanocrystal is a novel lymphatic targeting delivery system. In our study, MTO self-assembly nanocrystal (MTO NC) was successfully prepared to improve lymphatic targeting ability and reduce its toxicity. MTO NCs had small size, stable potential, and uniform distribution. The average particle size of MTO NCs was less than 100 nm with the 0.218 PDI and - 6.6 mV the Zeta potential value. TEM images showed that MTO NCs had a sphere-like morphology with smooth surface and uniform distribution; Atomic force microscopy (AFM) images gave a 3D surface of MTO NCs. Polarizing microscope micrograph (PLM) of MTO NCs in lymph nodes demonstrated the crystal structure of MTO NCs when it was exposed to physiological condition. Transmission electron microscopy showed the presence of MTO NCs in mice lymph nodes. Pharmacokinetic parameters of MTO strongly demonstrated that MTO NCs could target the lymph nodes after subcutaneous injection. Moreover, tissue distribution results indicated that MTO NCs were mainly absorbed by the lymphatics and reduced system toxicity. Finally, a lymphatic metastasis mice model was established to precede the pharmacodynamics of MTO NCs, and using MTO liposomes as a reference preparation, the inhibitory effect of MTO NCs on lymphatic metastasis was markedly higher. Briefly, MTO NCs, as a novel self-assembled lymphatic targeting system, can accumulate in the metastatic lymph nodes and lead anticancer drug to kill cancer cells and control lymphatic metastasis with extremely low systemic toxicity.

Blood and lymphatic systems work in close collaboration to ensure their respective physiological functions. The lymphatic vessel network is being extensively studied, but has been overlooked as compared to the blood vasculature mainly due to the problematic discrimination of lymphatic vessels from the blood ones. This issue has been fortunately resolved in the past decade leading to the emergence of a huge amount of data in lymphatic biology revealing many shared features with the blood vasculature. However, this likeliness between the two vascular systems may lead to a simplistic view of lymphatics and a direct transcription of what is known for the blood system to the lymphatic one, thereby neglecting the lymphatic specificities. In this context, this review aims to clarify the main differences between the two vascular systems focusing on recently discovered lymphatic features.

The classification of vascular neoplasms continues to evolve as we accumulate more genetic and clinical data, particularly for rare tumor types. Because of tumor rarity, changes to classification schema, overlapping histologic features, and in some cases, lack of morphologic evidence of vasoformation, vascular neoplasms present a diagnostic challenge. Here, we discuss recent developments in our understanding of vascular tumors, with a detailed discussion of epithelioid hemangioma, tufted angioma, kaposiform hemangioendothelioma, composite hemangioendothelioma, pseudomyogenic hemangioendothelioma, epithelioid hemangioendothelioma, and angiosarcoma.

Molecular heterogeneity of endothelial cells underlies their highly-specialized functions during changing physiological conditions within diverse vascular beds. For example, placental spiral arteries (SAs) undergo remarkable remodeling to meet the ever-growing demands of the fetus-a process which is deficient in preeclampsia. The extent to which maternal endothelial cells coordinate with immune cells and pregnancy hormones to promote SA remodeling remains largely unknown. Here we found that remodeled SAs expressed the lymphatic markers PROX1, LYVE1, and VEGFR3, mimicking lymphatic identity. Uterine natural killer (uNK) cells, which are required for SA remodeling and secrete VEGFC, were both sufficient and necessary for VEGFR3 activation in vitro and in mice lacking uNK cells, respectively. Using Flt4Chy/+ mice with kinase inactive VEGFR3 and Vegfcfl/fl;Vav1-Cre mice, we demonstrated that SA remodeling required VEGFR3 signaling, and that disrupted maternal VEGFR3 signaling contributed to late-gestation fetal growth restriction. Collectively, we identified a novel instance of lymphatic mimicry by which maternal endothelial cells promote SA remodeling, furthering our understanding of the vascular heterogeneity employed for the mitigation of pregnancy complications such as fetal growth restriction and preeclampsia.

The mechanisms regulating endothelial cell response to hemodynamic forces required for heart valve development, especially valve remodeling, remain elusive. Tie1, an endothelial specific receptor tyrosine kinase, is up-regulated by oscillating shear stress and is required for lymphatic valve development. In this study, we demonstrate that valvular endothelial Tie1 is differentially expressed in a dynamic pattern predicted by disturbed flow during valve remodeling. Following valvular endocardial specific deletion of Tie1 in mice, we observed enlarged aortic valve leaflets, decreased valve stiffness and valvular insufficiency. Valve abnormalities were only detected in late gestation and early postnatal mutant animals and worsened with age. The mutant mice developed perturbed extracellular matrix (ECM) deposition and remodeling characterized by increased glycosaminoglycan and decreased collagen content, as well as increased valve interstitial cell expression of Sox9, a transcription factor essential for normal ECM maturation during heart valve development. This study provides the first evidence that Tie1 is involved in modulation of late valve remodeling and suggests that an important Tie1-Sox9 signaling axis exists through which disturbed flows are converted by endocardial cells to paracrine Sox9 signals to modulate normal matrix remodeling of the aortic valve.

Pancreatic cancer is associated with a poor prognosis, mainly due to lymph node invasion and lymph node recurrence after surgical resection, even after extended lymphadenectomy. The peripancreatic lymphatic system is highly complex and the specific lymphatic drainage of each part of the pancreas has not been established. The aim of this study was to determine the lymphatic drainage pathways specific to each part of the pancreas on live pigs using Patent Blue. The pancreases of 14 live pigs were injected in different parts of the gland. The technique was efficient and reproducible. The diffusion patterns were similar for each location and were reported. Our results in pigs allowed us to define specific nodal relay stations and lymphatic drainage for each part of the pancreas and confirm that independent anatomical-surgical pancreatic segments can be described. It is interesting to note that lymphatic drainage for the upper part of the proximal part of pancreas (duodenal lobe) occurred on the left side of the portal vein. This suggests that lymph node resection during cephalic duodenopancreatectomy in humans should be extended to the left side of the mesenteric vein, and probably to the right side of the superior mesenteric artery, as recently suggested. These results could help surgeons perform safe anatomical-segmental pancreatic resections with accurate lymphadenectomies and improve survival in patients with pancreatic cancer. Based on these results we will perform an innovative prospective study. Patent Blue will be injected into different parts of the gland in patients operated for pancreatic resection, and lymphatic diffusion of the dye will be recorded in relation to their origin from the theoretical pancreatic segments (ClinicalTrials.gov Identifier: NCT03597230).

All endothelial cells have the common characteristic that they line the vessels of the blood circulatory system. However, endothelial cells display a large degree of heterogeneity in the function of their location in the vascular tree. In this article, we have summarized the expression patterns of a number of well-accepted endothelial surface markers present in normal microvascular endothelial cells, arterial and venous endothelial cells, lymphatic endothelial cells, tumor endothelial cells, and endothelial precursor cells.

Cationic liposomes prepared from dimethyldioctadecylammonium bromide (DDAB) and trehalose 6,6'-dibehenate (TDB) are strong liposomal adjuvants. As with many liposome formulations, within the laboratory DDAB:TDB is commonly prepared by the thin film method which is difficult to scale and gives high batch-to-batch variability. In contrast, controllable technologies such as microfluidics offer robust, continuous and scale-independent production. Therefore, within this study, we have developed a microfluidic production method for cationic liposomal adjuvants that is scale-independent and produces liposomal adjuvants with analogous biodistribution and immunogenicity compared to those produced by the small-scale lipid hydration method. Subsequently, we further developed the DDAB:TDB adjuvant system to include a lymphatic targeting strategy using microfluidics. By exploiting a biotin-avidin complexation strategy, we were able to manipulate the pharmacokinetic profile and enhance targeting and retention of DDAB:TDB and antigen within the lymph nodes. Interestingly, re-directing these cationic liposomal adjuvants did not translate into notably improved vaccine efficacy.

Secondary lymphedema is a worldwide affliction that exacts a significant public health burden. This review examines the etiology, presentation, and management of secondary lymphedema. In addition, emerging adjunctive strategies are explored, specifically evidence from animal and pilot human studies regarding implantation of a collagen nanofibrillar scaffold (BioBridge; Fibralign Corporation, Union City, CA) in promoting lymphangiogenesis, preventing and treating lymphedema, and enhancing outcomes with lymphaticovenous anastomosis and vascularized lymph node transfer.

Lymphatic vessels maintain body homeostasis by recirculation of fluid and cells. Cell senescence induces lymphatic dysfunction. Impaired contractile function is caused by low muscle cell investiture and decrease of nitric oxide in aged lymphatic collectors, leading to poor drainage of lymph. Aging-induced loss of endothelial glycocalyx and production of inflammatory cytokines increases permeability of lymphatic vessels. In addition, aging-associated basal activation of mast cells delays immune response. In this review, we summarize the structural and pathological changes of aged lymphatic vessels, and discuss the underlying molecular mechanisms.

Podoplanin (PDPN) is utilized as a specific marker of type I alveolar cells of lung and lymphatic endothelial cells of every tissue. Therefore, sensitive and specific monoclonal antibodies detecting PDPN are necessary for immunohistochemical analyses, especially using formalin-fixed paraffin-embedded tissues. Recently, we developed an anti-bear PDPN (bPDPN) mAb, PMab-247, which is useful for Western blot, flow cytometry, and immunohistochemical analyses. In this study, immunohistochemical analyses showed that PMab-247 strongly detected bPDPN, which is expressed in type I alveolar cells and lymphatic endothelial cells of bear lung and podocytes of bear kidney. These findings suggest that PMab-247 could be useful for pathophysiological analyses using immunohistochemistry.

Recently, endothelial cell metabolism has emerged as an essential driver and regulator of both blood and lymph vessel development. Evidence rapidly builds that metabolism is not only necessary for endothelial cell function, but moreover controls several aspects of the (lymph)-angiogenic process. So far, the best-characterized metabolic pathways to have an impact on angiogenesis are glycolysis, fatty acid oxidation and glutamine metabolism. Glycolysis regulates tip cell behavior by providing ATP, fatty acid oxidation controls stalk cell proliferation by producing nucleotide biomass, and glutamine metabolism is critical for tip and stalk cell dynamics by supporting Krebs cycle anaplerosis, protein production and redox homeostasis, and links to asparagine metabolism. During lymphangiogenesis, glycolysis and fatty acid oxidation are key metabolic pathways. Glycolysis provides energy for growing lymph vessels, while fatty acid oxidation is a critical metabolic regulator of lymphangiogenesis, in part by promoting nucleotide synthesis as well as by mediating epigenetic changes of histone acetylation, which promotes transcription of key lymphatic genes, and hence venous-to-lymphatic endothelial cell differentiation. On the whole, increasing knowledge on the metabolic landscape of endothelial cells offers a fresh impetus to future treatment possibilities of vascular related diseases.

Lymphatic vascular development establishes embryonic and adult tissue fluid balance and is integral in disease. In diverse vertebrate organs, lymphatic vessels display organotypic function and develop in an organ-specific manner. In all settings, developmental lymphangiogenesis is considered driven by vascular endothelial growth factor (VEGF) receptor-3 (VEGFR3), whereas a role for VEGFR2 remains to be fully explored. Here, we define the zebrafish Vegf/Vegfr code in receptor binding studies. We find that while Vegfd directs craniofacial lymphangiogenesis, it binds Kdr (a VEGFR2 homolog) but surprisingly, unlike in mammals, does not bind Flt4 (VEGFR3). Epistatic analyses and characterization of a kdr mutant confirm receptor-binding analyses, demonstrating that Kdr is indispensible for rostral craniofacial lymphangiogenesis, but not caudal trunk lymphangiogenesis, in which Flt4 is central. We further demonstrate an unexpected yet essential role for Kdr in inducing lymphatic endothelial cell fate. This work reveals evolutionary divergence in the Vegf/Vegfr code that uncovers spatially restricted mechanisms of developmental lymphangiogenesis.

It has been suggested that many forms of secondary lymphedema in humans are driven by a progressive loss of lymphatic pump function after an initial risk-inducing event. However, the link between pump failure and disease progression has remained elusive due to experimental challenges in the clinical setting and a lack of adequate animal models. Using a novel surgical model of lymphatic injury, we track the adaptation and functional decline of the lymphatic network in response to surgery. This model mimics the histological hallmarks of the typical mouse tail lymphedema model while leaving an intact collecting vessel for analysis of functional changes during disease progression. Lymphatic function in the intact collecting vessel negatively correlated with swelling, while a loss of pumping pressure generation remained even after resolution of swelling. By using this model to study the role of obesity in lymphedema development, we show that obesity exacerbates acquired lymphatic pump failure following lymphatic injury, suggesting one mechanism through which obesity may worsen lymphedema. This lymphatic injury model will allow for future studies investigating the molecular mechanisms leading to lymphedema development.

BACKGROUND: Lymphatic formations that effectively eradicate the virus in the lymphatic system will be therapeutically advantagous in hepatitis B virus (HBV) infection. Lipid-based formulation is often used to deliver drug via the lymphatic system. Baicalin nanoemulsion may be a promising drug delivery system for improved treatment of HBV infection. OBJECTIVE: This study aimed to prepare, characterize, and evaluate a lipid-based nanoemulsion containing baicalin for lymphatic system absorption. METHOD: The presence of a nanoemulsion region was studied by pseudoternary phase diagrams. The physicochemical properties of a baicalin-loaded nanoemulsion were investigated. The oral bioavailability of the baicalin-loaded nanoemulsion was compared to that of a baicalin suspension. A chylomicron flow blocking model was used to examine the extent of lymphatic uptake. The lymph node distribution of baicalin was measured to investigate the lymphatic transport ability of the nanoemulsion compared to the suspension. RESULTS: Compared to the baicalin suspension, the AUC0-t and Cmax values of the baicalin nanoemulsion were increased by 10.5-fold and 3.12-fold, respectively. Compared with the saline-treated rats that were orally administered the baicalin nanoemulsion, the AUC0-t and Cmax values of the nanoemulsion for the rats pretreated with cycloheximide were reduced from 23.076 ± 1.244 mg/L h to 9.236 ± 0.940 mg/L h and from 3.010 ± 0.119 mg/L to 1.567 ± 0.220 mg/L, respectively. In comparing baicalin in W/O nanoemulsion with suspension, the Cmax value was found to be 11.5-fold higher in the lymph nodes of the rats treated with the nanoemulsion. CONCLUSION: The results indicated that a baicalin-loaded W/O nanoemulsion may be a promising drug delivery system for the treatment of chronic hepatitis B.

Podoplanin (PDPN), a type I transmembrane sialoglycoprotein, is expressed on lymphatic endothelial cells, podocytes of the kidneys, and type I alveolar cells of the lungs. PDPN, a platelet aggregation-inducing factor, comprises three platelet aggregation-stimulating (PLAG) domains (PLAG1, PLAG2, and PLAG3) in the N-terminus and PLAG-like domains in the middle of the PDPN protein. We have previously reported a mouse antipig PDPN (pPDPN) monoclonal antibody (mAb) clone, PMab-213, which was developed using the Cell-Based Immunization and Screening (CBIS) method. PMab-213 is very useful in flow cytometry, Western blotting, and immunohistochemical analyses; however, the binding epitope of PMab-213, which was developed by CBIS method, remains to be elucidated. Therefore, this study aimed to investigate the epitope of PMab-213 using enzyme-linked immunosorbent assay, flow cytometry, and immunohistochemical analyses. The results revealed that the critical epitopes of PMab-213 are Pro53, Arg54, Arg56, and Tyr60 of pPDPN.

OBJECTIVES: This study aimed to investigate the clearance pathways of lamotrigine (LTG)-loaded micelles by intranasal administration and intracerebral injection in the brain and whether nanoparticles can induce the inflammation promoted by interleukin-6 (IL-6), accelerating the phagocytosis of drug particles in the brain and drainage through lymphatics. METHODS: The drug concentrations in the deep cervical lymph node, superficial cervical lymph node, brain tissues and jugular vein, the pharmacokinetic parameters, and the concentrations of IL-6 in deep cervical lymph node and brain tissues were investigated following UPLC/MS, DAS3.0, ELISA statistically analysed. KEY FINDINGS: The AUC0- t of deep cervical lymph node after intranasal and intracerebral injection was 1.93, 2.77, 1.34 times and 3.06, 16.4, 3.34 times higher compared with the superficial cervical lymph node, jugular vein and brain tissue, respectively. After intranasal administration of lamotrigine-loaded micelles for 30 min, the IL-6 concentrations in deep cervical lymph node and brain tissue were significantly increased (P < 0.05). CONCLUSIONS: These results suggested that lamotrigine micelles were primarily cleared from the brain by lymphatics rather than blood clearance. Also, the nanoparticle induced the increase in IL-6 level after entering the brain suggested that nanoparticles might induce the inflammation promoted by IL-6 in the brain, accelerating the clearance of drug particles in the brain and drainage through lymphatics.

The lymphatic vasculature requires intraluminal valves to maintain forward lymph flow. Lymphatic valves form and are constantly maintained by oscillatory fluid flow throughout life, yet the earliest steps of how lymphatic endothelial cells are able to respond to fluid shear stress remain unknown. Here, we show that the adherens junction protein VE-cadherin is required for the upregulation of valve-specific transcription factors. Conditional deletion of VE-cadherin in vivo prevented valve formation in the embryo and caused postnatal regression of nearly all lymphatic valves in multiple tissues. Since VE-cadherin is known to signal through beta-catenin and the VEGFR/AKT pathway, each pathway was probed. Expression of a constitutively active beta-catenin mutant or direct pharmacologic activation of AKT in vivo significantly rescued valve regression in the VE-cadherin-deficient lymphatic vessels. In conclusion, VE-cadherin-dependent signaling is required for lymphatic valve formation and maintenance and therapies to augment downstream pathways hold potential to treat lymphedema in patients.

The lymphatic vascular system plays important roles in the control of tissue fluid homeostasis and immune responses. While VEGF-A-induced angiogenesis promotes hair follicle (HF) growth, the potential role of lymphatic vessels (LVs) in HF cycling has remained unknown. In this study, we found that LVs are localized in close proximity to the HF bulge area throughout the postnatal and depilation-induced hair cycle in mice and that a network of LVs directly connects the individual HFs. Increased LV density in the skin of K14-VEGF-C transgenic mice was associated with prolongation of anagen HF growth. Conversely, HF entry into the catagen phase was accelerated in K14-sVEGFR3 transgenic mice that lack cutaneous LVs. Importantly, repeated intradermal injections of VEGF-C promoted hair growth in mice. Conditioned media from lymphatic endothelial cells promoted human dermal papilla cell (DPC) growth and expression of IGF-1 and alkaline phosphatase, both activators of DPCs. Our results reveal an unexpected role of LVs in coordinating and promoting HF growth and identify potential new therapeutic strategies for hair loss-associated conditions.

Kidney fibrosis is associated with an increased lymphangiogenesis, characterized by the formation and expansion of new lymphatic vessels. However, the trigger and underlying mechanism responsible for the growth of lymphatic vessels in diseased kidney remain poorly defined. Here we report that tubule-derived sonic hedgehog (Shh) ligand is a novel lymphangiogenic factor that plays a crucial role in mediating lymphatic endothelial cell proliferation and expansion. Shh was induced in renal tubular epithelium in various models of fibrotic CKD, and this was accompanied by an expansion of lymphatic vessels in adjacent areas. In vitro, Shh selectively promoted the proliferation of human dermal lymphatic endothelial cells (HDLEC), but not human umbilical vein endothelial cells (HUVEC), as assessed by cell counting, MTT assay and BrdU incorporation. Shh also induced the expression of vascular endothelial growth factor receptor-3, cyclin D1 and proliferating cell nuclear antigen in HDLEC. Shh did not affect the expression of Gli1, the downstream target and readout of canonical hedgehog signaling, but activated extracellular signal-regulated kinase-1 and -2 (ERK-1/2) in HDLEC. Inhibition of Smoothened with small molecule inhibitor or blockade of ERK-1/2 activation abolished the lymphatic endothelial cell proliferation induced by Shh. In vivo, inhibition of Smoothened also repressed lymphangiogenesis and attenuated renal fibrosis. These studies identify Shh as a novel mitogen that selectively promotes lymphatic, but not vascular, endothelial cell proliferation, and suggest that tubule-derived Shh plays an essential role in mediating lymphangiogenesis after kidney injury.

We report a preterm neonate who had a large cervical cystic hygroma and right chylothorax. She was operated on day-21 and a near-complete resection of cystic hygroma was done. She developed refractory hypoxemia and shock post surgery and died after 24 hours. During autopsy, the chest cavity was found to be filled with chyle. Histopathological examination showed dilated lymphatics in the pleura, hepatic capsule, serosa of stomach and intestines, peri-pancreatic regions, peri-renal capsule and peri-adrenal tissues suggestive of generalised lymphatic dysplasia. Clinical exome sequencing did not reveal any pathogenic mutation in the genes involved in primary lymphatic dysplasia, noonan syndrome or rasopathies.

BACKGROUND: Retroperitoneal lymphangiomatosis (RL) is a rare form of primary lymphedema featuring aberrant retroperitoneal lymphatic proliferation. It causes recurrent cellulitis, repeated interventions, and poor life quality. This study aimed to investigate proper diagnositc criteria and surgical outcomes for RL with extremity lymphedema. METHODS: Between 2012 and 2018, 44 primary lower-extremity lymphedema cases received lymphoscintigraphy, magnetic resonance imaging, and single-photon electron computed tomography to detect RL. RL patients underwent vascularized lymph node transfers (VLNT) for extremity lymphedema and intra-abdominal side-to-end chylovenous bypasses (CVB) for chylous ascites. Complications, CVB patency, and quality of life were evaluated postoperatively. RESULTS: Six RL patients (mean age of 30.3 years) had chylous ascites with five had lower-extremity lymphedema. All CVBs remained patent, though one required re-anastomosis, giving a 100% patency rate. Four unilateral and one bilateral extremity lymphedema underwent six VLNTs with 100% flap survival. Patients reported improved quality of life (P = 0.023), decreased cellulitis incidence (P = 0.041), and improved mean lymphedema circumference (P = 0.043). All patients resumed a normal diet and activity. CONCLUSIONS: Evaluating primary lower-extremity lymphedema patients with MRI and SPECT could reveal a 13.6% prevalence of RL and guide treatment of refractory extremity lymphedema. Intra-abdominal CVB with VLNT effectively treated RL with chylous ascites and extremity lymphedema.

BACKGROUND: Patients with GATA2 deficiency present with nontuberculous mycobacterial infections, severe viral infections (particularly refractory human papillomavirus disease), lymphedema, myelodysplastic syndrome (MDS), and acute myeloid leukemia. Patients with GATA2 deficiency who undergo allogeneic hematopoietic stem cell transplantation prior to the development of life-threatening infections or cytogenetic abnormalities may have optimal clinical outcomes. OBJECTIVES: The aim of this article is to determine ways in which oncology nurses can identify GATA2 deficiency in patients early and optimize treatment decisions. METHODS: A case study is presented of a 33-year-old man with recurrent infections and MDS and his two sons, all of whom were found to have the same GATA2 mutation. FINDINGS: Oncology nurses play an important role in early detection and identification by interviewing patients and obtaining a complete and thorough family history.

A meshwork of intraluminal processes in lymph node (LN) sinuses originates during LN development. Lymph flows through the meshwork, which has an important role in immunology and pathology. However, the formation mechanism of intraluminal processes has not been sufficiently studied. Our objective is to assess whether this mechanism is by intussusception, as occurs in transcapillary pillar formation in blood vessel intussusceptive angiogenesis. For this purpose, LNs with developing intrasinusal processes were used (human foetuses, 13–18GW) for serial histologic sections and immunohistochemical procedures. The studies showed (a) sinuses originating from lymphatic sacs around expanded LN anlagen, (b) intra-sinus structures (lined by anti-podoplanin(+), VEGFR3(+), Prox-1(+), CD31(+) lymphatic endothelial cells) with characteristics (in serial sections and 3D images) similar to those considered the hallmarks of intussusceptive angiogenesis, including pillars (≤2.5mum, with a collagen core), interstitial tissue structures (ITSs) or larger pillars (>2.5mum, with a more cellular core) and folds (that form pillars when spanning), and (c) remodelled and fused pillars, ITSs and folds, which formed meshworks, compartmentalizing the sinuses into small intercommunicating spaces (segmentation). In conclusion, intussusception participates in the formation of the meshwork of processes in LN sinuses during LN development. This mechanism is also of interest because it contributes to the general knowledge of intussusceptive lymphangiogenesis (which has received less attention than intussusception in blood vessels), provides a basis for further studies and supports a new role for vessel intussusception (formation of an intraluminal meshwork with known action in fluid filtering, cell interactions and immunology).

A second multigeneration family with hereditary lymphedema (LE) secondary to a variant in the planar polarity gene, CELSR1, is described. Dominant inheritance of the variant was discovered using whole-exome sequencing and confirmed by Sanger sequencing. In contrast to heterozygous males, all heterozygous females showed LE during physical examination albeit variable in severity and age of onset. Lymphscintigraphy in affected females showed previously undescribed lymphatic abnormalities consistent with lymphangiectasia, valve dysfunction, and thoracic duct reflux.

We present our “String of Pearls” technique for upper and lower extremity lymphedema based off the right gastroepiploic artery. The entire laprascopically harvested omentum is placed through a longitudinal incision at the lymphedematous area, and anastomosed proximally, with additional distal venous outflow. This approach preserves the native lymphaticovenous architecture, distributes free lymphatic tissue along the axis of the extremity, and allows for scar release. The additional vein serves to restore bidirectional physiologic drainage inherent in the omentum and providing further lymphaticovenous drainage. We present two cases of upper and lower extremity lymphedema as a result of malignancy treated using this method. The first case was a result of breast cancer in a 55-year-old female with orthotopically placed omentum, and the second case a result of malignant nodular fasciitis in the distal lower extremity in a 56-year-old female with distally placed omentum. No complications occurred. At 3 months follow up, there is a 25% and 28% reduction in lower and upper extremity volume, respectively, with no recurrent cellulitis episodes. The safety and feasibility of placement of the entire omentum longitudinally with additional venous anastomosis are apparent. However, long-term studies are required.

OBJECTIVES: The goal of this study was to define the parameters of movement of indocyanine green in the upper extremity of normal control and hand transplant recipients. The purpose was to establish a non-invasive method of determining the level of lymphatic function in hand transplant recipients. In hand transplantation (and replantation), the deep lymphatic vessels are rarely repaired, resulting in altered lymphatic connections. In most cases, the relatively rapid inosculation of superficial lymphatic networks and drainage via the venous systems results in sufficient interstitial fluid and lymph drainage of the graft to prevent edema. However, our group and others have determined that some transplant recipients demonstrate chronic edema which is associated with lymphatic stasis. In one case, a patient with chronic edema has developed chronic rejection characterized by thinning of the skin, loss of adnexal structures, and fibrosis and contracture of the hand. METHODS: Lymphatic function was evaluated by intradermal administration of near-infrared fluorescent dye, indocyanine green, and dynamic imaging with an infrared camera system (LUNA). To date, the assessment of lymphatic drainage in the upper extremity by clearance of indocyanine green dye has been studied primarily in oncology patients with abnormal lymphatic function, making assessment of normal drainage problematic. To establish normal parameters, indocyanine green lymphatic clearance functional tests were performed in a series of normal controls, and subsequently compared with indocyanine green clearance in hand transplant recipients. RESULTS: The results demonstrate varied patterns of lymphatic drainage in the hand transplant patients that partially mimic normal hand lymphatic drainage, but also share characteristics of lymphedema patients defined in other studies. The study revealed significant deceleration of the dye drainage in the allograft of a patient with suspected chronic rejection and edema of the graft. Analysis of other hand transplant recipients revealed differing levels of dye deceleration, often localized at the level of surgical anastomosis. CONCLUSION: These studies suggest intradermal injection of indocyanine green and near-infrared imaging may be a useful clinical tool to assess adequacy of lymphatic function in hand transplant recipients.

Sotos syndrome is an overgrowth-intellectual disability (OGID) syndrome caused by NSD1 pathogenic variants and characterized by a distinctive facial appearance, an intellectual disability, tall stature and/or macrocephaly. Other associated clinical features include scoliosis, seizures, renal anomalies, and cardiac anomalies. However, many of the published Sotos syndrome clinical descriptions are based on studies of children; the phenotype in adults with Sotos syndrome is not yet well described. Given that it is now 17 years since disruption of NSD1 was shown to cause Sotos syndrome, many of the children first reported are now adults. It is therefore timely to investigate the phenotype of 44 adults with Sotos syndrome and NSD1 pathogenic variants. We have shown that adults with Sotos syndrome display a wide spectrum of intellectual ability with functioning ranging from fully independent to fully dependent. Reproductive rates are low. In our cohort, median height in adult women is +1.9 SD and men +0.5 SD. There is a distinctive facial appearance in adults with a tall, square, prominent chin. Reassuringly, adults with Sotos syndrome are generally healthy with few new medical issues; however, lymphedema, poor dentition, hearing loss, contractures and tremor have developed in a small number of individuals.

INTRODUCTION: Pelvic lymphadenectomy (PL) causes changes to the inguinal lymph nodes with progressive loss of immune and lymphatic pump function. Efferent lymphatic vessel-to-venous anastomosis (ELVA) has been reported to address this problem. The aim of this report was to describe the feasibility of the SPECT/CT combined with ultrasound fusion imaging (UFI) to target the groin efferent lymph node (GELN) for ELVA. PATIENTS AND METHODS: Twelve patients with lower limb lymphedema after PL were scheduled for peripheric lymphaticovenular anastomosis (LVA) combined with ELVA. All-patients were clinically ISL-stage1, with good visualization of the inguinal lymph nodes at preoperative lymphoscintigraphy. The mean patient age was 55.4 years and the mean BMI was 25.5. The mean limb circumference (MLC) was calculated before surgery and 1 year after surgery. The LymQoL-Leg questionnaire was administered before surgery and 6 months after surgery. Before surgery, the GELN was identified by SPECT/CT and its location was marked on the skin by UFI virtual navigation. Peripheric LVA sites were planned by ultrasound and indocyanine green (ICG) lymphography. Pre and postoperative MLC and LymQoL-Leg scores were compared. RESULTS: In all-patients, the SPECT/CT succeeded at detecting and targeting the GELN. In all-patients, real-time anatomical coregistration with US was feasible, and it was possible to mark on the groin skin the depth and position of the GELN on the skin at the groin. During surgery, in every patient, we found the GELN marked before surgery and performed ELVA. We also performed two or three peripheric LVAs in every patient. The mean value of MLC decreased (38.2 ± 2.13 cm vs. 36.33 ± 2.14 cm; p = .04) and the mean score of the LymQoL-Leg questionnaire improved (9.3 ± 1.7 vs. 7.7 ± 1.1; p = .02). CONCLUSION: SPECT/CT combined with UFI is feasible for the preoperative identification of GELN for ELVA.

BACKGROUND: The World Health Organization (WHO) currently recommends height or age-based dosing as alternatives to weight-based dosing for mass drug administration lymphatic filariasis (LF) elimination programs. The goals of our study were to compare these alternative dosing strategies to weight-based dosing and to develop and evaluate new height-based dosing pole scenarios. METHODOLOGY/PRINCIPAL FINDINGS: Age, height and weight data were collected from >26,000 individuals in five countries during a cluster randomized LF clinical trial. Weight-based dosing for diethylcarbamazine (DEC; 6 mg/kg) and ivermectin (IVM; 200 ug/kg) with tablet numbers derived from a table of weight intervals was treated as the “gold standard” for this study. Following WHO recommended age-based dosing of DEC and height-based dosing of IVM would have resulted in 32% and 27% of individuals receiving treatment doses below those recommended by weight-based dosing for DEC and IVM, respectively. Underdosing would have been especially common in adult males, who tend to have the highest LF prevalence in many endemic areas. We used a 3-step modeling approach to develop and evaluate new dosing pole cutoffs. First, we analyzed the clinical trial data using quantile regression to predict weight from height. We then used weight predictions to develop new dosing pole cutoff values. Finally, we compared different dosing pole cutoffs and age and height-based WHO dosing recommendations to weight-based dosing. We considered hundreds of scenarios including country- and sex-specific dosing poles. A simple dosing pole with a 6-tablet maximum for both DEC and IVM reduced the underdosing rate by 30% and 21%, respectively, and was nearly as effective as more complex pole combinations for reducing underdosing. CONCLUSIONS/SIGNIFICANCE: Using a novel modeling approach, we developed a simple dosing pole that would markedly reduce underdosing for DEC and IVM in MDA programs compared to current WHO recommended height or age-based dosing.

Lymphatic thrombosis is rarer than venous thrombosis. This case report describes a patient with secondary lymphedema, who was found to have lymphatic thrombosis during lymphaticovenous anastomosis (LVA). A 51-year-old woman underwent hysterectomy and pelvic lymph node dissection for uterine cancer when she was 48 years old, and lymphedema developed in the left leg soon after the operation. She was diagnosed with lymphedema based on lymphoscintigraphic finding. Preoperative echography showed 2 hypoechoic circles measuring about 0.5 mm in diameter that did not collapse with pressure from the probe, although the veins collapsed with pressure. We diagnosed the 2 hypoechoic circles as lymphatic vessels based on the location and longitudinal continuity. During LVA, we identified 2 parallel white vessels beneath the superficial fascia. After they were cut, white material was extruded. A diagnosis of lymphatic thrombosis was made, and we ligated the lymphatic vessels, closed the wound at this site, and performed LVA at other sites (4 sites in the left and 1 site in the right leg). The postoperative course was uneventful. Histopathological examination showed hyperplasty of fibroblasts and organization in the thrombus. Lymphatic thrombosis is sometimes found in the lymphedema-affected extremities. On retrospective consideration, lymphatic thrombosis can be detected with preoperative echography.

BACKGROUND: Early lymphedema detection may reduce the symptoms and improve clinical outcomes. However, the lack of reliable serum biomarkers capable of predicting lymphedema development is a current medical problem. In this study, we investigated if serum levels of hyaluronic acid (HA) and leukotriene B4 (LTB4), two molecules involved in lymphedema development, may work as predictors of this condition. METHODS AND RESULTS: A mouse model of acquired lymphedema was generated through ablation of tail dermal lymphatic network. Tail diameter was measured daily, and HA and LTB4 serum levels were analyzed before and during the development of lymphedema. We found increased serum levels of HA and reduced levels of LTB4 at early days before the appearance of lymphedema signs. Similar results were observed in the lymphedema tissue. Increased local and systemic inflammation was also detected at early time points. Moreover, the ratio LTB4/HA arises as the strongest predictor for lymphedema development. In fact, we found an inverse correlation in our model, where reduced LTB4/HA levels showed increased lymphedema signs. CONCLUSIONS: These findings suggest that serum ratio of LTB4/HA may be a useful biomarker to predict acquired lymphedema development, with potential to be used in clinical conditions such as breast cancer patients.

BACKGROUND: Yellow nail syndrome is a rare condition associated with a triad of symptoms: yellow nails, lung lesions, and lymphedema. We report a case of yellow nail syndrome caused by titanium exposure from multiple artificial joint replacements. CASE PRESENTATION: A 78-year-old Asian woman presented to our outpatient department with chief complaints of cough, fever, and nausea. The patient was hospitalized for observation because of the presence of hypoxemia and bilateral pleural effusion. Her medical history included knee joint replacement and two spinal fusion surgeries. Her physical examination conducted following hospitalization revealed yellow nails on both hands and feet. This finding, combined with the observation of bilateral pleural effusion, raised suspicion for yellow nail syndrome. Blood analysis yielded negative results, as did the tests for sputum culture, interferon liberation, pleural effusion culture, and pleural effusion cytology. Pleural histopathological analysis and imaging yielded negative results. Considering the possibility of titanium exposure from artificial joints based on the patient's medical history, we examined a chest radiograph obtained before the second spinal fusion surgery; however, no pleural effusion was observed. Pleural effusion was observed, however, following the surgery. On the basis of these findings, the patient was diagnosed with yellow nail syndrome due to titanium exposure. CONCLUSIONS: Clinicians should examine the nails of patients with unexplained pleural effusion. Moreover, they should inquire about titanium exposure when obtaining the patient's medical history.

BACKGROUND: Upper extremity lymphedema occurs in 25 to 40% of patients after axillary lymph node dissection (ALND). Immediate lymphatic reconstruction (ILR) or the lymphatic micro- surgical preventative healing approach has demonstrated a significant decrease in postoperative rates of lymphedema (LE) from 4 to 12%. Our objective was to map the Mascagni -Sappey pathway, the lateral upper arm draining lymphatics, in patients undergoing ILR to better characterize the drainage pattern of this lymphosome to the axilla. METHODS: A retrospective review of our institutional lymphatic database was conducted and consecutive breast cancer patients undergoing ILR were identified from November 2017 through June 2018. Patient demographics, clinical characteristics, and intraoperative records were retrieved and analyzed. RESULTS: Twenty-nine consecutive breast cancer patients who underwent ILR after ALND were identified. Patients had a mean age of 54.6years and body mass index (BMI) of 26.6 kg/m2. Fluorescein isothiocyanate (FITC) was injected at the medial upper arm and isosulfan blue was injected at the cephalic vein, or lateral upper arm, prior to ALND. After ALND, an average 2.5 divided lymphatics were identified, and a mean 1.2 lymphatics were bypassed. In all patients, divided FITC lymphatics were identified. However, in only three patients (10%), divided blue lymphatics were identified after ALND. CONCLUSION: In this study, variable drainage of the lateral upper arm to the axillary bed was noted. This study is the first to provide a description of intraoperative findings, demonstrating variable drainage patterns of upper extremity lymphatics to the axilla. Moreover, we noted that the lateral- and medial-upper arm lymphosomes have mutually exclusive pathways draining to the axilla. Further study of lymphatic anatomy variability may elucidate the pathophysiology of lymphedema development and influence approaches to immediate lymphatic reconstruction.

The clinical diagnostic criteria for Proteus syndrome were defined before the discovery of the AKT1 c.49G>A; p.(Glu17Lys) causal variant and used a combination of general and specific phenotypic attributes that could be combined to make a clinical diagnosis. The most heavily weighted specific criterion was the cerebriform connective tissue nevus (CCTN). Here, we describe two individuals with connective tissue nevi (CTNs) and some general attributes of Proteus syndrome who were found to have mosaic PIK3CA variants. CTNs on the soles of individuals with PIK3CA-related overgrowth typically exhibit thickening of the soft tissues with at most a wrinkled surface, but these two patients had firm plaques with ridges and furrows characteristic of CCTNs, which was histologically confirmed in one. These data show that CCTNs are not specific to Proteus syndrome and that clinicians should be cautious in diagnosing individuals with Proteus syndrome based on the CCTN alone. Rather, a complete evaluation should include careful assessment of other attributes of the diagnostic criteria and, whenever possible, genetic analysis of affected tissue.

BACKGROUND: The island of Hispaniola, shared by Haiti and the Dominican Republic (DR), is the only remaining malaria-endemic island in the Caribbean and accounts for 95% of the lymphatic filariasis (LF) burden in the Americas. Both countries aim to eliminate the diseases by 2020. Migration from Haiti, where both diseases are more prevalent, may promote transmission in the DR. Historically, Haitian migrant labourers live in rural Dominican agricultural ‘company towns’ called bateyes, many of which received mass drug administration (MDA) for LF elimination. This study sought to determine the prevalence of malaria and LF in bateyes of the DR and to describe related risk factors for disease. METHODS: From March to April 2016, a cross-sectional, cluster survey was conducted across Dominican bateyes stratified into three regions: southwest, north and east. A household questionnaire (n = 776), captured demographics, ethnic origin, mobility patterns, malaria intervention coverage, and knowledge, and recent fever and treatment-seeking. Two individuals per household (n = 1418) were tested for malaria parasites by microscopy and rapid diagnostic test (RDT) and LF antigen by filariasis test strip (FTS). Population-level estimates and confidence intervals (CI) were computed adjusting for the survey design. Two-sided t-tests compared differences in knowledge scores. RESULTS: No (0%) blood sample was Plasmodium-positive by microscopy or RDT. Six individuals were FTS-positive (0.5%; 95% CI: 0.2–1.5), but none (0%) of these were microfilariae-positive. Most batey residents were born in the DR (57.8%), documented (85.0%), and permanent residents (85.1%). Very few respondents (9.4%) reported travel to Haiti in the past year. Overall, half (53.8%) of respondents owned a bed net, and 82.3% of net owners reported using it the previous night. Indoor residual spraying (IRS) differed by region (range: 4.7%-61.2%). Most of those with recent fever sought care (56.0%), yet only 30.5% of those seeking care were tested for malaria. Compared to Dominican-born populations, Haitian-born respondents more frequently reported recent fever, did not seek care for the fever, had not heard of malaria, and could not name symptoms or prevention methods. CONCLUSIONS: Malaria and LF transmission appear absent or extremely low in Dominican bateyes, which are a mixture of Haitian and Dominican residents. Travel to Haiti is rare, meaning risk of malaria and LF importation is low. Addressing identified gaps in intervention coverage, malaria knowledge, treatment seeking and service delivery will improve the quality of surveillance for these diseases, particularly among marginalized populations and promote island-wide elimination.

Pulmonary lymphangioleiomyomatosis (LAM) is a rare genetic multisystem disease characterized by the nodular proliferation of smooth muscle-like LAM cells, progressive cystic changes of the lung, lymphatic abnormalities, and renal angiomyolipomas (AMLs). LAM can arise sporadically or in women with the autosomal dominant disorder, tuberous sclerosis complex (TSC), in which hamartomatous tumors of brain, heart, skin, kidney, and lung are found. LAM and TSC are caused by mutations in the TSC1 or TSC2 tumor suppressor genes leading to elevated mechanistic/mammalian target of rapamycin complex activity. Recent data indicate that T cells within LAM nodules and within renal AMLs exhibit features of T-cell exhaustion, with coinhibitory receptor programmed cell death protein 1 (PD-1) expression on tumor-infiltrating T cells. Treatment of animal models of TSC and LAM with anti-PD-1 antibodies or with the combination of anti-PD-1 and anti-CTLA4 antibodies has led to remarkable results, suppressing TSC2-null tumor growth and inducing tumor rejection. Here we review our current knowledge about the potential for immunotherapy for the treatment of LAM and TSC and highlight critical unknowns and key next steps.

We report the first case of nonimmune hydrops fetalis (NIHF) associated with a recessive, in-frame deletion of V205 in the G protein-coupled receptor, Calcitonin Receptor-Like Receptor (hCALCRL). Homozygosity results in fetal demise from hydrops fetalis, while heterozygosity in females is associated with spontaneous miscarriage and subfertility. Using molecular dynamic modeling and in vitro biochemical assays, we show that the hCLR(V205del) mutant results in misfolding of the first extracellular loop, reducing association with its requisite receptor chaperone, receptor activity modifying protein (RAMP), translocation to the plasma membrane and signaling. Using three independent genetic mouse models we establish that the adrenomedullin-CLR-RAMP2 axis is both necessary and sufficient for driving lymphatic vascular proliferation. Genetic ablation of either lymphatic endothelial Calcrl or nonendothelial Ramp2 leads to severe NIHF with embryonic demise and placental pathologies, similar to that observed in humans. Our results highlight a novel candidate gene for human congenital NIHF and provide structure-function insights of this signaling axis for human physiology.

A whole exome sequencing approach was recently used to detect a CELSR1 truncating variant associated with lymphedema in a large pedigree. Since this first report, no other similar associations have been reported in the literature. Here, we present the genetic results of 95 probands tested using a next generation sequencing panel that covered all known lymphedema-associated genes, including CELSR1. Five out of 95 probands (5.3%) were found to carry novel loss-of-function variants in CELSR1. Family segregation studies were possible in four out of five probands and showed possible sex-specific differences: CELSR1 variants showed almost complete penetrance in females and were associated with early-onset lymphedema, whereas in males they showed incomplete penetrance and were associated with late onset of the condition. Since the percentage of lymphedema patients carrying CELSR1 variants is not negligible, we do not hesitate to recommend including this gene in routine genetic testing.

BACKGROUND: Ultrasound investigation potentials in lymphedema are still to be fully used in everyday practice. Aim of the present study is to report the sonographic characterization of the dermo-epidermal complex (DEC) and of the subcutaneous (SUBC) tissue, assessing the feasibility of a related mapping, in upper limb secondary lymphedema. METHODS: In this retrospective study 287 patients affected by monolateral upper limb post- mastectomy lymphedema (M5/F282; mean age 64 ± 4.24) were enrolled and scanned by ultrasound, considering the healthy contralateral limb as control. In order to standardize the assessment, the limb was divided in sectors: 4 anterior, 4 posterior below the elbow, 4 anterior and 4 posterior above the elbow, plus the hand. DEC and SUBC regions B-mode appearance were reported, both in the healthy and in the pathological arms. DEC thickness was measured and compared among the same sectors of the healthy and pathological limbs. RESULTS: DEC and SUBC sonographic appearance was differentiated in fluid and sclerotic. DEC included a third category characterized by differentiation loss. The different sectors showed significantly different lymphatic involvement in the affected limb. In the comparison with the contralateral unaffected segments a significantly thicker DEC was reported in the forearm affected by lymphedema (p < .005), while no significant difference was reported at the arm level. CONCLUSIONS: Traditional ultrasonography can provide a secondary upper limb lymphedema characterization with related mapping and useful data for a better lymphatic physiopathology understanding and for a properly addressed therapeutic protocol.

Certain systemic viral infections can be related to development of vascular complications, such as deep venous thrombosis and lymphedema of lower and upper limbs. These links have been well-established in patients with human immunodeficiency virus (HIV), hepatitis C, or influenza. Recently introduced into the American continent (2013), chikungunya virus is an arbovirus transmitted by mosquitoes of the Aedes genus and is the etiologic agent of chikungunya fever (CF), but its relationship to these vascular complications has not yet been consolidated. However, the CF outbreak that occurred during 2015 and 2016 resulted in the first cases described in the medical literature of acute and chronic vascular complications secondary to infection by this arbovirus. In this report, we describe the case of a patient who developed lymphedema of upper and lower limbs after an episode of CF.

BACKGROUND: The Global Programme to Eliminate Lymphatic Filariasis (GPELF) was launched in 2000 with the goal of eliminating LF as a public health problem by 2020. Despite considerable progress, around 60% is remaining with the deadline looming a year away. Consequently, there is a continued need for investment in both the mass drug administration (MDA) and morbidity management programmes, which this paper aims to demonstrate by estimating the health and economic burden of LF prior to MDA programmes starting in GPELF areas. METHODS: A previously developed model was used to estimate the number of individuals infected and those with symptomatic disease, along with the attributable number of disability-adjusted life years (DALYs). The economic burden was calculated by quantifying the costs incurred by the healthcare system in managing clinical cases, the patients' out-of-pocket costs, and their productivity costs. RESULTS: Prior to the MDA programme ∼129 million were infected with LF, of which 43 million had clinical disease, corresponding to a DALY burden of 5.25 million. The average annual economic burden per chronic case was US$115, majority of which resulted from productivity costs. The total economic burden of LF was estimated at US$5.8 billion annually. CONCLUSION: These results demonstrate the magnitude of the LF burden and highlight the continued need to support the GPELF. Patients with clinical disease bore the majority of the economic burden but will not benefit much from the current MDA programme aimed at reducing transmission. This assessment further highlights the need to scale up morbidity management programmes.

Paediatric chylous ascites in tropics is commonly caused by infections and trauma. We describe the clinical characteristics of an uncommon inherited cause of chylous ascites, Hennekam syndrome, treated by nutritional modification.

Primary intestinal lymphangiectasia (PIL) is a rare protein-losing gastroenteropathy which is defined as dilation of existing mucosal, submucosal, or subserosal lymphatics within the gastrointestinal tract. That causes loss of lymph fluid into the gastrointestinal tract, leading to the development of hypoproteinemia, edema, lymphocytopenia, hypogammaglobinemia, and immunologic anomalies. It is usually diagnosed in patients younger than 3 years old and is rarely first diagnosed in adulthood. Here we have a case report in a 23-month-old female presented with the complaint of peripheral edema and diarrhea. The diagnosis of PIL was made through upper gastrointestinal endoscopy and pathology histologic analysis. Patient placed on oral supplements of medium-chain triglycerides, a high protein diet, supplements of fat-soluble vitamins and responded well.

BACKGROUND: The current belief is that the calcium channel blocker (CCB)-induced edema is due to a preferential arterial over venous dilatation leading to increased fluid filtration. We challenged this conviction by measuring the lymphatic removal of interstitial fluid during chronic systemic treatment with the CCB, amlodipine. Lymphatic vessels could potentially be an off-target effect of the drugs and play a role in CCB edema. METHODS AND RESULTS: Sixteen healthy postmenopausal women completed a 12-week double-blinded randomized placebo-controlled crossover trial. Lymphatic function was assessed by near-infrared fluorescence imaging. The lymphatic function during amlodipine treatment compared with placebo did not show any difference in pumping pressure (53.9 ± 13.9 mmHg vs. 54.7 ± 9.4 mmHg, p = 0.829), contraction frequency (0.4 ± 0.2/min vs. 0.4 ± 0.3/min, p = 0.932), refill time (440 ± 438 seconds vs. 442 ± 419 seconds, p = 0.990), or propagation velocity of lymph packets (18 ± 10 mm/s vs. 15 ± 7 mm/s, p = 0.124). However, the subjects who developed edema during CCB treatment had a 20% lower baseline lymphatic pumping pressure (48.9 ± 4.4 mmHg, n = 7) than the subjects not affected by treatment (59.1 ± 1.2 mmHg, n = 9, p = 0.025). Contraction frequency, refill time, and lymph packet velocity showed no differences in baseline values between the two groups. CONCLUSION: Our results suggest that CCB does not directly impair lymphatic function. However, our results show that a reduced lymphatic function predisposes to CCB edema, which may explain why some patients develop edema during treatment.

The hands-on supermicrosurgery course provided participants a valuable learning experience of in-depth practices of supermicrosurgical skills with experts. Seven live surgeries were successfully demonstrated at 8th World Symposium for Lymphedema Surgery. Variable donor sites for vascularized lymph node transfer were the submental, supraclavicular, groin, and omental; while the recipient sites included the wrist and axilla in upper limb; and popliteal and groin in the lower limb. The therapeutic and preventive lymphovenous anastomosis was also satisfactorily performed.

BACKGROUND: Lymphedema of the lower limbs is a chronic disease caused by damage to the lymphatic system that influences people's mobility, functionality, and quality of life. Questionnaires and physical test are very practical, easy to apply, and low cost methods that provide important data for evaluation of these patients. OBJECTIVES: To evaluate the influence of unilateral lower limb lymphedema on functionality and quality of life, correlating 3 assessment tools. METHODS: This was a descriptive study investigating 25 patients of both sexes with unilateral lymphedema in a lower limb. Limb volume was assessed using circumferential tape measurements, the Medical Outcomes Study Short Form-36 Health Survey (SF-36) was used to assess quality of life, the Lymphoedema Functioning, Disability and Health Questionnaire for Lower Limb Lymphoedema (Lymph-ICF-LL) was used to assess physical, mental, and social skills related to lymphedema, and the Timed Up and Go (TUG) test was used for functional assessment. RESULTS: Lymphedema was present throughout the affected lower limb of participants. The domains most affected by lymphedema were physical aspects (25.0 ± 31.4) and emotional aspects (36.0 ± 42.9) from the SF-36 and the mobility domain (6.0 ± 2.6) from the Lymph -ICF-LL. Patients performed the TUG in 9.88 ± 1.98 seconds. The TUG was correlated with the questionnaires and the questionnaires were correlated with each other. CONCLUSIONS: People with unilateral lower limb lymphedema exhibited negative impacts on quality of life and functionality, as evaluated by questionnaires, which were correlated with each other. TUG performance was within normal limits, but results correlated with the questionnaires used.

Lipiodol is an iodinated poppy seed oil first synthesized in 1901. Originally developed for therapeutic purposes, it has mainly become a diagnostic contrast medium since the 1920s. At the end of the 20th century, Lipiodol underwent a transition back to a therapeutic agent, as exemplified by its increasing use in lymphangiography and lymphatic interventions. Nowadays, indications for lymphangiography include chylothorax, chylous ascites, chyluria, and peripheral lymphatic fistula or lymphoceles. In these indications, Lipiodol alone has a therapeutic effect with clinical success in 51% to 100% of cases. The 2 main access sites to the lymphatic system for lymphangiography are cannulation of lymphatic vessels in the foot (transpedal) and direct puncture of (mainly inguinal) lymph nodes (transnodal). In case of failure of lymphangiography alone to occlude the leaking lymphatic vessel as well as in indications such as protein-losing enteropathy, postoperative hepatic lymphorrhea, or plastic bronchitis, lymphatic vessels can also be embolized directly by injecting a mixture of Lipiodol and surgical glues (most commonly in thoracic duct embolization). The aim of this article is to review the historical role of Lipiodol and the evolution of its clinical application in lymphangiography over time until the current state-of-the-art lymphatic imaging techniques and interventions.

Secondary lymphedema is a worldwide affliction that exacts a significant public health burden. This review examines the etiology, presentation, and management of secondary lymphedema. In addition, emerging adjunctive strategies are explored, specifically evidence from animal and pilot human studies regarding implantation of a collagen nanofibrillar scaffold (BioBridge; Fibralign Corporation, Union City, CA) in promoting lymphangiogenesis, preventing and treating lymphedema, and enhancing outcomes with lymphaticovenous anastomosis and vascularized lymph node transfer.

BACKGROUND: This high volume, single center study investigated the prevalence, bacterial epidemiology, and responsiveness to antibiotic therapy of cellulitis in extremity lymphedema. METHODS: From 2003 to 2018, cellulitis events from a cohort of 420 patients with extremity lymphedema were reviewed. Demographics, lymphedema grading, symptoms, inflammatory markers, cultures and antibiotic therapy regimens were compiled from cellulitis episodes data. Univariate and multivariate analyses were performed for detailed analysis. RESULTS: A total of 131 separate episodes of cellulitis were recorded from 43 (81.1%) lower limb and 10 (19.9%) upper limb lymphedema patients. The prevalence and recurrence rates for cellulitis in lymphedema patients were 12.6% (53 of 420) and 56.6% (30 of 53), respectively. The most common findings were increased limb circumference (127 of 131; 96.9%) and abnormal C-reactive protein (CRP) level (86 of 113; 76.1%). Blood cultures were obtained in 79 (60.3%) incidents, with 9 (11.4%) returning positive. Streptococcus agalactiae was the most isolated bacterium (5 of 9; 55.5%). CONCLUSIONS: The cellulitis prevalence and recurrence rate in extremity lymphedema were 12.6%, and 56.6%, respectively. Strongest indicators of cellulitis were increased affected limb circumference and elevated CRP level. Empiric antibiotic therapy began with coverage for Steptococcus species before broadening to anti-Methicillin-resistant Staphylococcus aureus and anti-Gram negatives if needed for effective treatment of extremity lymphedema cellulitis.

Sinus histiocytosis with massive lymphadenopathy, also known as Rosai-Dorfman disease (RDD), is a self-limited histiocytic disorder of unclear etiology which most commonly presents with cervical lymphadenopathy. Purely extranodal presentation of RDD is uncommon, and isolated intralymphatic/intravascular confinement of this entity has not previously been described. We report a 16-yr-old female who presented with vaginal swelling and mass-like enlargement of the right labia. The mass had been present for nearly a year without pain or tenderness. Clinically, the lesion was thought to be a Bartholin gland cyst. Following surgical resection, histologic examination demonstrated a hypocellular myxedematous stroma with a mixture of ectatic thin and thick-walled vessels within which there were numerous collections of histiocytes, lymphocytes, and plasma cells. The histopathologic differential diagnosis included localized vulvar lymphedema, a specialized genital tract neoplasm, and childhood asymmetric labium majus enlargement. The histiocytes showed occasional plasma cells and lymphocytes within their cytoplasm, consistent with emperipolesis. Immunohistochemical studies showed that the histiocytes expressed CD163 and S100, while ERG and D2-40 highlighted their intralymphatic confinement, ultimately leading to the diagnosis of intralymphatic RDD. Intralymphatic RDD may present as vulvar lymphedema and can potentially mimic other myxedematous neoplasms of the vulvovaginal region.

BACKGROUND: Phelan-McDermid syndrome (PMS) is a rare genetic disorder characterized by global developmental delay, intellectual disability (ID), autism spectrum disorder (ASD), and mild dysmorphisms associated with several comorbidities caused by SHANK3 loss-of-function mutations. Although SHANK3 haploinsufficiency has been associated with the major neurological symptoms of PMS, it cannot explain the clinical variability seen among individuals. Our goals were to characterize a Brazilian cohort of PMS individuals, explore the genotype-phenotype correlation underlying this syndrome, and describe an atypical individual with mild phenotype. METHODOLOGY: A total of 34 PMS individuals were clinically and genetically evaluated. Data were obtained by a questionnaire answered by parents, and dysmorphic features were assessed via photographic evaluation. We analyzed 22q13.3 deletions and other potentially pathogenic copy number variants (CNVs) and also performed genotype-phenotype correlation analysis to determine whether comorbidities, speech status, and ASD correlate to deletion size. Finally, a Brazilian cohort of 829 ASD individuals and another independent cohort of 2297 ID individuals was used to determine the frequency of PMS in these disorders. RESULTS: Our data showed that 21% (6/29) of the PMS individuals presented an additional rare CNV, which may contribute to clinical variability in PMS. Increased pain tolerance (80%), hypotonia (85%), and sparse eyebrows (80%) were prominent clinical features. An atypical case diagnosed with PMS at 18 years old and IQ within the normal range is here described. Among Brazilian ASD or ID individuals referred to CNV analyses, the frequency of 22q13.3 deletion was 0.6% (5/829) and 0.61% (15/2297), respectively. Finally, renal abnormalities, lymphedema, and language impairment were found to be positively associated with deletion sizes, and the minimum deletion to cause these abnormalities is here suggested. CONCLUSIONS: This is the first work describing a cohort of Brazilian individuals with PMS. Our results confirm the impact of 22q13 deletions on ASD and several comorbidities, such as hypotonia. The estimation of a minimal deletion size for developing lymphedema and renal problem can assist prediction of prognosis in PMS individuals, particularly those diagnosed in early infancy. We also identified one atypical individual carrying SHANK3 deletion, suggesting that resilience to such mutations occurs. This case expands the clinical spectrum of variability in PMS and opens perspectives to identify protective mechanisms that can minimize the severity of this condition.

Indocyanine green (ICG) lymphography is a useful imaging modality for evaluation of lymphedema and detection of lymphatic vessels. It also allows us to ensure patency of the anastomosed vessels intraoperatively. However, strong light from the operating microscope usually disturbs ICG fluorescence imaging. Only some built-in ICG camera systems with specific operating microscopes make real-time ICG lymphography possible in lymphaticovenular anastomosis (LVA). We applied a new high-resolution ICG videolymphography system, which is separated from the operating microscope. Because the system can divide near-infrared fluorescence light of ICG from visible light of the operating microscope, real-time ICG videolymphography-navigated LVA under operating microscope illumination is possible regardless types of operating microscopes. The study involved 10 patients with upper extremity lymphedema characterized by International Society of Lymphology stage 2 and treated by 3 lymphaticovenular anastomoses at the forearm (30 lymphaticovenular anastomoses incorporating 30 lymphatic vessels) under real-time ICG videolymphography. The rate of intraoperative detection of lymphatic vessels using real-time ICG videolymphography was 86.7% (0.25–0.85 mm in diameter), and that of lymph flow through the lymphaticovenular anastomoses was 76.7%. None of lymphatic vessels and no flow were detected under the microscope light by means of another non-built-in ICG lymphography camera. Real-time ICG videolymphography in LVA is beneficial, because the surgeon could find lymphatic vessels easily by checking dual images of original view and ICG fluorescent view and ensure accuracy of the LVA in a suture by a suture without any pauses of the surgical procedures.

BACKGROUND: The primary aim of this study was to determine the detailed anatomy of the lymphatics in the lower extremity using fresh human cadavers with indocyanine green fluorescence lymphography. The secondary aim was to apply the anatomical results to establish a new protocol for lymphography based on feasible allocations for tracer injection sites. METHODS: One hundred lower extremities from 53 fresh human cadavers were used for this study. The authors injected indocyanine green solution subcutaneously at 19 points around the foot along the borderline between the dorsum and planta according to anatomical landmarks. Immediately after the indocyanine green injections, gentle hand massage was applied at each injection site to facilitate indocyanine green uptake into the lymphatic vessels. Fluorescent images of the lymphatics were obtained using a near-infrared camera system. Imaging data of the lymphatics were analyzed to find correlations between the injection sites and the identified lymphatic vessels. RESULTS: The lymphatic system in the lower extremity was divided into four distinct lymphatic groups: anteromedial, anterolateral, posterolateral, and posteromedial. The lymphatic vessels in all except the posterolateral group connected to the inguinal nodes, and those in the posterolateral group connected to the popliteal nodes. The authors successfully elucidated correlations between the injection sites in the foot and each lymphatic group. CONCLUSION: The new classification of the four lymphatic groups in the lower extremity and identification of their origins in the foot enabled the authors to propose a new protocol for lymphography that includes four injection sites in specific circumflex locations.

BACKGROUND: Lymphedema (LE) has been called the forgotten vascular disease, given such scant knowledge about LE-associated comorbidities or causes. Such knowledge of the comorbidities and treatment of LE may assist in diagnostic decisions and health care planning. METHODS: To determine the proportion of LE patients with various LE-associated comorbidities as well as the rate of associated treatment, deidentified Health Insurance Portability and Accountability Act-compliant commercial administrative claims from the Blue Health Intelligence (BHI) research database (165 million Blue Cross Blue Shield members) were queried. We analyzed a BHI study sample of 26,902 patients with LE who had been enrolled with continuous medical benefits for 12 months before and after the index date for the complete years 2012 through 2016. Patients were first identified by comorbidity and then grouped into those receiving no treatment for LE and those receiving any treatment for LE. Any treatment was defined as receiving manual lymphatic drainage, physical therapy, compression garments, or a pneumatic compression device. The purpose of this study was to determine the proportion of LE patients comorbid with various known LE-associated conditions and the treatment rates of LE patients with each comorbidity. RESULTS: Among the 84,579,269 BHI patients enrolled during the study window, 81,366 patients were identified with LE. From this LE group, our study focused on the 26,902 patients who were enrolled with continuous medical and pharmacy benefits for 12 months before and after the index date. Among these 26,902 LE patients, breast cancer was the most frequent comorbidity with LE (32.1%), and these patients almost universally received any treatment (94.2%); other cancer types, such as melanoma (2.1%) and prostate cancer (0.7%), were less frequent and received any treatment less often, 75% and 82% of the time, respectively. Venous leg ulcer was the most common non-cancer-linked comorbidity for LE (9.6%), but only 81.7% of venous leg ulcer patients received any treatment for LE. CONCLUSIONS: To our knowledge, this is the largest study to date detailing the comorbidities associated with LE and LE treatment rates within each. Our findings suggest that a sizable proportion of cancer-related LE patients do not receive appropriate treatment. Furthermore, this study highlights the role of advanced venous disease as an LE comorbidity that is frequently untreated and its associated gap in treatment.

Stress-lymphoscintigraphy evaluates the effects of exercise on lymph drainage. Results may predict outcomes of complex physical therapy as a first-line lymphedema treatment. Microsurgical approaches are best in patients with lymphangectasia or dermal backflow. Lymphatic pathway mapping helps plan lympho-venous anastomoses. For improved lymphatic visualization, we suggest a scan at rest, after a quick exercise (stepping or weight lifting for 2 min) and delayed scanning after prolonged symptom limited exercise (walking or hand squeezing for 30–40 min). In advanced lymphedema, identification of lymph pathways and residual regional lymph nodes might be difficult. In these patients, lymph node transplant is suggested.

OBJECTIVE: To evaluate the obstetric outcomes of fetuses with cystic hygroma other than karyotype abnormalities and structural malformations. MATERIAL AND METHODS: We conducted a retrospective study based on the review of medical records of pregnant women in whom ultrasonographic diagnosis of fetal cystic hygroma was established in the first trimester from January 2014 to October 2018. All patients were offered genetic counselling and prenatal invasive diagnostic procedures to obtain fetal karyotype. For ongoing pregnancies fetal echocardiography and detailed second trimester sonographic anomaly screening was performed by a perinatologist/pediatric cardiologist. Demographic characteristics of the women and results of the karyotype analysis were obtained from the database of our hospital and correlated with the obstetric outcome. RESULTS: Within five-year period, there were 106 cases of fetal cystic hygroma. Of those, fetal cardiac malformations were detected in 4 and migrognathia in one. 85 women underwent fetal invasive procedures and in 52 of the cases, karyotype abnormalities were detected. Fetal outcomes of 33 cases with normal karyotype and 21 cases in whom karyotyping analysis were not performed due to patient refusal were enrolled into the study. Obstetric outcomes of 21 women who refused karyotyping consisted of 13 livebirths, 7 missed abortions and one fetal death, whereas those of 33 women with normal karyotype were; 12 livebirths, 12 missed abortions, 2 hydrops fetalis and 5 fetal deaths. 19 of 33 fetuses with normal karyotype and 8 of 21 fetuses in whom karyotyping were not performed were terminated. CONCLUSION: The presence of cystic hygroma carries a high risk for fetal karyotype abnormalities and cardiac malformations. Postnatal outcomes of the fetuses with cystic hygroma appeared to be correlated with the absence of structural malformations and karyotype abnormalities.

OBJECTIVE: Lymphedema is classified as primary or secondary according to the underlying cause. Primary lymphedema is hereditary and is considered a consequence of an inherited abnormality of the lymphatic system. Secondary lymphedema, however, is a consequence of lymphatic failure resulting from trauma, parasitic infection, or iatrogenic obstruction. Primary lymphedema is divided into three broad groups, namely, lymphedema congenita, lymphedema praecox, and lymphedema tarda. With the exception of lymphedema tarda, it is thought that age-related deterioration in lymphatic pump function is caused by oxidative stress. The aim of this study was to evaluate and to classify indocyanine green (ICG) lymphography findings in patients with lower limb lymphedema to ascertain whether there is a pattern to age-related deterioration. METHODS: There were 56 patients (104 edematous lower limbs) who had undergone ICG lymphography and for whom the lower extremity lymphedema (LEL) index had been calculated enrolled in this study. Specific inclusion criteria were used to exclude other causes of edema. ICG lymphography images were recorded in the plateau phase (12–18 hours after injection), when no further changes of images would be expected. The LEL index was calculated by summation of the squares of the circumference for five areas in each lower extremity divided by the body mass index. RESULTS: The clinical lymphedema pattern was determined as bilateral in 48 patients and unilateral in 8 patients. Patients with bilateral lymphedema were significantly older than those with unilateral lymphedema (76.40 ± 8.03 years vs 53.13 ± 14.12 years; P < .01). The ICG lymphography pattern was categorized as linear, low enhancement (LE), distal dermal backflow (DB), or extended DB in bilateral lymphedema. ICG lymphography showed the DB pattern on both the thigh and lower leg regions in all eight legs with unilateral lymphedema. There were also significant between-group differences in the LEL index (linear vs distal DB, P < .05; linear vs extended DB, P < .01; linear vs unilateral, P < .01; LE vs extended DB, P < .01; LE vs unilateral, P < .01; distal DB vs extended DB, P < .05; and distal DB vs unilateral, P < .01). CONCLUSIONS: In this study, unilateral lymphedema, with its younger age at onset, severity, and unilateral dominance, corresponded to lymphedema tarda. In contrast, bilateral lymphedema corresponded to senile lymphedema, which is distinct from primary lymphedema in general and lymphedema tarda in particular. Age-related deterioration in lymphatic pump function rather than iatrogenic obstruction or genetic abnormality is likely to account for the characteristic older age at onset of lymphedema and its progression from the distal region.

Contrast-enhanced ultrasound (CEUS) utilising microbubbles shows great potential for visualising lymphatic vessels and identifying sentinel lymph nodes (SLNs) which are valuable for axillary staging in breast cancer patients. However, current CEUS imaging techniques have limitations that affect the accurate visualisation and tracking of lymphatic vessels and SLN. (i) Tissue artefacts and bubble disruption can reduce the image contrast. (ii) Limited spatial and temporal resolution diminishes the amount of information that can be captured by CEUS. (iii) The slow lymph flow makes Doppler-based approaches less effective. This work evaluates on a lymphatic vessel phantom the use of high frame rate (HFR) CEUS for the detection of lymphatic vessels where flow is slow. Specifically, the work particularly investigates the impact of key factors in lymphatic imaging, including ultrasound pressure and flow velocity as well as probe motion during vessel tracking, on bubble disruption and image contrast. Experiments were also conducted to apply HFR CEUS imaging on vasculature in a rabbit popliteal lymph node (LN). Our results show that (i) HFR imaging and singular value decomposition (SVD) filtering can significantly reduce tissue artefacts in the phantom at high clinical frequencies; (ii) the slow flow rate within the phantom makes image contrast and signal persistence more susceptible to changes in ultrasound amplitude or mechanical index (MI), and an MI value can be chosen to reach a compromise between images contrast and bubble disruption under slow flow condition; (iii) probe motion significantly decreases image contrast of the vessel, which can be improved by applying motion correction before SVD filtering; (iv) the optical observation of the impact of ultrasound pressure on HFR CEUS further confirms the importance of optimising ultrasound amplitude and (v) vessels inside rabbit LN with blood flow less than 3 mm/s are clearly visualised.

The formation of new lymphatic vessels, or lymphangiogenesis, is a critical step of the tissue repair program. In pathological conditions involving chronic inflammation or tumorigenesis, this process is often dysregulated and can contribute to disease progression. Yet, lymphangiogenesis is still incompletely understood. In this study, we identified A2a adenosinergic signaling as an important regulator of inflammatory and tumor-associated lymphangiogenesis. Using Adora2a (A2a)-deficient mice, we demonstrated that A2a signaling was involved in the formation of new lymphatic vessels in the context of peritoneal inflammation. We also demonstrated that tumor-associated and sentinel lymph node lymphangiogenesis were impaired in A2a-deficient mice, protecting them from lymph node metastasis. Notably, A2a signaling in both hematopoietic and non-hematopoietic cells contributed to sentinel lymph node metastasis. In A2a-deficient tumor-draining lymph nodes, impaired lymphangiogenesis was associated with a reduced accumulation of B cells and decreased VEGF-C levels. Supporting a role for non-hematopoietic A2a signaling, we observed that primary murine lymphatic endothelial cells (LEC) predominantly expressed A2a receptor and that A2a signaling blockade altered LEC capillary tube formation in vitro. Finally, we observed that Adora2a, Nt5e and Entpd1 gene expression positively correlated with Lyve1, Pdpn and Vegfc in several human cancers, thereby supporting the notion that adenosine production and A2a receptor activation might promote lymphangiogenesis in human tumors. In conclusion, our study highlights a novel pathway regulating lymphangiogenesis and further supports the use of A2a or adenosine blocking agents to inhibit pathological lymphangiogenesis in cancers and block the dissemination of tumor cells through the lymphatic system.

IMPORTANCE: Most lymphedema studies include a heterogeneous population and focus on patients treated with adjuvant chemotherapy. OBJECTIVE: To examine factors associated with lymphedema after neoadjuvant chemotherapy (NAC) and axillary lymph node dissection in women with node-positive breast cancer. DESIGN, SETTING, AND PARTICIPANTS: This cohort study included data from 701 women 18 years or older with cT0-T4N1–2M0 breast cancer with documented axillary nodal metastasis at diagnosis who were enrolled in the American College of Surgeons Oncology Group Z1071 (Alliance for Clinical Trials in Oncology) trial, which took place from January 1, 2009, to December 31, 2012. Data analysis was performed from January 11, 2018, to November 9, 2018. INTERVENTIONS: All participants received NAC, breast operation, and axillary lymph node dissection. Participants underwent prospective arm measurements and symptom assessment after NAC completion and at 6-month intervals to 36 months postoperatively. MAIN OUTCOMES AND MEASURES: Factors associated with lymphedema were defined as self-reported arm heaviness or swelling (lymphedema symptoms) or an arm volume increase of 10% or more (V10) or 20% or more (V20). RESULTS: A total of 486 patients (mean [SD] age, 50.1 [10.8] years) were included in this study. Median follow-up for the 3 measures was 2.2 to 3.0 years. Cumulative lymphedema incidence at 3 years was 37.8% (95% CI, 33.1%-43.2%) for lymphedema symptoms, 58.4% (95% CI, 53.2%-64.1%) for V10, and 36.9% (95% CI, 31.9%-42.6%) for V20. Increasing body mass index (hazard ratio [HR], 1.04; 95% CI, 1.01–1.06) and NAC for 144 days or longer (HR, 1.48; 95% CI, 1.01–2.17) were associated with lymphedema symptoms. The V20 incidence was higher among patients who received NAC for 144 days or longer (HR, 1.79; 95% CI, 1.19–2.68). The V10 incidence was highest in patients with 30 nodes or more removed (HR, 1.70; 95% CI, 1.15–2.52) and increased with number of positive nodes (HR, 1.03; 95% CI, 1.00–1.06). On multivariable analysis, obesity was significantly associated with lymphedema symptoms (HR, 1.03; 95% CI, 1.01–1.06), and NAC length was significantly associated with V20 (HR, 1.74; 95% CI, 1.15–2.62). CONCLUSIONS AND RELEVANCE: In this study, longer NAC duration and obesity were associated with increased lymphedema incidence, suggesting that patients in these groups may benefit from enhanced prospective lymphedema surveillance.

Among mammary tumor-infiltrating immune cells, the highest expression of podoplanin (PDPN) is found in a subset of tumor-associated macrophages (TAMs). We hereby demonstrate that PDPN is involved in the attachment of this TAM subset to lymphatic endothelial cells (LECs). Mechanistically, the binding of PDPN to LEC-derived galectin 8 (GAL8) in a glycosylation-dependent manner promotes the activation of pro-migratory integrin beta1. When proximal to lymphatics, PDPN-expressing macrophages (PoEMs) stimulate local matrix remodeling and promote vessel growth and lymphoinvasion. Anti-integrin beta1 blockade, macrophage-specific Pdpn knockout, or GAL8 inhibition impairs TAM adhesion to LECs, restraining lymphangiogenesis and reducing lymphatic cancer spread. In breast cancer patients, association of PoEMs with tumor lymphatic vessels correlates with incidences of lymph node and distant organ metastasis.

Colon cancer is one of the first tumor types where a functional link between inflammation and tumor onset has been described; however, the microenvironmental cues affecting colon cancer progression are poorly understood. Here we demonstrate that the expression of the ECM molecule EMILIN-1 halts the development of AOM-DSS induced tumors. In fact, upon AOM-DSS treatment the Emilin1(-/-) (E1(-/-)) mice were characterized by a higher tumor incidence, bigger adenomas and less survival. Similar results were obtained with the E933A EMILIN-1 (E1-E933A) transgenic mouse model, expressing a mutant EMILIN-1 unable to interact with alpha4/alpha9beta1 integrins. Interestingly, upon chronic treatment with DSS, E1(-/-) and E1-E933A mice were characterized by the presence of increased inflammatory infiltrates, higher colitis scores and more severe mucosal injury respect to the wild type (E1(+/+)) mice. Since alterations of the intestinal lymphatic network are a well-established feature of human inflammatory bowel disease and EMILIN-1 is a key structural element in the maintenance of the integrity of lymphatic vessels, we assessed the lymphatic vasculature in this context. The analyses revealed that both E1(-/-) and E1-E933A mice displayed a higher density of LYVE-1 positive vessels; however, their functionality was severely compromised after colitis induction. Taken together, these results suggest that the loss of EMILIN-1 expression may cause the reduction of the inflammatory resolution during colon cancer progression due to a decreased lymph flow and impaired inflammatory cell drainage.

Secondary lymphedema is associated with impaired lymph fluid drainage and remains incurable. Alternatively, cell-based therapy may pave the way for lymphedema treatment. We found 11 animal and seven human studies had been conducted from 2008 to 2018. Most studies showed great potential for this treatment modality. Emerging studies have focused on novel techniques, such as coupling cell therapy with lymph node transfer, or adding growth factors to cell therapy.

BACKGROUND: Breast cancer-related lymphedema (BCRL) is hard to control. Management may include lymphatic drainage, skin care, bandaging, or even surgery. Since acupuncture has been proven to affect the neurophysiology and neuroendocrine systems, it has the potential to control BCRL. AIM: To evaluate the effect of acupuncture in BCRL in randomized controlled trials. DESIGN: A literature search was performed, following the PRISMA (Preferred Reporting Items for Systematic Reviews and Meta-Analyses) statement and without language restrictions. DATA SOURCES: Five databases were searched from inception tthrough September 2018. Only studies that fulfilled the eligibility criteria of evaluating the effect of acupuncture on lymphedema in breast cancer were included. The methodological quality of these trials was assessed using the Cochrane criteria, and meta-analysis software (RevMan 5.3) was used for analysis. RESULTS: We examined 178 breast cancer patients from 6 trials. All included randomized controlled trials had medium to high quality, based on the modified Jadad scale. The systematic review showed that acupuncture is safe and has a trend to improve symptoms, but trials did not consistently measure outcomes. The meta-analysis showed that acupuncture produced no significant improvement in the extent of lymphedema as compared with the control intervention (-1.90; 95% confidence interval = -5.39 to 1.59, P = .29). None of the studies reported severe adverse events. CONCLUSIONS: Acupuncture is safe and has a trend to improve the lymphedema related to breast cancer, yet it did not significantly change arm circumference in BCRL. Future studies should include both subjective and objective measurements and large-scale studies are warranted.

BACKGROUND: Near-infrared (NIR) fluorescence imaging with indocyanine green (ICG) has been recently introduced for lymphatic mapping in several tumors. We aimed at investigating whether this technology may improve the intraoperative visualization of lymph nodes during robotic gastrectomy for gastric cancer. METHODS: Between June 2014 and June 2018, a total of 94 patients underwent robotic gastrectomy with D2 lymph node dissection for gastric cancer. In 37 patients, ICG was injected endoscopically into the submucosal layer around the tumor the day before surgery. After propensity score matching, the results of these 37 patients were compared with the results of 37 control patients who had undergone robotic gastrectomy without ICG injection. RESULTS: Among the 37 patients within the ICG group, no adverse events related to ICG injection or intraoperative NIR imaging occurred. After completion of D2 lymph node dissection, no residual fluorescent lymph nodes were left in the surgical field. A mean of 19.4 ± 14.7 fluorescent lymph nodes was identified per patient. The mean total number of harvested lymph nodes was significantly higher in the ICG group than in the control group (50.8 vs 40.1, P = 0.03). In the ICG group, 23 patients had metastatic lymph nodes. The accuracy, sensitivity, and specificity of ICG fluorescence for metastatic lymph nodes were 62.2%, 52.6%, and 63.0%, respectively. CONCLUSION: Our study indicates that NIR imaging with ICG may provide additional node detection during robotic surgery for gastric cancer. Unfortunately, this technique failed to show good selectivity for metastatic lymph nodes.

Although many solid tumors use the lymphatic system to metastasize, there are few treatment options that directly target cancer present in the lymphatic system, and those that do are highly invasive, uncomfortable, and/or have limitations. In this review we provide a brief overview of lymphatic function and anatomy, discusses changes that befall the lymphatics in cancer and the mechanisms by which these changes occur, and highlight limitations of lymphatic drug delivery. We then go on to summarize relevant techniques and new research for targeting cancer populations in the lymphatics and enhancing drug delivery intralymphatically, including intralymphatic injections, isolated limb perfusion, passive nano drug delivery systems, and actively targeted nanomedicine.

Squamous cell carcinoma (SCC) is a unique clinical and histological category that accounts for about 30% of total lung cancer. To identify risk factors for lymph node metastasis and analyze the molecular features of these metastases in lung SCC, a retrospective study was performed for 170 lung SCC patients who underwent surgical treatment. The overall survival of these patients with or without lymph node metastasis (LM/NLM) was analyzed using the Kaplan-Meier method. We also used the TCGA database to compare the differentially expressed genes (DEGs) in patients with stage T1–2 and T3–4 lung SCC. Data from both our retrospective study and the TCGA database demonstrated a correlation between age and stage T1–T2 LM (P = .002). There were significant differences between the LM and NLM groups in both mean survival time and median survival time for different T-stages (P = .031). There were 176 upregulated and 177 downregulated DEGs between the LM and NLM groups in the stage T1–2 group and 93 upregulated and 34 downregulated DEGs in the stage T3–T4 group. These differentially expressed genes were predicted to participate in five cellular components, five molecular functions, and five biological processes. There were 20 genes, including GCG, CASR, NPY, CGA, TAC1, ALB, APOA1, CRH, CHRH, TRH, and GHSR, located at the core of the protein-protein interaction network in the stage T1–2 group and 11 genes, including F2, CASR, GRM1, GNRHR, GRPR, NTSR1, PROKR2, UTS2D, PTH, ALB, and FGA, in the stage T3–4 group. Overall, LM plays a key role in the treatment response and prognosis of SCC patients. Several risk factors, including age and stage, were identified for LM. There was a previously undiscovered enrichment of significant novel genes in lung SCC between the LM and NLM groups, which may have the potential for predicting prognosis and targeting.

BACKGROUND: The Borrmann classification system is used to describe the macroscopic appearance of advanced gastric cancer, and Borrmann type IV disease is independently associated with a poor prognosis. AIM: To evaluate the prognostic significance of lymphatic and/or blood vessel invasion (LBVI) combined with the Borrmann type in advanced proximal gastric cancer (APGC). METHODS: The clinicopathological and survival data of 440 patients with APGC who underwent curative surgery between 2005 and 2012 were retrospectively analyzed. RESULTS: In these 440 patients, LBVI+ status was associated with Borrmann type IV, low histological grade, large tumor size, and advanced pT and pN status. The 5-year survival rate of LBVI+ patients was significantly lower than that of LBVI- patients, although LBVI was not an independent prognostic factor in the multivariate analysis. No significant difference in the prognosis of patients with Borrmann type III/LBVI+ disease and patients with Borrmann type IV disease was observed. Therefore, we proposed a revised Borrmann type IV (r-Bor IV) as Borrmann type III plus LBVI+, and found that r-Bor IV was associated with poor prognosis in patients with APGC, which outweighed the prognostic significance of pT status. CONCLUSION: LBVI is related to the prognosis of APGC, but is not an independent prognostic factor. LBVI status can be used to differentiate Borrmann types III and IV, and the same approach can be used to treat r-Bor IV and Borrmann type IV.

The biology of primary lytic Kaposi's sarcoma-associated herpesvirus (KSHV) infection is still not well understood, which is largely attributed to the lack of cell lines permissive to robust lytic KSHV infection in vitro. Our study demonstrates that primary human dermal lymphatic microvascular endothelial cells (HDLMEC) support lytic KSHV replication following de novo infection, resulting in robust KSHV production, indicating that HDLMECs are suitable for studying the regulation of primary lytic KSHV infection. Importantly, by utilizing lytically infected HDLMECs, we show for the first time that the KSHV latent genes LANA and viral cyclin are required for lytic replication during de novo lytic infection, a function of these latent genes that has not yet been recognized. Since Kaposi's sarcoma is considered to be originated from infected lymphatic endothelial cells, HDLMECs represent a valuable in vitro cell culture model for investigating lytic KSHV infection, which has been understudied in KSHV pathogenesis.

Lymphatic vessels are essential for tissue fluid homeostasis, immune cell trafficking, and intestinal lipid absorption. The lymphatics have long been recognized to serve as conduits for distant tumor dissemination. However, recent findings suggest that the regional lymphatic vasculature also shapes the immune microenvironment of the tumor mass and potentiates immunotherapy. This review discusses the role of lymphatic vessels in tumor metastasis and tumor immunity.

BACKGROUND: Although additive radical surgery is recommended for patients with non-curative endoscopic resection for early gastric cancer (EGC), lymph node (LN) metastasis or remnant tumor is detected in only about 10% of patients. Therefore, we aimed to identify patients who required surgery by identifying significant risk factors for LN metastasis and evaluate long-term outcomes in patients with non-curative endoscopic resection. METHODS: We retrospectively analyzed the database of Seoul National University Hospital to identify patients who underwent endoscopic resection for EGC from June 2005 to December 2016. RESULTS: Three hundred and twenty-nine patients did not meet the criteria for curative resection after endoscopic resection. Among them, 140 patients underwent additional surgery and 171 patients refused surgery and regularly received follow-up. In the surgery group, LN metastasis was found in 12.1% of patients. Logistic regression analysis revealed that the rate of LN metastasis was significantly higher in patients with lymphatic invasion (LI) (odds ratio [OR] 5.84, p = 0.014) and venous invasion (VI) (OR 5.66, p = 0.006). We analyzed LN metastasis based on LI and VI in the surgical group. LN metastasis was significantly increased in the positive LI and VI groups compared with the negative LI and VI groups (OR 68.32; 95% confidence interval, 4.74–984.82; p = 0.002). CONCLUSIONS: Both LI and VI were significant predictors of LN metastasis. The risk of LN metastasis was augmented when both LI and VI were positive. Therefore, LI and VI should be evaluated separately in patients with non-curative endoscopic resection. Additive surgery should be recommended for patients with LI and/or VI.

OBJECTIVE: To compare two published risk stratification models (Milwaukee Model vs. Mayo Criteria) to predict lymphatic dissemination (LD) in endometrioid endometrial cancer (EC). METHODS: Patients with stage I-III EC undergoing surgery from 1/1/2004–9/30/2013 were retrospectively reviewed and classified as low-risk vs at-risk for LD using two independent risk models. LD was defined as positive nodes at surgery or lymph node recurrence within 2years of surgery after negative lymph node dissection (LND) or when LND was not performed. False positive (FP) and false negative (FN) rates for each risk model were calculated. RESULTS: Among 1103 patients, 81 (7.3%) had LD (72 positive LN and 9 LN recurrences), and most (90.2%) had stage I EC. The Milwaukee Model yielded a low at-risk rate for LD (38.1%) but a high FN rate (13.6%, 95% CI 7.0–23.0). The traditional Mayo Criteria using a cut-off of 2cm for tumor diameter (TD) had a higher at-risk rate for LD (69.5%) but a FN rate of 0% (95% CI, 0–4.5). Modifying the Mayo Criteria using a TD cutoff of ≤3cm identified fewer women at-risk (56.8% vs. 69.5%) and had a lower FP rate (53.6% vs. 67.1%), but had a higher FN rate (3.7%, 95% CI, 0.8–10.4). CONCLUSIONS: The Milwaukee Model had the lowest at-risk rate of LD but an unacceptable FN rate. Modifying the Mayo Criteria by increasing the TD cutoff from the traditional ≤2cm to ≤3cm would spare an estimated 13.5% of patients LND, but the accompanying FN rate is unacceptably high. The traditional Mayo Criteria for low-risk EC remains the most sensitive in determining which patients LND can be omitted.

BACKGROUND: Breast cancer survivors are at risk of developing breast cancer-related lymphedema (BCRL) after surgical treatment, which may have a negative effect on quality of life. The purpose of this study was to investigate the clinical role of bioelectrical impedance analysis (BIA) and the relationship between the development of BCRL in breast cancer survivors who have undergone axillary surgery. METHODS: A total of 228 patients with breast cancer were enrolled in the study between May 2016 and January 2017. BCRL was assessed by measuring the circumference of both arms at 15 cm below the acromion process and the olecranon process. Patients were classified as BCRL (n = 22) and non-BCRL (n = 206) based on the difference of the arm circumference of 2 cm. Data including lymphedema, anthropometry, BIA measurements, food frequency questionnaire, type of surgery, total number of dissected lymph nodes, and post-operative treatment were collected. RESULTS: Of the breast cancer survivors, 10.4% had BCRL by the definition. The BCRL group contained 22 patients, while the non-BCRL group contained 206 patients. Compared to the non-BCRL group, the BCRL group had a higher body mass index, a larger percentage of ideal body weight, more dissected lymph nodes, and higher single frequency BIA (SFBIA) ratio (P = 0.027, P = 0.031, P < 0.001, and P < 0.001, respectively). The SFBIA ratio provided 63.64% sensitivity and 95.15% specificity in estimating the risk of BCRL. CONCLUSION: Our data provides evidence to support that the use of SFBIA ratio can serve as an alternative method to monitor and/or diagnose BCRL. TRIAL REGISTRATION: This trial was retrospectively registered at ClinicalTrials.gov identifier (NCT03391206) on the 5 January 2018.

BACKGROUND: The role of TGF-beta1 in lymph node metastasis and lymphangiogenesis, one of the most important steps of gastric cancer dissemination, is largely unknown. The goal of this study was to investigate the role of TGF-beta1 signaling and its molecular mechanisms involved in lymphangiogenesis of gastric cancer. METHODS: Two gastric cell line models, MKN45 and KATOIII, were selected for this study. The protein expression of TGF-beta1 pathway molecules and VEGF-C were examined with western blot, or ELISA according to TGF-beta1 treatment. To explore whether Smad3 binds to the specific DNA sequences in the VEGFC promoter, we performed an electrophoretic mobility shift assay. Lymphatic tube forming assay and gastric cancer xenograft mouse models were also used to elucidate the effect of TGF-beta1 on lymphangiogenesis. RESULTS: TGF-beta1 induced the activation of Smad2/3 and Smad pathway-modulated VEGF-C expression in gastric cancer cell line models. Phosphorylated and activated Smad3 in the nucleus bound to the promoter of VEGFC in KATO III cells. Of note, in MKN45 cells, the Smad-independent AKT pathway was also activated in response to TGF-beta1 and induced VEGF-C expression. Inhibition of TGF-beta1 signaling down-regulated the expression of VEGF-C. We also confirmed, through tube forming assay and tumor xenograft mouse model, that TGF-beta1 increased lymphatic formation, while TGF-beta1 inhibition blocked lymphangiogenesis. CONCLUSION: Smad-dependent and -independent TGF-beta1 pathways induce VEGF-C, which make lymphangiogenesis around tumor. These findings suggest that TGF-beta might be a potential therapeutic target for preventing gastric cancer progression and dissemination.

OBJECTIVE: Breast cancer-related lymphedema is an important health problem. The aim of this study is to ensure early diagnosis of patients at risk of developing lymphedema and revealing the predisposing factors. MATERIALS AND METHODS: Measurements in the pre-operative period and in postoperative months 3, 6, 9 and 12 and years 2 and 3 were performed prospectively with bio-impedance spectroscopy for patients treated for breast cancer between November, 2013 and November, 2016. Demographic and clinical-pathological data of the patients were investigated to assess the factors that affect the development of lymphedema. RESULTS: 245 measurements were obtained from the 67 patients who participated in the study. 18 (26.8%) patients were diagnosed with lymphedema and 16 (89%) of these patients were clinically diagnosed with stage 0 and 2 (1%) patients with stage 1 lymphedema. The median age was 50.7 (32–77) years. Performing axillary dissection and positivity in more than 3 nodes were found to be statistically significant with a percentage of 63.3% (n = 15) and 64.7% (n = 11) p = 0.049 and p < 0.001, respectively. CONCLUSION: Periodic measurements with bio impedance spectroscopy can be an effective method to diagnose early stage lymphedema after breast cancer, and enable selecting the group of patients who would benefit from early treatment.

IMPORTANCE: To our knowledge, no randomized clinical trials have assessed the effects of the combination of weight loss and home-based exercise programs on lymphedema outcomes. OBJECTIVE: To assess weight loss, home-based exercise, and the combination of weight loss and home-based exercise with clinical lymphedema outcomes among overweight breast cancer survivors. DESIGN, SETTING, AND PARTICIPANTS: This randomized clinical trial (Women in Steady Exercise Research [WISER] Survivor clinical trial) of 351 overweight breast cancer survivors with breast cancer-related lymphedema (BCRL) was conducted in conference rooms at academic and community hospitals and in the homes of participants from March 12, 2012, to May 28, 2016; follow-up was conducted for 1 year from the start of the intervention. Statistical analysis by intention to treat was performed from September 26, 2018, to October 28, 2018. INTERVENTIONS: A 52-week, home-based exercise program of strength/resistance training twice per week and 180 minutes of walking per week, a weight loss program of 20 weeks of meal replacements and 52 weeks of lifestyle modification counseling, and a combination of the home-based exercise and weight loss programs. MAIN OUTCOMES AND MEASURES: The 12-month change in the percentage of interlimb volume difference. RESULTS: Of 351 participants, 90 were randomized to the control group (facility-based lymphedema care with no home-based exercise or weight loss intervention), 87 to the exercise intervention group, 87 to the weight loss intervention group, and 87 to the combined exercise and weight loss intervention group; 218 (62.1%) were white, 122 (34.8%) were black, and 11 (3.1%) were of other races or ethnicities. Median time since breast cancer diagnosis was 6 years (range, 1–29 years). Mean (SD) total upper extremity score changes from the objective clinical evaluation were -1.40 (11.10) in the control group, -2.54 (13.20) in the exercise group, -3.54 (12.88) in the weight loss group, and -3.84 (10.09) in the combined group. Mean (SD) overall upper extremity score changes from the self-report survey were -0.39 (2.33) in the control group, -0.12 (2.14) in the exercise group, -0.57 (2.47) in the weight loss group, and -0.62 (2.38) in the combined group. Weight loss from baseline was -0.55% (95% CI, -2.22% to 1.11%) in the control group, -8.06% (95% CI, -9.82% to 6.29%) in the combined group, -7.37% (95% CI, -8.90% to -5.84%) in the weight loss group, and -0.44% (95% CI, -1.81% to 0.93%) in the exercise group. CONCLUSIONS AND RELEVANCE: Study results indicate that weight loss, home-based exercise, and combined interventions did not improve BCRL outcomes; a supervised facility-based program of exercise may be more beneficial than a home-based program for improving lymphedema outcomes. TRIAL REGISTRATION: ClinicalTrials.gov identifier: NCT01515124.

BACKGROUND: Magnetic resonance lymphangiography (MRL) has been proven to be able to visualize pathological lymphatic networks and accompanying complications through subcutaneous injection of commonly used contrast agents. However, no comprehensive prior studies have previously been reported regarding MRL for the evaluation of upper extremity lymphedema in patients with breast cancer-related lymphedema (BCRL). In this study, we establish a novel MRL protocol to characterize the normal and abnormal characteristics of different clinical stages of BCRL in patients using high-spatial-resolution MRL. METHODS: Fifty females with unilateral upper extremity BCRL underwent MRL. Lymphatic vessel morphology in normal and affected limbs was compared. The appearance, distribution pattern, morphologic characteristics, and maximum transversal diameter of the lymphatic vessels, dermal backflow, and regeneration of lymphatic vessels were analyzed. RESULTS: Lymph fluid was retained in the subcutis of the affected limbs, and no edema was observed in the subfascial compartment. In stage 1, tortuous and dilated lymphatic vessels exhibited a beaded appearance, and their diameters were larger than those in the contralateral forearm (P < 0.05). In stage 2, the dilated lymphatic vessels exhibited larger diameters. “Dermal backflow” and tiny regenerated lymphatic vessels appeared. The thickened subcutaneous tissue showed a honeycomb pattern induced by soft tissue fibrosis and adipose hypertrophy. In stage 3, disordered and unrecognizable affected lymphatic vessels were observed with many small regenerated lymphatics and confluent dermal backflow; the tissue fibrosis was more serious. CONCLUSIONS: Each stage presents different characteristics, and the deformity degree of the lymphatic network is consistent with the severity of the disease. Magnetic resonance lymphangiography could provide adequate information for clinical staging in patients with BCRL.

BACKGROUND: To assess the risk of lymphedema associated with use of calcium channel blockers among breast cancer patients. METHODS: A nested case-control study of adult female breast cancer patients receiving an antihypertensive agent was conducted using administrative claims data between 2007 and 2015. Cases were patients with lymphedema who were matched to 5 controls based on nest entry date (±180 days), age (±5 years), number of hypertensive drug classes, Charlson Comorbidity Index (CCI), thiazide exposure, and insurance type. Exposure to calcium channel blockers (CCB) and covariates were identified in the 180-day period prior to event date. Conditional logistic regression was used to assess the impact of exposure among cases and controls. RESULTS: A total of 717 cases and 1681 matched controls were identified. After matching on baseline characteristics, mastectomy (7.8% vs 4.8%; p = 0.0039), exposure to radiation therapy (27.1% vs 21.7%; p = 0.0046), taxane-based chemotherapy (11.7%vs 7.4%; p = 0.0007), anthracycline-based chemotherapy (6.0% vs 3.6%; p = 0.0073) CCB use (28.3% vs 23.3%; p = 0.0087), and CCI (19.8% vs 12.7%; p < 0.0001 [score of 4 or above]) were all higher in cases during the 180 days prior to the event date. In the adjusted analysis, CCB exposure was significantly associated with increased risk of lymphedema (odds ratio: 1.320; 95% confidence interval 1.003 to 1.737). CONCLUSIONS: CCB use was significantly associated with development of lymphedema in breast cancer patients. IMPACT: CCBs should be avoided or used with caution in breast cancer patients to reduce the risk for developing lymphedema.

Tumor-associated lymphangiogenesis correlates with lymph node metastasis and poor outcome in several human malignancies. In addition, the presence of functional lymphatic vessels regulates the formation of tumor inflammatory and immune microenvironments. Although lymphatic structures are often found deeply integrated into the fabric of adipose tissue, the impact of lymphangiogenesis on tumor-associated adipose tissue (AT) has not yet been investigated. Using K14-vascular endothelial growth factor receptor (VEGFR)3-Ig mice that constitutively express soluble VEGFR3-Ig in the skin, scavenging VEGF-C and VEGF-D, the role of lymphangiogenesis in the generation of an inflammatory response within tumor-associated AT was studied. Macrophages expressing lymphatic vessel endothelial hyaluronan receptor-1 were found within peritumoral adipose tissue from melanoma-bearing K14-VEGFR3-Ig mice, which were further enriched with alternatively activated macrophages based on surface marker CD301/C-type lectin domain family 10 member A expression. The blockade of lymphangiogenesis also resulted in accumulation of the cytokine IL-6, which correlated with enhanced macrophage proliferation of the alternatively activated phenotype. Furthermore, melanomas co-implanted with freshly isolated adipose tissue macrophages grew more robustly than melanomas growing alone. In human cutaneous melanomas, adipocyte-selective FABP4 transcripts closely correlated with gene signatures of CLEC10A and were associated with poor overall survival. These data suggest that the blockade of pathways regulating lymphatic vessel formation shapes an inflammatory response within tumor-associated AT by facilitating accumulation of tumor-promoting alternatively activated macrophages.

OBJECTIVE: To evaluate the effect of moxibustion on relieving breast cancer-related lymphedema. MATERIALS AND METHODS: A randomized controlled trial was conducted in our institution from March 2016 to March 2017. All patients (N = 48) with cancer-related lymphedema were allocated to 2 groups: a treatment group, in which moxibustion was performed, and a control group, in which pneumatic circulation was performed with compression garments worn every day. Therapeutic efficacy was evaluated by measuring arm circumference (wrist crease, 10 cm proximal to wrist crease, elbow crease, and 10 cm proximal to elbow crease) and determining the Revised Piper Fatigue Scale score and Visual Analog Scale score for swelling before and after treatment. RESULTS: All patients were treated for 4 consecutive weeks. Compared with 0 week after treatment, the affected-side arm circumference after 4 weeks' treatment decreased in both treatment and control groups; the difference value in the treatment group was superior to that in the control group. Compared with the controls, moxibustion resulted in a lower Visual Analog Scale score. The Revised Piper Fatigue total scores were improved in both the moxibustion and control group, and there was no significant difference between the 2 groups. Moxibustion reduced the behavioral, sensory, emotional, and cognitive Revised Piper Fatigue scores, but only the behavioral and sensory scores improved in the control group. CONCLUSION: Moxibustion has potential effect on breast cancer-related lymphedema. We present promising preliminary data for larger randomized trials to enable accurate evaluation of moxibustion as a lymphedema treatment.

BACKGROUND: Cutaneous melanoma is a highly malignant tumor which tends to metastasize in the early stage and leads to poor prognosis. Hematogenous and lymphatic metastasis are common in the dissemination of melanoma. Rapamycin, an mTOR inhibitor, was reported to have anti-angiogenic and anti-lymphangiogenic properties. AIM: The aim of this study was to investigate if rapamycin can inhibit the formation of blood vessels and lymphatic vessels in melanoma. METHODS: A melanoma xenograft model was generated by subcutaneously transplanting A375 human melanoma cells into the back of immunodeficient mice. Two weeks after cell transplantation, rapamycin was injected intraperitoneally every other day seven times. Then, tumors were harvested. Hematoxylin-eosin (H-E) staining, immunohistochemical staining, Western blot, and quantitative PCR were performed to observe the pathological structure of the tumor, the distribution of blood vessels and lymphatic vessels, and the expression of mTOR signal pathway, VEGF-A/VEGFR-2, and VEGF-C/VEGFR-3. RESULTS: The results showed that CD34(+) blood vessels and LYVE-1(+) lymphatic vessels decreased in the peritumor and intratumor region in rapamycin-treated tumors. Expression of p-4EBP1 and p-S6K1 proteins was downregulated. Expression of both proteins and mRNAs of VEGF-A/VEGFR-2 and VEGF-C/VEGFR-3 was downregulated. CONCLUSION: In conclusion, rapamycin suppresses angiogenesis and lymphangiogenesis in melanoma by blocking the mTOR signal pathway and subsequently downregulating the expression of VEGF-A/VEGFR-2 and VEGF-C/VEGFR-3. Therefore, targeted therapy via mTOR signal pathway may control the hematogenous and lymphatic metastasis of melanoma, and even prolong patients' survival time.

Patients with leg lymphedema sometimes suffer under constraint feeling leg heaviness and pain, requiring lifelong treatment and psychosocial support after surgeries or radiation therapies for gynecologic cancers. We herein review the current issues (a review of the relevant literature) associated with recently developed diagnostic procedures and treatments for secondary leg lymphedema, and discuss how to better manage leg lymphedema. Among the currently available diagnostic tools, indocyanine green lymphography (ICG-LG) can detect dermal lymph backflow in asymptomatic legs at stage 0. Therefore, ICG-LG is considered the most sensitive and useful tool. At symptomatic stage ≥1, ultrasonography, magnetic resonance imaging-lymphography/computed tomography-lymphography (MRI-LG/CT-LG) and lymphosintiography are also useful. For the treatment of lymphedema, complex decongestive physiotherapy (CDP) including manual lymphatic drainage (MLD), compression therapy, exercise and skin care, is generally performed. In recent years, CDP has often required effective multi-layer lymph edema bandaging (MLLB) or advanced pneumatic compression devices (APCDs). If CDP is not effective, microsurgical procedures can be performed. At stage 1–2, when lymphaticovenous anastomosis (LVA) is performed, lymphaticovenous side-to-side anastomosis (LVSEA) is principally recommended. At stage 2–3, vascularized lymph node transfer (VLNT) is useful. These ingenious procedures can help maintain the patient's quality of life (QOL) but unfortunately cannot cure lymphedema. The most important concern is the prevention of secondary lymphedema, which is achieved through approaches such as skin care, weight control, gentle limb exercises, avoiding sun and heat, and elevation of the affected leg.

BACKGROUND: Increasing scientific evidences suggest that body weight is a risk factor for breast cancer-related lymphedema (LE) in breast cancer patients, but many existing studies have yielded inconclusive results. This meta-analysis aims to provide a more precise estimation of the effects of body mass index (BMI) on LE in breast cancer patients. METHODS: Two authors searched independently in the main English-language databases, including PubMed, Embase, and Cochrane Central Register of Controlled Trials, and the main Chinese databases, including China National Knowledge Infrastructure and WanFang Data from inception through June 1, 2018 in human. Odds ratios with 95% confidence interval were calculated to evaluate the effect of BMI on LE. RESULTS: Twelve studies were identified with a total of 8,039 breast cancer patients, including 2102 patients who were suffered from LE; therefore, the total incidence of LE was 26.15%. The meta-analysis results reveal that the odds ratios were 1.42 [95% confidence interval (CI), 1.20 to 1.68] for BMI 25–30 kg/m(2) versus BMI <25 kg/m(2) group, 1.39 (95% CI, 1.21 to 1.60) for BMI ≥30 kg/m(2) versus BMI 25–30 kg/m(2) group, and 1.84 (95% CI, 1.47 to 2.32) for BMI ≥30 kg/m(2) versus BMI <25 kg/m(2) group. CONCLUSIONS: Our results will generate awareness of LE, especially obese patients should pay more attention to LE after breast cancer than overweight patients. Thus, it is necessary and meaningful to distinguish obese from overweight patients.

Malignant tumors are a major threat to human life and high lymphatic vessel density is often associated with metastatic tumors. With the discovery of molecules targeted at the lymphatic system such as lymphatic vessel endothelial hyaluronan receptor 1 (LYVE-1) and Podoplanin, many studies have been performed to determine the role of lymphatic endothelial cells (LECs) in tumor metastasis. However, disadvantages such as non-specificity and high cost limit their research and diagnostic applications. In this study, Fe3O4@KCTS, a core-shell type of magnetic nanoparticles, was prepared by activating Fe3O4 with carbodiimide and cross-linking it with alpha-ketoglutarate chitosan (KCTS). The LYVE-1 and Podoplanin antibodies were then incorporated onto the surface of these magnetic nanoparticles and as a result, dual-targeting magnetic nanoprobes were developed. The experimental tests of this study demonstrated that a dual-targeting magnetic nanoprobe with high-purity LECs from tumor tissues was successfully developed, providing a basis for clinical application of LECs in colorectal cancer treatment as well as in early clinical diagnosis using bimodal imaging.

BACKGROUND: Many studies have explored the prognostic value of T-cell lymphoma invasion and metastasis inducing factor 1 (Tiam1) and its association with lymphatic metastasis in malignant solid tumors, but the conclusions remain controversial. Therefore, we performed a meta-analysis to systematically assess the prognostic value of Tiam1 expression and its association with lymphatic metastasis in malignant solid tumors. METHODS: We searched eligible studies in PubMed, Web of Science and EMBASE databases (from inception up to October 2018). The combined HR with 95% CI was used to estimate the prognostic value of Tiam1 expression. The correlation between Tiam1 expression and lymphatic metastasis was assessed using the combined odds ratio (OR) with 95% CI. RESULTS: A total of 17 studies with 2,228 patients with solid tumors were included in this meta-analysis. The overall estimated results showed that high Tiam1 expression was significantly associated with shorter overall survival (HR = 2.08, 95% CI: 1.62–2.68, P < 0.01), and disease-free survival (HR = 1.86, 95% CI: 1.49–2.32, P < 0.01). Besides, we also found that there was a close relationship between high Tiam1 expression and positive lymphatic metastasis (OR = 2.63; 95% CI: 1.79–3.84, P < 0.01). CONCLUSION: High Tiam1 expression was significantly associated with shorter survival and positive lymphatic metastasis in patients with malignant solid tumors. Therefore, Tiam1 may be a promising prognostic biomarker and an effective therapeutic target for malignant solid tumors.

Lymphedema is a non-curative chronic swelling caused by impairment of the lymphatic system, affecting up to 250 million patients worldwide. The patients suffer from low quality of life because of discomfort and reduced range of motion due to the swelling. Severe swellings can be immediately mediated with special massaging technique known as the Manual Lymphatic Drainage (MLD). Limitations of MLD involves long travel distances, the cost of regular treatment sessions, and the lack of lymphedema specialists. Since MLD is performed very gently, described as caressing a baby's head, soft wearable robotics with its inherent compliance and safety is the perfect solution to creating a light and safe wearable lymphedema massaging device. In this paper, origami-inspired soft fabric pneumatic actuator is developed that creates not only normal force, but also shear force which is essential in the performance of MLD. The shear is created by the unfolding of the Z-shaped fold-lines as the actuator is inflated. One Z-folded actuator module of 30 × 60 mm dimension with a single fold of 15 mm fold height creates maximum shear force of about 1.5 N and stroke displacement of about 30 mm when subjected to compression loading of 5 N. The range of forces exerted can be tuned by varying the tension of the compressive clothing covering the actuators, and the stroke displacement can be varied by changing the parameter of the actuator module itself, such as the fold height and the number of the folds. The modules can also be repeatedly actuated under compressive clothing, and therefore, the developed actuator modules have high potential as a wearable massaging device.

Hereditary hemorrhagic telangiectasia (HHT) is a vascular dysplasia characterized by recurrent and spontaneous epistaxis (nose bleeds), telangiectases on skin and mucosa, internal organ arteriovenous malformations, and dominant autosomal inheritance. Mutations in Endoglin and ACVRL1/ALK1, genes mainly expressed in endothelium, are responsible in 90% of the cases for the pathology. These genes are involved in the transforming growth factor-beta(TGF-beta) signaling pathway. Epistaxis remains as one of the most common symptoms impairing the quality of life of patients, becoming life-threatening in some cases. Different strategies have been used to decrease nose bleeds, among them is antiangiogenesis. The two main angiogenic pathways in endothelial cells depend on vascular endothelial growth factor and fibroblast growth factor (FGF). The present work has used etamsylate, the diethylamine salt of the 2,5-dihydroxybenzene sulfonate anion, also known as dobesilate, as a FGF signaling inhibitor. In endothelial cells, in vitro experiments show that etamsylate acts as an antiangiogenic factor, inhibiting wound healing and matrigel tubulogenesis. Moreover, etamsylate decreases phosphorylation of Akt and ERK1/2. A pilot clinical trial (EudraCT: 2016-003982-24) was performed with 12 HHT patients using a topical spray of etamsylate twice a day for 4 weeks. The epistaxis severity score (HHT-ESS) and other pertinent parameters were registered in the clinical trial. The significant reduction in the ESS scale, together with the lack of significant side effects, allowed the designation of topical etamsylate as a new orphan drug for epistaxis in HHT (EMA/OD/135/18).

Cerebellar malformations are diverse congenital anomalies frequently associated with developmental disability. Although genetic and prenatal non-genetic causes have been described, no systematic analysis has been performed. Here, we present a large-exome sequencing study of Dandy-Walker malformation (DWM) and cerebellar hypoplasia (CBLH). We performed exome sequencing in 282 individuals from 100 families with DWM or CBLH, and we established a molecular diagnosis in 36 of 100 families, with a significantly higher yield for CBLH (51%) than for DWM (16%). The 41 variants impact 27 neurodevelopmental-disorder-associated genes, thus demonstrating that CBLH and DWM are often features of monogenic neurodevelopmental disorders. Though only seven monogenic causes (19%) were identified in more than one individual, neuroimaging review of 131 additional individuals confirmed cerebellar abnormalities in 23 of 27 genetic disorders (85%). Prenatal risk factors were frequently found among individuals without a genetic diagnosis (30 of 64 individuals [47%]). Single-cell RNA sequencing of prenatal human cerebellar tissue revealed gene enrichment in neuronal and vascular cell types; this suggests that defective vasculogenesis may disrupt cerebellar development. Further, de novo gain-of-function variants in PDGFRB, a tyrosine kinase receptor essential for vascular progenitor signaling, were associated with CBLH, and this discovery links genetic and non-genetic etiologies. Our results suggest that genetic defects impact specific cerebellar cell types and implicate abnormal vascular development as a mechanism for cerebellar malformations. We also confirmed a major contribution for non-genetic prenatal factors in individuals with cerebellar abnormalities, substantially influencing diagnostic evaluation and counseling regarding recurrence risk and prognosis.

A 70-year-old male patient was admitted for multiple cutaneous vascular malformations appeared progressively on the face and the trunk for 20 years (Fig. 1). At first sight, a diagnosis of blue rubber bleb nevus syndrome was suggested. Histological analysis of cutaneous lesions revealed cavernous venous malformations. A full-body computed tomography scan (CT-scan) showed three cerebral nodules in the frontal lobe of 7, 4 and 3 mm respectively, several hepatic lesions of the segments IV, V and VII, and a 25 mm para-aortic formation. This article is protected by copyright. All rights reserved.

RATIONALE: Klippel-Trenaunay syndrome (KTS) is a congenital disorder characterized by cutaneous port wine capillary malformations, varicose veins with hemihypertrophy of soft tissue and bone.Pelvic and retroperitoneal vascular malformations have been described up to the 30% of patients with KTS while hemangiomas of the urinary tract have been reported in 6% PATIENT CONCERNS: A 30-year-old man with KTS was referred to our center for primary erectile dysfunction (ED) associated with varicosities of unusual distribution and asymmetry of the lower limbs. Furthermore, he suffered from hypertension and autosomal dominant polycystic kidney disease.During penile prosthesis implantation, a significant intraoperative bleeding (1 liter) due to large scrotal venous malformations and profuse bleeding from both corpora was recorded. One month later, the day after the first prosthesis training session, the patient returned with swelling in the penoscrotal region. A large inguino-scrotal hematoma was drained. There was a complete bilateral dehiscence of corpora cavernosa with a spread venous bleeding in the scrotum. DIAGNOSES: CT scan showed hypertrophy of the right hypogastric artery with severe vascular malformations: the right pudendal artery was massively dilated with early visualization of venous drainage without evidence of arteriovenous fistulae; regular bulbocavernous capillary blush; right upper gluteus artery hypertrophic and dilated. Multiple twisting and aneurysms of the right internal pudenda artery were bleeding from multiple points. Cystoscopy showed a fistula between the proximal urethra and the penoscrotal dartos. Coagulation tests revealed the presence of factor XIII deficiency INTERVENTIONS: The patient underwent several procedures including percutaneous scleroembolization of the internal pudendal arteries, removal of the penile implant, recombinant factor XIII (FXIII) administration, and cord blood platelet gel application. OUTCOMES: The patient was discharged after almost 3 months in hospital, hemodynamically stable. LESSONS: Experience regarding management of ED in KTS patient is limited and in case of concomitant factor XIII deficiency, the clinical scenario can be life-threatening. A multidisciplinary approach including a urologist, an interventional radiologist and a hematologist in our experience represented the key approach in case of severe bleeding following surgery for ED.

The hyper-activation of the phosphoinositide (PI) 3-kinase signaling pathway is a hallmark of many cancers and overgrowth syndromes, and as a result, there has been intense interest in the development of drugs that target the various isoforms of PI 3-kinase. Given the key role PI 3-kinases play in many normal cell functions, there is significant potential for the disruption of essential cellular functions by PI 3-kinase inhibitors in normal tissues; so-called on-target drug toxicity. It is, therefore, no surprise that progress within the clinical development of PI 3-kinase inhibitors as single-agent anti-cancer therapies has been slowed by the difficulty of identifying a therapeutic window. The aim of this review is to place the cellular, tissue and whole-body effects of PI 3-kinase inhibition in the context of understanding the potential for dose limiting on-target toxicities and to introduce possible strategies to overcome these.

The efficacy of lauromacrogol injection therapy and intralesional triamcinolone for infantile hemangiomas (IH) has been well documented recently, but with an increase in serious or rare adverse reactions. The aim of this study is to investigate the safety concerns regarding intralesional injection of lauromacrogol combined with triamcinolone for IH and to study its effect on infant growth and development. A total of 1039 IH patients who were subjected to intralesional injection of lauromacrogol combined with triamcinolone in the Plastic Surgery Department of Shandong Provincial Hospital between 1 January 2015 and 31 May 2018 were enrolled in this study. When the dose of lauromacrogol and triamcinolone was less than 3.5 and 2.0 mg/kg respectively, no serious side-effects were observed. The adverse event rate reported was 7.7%. Among the 405 patients not subjected to propranolol before the last injection, the study included three modes of treatment response: regression (82.7%), stabilization (13.8%) and failure (3.5%). By comparing height and weight to the reference standards and also by comparisons between the same-sex groups, our results confirmed that there was no significant effect on children's height and weight, regardless of whether the injection therapy was combined with oral propranolol at the appropriate dose and with more than 4-week intervals. Intralesional injection of lauromacrogol combined with triamcinolone in the treatment of IH was highly safe and effective.

BACKGROUND: Sturge-Weber syndrome (SWS) is caused by a somatic mutation in GNAQ leading to capillary venous malformations in the brain presenting with various neurological, ophthalmic, and cognitive symptoms of variable severity. This clinical variability makes accurate prognosis difficult. We hypothesized that the greater extent of physical factors (extent of skin, eye, and brain involvement), presence of possible genetic factors (gender and family history), and age of seizure onset may be associated with greater symptom severity and need for surgery in patients with SWS. METHODS: The questionnaire was collected from 277 participants (age: two months to 66 years) with SWS brain involvement at seven US sites. RESULTS: Bilateral brain involvement was associated with both learning disorder and intellectual disability, whereas port-wine birthmark extent was associated with epilepsy and an increased likelihood of glaucoma surgery. Subjects with family history of vascular birthmarks were also more likely to report symptomatic strokes, and family history of seizures was associated with earlier seizure onset. Learning disorder, intellectual disability, strokelike episodes, symptomatic stroke, hemiparesis, visual field deficit, and brain surgery were all significantly associated with earlier onset of seizures. CONCLUSION: The extent of brain and skin involvement in SWS, as well as the age of seizure onset, affect prognosis. Other genetic factors, particularly variants involved in vascular development and epilepsy, may also contribute to neurological prognosis, and further study is needed.

Megalencephaly (MEG) is a developmental abnormality of brain growth characterized by early onset, often progressive, brain overgrowth. Focal forms of megalencephaly associated with cortical dysplasia, such as hemimegalencephaly and focal cortical dysplasia, are common causes of focal intractable epilepsy in children. The increasing use of high throughput sequencing methods, including high depth sequencing to more accurately detect and quantify mosaic mutations, has allowed us to identify the molecular etiologies of many MEG syndromes, including most notably the PI3K-AKT-MTOR related MEG disorders. Thorough molecular and clinical characterization of affected individuals further allow us to derive preliminary genotype-phenotype correlations depending on the gene, mutation, level of mosaicism, and tissue distribution. Our review of published data on these disorders so far shows that mildly activating variants (that are typically constitutional or germline) are associated with diffuse megalencephaly with intellectual disability and/or autism spectrum disorder; moderately activating variants (that are typically high-level mosaic) are associated with megalencephaly with pigmentary abnormalities of the skin; and strongly activating variants (that are usually very low-level mosaic) are associated with focal brain malformations including hemimegalencephaly and focal cortical dysplasia. Accurate molecular diagnosis of these disorders is undoubtedly crucial to more optimally treat children with these disorders using PI3K-AKT-MTOR pathway inhibitors.

Generalized lymphatic anomaly (GLA) is characterized by diffuse or multicentric proliferation of dilated lymphatic vessels resembling common lymphatic malformation, and thoracic lesions can be related to a poor prognosis. Sirolimus, an inhibitor of the mammalian target of rapamycin, is effective against vascular anomalies with few severe adverse drug reactions. Here, we report the case of a patient with intractable hemothorax pleural effusion due to GLA who was treated with sirolimus and experienced disseminated intravascular coagulation. Although a standard treatment for GLA has not been established, pleural fluid might be reduced using the Kampo medicine Eppikajyutsuto.

Kaposiform hemangioendothelioma (KHE) and tufted angioma (TA) primarily occurring in infants are difficult to distinguish. This study evaluated ultrasonography (US) and magnetic resonance imaging (MRI) features of KHE and TA. Pathologically proven TA (n = 21) and KHE (n = 40 [11 KHE + Kasabach-Merritt phenomenon [KMP]]) occurring between January 2015 and December 2017 were reviewed. US (n = 61) and MRI (n = 50) findings were retrospectively evaluated. On US, KHE and TA lesions were subcutaneous, while 40% of KHE exhibited an infiltrative pattern extending into adjacent muscles. Of TA lesions, 42.9% were hyperechoic and 96.15% of KHE lesions exhibited mixed echogenicity. Of TA lesions, 76.2% exhibited well-defined margins and all KHE lesions exhibited ill-defined margins. The depth and vascular density of KHE and KHE + KMP were significantly increased compared with TA. The arterial peak systolic blood flow velocity of KHE + KMP was significantly higher than that in TA and KHE. KHE and KHE + KMP were significantly harder than TA on elastography. 3-D color Doppler revealed branch-shape blood flow for KHE and KHE + KMP lesions; for TA, it revealed a dot-like and striped pattern. Considering MRI findings, KHE and KHE + KMP were more likely to exhibit diffuse heterogeneous enhancement after contrast than TA. KHE was infiltrative and more likely to be thick, hypoechoic, ill-defined, richly vascular and hard than TA on US. KHE lesions were subcutaneous and reticular, with heterogeneous enhancement on MRI. Awareness of these features should prompt radiologists in the differential diagnosis of pediatric masses.

CLAPO syndrome (Capillary vascular malformation of the lower lip, Lymphatic malformations of the head and neck, Asymmetry and Partial or generalized Overgrowth) is a nonfrequent pathology. This syndrome is characterized by the capillary malformation (CM) of the lower lip, a very important clinical sign when diagnosing CLAPO. The aim of our report is to demonstrate that rapamycin could be a reliable and safe targeted therapy in lymphatic malformations (LMs). This drug is useful in reducing the LM's size before final surgical treatment. The clinical and radiological evolution of a patient with CLAPO syndrome is reported in this article, before and after the treatment with rapamycin.

BACKGROUND: The term “intramuscular hemangioma capillary type” (IHCT) refers to a fast-flow vascular lesion that is classified as a tumor, although its phenotype overlaps with arteriovenous malformation (AVM). The purpose of this study was to identify somatic mutations in IHCT. METHODS: Affected tissue specimens were obtained during a clinically indicated procedure. The diagnosis of IHCT was based on history, physical examination, imaging and histopathology. Because somatic mutations in cancer-associated genes can cause vascular malformations, we sequenced exons from 446 cancer-related genes in DNA from 7 IHCT specimens. We then performed mutation-specific droplet digital PCR (ddPCR) to independently test for the presence of a somatic mutation found by sequencing and to screen one additional IHCT sample. RESULTS: We detected somatic mutations in 6 of 8 IHCT specimens. Four specimens had a mutation in MAP2K1 (p.Q58_E62del, p.P105_I107delinsL, p.Q56P) and 2 specimens had mutations in KRAS (p.K5E and p.G12D, p.G12D and p.Q22R). Mutant allele frequencies detected by sequencing and confirmed by ddPCR ranged from 2 to 15%. CONCLUSIONS: IHCT lesions are phenotypically similar to AVMs and contain the same somatic MAP2K1 or KRAS mutations, suggesting that IHCT is on the AVM spectrum. We propose calling this lesion “intramuscular fast-flow vascular anomaly.”

Klippel-Trenaunay syndrome is characterized by a persistent lateral embryonic vein. This so-called marginal vein has a large diameter causing venous stasis and venous hypertension because of the absence of valves along its entire length. The extensive diameter of the vein impedes successful treatment by sclerotherapy. Surgical removal is considered technically challenging for potential severe intraoperative blood loss due to the large perforators to the deep veins. Here we show that endovenous treatments (laser ablation, cyanoacrylate adhesive) might be feasible options in therapy for lateral embryonic veins.

Hemorrhagic hereditary telangiectasia (HHT) type 2 patients have increased activation of the phosphatidylinositol 3-kinase (PI3K) signaling pathway in telangiectasia. The main objective is to evaluate the activation of the PI3K pathway in cutaneous telangiectasia of HHT1 patients. A cutaneous biopsy of a digital hand telangiectasia was performed in seven HHT1 and eight HHT2 patients and compared with six controls. The study was approved by the Clinical Research Ethics Committee of our center. A histopathological pattern with more dilated and superficial vessels that pushed up the epidermis was identified in HHT patients regardless of the type of mutation and was associated with older age, as opposed to the common telangiectasia pattern. The mean proliferation index (Ki-67) was statistically higher in endothelial cells (EC) from HHT1 than in controls. The percentage of positive EC for pNDRG1, pAKT, and pS6 in HHT1 patients versus controls resulted in higher values, statistically significant for pNDRG1 and pS6. In conclusion, we detected an increase in EC proliferation linked to overactivation of the PI3K pathway in cutaneous telangiectasia biopsies from HHT1 patients. Our results suggest that PI3K inhibitors could be used as novel therapeutic agents for HHT.

Lymphatic malformations (LMs) are disfiguring congenital anomalies characterized by aberrant growth of lymphatic vessels. They are broadly categorized histopathologically as macrocystic and microcystic. Although sclerotherapy has shown some success in the treatment of macrocystic malformations, there has been less progress with developing treatment strategies for microcystic malformations. In this study, we characterized lymphatic endothelial cells isolated from lymphatic and lymphaticovenous malformations. When compared to cells from normal lymphatic vessels, we found that the primary cultured malformed cells are morphologically different and also exhibited differences in binding, proliferation, migration and tube formation. Transcriptome analysis identified several genes whose expression was substantially higher in malformed compared to normal lymphatic endothelium, including DIRAS3 and FOXF1. Further analysis of LM tissue samples revealed distinguishing gene expression patterns that could pave the way to understanding the molecular pathogenesis of LMs. Based on gene expression signatures, we propose a new hypothesis that the subtype of localized LMs could be formed because of disruptions in lymph node development.

Infantile hemangioma is one of the most common tumors in infancy. Delivery may be a clue for the trigger of infantile hemangioma formation in the head and face areas. In this study, we tried to plot localization of infantile hemangioma as well as capillary malformation on the head and face, and compared them to identify their characteristics and risk factors. The distribution of 104 lesions in 100 patients with infantile hemangioma was as follows: 32 lesions on the head, 12 on the forehead, 57 on the cheek and three in the jaw area. We could not find a statistically significant correlation of the distribution with three clinical subtypes (superficial, deep and mixed), sex or size of the lesions. However, the lesions in the jaw or chin areas were significantly less frequent than other areas (P = 0.0008 or 0.03, respectively). This tendency was not found in 40 patients with capillary malformation. Mechanical stress to jaw or chin areas may be less than other areas in normal cephalic delivery. Considering the emergence after birth and age-dependent involution of infantile hemangioma, we speculate that physiological events including perinatal hypoxia or mechanical stress during delivery as the trigger of hemangioma formation. Taken together, our results may reveal the contribution of mechanical stress to the trigger of infantile hemangioma, not capillary malformation, and may facilitate clinical differentiation between the two diseases by their localization. Further studies with an increased number of patients will be necessary to validate the finding.

PURPOSE: Hereditary hemorrhagic telangiectasia (HHT), a rare genetic vascular disorder, has been rarely reported in South Korea. We investigated the current prevalence and presenting patterns of genetically confirmed HHT in South Korea. MATERIALS AND METHODS: We defined HHT patients as those with proven mutations on known HHT-related genes (ENG, ACVRL1, SMAD4, and GDF2) or those fulfilling 3 or 4 of the Curacao criteria. A computerized systematic search was performed in PubMed and KoreaMed using the following search term: (“hereditary hemorrhagic telangiectasia” AND “Korea”) OR (“Osler-Weber-Rendu” AND “Korea”). We also collected government health insurance data. HHT genetic testing results were collected from three tertiary hospitals in which the genetic tests were performed. We integrated patient data by analyzing each case to obtain the prevalence and presenting pattern of HHT in South Korea. RESULTS: We extracted 90 cases from 52 relevant articles from PubMed and KoreaMed. An additional 22 cases were identified from the three Korean tertiary hospitals after excluding seven cases that overlapped with those in the published articles. Finally, 112 HHT patients were identified (41 males and 71 females, aged 4–82 years [mean ± standard deviation, 45.3 ± 20.6 years]). The prevalence of HHT in South Korea is about 1 in 500,000, with an almost equal prevalence among men and women. Forty-nine patients underwent genetic testing, of whom 28 had HHT1 (ENG mutation) and 19 had HHT2 (ACVRL1 mutation); the other two patients were negative for ENG, ACVRL1, and SMAD4 mutations. CONCLUSION: The prevalence of HHT is underestimated in Korea. The rate of phenotypic presentation seems to be similar to that found worldwide. Korean health insurance coverage is limited to representative genetic analysis to detect ENG and ACVRL1 mutations. Further genetic analyses to detect HHT3, HHT4, and other forms of HHT should be implemented.

OBJECTIVES: Hereditary hemorrhagic telangiectasia (HHT) is a rare autosomal dominant genetic disorder characterized by pathogenic blood vessel development and maintenance. HHT type 1 (HHT1) and type 2 (HHT2) are caused by variants in endoglin (ENG) and activin receptor-like kinase-1 (ACVRL1), respectively. The aim of this study was to identify the spectrum of pathogenic variants in ENG and ACVRL1 in Austrian HHT families. METHODS: In this prospective study, eight Austrian HHT families were screened for variants in ENG and ACVRL1 by polymerase chain reaction amplification and sequencing of DNA isolated from peripheral blood. RESULTS: Heterozygous variants were identified in all families under study. HHT1 was caused by a novel c.816 + 1G>A splice donor variant, a novel c.1479C>A nonsense (p.Cys493X) variant and a published c.1306C>T nonsense (p.Gln436X) variant in ENG. Variants found in ACVRL1 were novel c.200G>C (p.Arg67Pro) and known c.772G>A (p.Gly258Ser) missense variants in highly conserved residues, a known heterozygous c.100dupT frameshift (p.Cys34Leufs*4) and the known c.1204G>A missense (p.Gly402Ser) and c.1435C>T nonsense (p.Arg479X) variants as causes of HHT2. CONCLUSION: Novel and published variants in ENG (37.5%) and ACVRL1 (62.5%) were exclusively identified as the cause of HHT in an Austrian patient cohort. Identification of novel causative genetics variants should facilitate the development of tailored therapeutical applications in the future treatment of autosomal dominant HHT.

PURPOSE OF REVIEW: Currently, there is no ideal management for orbital lymphatic malformations. Significant advances have been made since the discovery of new agents in the treatment. The purpose of this manuscript is to review the recent evidence on new sclerotherapy agents and systemic medications. RECENT FINDINGS: Traditional sclerosants are OK-432, sodium tetradecyl sulphate and ethanol. More recent developments are the use of doxycycline, bleomycin, and pingyangmycin. Sirolimus as a systemic medication has revolutionized the medical management of lymphatic malformations. Other oral drugs such as propranolol and sildenafil are controversial. Future treatment involves targeting lymphangiogenic pathways including inhibition of vascular endothelial growth factors and the phosphatidylinositol 4,5-bisphosphate 3-kinase catalytic subunit. SUMMARY: The development of new agents allows multimodal management either as monotherapy or combined therapy to achieve better outcomes in this difficult to manage disease.

The treatment of lymphatic anomaly, a rare devastating disease spectrum of mostly unknown etiologies, depends on the patient manifestations (1). Identifying the causal genes will allow for developing affordable therapies in keeping with precision medicine implementation (2). Here we identified a recurrent gain-of-function ARAF mutation (c.640T>C:p.S214P) in a 12-year-old boy with advanced anomalous lymphatic disease unresponsive to conventional sirolimus therapy and in another, unrelated, adult patient. The mutation led to loss of a conserved phosphorylation site. Cells transduced with ARAF-S214P showed elevated ERK1/2 activity, enhanced lymphangiogenic capacity, and disassembly of actin skeleton and VE-cadherin junctions, which were rescued using the MEK inhibitor trametinib. The functional relevance of the mutation was also validated by recreating a lymphatic phenotype in a zebrafish model, with rescue of the anomalous phenotype using a MEK inhibitor. Subsequent therapy of the lead proband with a MEK inhibitor led to dramatic clinical improvement, with remodeling of the patient's lymphatic system with resolution of the lymphatic edema, marked improvement in his pulmonary function tests, cessation of supplemental oxygen requirements and near normalization of daily activities. Our results provide a representative demonstration of how knowledge of genetic classification and mechanistic understanding guides biologically based medical treatments, which in our instance was life-saving.

BACKGROUND: Most patients with phosphoinositide-3-kinase, catalytic, alpha polypeptide (PIK3CA)-related overgrowth spectrum become symptomatic early in life and need treatment before puberty. Recently, the specific inhibition of PIK3CA pathways has been proposed as a therapeutic option for these patients improving their surgical options and quality of life. Alpelisib, a specific alpha fraction inhibitor, has shown promising results. CASE: A 17-year-old girl presented with severe involvement of her external genitalia with a combined vascular malformation in the context of congenital, lipomatous, overgrowth, vascular malformations, epidermal nevi and spinal/skeletal anomalies and/or scoliosis syndrome, needing frequent blood transfusions for anemia due to vaginal bleeding and use of a crutch for walking. After failure of treatment with rapamycin, compassionate treatment with alpelisib was started with excellent response. SUMMARY AND CONCLUSION: PIK3CA inhibitors might become a new option of treatment for PIK3CA-related overgrowth spectrum patients.

Lycium barbarum polysaccharide (LBP) has a variety of pharmacological and biological activities such as anti-inflammatory, antioxidation, anti-apoptosis, immune regulation and other pharmacological effects; however, the effect of LBP on infantile hemangioma (IH) was less reported. Primary human hemangioma endothelial cells (HemECs) were isolated from fresh surgical specimens of patients. HemECs was treated with LBP and the changes in proliferative and apoptotic signaling pathways were investigated by performing cell counting kit-8, cloning formation experiment, in vitro angiogenesis experiment, flow cytometry, Western blot, immunofluorescence, HE stain and real-time quantitative polymerase chain reaction. We found that LBP potently inhibited the proliferation of HemECs and achieved a low-micromolar IC50 (45 and 40 mug/ml, the half maximal inhibitory concentration) value and less angiogenesis, however, the IC50 had no effect on human umbilical vein endothelial cells (HUVECs) viability. LBP treatment induced apoptosis in HemECs, which was supported by positive Annexin-V-FITC staining, the activation of cleaved caspase-3 and Bcl-2-associated X protein (Bax) and the inhibition of B-cell lymphoma/leukemia-2 (Bcl-2). Moreover, the result demonstrated that LBP suppressed the expressions of proliferating cell nuclear antigen (PCNA), Ki67, vascular endothelial growth factor (VEGF), VEGFR2 and phosphoinositide 3-kinase (PI3K)/protein kinase B (Akt) signal pathway. PI3K-specific agonist (IGF-1) had promotive effects on HemECs proliferation, which was reversed by LBP. Our study suggests that the effectiveness of LBP in IHs may be associated with its potent anti-proliferative and apoptotic activities in HemECs. Thus, our findings may provide an effective medicine for IHs treatment.

BACKGROUND: Aquaporins (AQPs) are a family of water channels expressed in various body tissues. Beyond osmotic balance, AQPs have recently been confirmed to be involved in processes related to cancer (tumour proliferation, angiogenesis, etc.). OBJECTIVES: To analyse the presence of these proteins in the endothelium of several vascular tumours, both benign and malignant, in order to establish whether AQPs may be used as a marker or future therapeutic target. MATERIALS AND METHODS: We studied AQP1 expression in 39 patients with vascular tumours, classified into six groups according to ISSVA classification: haemangiomas, benign vascular tumours different from infantile haemangiomas, angiosarcomas, classic Kaposi's sarcoma (KS), and epidemic KS. RESULTS: AQP1 expression was present in 28 of 39 patients, representing 92.9% benign lesions, whereas no expression was found in 72% of malignant lesions. AQP1 expression was associated with benign lesions with an OR of 34.5 (95% CI: 5–250); p < 0.0005, and was most frequently identified with a focal endothelial pattern (38%). A kappa index of 0.823 (95% CI: 0.678–0.971) was determined regarding the patterns of expression overall. CONCLUSION: The expression of AQP1 was greater in benign lesions than malignant lesions and this difference was statistically significant, thus AQP1 expression could serve as a marker for benignity of vascular tumours. In addition, the expression pattern of AQP1 was different according to the type of vascular tumour.

Port wine stain (PWS) is a congenital vascular malformation involving human skin. Approximately 15–20% of children a facial PWS involving the ophthalmic (V1) trigeminal dermatome are at risk for Sturge Weber syndrome (SWS), a neurocutaneous disorder with vascular malformations in the cerebral cortex on the same side of the facial PWS lesions. Recently, evidence has surfaced that advanced our understanding of the pathogenesis of PWS/SWS, including discoveries of somatic genetic mutations (GNAQ, PI3K), MAPK and PI3K aberrant activations, and molecular phenotypes of PWS endothelial cells. In this review, we summarize current knowledge on the etiology and pathology of PWS/SWS based on evidence that the activation of MAPK and/or PI3K contributes to the malformations, as well as potential futuristic treatment approaches targeting these aberrantly dysregulated signaling pathways. Current data support that: (1) PWS is a multifactorial malformation involving the entire physiological structure of human skin; (2) PWS should be pathoanatomically re-defined as “a malformation resulting from differentiation-impaired endothelial cells with a progressive dilatation of immature venule-like vasculatures”; (3) dysregulation of vascular MAPK and/or PI3K signaling during human embryonic development plays a part in the pathogenesis and progression of PWS/SWS; and (4) sporadic low frequency somatic mutations, such as GNAQ, PI3K, work as team players but not as a lone wolf, contributing to the development of vascular phenotypes. We also address many crucial questions yet to be answered in the future research investigations.

BACKGROUND: The objective of this study is to examine the hypothesis that cystic hygroma (CH) with normal karyotype can manifest as a Mendelian inherited trait, and that a genetic similitude with hereditary lymphedema exists. To reach this goal, we investigated the prevalence of genetic variants in angiogenesis and lymphangiogenesis genes in a cohort of euploid fetuses with CH that almost resolved before delivery. A short review of cases from literature is also reported. METHODS AND RESULTS: Five fetuses were screened using a next-generation sequencing approach by targeting 33 genes known to be associated with vascular and lymphatic malformations. The genetic evaluation revealed two novel variants in KDR and KRIT1 genes. CONCLUSION: A review of the literature to date revealed that an association exists between CH and hereditary lymphedema and, similar to lymphedema, CH can be inherited in autosomal recessive and autosomal dominant manner, with the latter most likely associated with a better prognosis. About KDR and KRIT1 genes, no other similar associations are reported in the literature and caution is needed in their interpretation. In conclusion, we thought that a genetic test for the outcome of familial CH could be of enormous prognostic value.

Propranolol is an approved non-selective beta-adrenergic blocker that is first line therapy for infantile hemangioma. Despite the clinical benefit of propranolol therapy in hemangioma, the mechanistic understanding of what drives this outcome is limited. Here, we report successful treatment of pericardial edema with propranolol in a patient with Hypotrichosis-Lymphedema-Telangiectasia and Renal (HLTRS) syndrome, caused by a mutation in SOX18. Using a mouse pre-clinical model of HLTRS, we show that propranolol treatment rescues its corneal neo-vascularisation phenotype. Dissection of the molecular mechanism identified the R(+)-propranolol enantiomer as a small molecule inhibitor of the SOX18 transcription factor, independent of any anti-adrenergic effect. Lastly, in a patient-derived in vitro model of infantile hemangioma and pre-clinical model of HLTRS we demonstrate the therapeutic potential of the R(+) enantiomer. Our work emphasizes the importance of SOX18 etiological role in vascular neoplasms, and suggests R(+)-propranolol repurposing to numerous indications ranging from vascular diseases to metastatic cancer.

BACKGROUND: Kaposiform lymphangiomatosis (KLA) has recently been distinguished as a novel subtype of generalized lymphatic anomaly (GLA) with foci of spindle endothelial cells. All cases of KLA involve multiple organs and have an unfavorable prognosis. However, the molecular pathogenesis is unknown, and there are no useful biomarkers. In the present study, we performed genetic analysis to elucidate the cause of this disease and detect biomarkers for it. METHODS: We performed whole-exome sequencing of DNA samples from leukocytes and a biopsy specimen and analyzed cell-free DNA (cfDNA) from plasma and pleural effusion of patients to identify the NRAS c.182A > G (p.Q61R) mutation using the droplet digital polymerase chain reaction (ddPCR). RESULTS: All KLA patients (patients 1–5) had invasive and aggressive features (hemorrhagic pleural effusions, coagulation disorder, and thrombocytopenia) and characteristic findings of KLA in their pathological examinations. In whole exome sequencing for patient 1, c.182A > G missense variant (p.Q61R) in NRAS was identified in fresh frozen samples of a mass on the left chest wall at a frequency of 5% of total alleles but not in his blood leukocytes. Furthermore, the same mutation was detected in cfDNA isolated from plasma and pleural effusion by using ddPCR. ddPCR analysis of plasma/pleural effusion samples from an additional four KLA patients showed that the same mutation was detected in isolated cfDNA in three of the four, as well as in a tissue sample from one of the three plasma/effusion-positive patients that had been obtained to confirm the mutation. CONCLUSION: These results provide the first evidence that NRAS oncogenic variant was identified in DNA samples from KLA patients from not only two affected lesions but also plasma and pleural effusion.

INTRODUCTION: Lymphatic anomalies (LAs) refer to a group of diseases involving systemic dysplasia of lymphatic vessels. These lesions are classified as cystic lymphatic malformation (macrocystic, microcystic or mixed), generalized lymphatic anomaly, and Gorham-Stout disease. LAs occur mainly in childhood, and present with various symptoms including chronic airway problems, recurrent infection, and organ disorders. Individuals with LAs often experience progressively worsening symptoms with a deteriorating quality of life. Although limited treatment options are available, their efficacy has not been validated in prospective clinical trials, and are usually based on case reports. Thus, there are no validated standards of care for these patients because of the lack of prospective clinical trials. METHODS: This open-label, single-arm, multicenter, prospective study will assess the efficacy and safety of a mammalian target of the rapamycin inhibitor sirolimus in the treatment of intractable LAs. Participants will receive oral sirolimus once a day for 52 weeks. The dose is adjusted so that the nadir concentration remains within 5–15 ng/ml. The primary endpoint is the response rate of radiological volumetric change of the target lesion confirmed by central review at 52 weeks after treatment. The secondary endpoints are the response rates at 12 and 24 weeks, respiratory function, pleural effusion, ascites, blood coagulation parameters, bleeding, pain, quality of life, activities of daily living, adverse events, side effects, laboratory examinations, vital signs, and pharmacokinetic data. RESULTS: This is among the first multicenter studies to evaluate sirolimus treatment for intractable LAs, and few studies to date have focused on the standard assessment of the efficacy for LAs treatment. Our protocol uses novel, uncomplicated methods for radiological assessment, with reference to the results of our previous retrospective survey and historical control data from the literature. CONCLUSIONS: We propose a multicenter study to investigate the efficacy and safety of sirolimus for intractable LAs (SILA study; trial registration UMIN000028905). Our results will provide pivotal data to support the approval of sirolimus for the treatment of intractable LAs.

PURPOSE: To assess the efficacy of sclerotherapy with sodium tetradecyl sulfate (STS; Fibrovein 1%) in superficial periocular venous and lymphatic malformations. METHODS: Eleven patients with low-flow venous and lymphatic malformations with extension predominantly to the eyelids, conjunctiva, and anterior orbit were selected. Sclerotherapy with STS was undertaken as an office-based procedure without any radiological guidance. Injections were repeated every 4 weeks until desired response occurred. Therapeutic effect was assessed objectively by change in the size of the lesions in serial photography. RESULTS: The lesions completely resolved in 4 cases with small eyelid and fornix lesions. In other 7 cases there was partial resolution to less than half of primary size. We did not have any significant complications. CONCLUSION: Sclerotherapy with STS is an easy and effective modality for treatment of venous-lymphatic malformations and can be undertaken as an office-based procedure in lesions which are limited to eyelids and anterior orbit.

BACKGROUND: Capillary malformation-arteriovenous malformation is an autosomal dominant disorder, characterised by capillary malformations and increased risk of fast-flow vascular malformations, caused by loss-of-function mutations in the RASA1 or EPHB4 genes. Around 25% of the patients do not seem to carry a germline mutation in either one of these two genes. Even if other genes could be involved, some individuals may have mutations in the known genes that escaped detection by less sensitive techniques. We tested the hypothesis that mosaic mutations could explain some of previously negative cases. METHODS: DNA was extracted from peripheral blood lymphocytes, saliva or vascular malformation tissues from four patients. RASA1 and EPHB4 coding regions and exon/intron boundaries were analysed by targeted custom gene panel sequencing. A second panel and/or Sanger sequencing were used to confirm the identified mutations. RESULTS: Four distinct mosaic RASA1 mutations, with an allele frequency ranging from 3% to 25%, were identified in four index patients with classical capillary malformation-arteriovenous malformation phenotype. Three mutations were known, one was novel. In one patient, a somatic second hit was also identified. One index case had three affected children, illustrating that the mosaicism was also present in the germline. CONCLUSION: This study shows that RASA1 mosaic mutations can cause capillary malformation-arteriovenous malformation. Thus, highly sensitive sequencing techniques should be considered as diagnostic tools, especially for patients with no family history. Even low-level mosaicism can cause the classical phenotype and increased risk for offspring. In addition, our study further supports the second-hit pathophysiological mechanism to explain the multifocality of vascular lesions in this disorder.

BACKGROUND: Limited data exist about the clinical presentation, ideal therapy and outcomes of patients with hereditary hemorrhagic telangiectasia (HHT) who develop venous thromboembolism (VTE). METHODS: We used the data in the RIETE Registry to assess the clinical characteristics, therapeutic approaches and clinical outcomes during the course of anticoagulant therapy in patients with HHT according to initial presentation as pulmonary embolism (PE) or deep venous thrombosis (DVT). RESULTS: Of 51,375 patients with acute VTE enrolled in RIETE from February 2009 to January 2019, 23 (0.04%) had HHT: 14 (61%) initially presented with PE and 9 (39%) with DVT alone. Almost half (47.8%) of the patients with VTE had a risk factor for VTE. Most PE and DVT patients received low-molecular-weight heparin for initial (71 and 100%, respectively) and long-term therapy (54 and 67%, respectively). During anticoagulation for VTE, the rate of bleeding events (major 2, non-major 6) far outweighed the rate of VTE recurrences (recurrent DVT 1): 50.1 bleeds per 100 patient-years (95%CI: 21.6–98.7) vs. 6.26 recurrences (95%CI: 0.31–30.9; p = 0.020). One major and three non-major bleeding were epistaxis. No patient died of bleeding. One patient died shortly after being diagnosed with acute PE. CONCLUSIONS: During anticoagulation for VTE in HHT patients, there were more bleeding events than VTE recurrences. Most bleeding episodes were non-major epistaxis.

We report a rare pattern of extensive bone abnormalities on the Tc-99m MDP bone scintigraphy in a patient with Gorham disease. This rare condition is the result of vascular and lymphatic channel proliferation in bony structures which induce bone resorption. Our case is a 28-year-old man with a history of biopsy-proven soft tissue hemangioma in the left thigh, encountered with a recent diagnosis of multiple vertebral hemangiomata in the axial skeleton and progressive bony destructions in the pelvis on CT and MRI images, referred for bone scintigraphy. Multiple photopenic hemangiomata were noted on bone scan.

Lymphatic malformations (LM) are rare congenital low flow vascular malformations.(1) The goal of LM management is to maintain functionality, control associated symptoms, and preserve aesthetic integrity. It requires a multidisciplinary approach, considering surgery, sclerotherapy, observation and new, promising medical treatments.(2,3) Overtime, except in case of small well circumscribed lesions, it is obvious that complete surgical removal of the LM is almost always impossible because of infiltrative lesions.(4,5) Moreover, partial excision is classically considered to be associated with the risk of local recurrence.(6) Worsening of LM after surgery, especially occurrence of superficial lymphangiectasia (SL), is sometimes discussed. SL are bothersome for patients because of intermittent swelling, haemorrhage and leakage of lymph from superficial vesicles. The aim with this retrospective study was to assess whether surgery might be a triggering factor for SL occurrence. This article is protected by copyright. All rights reserved.

BACKGROUND/OBJECTIVES: The International Society for the Study of Vascular Anomalies (ISSVA) classification separates vascular anomalies into vascular malformations and vascular tumors. However, misdiagnoses and misperceptions still persist around the use of the term “hemangioma.” We assessed whether the term “haemangioma” (British spelling) was used as part of ISSVA terminology in the literature. METHODS: We searched PubMed for all English-language publications containing the British spelling “haemangioma” in the title or abstract from January 1, 2015 to December 31, 2016. Each paper was judged by two independent reviewers, with conflicts resolved by senior review. RESULTS: By the standard of the 2014 ISSVA classification, 126/195 (64.6%) publications used incorrect terminology for vascular anomalies. This was reduced to 118/195 (60.5%) when using the 2018 ISSVA classification. The most commonly misused terms were cavernous haemangioma (27.1%), haemangioma without further specification (26.3%), and hepatic/liver haemangioma (12.7%). Age was a significant predictor of accuracy of terminology (P = 0.01), with a higher accuracy in children. Correct usage also varied by the site of the vascular anomaly, being highest for lesions of the skin (76.5%) followed by muscle (58.3%), soft tissue (23.5%), bone (21.4%), viscera (7.7%), and eye (0.0%) (P = 0.02). CONCLUSIONS: The term “haemangioma” is frequently used incorrectly by the standards of the 2014 and 2018 ISSVA classifications. Correct terminology is important as the natural history and treatment options vary depending on the type of vascular anomaly.

BACKGROUND: We have recently demonstrated the presence of a NANOG(+)/pSTAT3(+)/OCT4(+)/SOX2(+)/SALL4(+)/CD44(+) embryonic stem cell (ESC)-like subpopulation localized to the endothelium and a NANOG(+)/pSTAT3(+)/SOX2(+)/CD44(+) subpopulation outside of the endothelium, within subcutaneous VM (SCVM) and intramuscular VM (IMVM). We have also shown the expression of components of the renin-angiotensin system (RAS): (pro)renin receptor (PRR); angiotensin converting enzyme (ACE), angiotensin II receptor 1 (ATIIR1) and angiotensin II receptor 2 (ATIIR2), in both SCVM and IMVM. This study investigated whether the ESC-like subpopulations within SCVM and IMVM expressed the RAS. METHODS: Formalin-fixed paraffin-embedded sections of two representative samples from each of the seven SCVM and seven IMVM patients included in our previous studies underwent dual immunofluorescence (IF) immunohistochemical (IHC) staining for ESC marker OCT4 or SOX2 with PRR, ACE, ATIIR1, and ATIIR2. RESULTS: IF IHC staining demonstrated the expression PRR by the OCT4(+) cells on the endothelium and outside the endothelium in SCVM and IMVM. ACE was expressed by the SOX2(+) cells, predominantly in the endothelium in SCVM and IMVM. ATIIR1 was expressed by the SOX2(+) cells on the endothelium and outside the endothelium in SCVM and IMVM. ATIIR2 was expressed by the OCT4(+) endothelium and outside the endothelium in SCVM and IMVM. CONCLUSIONS: Components of the RAS are expressed by ESC-like subpopulations within both SCVM and IMVM. These primitive subpopulations may be a novel therapeutic target by manipulation of the RAS using existing medications.

Long before the classification of vascular anomalies from the International Society for the Study of Vascular Anomalies (ISSVA) provided a framework to differentiate vascular anomalies, otherwise known as vascular birthmarks, it was recognized that patients with such lesions can present with acute life-threatening hemostatic and/or thrombotic complications, as well as chronic long-standing bleeding or thrombotic issues. Scenarios such as a rapidly growing vascular lesion with severe acute thrombocytopenia, a visceral hemorrhagic lesion, a lesion associated with repetitive and painful superficial thrombosis, and cases of unprovoked or post-procedural fatal pulmonary embolism highlight the wide spectrum of manifestations of abnormal coagulation in patients with vascular anomalies. The separation of vascular anomalies into two distinct groups, vascular tumors and vascular malformations, was followed by the characterization that their respective coagulopathies were due to either a derangement of platelets or to a disequilibrium of the patient's coagulation/fibrinolytic process. This configuration of coagulopathies will be the foundation for this two-chapter review series. In the initial review, coagulopathies where thrombocytopenia is the main feature will be characterized, whereas the second review will focus on vascular malformations that have a coagulation disorder secondary to some degree of coagulation consumption and/or fibrinolytic pathway derangement.

Numerous studies have established population pharmacokinetics (PPK) models of sirolimus in various populations. However, a PPK model of sirolimus in Chinese patients with pediatric kaposiform hemangioendothelioma (PKHE) has yet to be established; therefore, this was the purpose of the present study. The present study was a retrospective analysis that utilized the trough concentration data obtained from traditional therapeutic drug monitoring-based dose adjustments. A total of 17 Chinese patients with PKHE from a real-world study were characterized by non-linear mixed-effects modeling. The impact of demographic features, biological characteristics and concomitant medications was assessed. The developed final model was evaluated via bootstrap and a prediction-corrected visual predictive check. A one-compartment model with first-order absorption and elimination was used for modeling of data for PKHE. The typical values of apparent oral clearance (CL/F) and apparent volume of distribution (V/F) in the final model were 3.19 l/h and 165 liters, respectively. Age, alanine transaminase levels and sex were included as significant covariates for CL/F, while the duration of treatment with sirolimus was a significant covariate for V/F. In conclusion, the present study developed and validated the first sirolimus PPK model for Chinese patients with PKHE.

BACKGROUND: Fibroadipose vascular anomaly (FAVA) is a recently-defined vascular malformation often involving the extremities and presenting in childhood. Patients may present to orthopaedic surgeons with pain, swelling, joint contractures, and leg length discrepancy. There is no established therapy or treatment paradigm. We report on outcomes following surgical excision for patients with this condition. METHODS: Between 2007 and 2016, all 35 patients that underwent excision of lower-extremity FAVA were retrospectively reviewed using a combination of medical records, radiologic findings, and telemedicine reviews. RESULTS: Mean age at initial presentation was 12.3 ± 6.8 years. Mean follow-up from time of definitive diagnosis at our institution was 66 months (range: 12 to 161 mo). Mean follow-up after surgery was 35 months (range: 6 to 138 mo). Females were affected more than males (71% vs. 29%). The most common location of FAVA was in the calf (49%), followed by the thigh (40%). The most commonly involved muscle was gastrocnemius (29%), followed by the quadriceps (26%). At latest follow-up after surgery, there was an improvement in the proportion of patients with pain at rest (63% vs. 29%), pain with activity (100% vs. 60%), as well as analgesia use (94% vs. 37%). Fourteen patients (40%) had symptomatic residual disease or recurrence of FAVA requiring further treatment. Six patients (17%) required further surgery and 6 (17%) required further interventional radiologic procedures. Three patients (9%) required eventual amputation for intractable pain and loss of function. Lesions with direct nerve involvement were associated with persistent neuropathic symptoms at latest follow-up (P = 0.002) as well as symptomatic residual disease and/or recurrence requiring further treatment (P = 0.01). Seventeen patients (49%) had 19 preoperative joint contractures. Eighteen of the 19 contractures (95%) had sustained improvement at latest follow-up. CONCLUSIONS: In carefully selected patients, surgical excision of FAVA results in improvement of symptoms. However, symptomatic residual disease and/or recurrence are not uncommon. Direct nerve involvement is associated with a worse outcome. LEVEL OF EVIDENCE: Level IV-case series.

BACKGROUND: Lymphatic malformations (LMs) are congenital low-flow vascular anomalies resulting from abnormal embryogenesis. Clinical researches have shown that rapamycin, a specific inhibitor of mTOR, is effective in treating LMs. It suggests the abnormality of mTOR signal pathway in LMs. METHODS AND RESULTS: From January 2009 to December 2018, 10 patients who accepted the resection of LMs were enrolled into the study. Samples of each subtype of LMs (macrocystic, microcystic, and mixed subtypes) were further investigated. Expression of molecules in mTOR signal pathway-mTORC1, p70 S6, p-p70 S6, elF4EBP1, and p-elF4EBP1-in LMs were investigated by immunohistochemical staining. Location of mTORC1, p70 S6, and elF4EBP1 in LMs were shown by immunofluorescence co-staining. Phosphorylation level of mTOR signal pathway in LMs was examined by Western blotting. Immunohistochemical staining showed the expression of mTORC1, p70 S6, p-p70 S6, eIF4EBP1, and p-eIF4EBP1 in LMs. Immunofluorescence staining further verified the co-expression of mTORC1, p70 S6, and eIF4EBP1 in the lymphatic endothelium of LMs. Western blotting analysis revealed obviously higher phosphorylation level of mTOR signal pathway in LMs than that in normal skins (P < 0.05). CONCLUSIONS: The results showed that the mTOR signal pathway was overactivated in LMs. The study provides compelling evidence for treating LMs or syndromes with lymphatic anomalies by inhibiting mTOR signaling.

BACKGROUND: Arteriovenous malformations (AVMs) are a cardinal feature of hereditary hemorrhagic telangiectasia (HHT). However, whether to treat brain AVMs in patients with HHT remains questionable because of the possible risks. METHODS: We performed a retrospective study of patients with HHT who had been treated for brain AVMs at our institution from January 1, 2003, to December 31, 2016. An institutional database was queried for the phrases “hereditary hemorrhagic telangiectasia” and “HHT,” and those patients who had been treated during the study period were identified. Data were extracted regarding presentation, AVM characteristics, treatment modality, and treatment outcomes. RESULTS: We identified 14 patients (10 males, 4 females) with HHT who had had AVMs (n = 27) from the institutional database. The mean age of the patients was 43 years (range, 2–64). Of the 27 brain AVMs, 13 were Spetzler-Martin grade I, 12 were grade II, and 2 were grade III; none were grade IV or V. Treatment was by microsurgery only (11 AVMs in 10 patients), embolization followed by microsurgery (2 AVMs in 2 patients), and radiosurgery only (12 AVMs in 2 patients). AVM obliteration was achieved in 100% of the patients. No new fixed neurologic deficits developed after treatment of unruptured HHT AVMs. CONCLUSIONS: The risk of treatment of brain AVMs in patients with HHT is quite low for appropriately selected patients with treatment individualized to radiosurgery, microsurgery, or a combination of embolization and microsurgery.

PTEN is a tumor suppressor gene that classically dampens the PI3K/AKT/mTOR growth-promoting signaling cascade. PTEN dysfunction causes dysregulation of this and other pathways, resulting in overgrowth. Cowden syndrome, a hereditary cancer predisposition and overgrowth disorder, was the first Mendelian condition associated with germline PTEN mutations. Since then, significant advances by the research and medical communities have elucidated how clinical phenotypic manifestations result from the underlying germline PTEN mutations. With time, it became evident that PTEN mutations can result in a broad phenotypic spectrum, causing seemingly disparate disorders from cancer to autism. Hence, the umbrella term of PTEN hamartoma tumor syndrome (PHTS) was coined. Timely diagnosis and understanding the natural history of PHTS are vital because early recognition enables gene-informed management, particularly as related to high-risk cancer surveillance and addressing the neurodevelopmental symptoms. Expected final online publication date for the Annual Review of Medicine, Volume 71 is January 27, 2020. Please see http://www.annualreviews.org/page/journal/pubdates for revised estimates.

Kaposiform hemangioendothelioma (KHE) and tufted angioma (TA) are rare infiltrative vascular tumors. Currently, no standard treatment regimens exist for KHE/TA. The purpose of our study was to evaluate the efficacy and safety of topical application of tacrolimus for superficial KHE/TA. We examined six patients with superficial KHE/TA. All patients were treated with tacrolimus 0.1% ointment twice daily for at least 12 months. The response rate was 100%, including three nearly complete remissions. Only one patient experienced local pruritus during treatment. The data constituted an intriguing rationale for clinical trials of topical tacrolimus in the treatment of superficial KHE/TA.

OBJECTIVES: To identify clinical factors associated with complications of periocular infantile hemangioma (IH) and monitor improvement in complication rates post-treatment. METHODS: Retrospective cohort study. Eighty-nine patients diagnosed with periocular IH at a pediatric dermatology clinic of a tertiary care center between 2001 and 2013 were included with parental approval. Parents were interviewed by telephone between July and September of 2015, then again in January 2018 to inquire about ophthalmologic follow-up. Electronic medical records were reviewed from January 2001 through January 2018. RESULTS: Sixty percent of patients demonstrated ocular sequelae, including astigmatism (33%), visual axis obstruction (29%), nasolacrimal duct obstruction (7%), ptosis (4%), amblyopia (3%), and strabismus (1%). Compared with superficial IH, deep and mixed IH had higher odds, 3.4 (P = 0.025) and 3.8 (P = 0.034), respectively, of developing ocular sequelae. All patients with astigmatism prior to involution of IH received systemic therapy, with a significant post-treatment decrease in the proportion of patients with astigmatism (40% to 18%, P = 0.027). Three-quarters of patients experienced complete IH involution by time of enrollment in kindergarten. Fifty-one (57.3%) patients received formal ophthalmologic evaluation confirmed through chart review or phone interview, with average follow-up duration of 51.2 months (range: 1.9, 99.3). CONCLUSION: Deep and mixed IH were more likely to demonstrate ocular complications than superficial IH. Rate of astigmatism decreased with systemic therapy. Our study suggests that patients with periocular IH have a lower rate of amblyopia now compared with the prepropranolol era and emphasizes the importance of early treatment of periocular IH to prevent permanent visual sequelae.