Update April 2020

Hepatic tissue repair plays a critical role in determining the outcome of hepatic ischemia-reperfusion (I/R) injury. Hepatic lymphatics participate in the clearance of dead tissues and contribute to the reparative process after acute hepatic injury; however, it remains unknown whether lymphangiogenesis in response to hepatic inflammation is involved in liver repair. Herein, we determined if hepatic lymphangiogenesis improves liver repair after hepatic I/R injury. Using a mouse model of hepatic I/R injury, we investigated hepatic lymphatic structure, growth, and function in injured murine livers. Hepatic I/R injury enhanced lymphangiogenesis around the portal tract and this was associated with increased expression of pro-lymphangiogenic growth factors including vascular endothelial growth factor (VEGF)-C and VEGF-D. Recombinant VEGF-D treatment facilitated liver repair in association with the expansion of lymphatic vessels and increased expression of genes related to the reparative macrophage phenotype. Treatment with a VEGF receptor 3 (VEGFR3) inhibitor suppressed liver repair, lymphangiogenesis, drainage function, and accumulation of VEGFR3-expressing reparative macrophages. VEGF-C and VEGF-D upregulated expression of genes related to lymphangiogenic factors and the reparative macrophage phenotype in cultured macrophages. These results suggest that activation of VEGFR3 signaling increases lymphangiogenesis and the number of reparative macrophages, both of which play roles in liver repair. Expanded lymphatics and induction of reparative macrophage accumulation may be therapeutic targets to enhance liver repair after hepatic injury.

Next-generation sequencing has enabled patient selection for targeted drugs, some of which have shown remarkable efficacy in cancers that have the cognate molecular signatures. Intriguingly, rapidly emerging data indicate that altered genes representing oncogenic drivers can also be found in sporadic non-malignant conditions, some of which have negligible and/or low potential for transformation to cancer. For instance, activating KRAS mutations are discerned in endometriosis and in brain arteriovenous malformations, inactivating TP53 tumor suppressor mutations in rheumatoid arthritis synovium, and AKT, MAPK, and AMPK pathway gene alterations in the brains of Alzheimer's disease patients. Furthermore, these types of alterations may also characterize hereditary conditions that result in diverse disabilities and that are associated with a range of lifetime susceptibility to the development of cancer, varying from near universal to no elevated risk. Very recently, the repurposing of targeted cancer drugs for non-malignant conditions that are associated with these genomic alterations has yielded therapeutic successes. For instance, the phenotypic manifestations of CLOVES syndrome, which is characterized by tissue overgrowth and complex vascular anomalies that result from the activation of PIK3CA mutations, can be ameliorated by the PIK3CA inhibitor alpelisib, which was developed and approved for breast cancer. In this review, we discuss the profound implications of finding molecular alterations in non-malignant conditions that are indistinguishable from those driving cancers, with respect to our understanding of the genomic basis of medicine, the potential confounding effects in early cancer detection that relies on sensitive blood tests for oncogenic mutations, and the possibility of reverse repurposing drugs that are used in oncology in order to ameliorate non-malignant illnesses and/or to prevent the emergence of cancer.

PURPOSE: Most organs of the human body are supplied with a dense network of blood and lymphatic vessels. However, some tissues are either hypovascular or completely devoid of vessels for proper function, such as the ocular tissues sclera and cornea, cartilage and tendons. Since many pathological conditions are affecting the human sclera, this review is focussing on the lymphangiogenic and hemangiogenic privilege in the human sclera. METHODS: This article gives an overview of the current literature based on a PubMed search as well as observations and experience from clinical practice. RESULTS: The healthy human sclera is the outer covering layer of the eye globe consisting mainly of collagenous extracellular matrix and fibroblasts. Physiologically, the sclera shows only a superficial network of blood vessels and a lack of lymphatic vessels. This vascular privilege is actively regulated by balancing anti- and proangiogenic factors expressed by cells within the sclera. In pathological situations, such as open globe injuries or ciliary body melanomas with extraocular extension, lymphatic vessels can secondarily invade the sclera and the inner eye. This mechanism most likely is important for tumor cell metastasis, wound healing, immunologic defense against intruding microorganism, and autoimmune reactions against intraocular antigens. CONCLUSIONS: The human sclera is characterized by a tightly regulated vascular network that can be compromised in pathological situations, such as injuries or intraocular tumors affecting healing outcomes Therefore, the molecular and cellular mechanisms underlying wound healing following surgical interventions deserve further attention, in order to devise more effective therapeutic strategies.

Arteriovenous malformations (AVMs) are abnormal connections of vessels that shunt blood directly from arteries into veins. Rupture of brain AVMs (bAVMs) can cause life-threatening intracranial bleeding. Even though the majority of bAVM cases are sporadic without a family history, some cases are familial. Most of the familial cases of bAVMs are associated with a genetic disorder called hereditary hemorrhagic telangiectasia (HHT). The mechanism of bAVM formation is not fully understood. The most important advances in bAVM basic science research is the identification of somatic mutations of genes in RAS-MAPK pathways. However, the mechanisms by which mutations of these genes lead to AVM formation are largely unknown. In this review, we summarized the latest advance in bAVM studies and discussed some pathways that play important roles in bAVM pathogenesis. We also discussed the therapeutic implications of these pathways.

BACKGROUND: Kaposiform lymphangiomatosis (KLA) is a rare lymphatic anomaly with significant morbidity and mortality. KLA is characterized by diffuse multifocal lesions comprised of focal areas of “kaposiform” spindled cells accompanying malformed lymphatic channels. The goal of this study was to identify activated signaling pathways in cells isolated from three KLA patients for the purpose of testing new therapies. PROCEDURE: Cells were obtained from the lungs of one patient isolated at autopsy and the spleen of two patients removed in surgery due to disease complications. A protein kinase array was performed on the KLA cell lysates and normal lymphatic endothelial cells. RESULTS: Higher activation of key signaling pathways in the KLA cells, including PRAS40, AKT1/2/3, and ERK-1/2, was identified by protein kinase array and confirmed by Western blot analysis. This indicated a role for highly activated PI3K-AKT and MAPK-ERK-1/2 signaling pathways in KLA cells. Cell proliferation studies assessed PI3K inhibitors (LY294002; BYL719), AKT inhibitor ARQ092, mTOR inhibitor rapamycin, and MAPK inhibitor U0126. These studies demonstrated that PI3K-AKT-mTOR and MAPK signaling are important mediators of KLA cell proliferation. BYL719 and rapamycin were more effective at inhibiting KLA cell proliferation than U0126. CONCLUSIONS: Our studies using cells from KLA patient lesions demonstrate that these cells are highly proliferative and the PI3K-AKT-mTOR and MAPK pathways are promising therapeutic targets. Development and clinical trials of PI3K, AKT, and MAPK inhibitors for cancer treatment and the data in this study lend support for early clinical trials assessing the efficacy of these inhibitors in KLA patients.

AIMS: To investigate the relationship between the ophthalmic and systemic phenotypes in patients with hereditary transthyretin amyloidosis with the S77Y mutation (ATTRS77Y). METHODS: In this cross-sectional study, patients with genetically confirmed ATTRS77Y amyloidosis were enrolled. All patients underwent complete neurological examination, including staging with the Neuropathy Impairment Score (NIS), Polyneuropathy Disability (PND) score; complete cardiological evaluation, including echocardiography, cardiac MRI and/or cardiac scintigraphy and complete ophthalmic evaluation, including slit lamp examination and fundus examination. Ocular ancillary tests (fluorescein and indocyanine green angiography, and anterior segment optical coherence tomography) were performed in cases with abnormal findings. The Kruskal-Wallis test was used for quantitative outcomes and Fisher's exact test for qualitative outcomes. Statistical significance was indicated by p < 0.05 (two tailed). RESULTS: The study sample was composed of 24 ATTRS77Y patients. The mean patient age was 58.4 ± 12.4 years. None of the patients presented with amyloid deposits in the anterior chamber, secondary glaucoma or vitreous amyloidosis. Retinal angiopathy was observed in four patients, complicated with retinal ischaemia in one patient. Conjunctival lymphangiectasia (CL) was detected in 13 patients (54%), associated with perilymphatic amyloid deposits. The presence of CL was statistically associated with more severe neurological disease (NIS = 43.3 ± 31.9 vs 18.9 ± 20.4; PND = 2.6 ± 1.0 vs 1.4 ± 0.7 in patients with and without CL, respectively; both p < 0.05) and amyloid cardiomyopathy (p = 0.002). CONCLUSION: In ATTRS77Y patients, CL is common and could serve as a potential biomarker for severe systemic disease. There were neither anterior chamber deposits, secondary glaucoma nor vitreous deposits in ATTRS77Y patients.

Recently peripheral lymphatic vessels were found to transport HDL from interstitial tissues to the blood circulation during reverse cholesterol transport. This function is critical to the clearance of cholesterol from atherosclerotic plaques. The role of organ-specific lymphatics in modulating HDL transport and composition is, however, incompletely understood. This study aimed to 1) determine the contribution of the lymphatics draining the intestine and liver (which are major sites of HDL synthesis) to total (thoracic) lymph HDL transport, and 2) verify whether the HDLs in lymph are derived from specific organs and are modified during trafficking in lymph. The mesenteric, hepatic, or thoracic lymph duct was cannulated in non-fasted Sprague-Dawley rats and lymph was collected over 5 hours under anaesthesia. Whole lymph and specific lymph lipoproteins were analysed for protein and lipid composition. The majority of thoracic lymph fluid, protein and lipid mass was sourced from the mesenteric, and to a lesser extent, hepatic lymph. Mesenteric and thoracic lymph were both CM/VLDL-rich, whereas hepatic lymph and plasma were HDL-rich. The protein and lipid mass in thoracic lymph HDL was mostly sourced from mesenteric lymph whereas the cholesterol mass was equally sourced from mesenteric and hepatic lymph. HDLs were compositionally distinct across the lymph sources and plasma. The composition of HDL also appeared to be modified during passage from mesenteric and hepatic to thoracic lymph. Overall, this study demonstrates that the lipoproteins in lymph are organ-specific in composition, and the intestine and liver are the main source of HDL in the lymph.

RASopathies caused by germline pathogenic variants in genes that encode RAS pathway proteins. These disorders include neurofibromatosis type 1 (NF1), Noonan syndrome (NS), cardiofaciocutaneous syndrome (CFC), and Costello syndrome (CS), and others. RASopathies are characterized by heterogenous manifestations, including congenital heart disease, failure to thrive, and increased risk of cancers. Previous work led by the NCI Pediatric Oncology Branch has altered the natural course of one of the key manifestations of the RASopathy NF1. Through the conduct of a longitudinal cohort study and early phase clinical trials, the MEK inhibitor selumetinib was identified as the first active therapy for the NF1-related peripheral nerve sheath tumors called plexiform neurofibromas (PNs). As a result, selumetinib was granted breakthrough therapy designation by the FDA for the treatment of PN. Other RASopathy manifestations may also benefit from RAS targeted therapies. The overall goal of Advancing RAS/RASopathy Therapies (ART), a new NCI initiative, is to develop effective therapies and prevention strategies for the clinical manifestations of the non-NF1 RASopathies and for tumors characterized by somatic RAS mutations. This report reflects discussions from a February 2019 initiation meeting for this project, which had broad international collaboration from basic and clinical researchers and patient advocates.

C-type lectin-like receptor 2 (CLEC-2) is considered as a potential drug target in settings of wound healing, inflammation, and infection. A potential barrier to this is evidence that CLEC-2 and its ligand podoplanin play a critical role in preventing lymphatic vessel blood filling in mice throughout life. In this study, this aspect of CLEC-2/podoplanin function is investigated in more detail using new and established mouse models of CLEC-2 and podoplanin deficiency, and models of acute and chronic vascular remodeling. We report that CLEC-2 expression on platelets is not required to maintain a barrier between the blood and lymphatic systems in unchallenged mice, post-development. However, under certain conditions of chronic vascular remodeling, such as during tumorigenesis, deficiency in CLEC-2 can lead to lymphatic vessel blood filling. These data provide a new understanding of the function of CLEC-2 in adult mice and confirm the essential nature of CLEC-2-driven platelet activation in vascular developmental programs. This work expands our understanding of how lymphatic blood filling is prevented by CLEC-2-dependent platelet function and provides a context for the development of safe targeting strategies for CLEC-2 and podoplanin.

Background: The mechanisms of lymphangiogenesis in the cholestatic liver after partial hepatectomy (PH) remain unclear. We aimed to demonstrate the relationship between lymphangiogenesis and liver regeneration after partial hepatectomy in the cholestatic liver. Methods and Results: C57BL/6 mice were subjected to 70% partial hepatectomy only (PH group, n = 20) and 70% partial hepatectomy with temporary common bile duct (BD) obstruction by clipping (BD+PH group, n = 20). Five mice per group were sacrificed at 1, 3, 5, and 7 days after the procedure. The liver function was examined by blood tests, and the liver regeneration rate was assessed by body weight and liver weight. Immunohistochemical staining of lymphatic vessel endothelial hyaluronan receptor-1 (LYVE-1) showed liver lymphangiogenesis. The gene expression of lymphangiogenesis-associated factors (e.g., vascular endothelial growth factor receptor-3 [VEGFR-3]) was examined by a real-time polymerase chain reaction. The liver function in the BD+PH group was worse than that in the PH group on postoperative day 1 (POD1) (aspartate aminotransferase: 6528 ± 1641 U/L vs. 2741 ± 368 U/L, p < 0.05, alanine aminotransferase: 4160 ± 1255 U/L vs. 2315 ± 357 U/L, total bilirubin: 1.36 ± 1.16 mg/dL vs. 0.09 ± 0.01 mg/dL), and the liver regeneration rate in the BD+PH group was worse on POD7 (4.57% vs. 5.91%, p < 0.05). The LYVE-1 expression in Glisson's capsule peaked on POD5 and POD7 in the PH and BD+PH groups, respectively. The peak gene expression of VEGFR-3 in the BD+PH group was delayed in comparison with the PH group. Conclusions: Lymphangiogenesis after partial hepatectomy in the cholestatic liver was suggested to be delayed due to impaired liver regeneration and the late expression of VEGFR-3.

Mutations that activate the RAS oncoproteins are common in cancer. However, aberrant upregulation of RAS activity often occurs in the absence of activating mutations in the RAS genes due to defects in RAS regulators. It is now clear that loss of function of Ras GTPase-activating proteins (RasGAPs) is common in tumors, and germline mutations in certain RasGAP genes are responsible for some clinical syndromes. Although regulation of RAS is central to their activity, RasGAPs exhibit great diversity in their binding partners and therefore affect signaling by multiple mechanisms that are independent of RAS. The RASSF family of tumor suppressors are essential to RAS-induced apoptosis and senescence, and constitute a barrier to RAS-mediated transformation. Suppression of RASSF protein expression can also promote the development of excessive RAS signaling by uncoupling RAS from growth inhibitory pathways. Here, we will examine how these effectors of RAS contribute to tumor suppression, through both RAS-dependent and RAS-independent mechanisms.

There is limited information about denosumab-related osteonecrosis of the jaw (DRONJ), unlike bisphosphonate-related ONJ (BRONJ). The mode of action is clearly different between denosumab and bisphosphonates. DRONJ occurs mainly following tooth extraction in cancer patients treated with the combination of denosumab and other drugs including chemotherapy. However, DRONJ animal models similar to these clinical situations have not been developed. The aims of this study were to 1) create a new model of high-prevalence chemotherapy/anti-RANKL antibody-related ONJ-like lesions to mimic patients receiving a high-dose denosumab/chemotherapy combination; and 2) compare the histopathological and immunopathological findings in the early stages of BRONJ-like and anti-RANKL antibody-related ONJ-like lesions. Cyclophosphamide (CY) and anti-mouse RANKL monoclonal antibody (mAb) or zoledronate combination therapy (CY/mAb and CY/ZA, respectively) was performed to create ONJ-like lesions in female C57BL/6J mice. Both maxillary first molars were extracted at 3weeks after drug administration. The animals were euthanized at either 2 or 4weeks after tooth extraction. Increased necrotic bone and empty lacunae with decreased living bone and osteocyte numbers were common histopathological findings in CY/mAb- and CY/ZA-induced impaired wound healing at 4weeks after tooth extraction, and they were diagnosed as ONJ-like lesions based on validation of BRONJ and DRONJ in humans. In areas of impaired healing at 2weeks post-extraction, decreases in angiogenesis and F4/80(+)LYVE-1(-) macrophages were noted as common immunopathological findings, although anti-angiogenesis was worse with CY/mAb than with CY/ZA. Interestingly, CY/mAb did not reduce F4/80(+)LYVE-1(+) cells and normal lymphangiogenesis remained, whereas CY/ZA profoundly suppressed the larger size of F4/80(+)LYVE-1(+) cells, similar to vessels with a concomitant decrease in lymphangiogenesis. Therefore, the distribution of the larger size of F4/80(+)LYVE-1(+) cells differed in the early stages between different antiresorptive-induced ONJ-like lesions in conjunction with lymphangiogenesis, although the histopathological findings were similar. These findings suggest that the pathogenesis of BRONJ and DRONJ may differ due to the distributions of F4/80(+)LYVE-1(+) tube-like-structured cells.

The mammalian target of rapamycin (mTOR) inhibitor sirolimus is an effective treatment for difficult-to-treat lymphatic anomalies. However, little is known about the expression of mTOR pathway components in lymphatic anomalies. Here we investigated the expression pattern of mTOR pathway components and their phosphorylated forms (mTOR, p-mTOR, 4EBP1, p-4EBP1, S6K1 and p-S6K1) in normal lymphatic vessels and lymphatic anomalies using immunohistochemistry. We studied 18 patients of lymphatic anomalies, including lymphatic malformation (LM, n = 14), Kaposiform lymphangiomatosis (KLA, n = 2) and Kaposiform hemangioendothelioma (KHE, n = 2). Normal lymphatic vessels expressed 4EBP1, S6K1 and p-S6K1, but not p-4EBP1, mTOR or p-mTOR. The mTOR was detected in all lymphatic anomalies, whereas its activation form p-mTOR was detected in half cases of KLA and KHE but not in LM. All lymphatic anomalies expressed S6K1 and its activated form p-S6K1. The expression of 4EBP1 was also found in all lymphatic anomalies, but its activation was detected in approximately half of them. The activation of mTOR was seen in tumor (KLA and KHE) but not in malformation (LM), whereas the activation of S6K1 and 4EBP1 was seen in all and half of lymphatic anomalies, respectively.

The influx and efflux of cells and antigens to and from the draining lymph nodes largely take place through the subcapsular, cortical and medullary sinus systems. Recent analyses in mice and humans have revealed unexpected diversity in the lymphatic endothelial cells, which form the distinct regions of the sinuses. As a semipermeable barrier, the lymphatic endothelial cells regulate the sorting of lymph-borne antigens to the lymph node parenchyma and can themselves serve as antigen-presenting cells. The leukocytes entering the lymph node via the sinus system and the lymphocytes egressing from the parenchyma migrate through the lymphatic endothelial cell layer. The sinus lymphatic endothelial cells also orchestrate the organogenesis of lymph nodes, and they undergo bidirectional signalling with other sinus-resident cells, such as subcapsular sinus macrophages, to generate a unique lymphatic niche. In this Review, we consider the structural and functional basis of how the lymph node sinus system coordinates immune responses under physiological conditions, and in inflammation and cancer.

Raloxifene hydrochloride (RH) suffers from low oral bioavailability due to its low water-solubility and first-pass metabolism. Therefore, a novel phospholipid complex of RH (RHPC) and a matrix dispersion based on phospholipid complex (RHPC-MD) were successfully prepared and optimized. Several methods were used to validate the formation of RHPC and RHPC-MD, such as differential scanning calorimetry, X-ray diffraction, scanning electron microscopy, transmission electron microscopy, infrared spectroscopy, particle size, and zeta potential, meanwhile, their octanol-water partition coefficient, solubility, and dissolution in vitro were also evaluated. To investigate the absorption mechanism of RHPC in vivo, the RHPC was administered to the chylomicron flow blockage rat model. Interestingly, as we expected, a significant reduction in RHPC absorption (67%) (**p < .01) in presence of cycloheximide (CXI) inhibitor was observed, thus confirming the RHPC could be absorbed by lymphatic transport in vivo. Pharmacokinetic studies revealed that the relative oral bioavailability of RHPC as well as RHPC-MD was 223% and 329%, respectively, when comparing with the commercial RH tablets. These outcomes suggested that the current study provided an attractive formulation to enhance the oral bioavailability of RH and stimulated to further research the absorption mechanism of RHPC in vivo.

We investigated the distribution and formation of new lymphatic vessels in gliomas. Specimens from seven glioma cases were analyzed by immunohistochemical staining for CD34, lymphatic endothelial hyaluronic acid receptor 1 (LYVE-1), prospero-related homeobox 1 (Prox1), nestin, and hypoxia-inducible factor 1alpha (HIF-1alpha). Three types of vessels were observed in glioma specimens: LYVE-1(+) lymphatic vessels, CD34(+) blood vessels, and LYVE-1(+)/CD34(+) blood vessels. Prox1(+)/LYVE-1(+) cells were distributed in some lymphatic vessels as well as among vascular endothelial cells and glioma cells. Nestin(+) cells were scattered throughout the gliomas, and some lymphatic cells also expressed nestin. HIF-1alpha(+) Prox1(+) cells were widely distributed within the glioma specimens. The present immunohistochemical analysis revealed upregulation of Prox1 and HIF-1alpha in some glioma tissues as well as the differentiation of nestin(+) tumor stem cells into LYVE-1(+) lymphatic vessels.

The majority of lymph generated in the body is returned to the blood circulation via the lymphovenous junction (LVJ) of the thoracic duct (TD). A lymphovenous valve (LVV) is thought to guard this junction by regulating the flow of lymph to the veins and preventing blood from entering the lymphatic system. Despite these important functions, the morphology and mechanism of this valve remains unclear. The aim of this study was to investigate the anatomy of the LVV of the TD. To do this, the TD and the great veins of the left side of the neck were harvested from 16 human cadavers. The LVJs from 12 cadavers were successfully identified and examined macroscopically, microscopically, and using microcomputed tomography. In many specimens, the TD branched before entering the veins. Thus, from 12 cadavers, 21 LVJs were examined. Valves were present at 71% of LVJs (15/21) and were absent in the remainder. The LVV, when present, was typically a bicuspid semilunar valve, although the relative size and position of its cusps were variable. Microscopically, the valve cusps comprised luminal extensions of endothelium with a thin core of collagenous extracellular matrix. This study clearly demonstrated the morphology of the human LVV. This valve may prevent blood from entering the lymphatic system, but its variability and frequent absence calls into question its utility. Further structural and functional studies are required to better define the role of the LVV in health and disease.

PURPOSE: Although there is no lymphatic system in the central nervous system (CNS), there seems to be a mechanism to remove macro molecules from the brain. Cerebrospinal fluid (CSF) and interstitial fluid (ISF) are thought to be parts of this pathway, but the details are not known. In this study, MR signal of the extracellular water was decomposed into components with distinct T2's, to obtain some information about distribution of waste material in the brain. METHODS: Images were acquired using a Curr, Purcell, Meiboom, Gill (CPMG) imaging sequence. In order to reduce T1 contamination and the signal oscillation, hard pulses were used as refocusing pulses. The signal was then decomposed into many T2 components using non-negative least squares (NNLS) in pixel-by-pixel basis. Finally, a color map was generated by assigning different color for each T2 component, then adding them together. RESULTS: From the multi-echo images, it was possible to decompose the decaying signal into separate T2 components. By adjusting the color table to create the color map, it is possible to visualize the extracellular water distribution, as well as their T2 values. Several observation points include: (1) CSF inside ventricles has very long T2 (∼2 s), and seems to be relatively homogeneous, (2) subarachnoid CSF also have long T2, but there are short T2 component at the brain surface, at the surface of dura, at the blood vessels in the subarachnoid space, etc., (3) in the brain parenchyma, short T2 components (longer than intracellular component but shorter than CSF) exists along the white matter, in the choroid plexus, etc. These can be considered as distribution of macromolecules (waste materials) in the brain. CONCLUSION: From T2 component analysis it is possible to obtain some insight into pathways for the transport of large molecules in the CNS, where no lymphatic system is present.

We investigated the pathogenesis of skin lesions due to hypothyroidism and hyperthyroidism in rats. We used 30 rats allocated into hypothyroidism, hyperthyroidism and control groups. Blood samples were evaluated for levels of thyroid stimulating hormone (TSH), tri-iodothyronine (T3) and thyroxine (T4). Skin samples were examined for melan-A, lymphatic vessel endothelial hyaluronic acid receptor 1 (LYVE1), cluster of differentiation 31 (CD31), protein gene product 9.5 (PGP9.5), calretinin, chromogranin, synaptophysin and pancytokeratin. Histopathological examination of the skin sections revealed thickened epidermis in the hyperthyroidism group due to an increased number of cells, and a decreased number of hair follicles and epithelial cell rows in the epidermis with an increased number of fat cells in the dermis of the rats in the hypothyroidism group. No significant difference was observed in the immunoreactions of pancytokeratin, PGP9.5, CD31 and synaptophysin among the groups. The hyperthyroidism and hypothyroidism groups exhibited a marked increase in melan-A immunoreaction. Expression of LYVE-1, chromogranin and calretinin was increased in the hyperthyroidism group and decreased in the hypothyroidism group. We found that melan-A, LYVE-1, chromogenin and calretinin play an important role in the pathogenesis of skin lesions caused by thyroid disorders.

Lymphatic vessels serve as conduits through which immune cells traffic. Because lymphatic vessels are also involved in lipid transport, their function is vulnerable to abnormal metabolic conditions such as obesity and hyperlipidemia. Exactly how these conditions impact immune cell trafficking, however, is not well understood. Here, we found higher numbers of LYVE-1-positive lymphatic endothelial cells and CD3-positive T cells in the lymph nodes of mice fed high-cholesterol or high-fat diets compared with those of mice fed a normal chow diet. To confirm the effect of fat content on immune cell trafficking, the lymphatic endothelial SVEC4–10 cell line was treated with palmitic acid at a 100 muM concentration. After 24 h, palmitic acid-treated cells exhibited increased expression of podoplanin and vascular growth-associated molecules (VEGFC, VEGFD, VEGFR3, and NRP2) and enhanced tube formation. Microarray analysis showed an increase in pro-inflammatory cytokine and chemokine transcription after palmitic acid treatment. Finally, transwell migration assay confirmed that T cell line moved toward medium previously cultured with palmitic acid-treated SVEC4–10 cells. Together, our results suggest that hyperlipidemia drives lymphatic vessel remodeling and T cell migration toward lymphatic endothelial cells.

OBJECTIVE: Impaired ALK1 (activin receptor-like kinase-1)/Endoglin/BMP9 (bone morphogenetic protein 9) signaling predisposes to arteriovenous malformations (AVMs). Activation of SMAD1/5 signaling can be enhanced by shear stress. In the genetic disease hereditary hemorrhagic telangiectasia, which is characterized by arteriovenous malformations, the affected receptors are those involved in the activation of mechanosensitive SMAD1/5 signaling. To elucidate how genetic and mechanical signals interact in arteriovenous malformation formation, we sought to identify targets differentially regulated by BMP9 and shear stress. Approach and Results: We identify Cx37 (Connexin37) as a differentially regulated target of ligand-induced and mechanotransduced SMAD1/5 signaling. We show that stimulation of endothelial cells with BMP9 upregulated Cx37, whereas shear stress inhibited this expression. This signaling was SMAD1/5-dependent, and in the absence of SMAD1/5, there was an inversion of the expression pattern. Ablated SMAD1/5 signaling alone caused arteriovenous malformation-like vascular malformations directly connecting the dorsal aorta to the inlet of the heart. In yolk sacs of mouse embryos with an endothelial-specific compound heterozygosity for SMAD1/5, addition of TNFalpha (tumor necrosis factor-alpha), which downregulates Cx37, induced development of these direct connections bypassing the yolk sac capillary bed. In wild-type embryos undergoing vascular remodeling, Cx37 was globally expressed by endothelial cells but was absent in regions of enlarging vessels. TNFalpha and endothelial-specific compound heterozygosity for SMAD1/5 caused ectopic regions lacking Cx37 expression, which correlated to areas of vascular malformations. Mechanistically, loss of Cx37 impairs correct directional migration under flow conditions. CONCLUSIONS: Our data demonstrate that Cx37 expression is differentially regulated by shear stress and SMAD1/5 signaling, and that reduced Cx37 expression is permissive for capillary enlargement into shunts.

PURPOSE OF REVIEW: Endothelial cells are of great importance in many types of diseases including the coronary artery diseases in heart and stroke in brain. In this review, we explore the heterogeneity among endothelial cells from an organism-wide, organ-specific, and healthy versus disease perspective. RECENT FINDINGS: Recent studies addressing the cellular heterogeneity between arterial versus venous endothelial cells (ECs) have revealed that arterial ECs have tighter junctions, a decreased immune response, anticoagulant properties while veins have both anticoagulant and procoagulant properties. Blood and lymphatic ECs are quite distinct from each other as well, with the lymphatic ECs being more involved in the immune response and lymphangiogenesis while blood vessel ECs being involved in angiogenesis and maintenance of perfusion throughout the body. ECs from various organs such as the heart, the lung, and especially the brain are quite heterogeneous and provide barriers that prevent small particles to pass through the endothelium when compared with the endothelium of the liver and the kidney that are quite porous. The heart ECs have higher angiogenesis and metabolic rates (oxidation and glycolysis) than lung, liver, and kidney ECs. Ex vivo liver and kidney ECs grow at a moderate pace, while the lung and brain ECs grow very slowly. ECs from within a tumor have fenestrae and large intracellular gaps and junctions leading to increased permeability and tumor cell overgrowth. There is a large degree of heterogeneity among organism-wide and organ-specific ECs as well as between healthy and disease-specific ECs. We believe this review will help highlight the EC heterogeneity and further advance our ability to treat cardiovascular disease and other conditions.

Evidence overwhelmingly suggests that the lymphatics play a critical role in the clearance of cerebrospinal fluid (CSF) from the cranial space. Impairment of CSF outflow into the lymphatics is associated with a number of pathological conditions including spaceflight-associated neuro-ocular syndrome (SANS), a problem that limits long-duration spaceflight. We used near-infrared fluorescence lymphatic imaging (NIRFLI) to dynamically visualize the deep lymphatic drainage pathways shared by CSF outflow and disrupted during head-down tilt (HDT), a method used to mimic the cephalad fluid shift that occurs in microgravity. After validating CSF clearance into the lymph nodes of the neck in swine, a pilot study was conducted in human volunteers to evaluate the effect of gravity on the flow of lymph through these deep cervical lymphatics. Injected into the palatine tonsils, ICG was imaged draining into deep jugular lymphatic vessels and subsequent cervical lymph nodes. NIRFLI was performed under HDT, sitting, and supine positions. NIRFLI shows that lymphatic drainage through pathways shared by CSF outflow are dependent upon gravity and are impaired under short-term HDT. In addition, lymphatic contractile rates were evaluated from NIRFLI following intradermal ICG injections of the lower extremities. Lymphatic contractile activity in the legs was slowed in the gravity neutral, supine position, but increased under the influence of gravity regardless of whether its force direction opposed (sitting) or favored (HDT) lymphatic flow toward the heart. These studies evidence the role of a lymphatic contribution in SANS.

Telocytes (TCs) have recently emerged as a peculiar type of stromal cells located in both perivascular and interstitial compartments of multiple anatomical sites in humans, other mammals and vertebrates. Pioneer electron microscopy studies have ultrastructurally defined TCs as “stromal cells with telopodes” (i.e., very long and thin cell processes with a moniliform morphology conferred by the irregular alternation of slender segments and small, bead-like, dilated portions), whereupon it has become apparent that TCs largely correspond to the CD34+ stromal/interstitial cells detectable by immunohistochemical assays. Besides CD34, TCs are also characterized by the expression of platelet-derived growth factor receptor (PDGFR)alpha. Interestingly, recent works recommended that lymphatic endothelial cell (LEC) markers should be routinely assessed to discriminate with certainty TCs from LECs, because these two cell types may exhibit similar morphological traits, especially when initial lymphatics are sectioned longitudinally and appear as vascular profiles with no obvious lumen. Furthermore, it has been argued that lymphatic microvessels immunostained for the small mucin-type transmembrane glycoprotein podoplanin (PDPN), which is widely used as lymphatic endothelial marker, can be easily misidentified as TCs. Nevertheless, surprisingly these assumptions were not based on double tissue immunostaining for TC and LEC markers. Therefore, the present morphological study was undertaken to precisely investigate the mutual spatial organization and putative relationships of TCs and lymphatic vessels in tissues from different human organs. For this purpose, we carried out a series of double immunofluorescence analyses simultaneously detecting the CD34 or PDGFRalpha antigen and a marker of LECs, either PDPN or lymphatic vessel endothelial hyaluronan receptor-1 (LYVE-1). In the connective tissue compartment of different organs, TCs were CD34+/PDGFRalpha+/PDPN-/LYVE-1- while LECs were CD34-/PDGFRalpha-/PDPN+/LYVE-1+, thus representing two definitely distinct, though spatially close, cell entities. The arrangement of telopodes to intimately surround the abluminal side of LECs suggests a possible role of TCs in the regulation of lymphatic capillary functionality, which is worth investigating further.

Lymphatic vessel endothelial hyaluronan receptor 1 (LYVE-1) mediates the docking and entry of dendritic cells to lymphatic vessels through selective adhesion to its ligand hyaluronan in the leucocyte surface glycocalyx. To bind hyaluronan efficiently, LYVE-1 must undergo surface clustering, a process that is induced efficiently by the large crosslinked assemblages of glycosaminoglycan present within leucocyte pericellular matrices, but is induced poorly by the shorter polymer alone. These properties suggested that LYVE-1 may have limited mobility in the endothelial plasma membrane, but no biophysical investigation of these parameters has been carried out to date. Here, using super-resolution fluorescence microscopy and spectroscopy combined with biochemical analyses of the receptor in primary lymphatic endothelial cells, we provide first evidence that LYVE-1 dynamics are indeed restricted by the submembranous actin network. We show that actin disruption not only increases LYVE-1 lateral diffusion but also enhances hyaluronan binding activity. However, unlike the related leucocyte HA receptor CD44 which uses ERM and ankyrin motifs within its cytoplasmic tail to bind actin, LYVE-1 displays little if any direct interaction with actin as determined by co-immunoprecipitation. Instead, as shown by super-resolution stimulated emission depletion (STED) microscopy in combination with fluorescence correlation spectroscopy, LYVE-1 diffusion is restricted by transient entrapment within submembranous actin corrals. These results point to an actin-mediated constraint on LYVE-1 clustering in lymphatic endothelium that tunes the receptor for selective engagement with hyaluronan assemblages in the glycocalyx that are large enough to cross-bridge the corral-bound LYVE-1 molecules and thereby facilitate leucocyte adhesion and transmigration.

Background: Acquired lymphedema is a common consequence of cancer surgery. Fibrosis is one of the main causes of chronic lymphedema since it hinders lymphatic regeneration and this causes a significant decrease in lymphatic flow and accumulation of excessive protein-rich fluid. The transforming growth factor-beta1 (TGF-beta1) signaling pathway is known in a process of wound repair and fibrosis. In our study, the purpose was to evaluate the efficacy of EW-7197, a peroral TGF-beta type I receptor kinase inhibitor, in treating acquired lymphedema. Methods and Results: For lymphedema mouse tail model, we used 10- to 12-week-old female C57BL/6 mice. The skin was circumferentially excised, making a circular band followed by cauterization of lymphatic collecting vessels. Two groups were made in this study: control and treatment. The treatment group (n = 12) received a solution consisting of 0.1 mL of artificial gastric juice and 20 mg/kg EW-7197 by gavage once daily. For evaluation, tail diameter measurement, fluorescence lymphography, and immunofluorescence images were used. EW-7197 treatment ameliorates acquired lymphedema in a mouse tail model by increasing lymphangiogenesis and interstitial flow of the lymphatics by inhibition of the fibrosis. The differences in maximal tail thicknesses between the control and treatment groups were statistically significant from 2 to 4 weeks after surgery. The treatment group showed a greater number of lymphatic vessels at the surgery site than the control group. The treatment group also showed more FITC coverage area at the surgery site. Conclusion: EW-7197 treatment ameliorates acquired lymphedema in a mouse tail model by increasing lymphangiogenesis and interstitial flow.

Although the mouse tail model of secondary lymphedema has been widely used in research, our knowledge regarding some of the characteristic changes in this model is lacking. Therefore, in the current study, we aimed to identify pathologic changes after surgery. Tail lymphedema was created in C57BL/6J mice by disconnecting both superficial and deep lymphatic vessels. The surgery resulted in chronic edema formation with the proliferation of subcutaneous adipose tissue, deposition of fibrotic tissue, and gradual increase in CD4(+) T lymphocyte infiltration. Furthermore, dramatic expansion and an increased number of lymphatic vessels were observed postoperatively. Lymphatic reflux was established at least 8 weeks after surgery, as evidenced by staining of the scar from the surgical excision. In addition, tissue fibrosis was irreversible, although CD4(+) T cell infiltration, tail swelling, and subcutaneous adipose hyperplasia were alleviated over time. We also show that necrosis could be effectively avoided by paying attention to several details in the modeling process. As animal models play a key role in exploring the pathophysiology of disease, our findings provide strong support for the study of lymphedema. The irreversibility of fibrosis suggests the importance of treating lymphedema by preventing fibrosis development.

Pancreas-sparing duodenectomy (PSD) is a known surgical technique used in patients with duodenal pathologies in the adult age group. We present a 3-year-old female patient with intestinal lymphangiectasia who underwent PSD. This is the first case in which this surgical technique was used in childhood. We believe that PSD can be used in the pediatric age group for benign pathologies. Introducing a stent to the common bile duct and the main pancreatic duct is not a requirement, especially if the ampulla is preserved as a “button” duodenal patch.

Background: Platelet-rich plasma (PRP) is an autologous concentrated preparation of platelets characterized by lymphangiogenetic and tissue-repairing effects. Although PRP has been safely used in many different fields, there is no clinical study regarding the use of PRP in lymphedema treatment in humans. We assessed the clinical outcomes of PRP in patients with lower extremity lymphedema (LEL) in a randomized controlled trial. Methods and Results: Patients with secondary LEL were randomly allocated to one of three groups consisting of treatment with PRP with complex decongestive physiotherapy (PRP+CDP group), low-level laser therapy with CDP (LLLT+CDP group), and only CDP (CDP group). Assessment of Lymphedema Quality of-Life Questionnaire (LYMQOL) for health-related quality of life, lower-extremity-circumference (LEC) for edema, tissue dielectric constant (TDC) for extremity volume, 6-minute walking test (6MWT) for functional capacity, and numeric rating scale (NRS) scoring for extremity fullness were evaluated both before and after treatment. Forty-five patients (68.8% female) with mean age 40.84 ± 15.81 years were included in the study. Significant differences in LYMQOL, LEC, NRS, and TDC values both before and after treatment were found in all groups; however, there were no statistically significant difference in values between the three groups. In the PRP+CDP group, LYMQOL values had a larger effect size than the other two groups. Significant differences in 6MWT values both before and after treatment were found in PRP+CDP and LLLT+CDP groups; however, there was no statistically significant difference in the CDP group. Conclusion: This is the first clinical study to evaluate the usage of PRP in patients with secondary LEL. PRP might be an additional treatment option of lymphedema management; however, more clinical trials in humans are needed to yield more evidence in the usage of PRP in patients with lymphedema.

The lymphatic system is arguably the most neglected bodily system. As a result, its contribution to human health and disease is not well understood. In this review, the clinical approaches based on new knowledge and developments of the lymphatic system are covered. The lymphatic system has 3 major functions: (1) the preservation of fluid balance; (2) a nutritional function, as intestinal lymphatics are responsible for fat absorption; and (3) host defense. Lymph vessels return the capillary ultrafiltrate and escaped plasma proteins from most tissues back, ultimately, to the blood circulation. Hence, lymphatics are responsible for maintaining tissue (and plasma) volume homeostasis. Impaired lymph drainage results in peripheral edema (lymphedema) and may have more far-reaching effects on cardiovascular disease, in particular hypertension and atherosclerosis. Lymphatics have an important immune surveillance function, as they represent the principal route of transport from tissues for antigen and immune cells. Intestinal lymphatics (lacteals) are responsible for most fat absorption, first documented by Gaspare Aselli in 1627, when the lymphatic system was discovered. A relationship between fat and lymphatics may exist well beyond the gut alone. Fat deposition is a defining clinical characteristic of lymphedema. Suction-assisted lipectomy of lymphedema has shown the swelling is not just fluid but is dominated by fat. Lymphatics are the preferred route for the metastatic spread of cancer. Accordingly, the lymphatic system may be important for defense against cancer by generating immune responses to malignant cell antigens. Preventing lymphatic entry and propagation of malignant metastasis would effectively render the cancer nonfatal. As one can see, the lymphatic circulation is fundamentally important to cardiovascular disease, infection and immunity, cancer, and, in all likelihood, obesity—4 of the major challenges to health care in the 21st century.

BACKGROUND: Lymphoedema caused by lymphatic filariasis (LF) or podoconiosis can result in physical disability and social exclusion, which is exacerbated by painful acute dermatolymphangioadenitis (ADLA) episodes. These conditions have a significant impact on patients, however, little is known about the indirect effects on their caregivers. This study, therefore, aimed to determine the impact on caregivers for patients with leg lymphoedema in a co-endemic district of Ethiopia. METHODOLOGY/PRINCIPAL FINDINGS: A cross-sectional survey of lymphoedema patients and their caregivers was conducted using semi-structured questionnaires in the Southern Nation Nationalities Peoples Region (SNNPR) of Ethiopia. Lymphoedema patient information on clinical severity (mild, moderate, severe), frequency of ADLAs, their socio-demographic characteristics and the identity of main caregiver(s) was collected. Caregiver information on socio-demographic characteristics, types of care provided, their quality of life (QoL) measured across nine domains, and productivity was collected, with key indicators compared in the presence and absence of patients' ADLAs. A total of 73 patients and 76 caregivers were included. Patients were grouped by mild/moderate (n = 42, 57.5%) or severe (n = 31, 42.5%) lymphoedema, and reported an average of 6.1 (CI+/- 2.18) and 9.8 (CI+/- 3.17) ADLAs respectively in the last six months. A total of 48 (65.8%) female and 25 (34.2%) male patients were interviewed. Caregivers were predominately male (n = 45, 59.2%), and spouses formed the largest caregiving group for both female and male patients. In the absence of an ADLA, most caregivers (n = 42, 55.2%) did not provide care, but only one caregiver did not provide care during an ADLA. In the absence of an ADLA, the average time (hour:minute) spent by mild/moderate (00:17, CI: +/- 00:08) and severe (00:10, CI: +/- 00:07) patient caregiver per task was minimal. The time mild/moderate (00:47, CI: +/- 00:11) and severe (00:51, CI: +/- 00:16) patient caregivers spent per task significantly increased in the presence of an ADLA. In addition, caregivers' QoL was negatively impacted when patients experienced an ALDA, and they had to forfeit an average of 6 to 7 work/school days per month. CONCLUSION/SIGNIFICANCE: Lymphoedema and ADLAs impact negatively on patients' and their caregivers' lives. This emphasises the importance of increasing access to effective morbidity management and disability prevention services to reduce the burden and help to address the Sustainable Development Goal (SDG) 5, target 5.4, which seeks to recognise and value unpaid care and domestic work.

Podoconiosis is a form of lymphoedema that occurs in tropical highland areas in genetically susceptible individuals who are exposed to irritant volcanic soils. The disease is preventable through consistent use of footwear and attention to foot hygiene; however, in endemic areas there is a strong barefoot tradition, and many cannot afford shoes. Patients with podoconiosis face significant physical disability, psychological comorbidity, reduced quality of life and experience frequent episodes of systemic illness due to acute dermatolymphangioadenitis. This review provides an overview of this important and neglected tropical skin disease and summarizes the latest research findings.

Paediatric lymphoedema (LE) is a rare condition, for which there is little data available regarding treatments. The aim of this study was to assess the short-term effect and acceptability of a 30-min session of manual lymphatic drainage (MLD) in children with well-documented LE of the lower limbs. Fifteen children were included (8 males; median age 11 years). Comparison of the sum of circumference values for the whole limb before and after MLD revealed a slight, but significant, reduction (from a median of 289.8 to 285.5 cm, p = 0.024), but the limb volumes did not decrease significantly (from a median of 4,870.3 to 4,772.3 ml, p = 0.394). Dermal thickness, measured by high-resolution ultrasound, decreased from 1.44 to 1.40 mm (p < 0.001). All children reported improvement in well-being, and found MLD useful. In conclusion, MLD is well accepted by children, but has poor impact on LE swelling. However, it decreases cutaneous oedema by mobilizing the lymph fluid.

PURPOSE: To assess the ability and reproducibility of Non-contrast Magnetic Resonance Lymphography (NMRL) in detecting and quantify lymphedema, using a semiquantitative scoring system. METHODS AND MATERIAL: This is a monocentric retrospective study of 134 consecutive patients with a clinical diagnosis of limb lymphedema who performed a Non-contrast Magnetic Resonance Lymphography (NMRL) at our Institution between November 2014 and February 2017. Lymphedema was classified based both on clinical and radiologic evaluation. An NMRL total score was obtained for each limb's segment and compared to the clinical grade, used as reference standard. NMRL intra-observer, inter-observer variability and intraclass correlation were calculated. NMRL sensitivity, specificity, and accuracy in identifying lymphedema were provided. Based on score distribution an NMRL four-stage system was developed. RESULTS: NMRL showed 92% sensitivity, 77% specificity and 82% accuracy in identifying lymphedema. An almost perfect agreement was obtained by expert operators, while substantial agreement was obtained by non-expert operators. Substantial agreement resulted also for the inter-observer variability (Cohen's Kappa K = 0.73, CI 95% [0.69–0.78]). The intra-class correlation showed an almost perfect relationship both by expert and non-expert operators. Excellent correlation between clinical grade and NMRL score and between clinical grade and NMRL stage were found for each segment. CONCLUSIONS: NMRL is a confident and reproducible exam with high sensitivity, good specificity and high accuracy in lymphedema detection; the semiquantitative NMRL score resulted a reliable and reproducible tool able to quantify lymphedema severity.

Background: Lymphedema is a chronic pathology characterized by progressive swelling due to lymphatic dysfunction (1). Literature contains few studies that focus on male genital lymphedema. A variety of surgical techniques as part of the male genital lymphedema therapeutic strategy has been described. Supramicrosurgical lymphatico-venular anastomosis s-LVA, based on connecting lymphatic collectors to venules, has evidenced efficient outcomes thus far. However, the peculiarity of the genital area may lead to an innovative and even more accurate surgical technique as a treatment of male genital lymphedema: lymphatic pre-collectors located superficially over the fascial layer can be used to perform the ultramicrosurgical anastomosis. Purpose of the study: In this paper, the authors report their experience of this new surgical concept based on anastomosing lymphatic precollectors to venules. Methods: We performed a retrospective study from 2014 to 2016. Six male patients with primary genital lymphedema underwent ultramicrosurgical lymphatico-venular anastomosis in Siena University Hospital, Italy. Results: Ultramicrosurgical lymphatico-venular anastomosis has evidenced positive outcomes in terms of prognosis, infectious complications, volume reduction, and quality of life. The average cellulitis rate dropped from 2.5 episodes a year to 0.5 episodes after surgical intervention. The mean satisfaction index passed from 1.33 before the intervention to 2.83. Conclusion: Ultramicrosurgical lymphatico-venular anastomosis represents a challenging physiological approach for male genital lymphedema with promising outcomes.

Fine needle diathermy (FND) is an effective method to destroy and regress pathologic corneal blood and lymphatic vessels. However, it is unknown whether FND itself causes a rebound corneal neovascularisation and whether that can be prevented by VEGF blockade. In female BALB/c mice, the suture-induced inflammatory corneal neovascularisation model was used to induce hem- and lymphangiogenesis. Thereafter, prevascularized mice were divided into 2 groups: the combination therapy group received FND cauterization and subsequent VEGF TrapR1R2 eye drops three times per day whereas the monotherapy group was treated only with FND. Three, 7 and 14 days after the treatment, corneas were collected and stained with FITC-conjugated CD31 and LYVE-1 followed by Cy3-conjugated secondary antibody to quantify corneal blood and lymphatic vessels. Relative mRNA expression of VEGF in the cornea was quantified by using qPCR. FND cauterization as monotherapy significantly obliterated (lymph)angiogenesis at early time points; however, this treatment led to secondary corneal hem- and lymphangiogenesis associated with significant upregulation of pro(lymph)angiogenic VEGF-A, VEGF-C, VEGF-D and infiltration of macrophages. Combining FND cauterization with VEGF TrapR1R2 treatment prevented the undesired effect of the FND procedure alone and significantly better regressed corneal blood and lymphatic vessels at 1 week after the treatment compared to monotherapy and control group (p < 0.01).

A 29-month-old girl had idiopathic massive pericardial effusion for over 6 months. Lymphangiography was performed for chronic and recurrent pericardial effusion and pulmonary lymphangiectasia, suspected based on CT findings. Magnetic resonance lymphangiography revealed chylolymphatic reflux from a tortuously dilated thoracic duct in the mediastinum to the pericardial space, suggesting primary chylopericardium with lymphangiectasia. Pericardial effusion resolved immediately after thoracic duct embolisation at the lower thoracic level. However, pericardial effusion recurred after 5 months, which resolved after additional embolisation of the abnormal lymphatic collateral vessels from the remnant upper thoracic duct. Here, we report an unusual case with chylous massive pericardial effusion diagnosed by magnetic resonance lymphangiography and treated with percutaneous embolisation.

Background Detailed visualization of the lymphatic vessels would greatly assist in the diagnosis and monitoring of lymphatic diseases and aid in preoperative planning of lymphedema surgery and postoperative evaluation. Purpose To evaluate the usefulness of photoacoustic imaging (PAI) for obtaining three-dimensional images of both lymphatic vessels and surrounding venules. Materials and Methods In this prospective study, the authors recruited healthy participants from March 2018 to January 2019 and imaged lymphatic vessels in the lower limbs. Indocyanine green (5.0 mg/mL) was injected into the subcutaneous tissue of the first and fourth web spaces of the toes and below the lateral malleolus. After confirmation of the lymphatic flow with near-infrared fluorescence (NIRF) imaging as the reference standard, PAI was performed over a field of view of 270 × 180 mm. Subsequently, the number of enhancing lymphatic vessels was counted in both proximal and distal areas of the calf and compared between PAI and NIRF. Results Images of the lower limbs were obtained with PAI and NIRF in 15 participants (three men, 12 women; average age, 42 years ±12 [standard deviation]). All participants exhibited a linear pattern on NIRF images, which is generally considered a reflection of good lymphatic function. A greater number of lymphatic vessels were observed with PAI than with NIRF in both the distal (mean: 3.6 vessels ±1.2 vs 2.0 vessels ±1.1, respectively; P < .05) and proximal (mean: 6.5 vessels ±2.6 vs 2.6 vessels ±1.6; P < .05) regions of the calf. Conclusion Compared with near-infrared fluorescence imaging, photoacoustic imaging provided a detailed, three-dimensional representation of the lymphatic vessels and facilitated an increased understanding of their relationship with the surrounding venules. (c) RSNA, 2020 Online supplemental material is available for this article. See also the editorial by Lillis and Krishnamurthy in this issue.

OBJECTIVE: The lymphatic system is a circulatory system that unidirectionally drains the interstitial tissue fluid back to blood circulation. Although lymph is utilized by leukocytes for immune surveillance, it remains inaccessible to platelets and erythrocytes. Activated cells release submicron extracellular vesicles (EV) that transport molecules from the donor cell. In rheumatoid arthritis, EV accumulate in the joint where they can interact with numerous cellular lineages. However, whether EV can exit the inflamed tissue to recirculate is unknown. Here, we investigated whether vascular leakage that occurs during inflammation could favor EV access to the lymphatic system. Approach and Results: Using an in vivo model of autoimmune inflammatory arthritis, we show that there is an influx of platelet EV, but not EV from erythrocytes or leukocytes, in joint-draining lymph. In contrast to blood platelet EV, lymph platelet EV lacked mitochondrial organelles and failed to promote coagulation. Platelet EV influx in lymph was consistent with joint vascular leakage and implicated the fibrinogen receptor alpha2bbeta3 and platelet-derived serotonin. CONCLUSIONS: These findings show that platelets can disseminate their EV in fluid that is inaccessible to platelets and beyond the joint in this disease.

Elimination of a disease is the holy grail in global health. The pathology of several neglected tropical diseases (NTDs) such as lymphatic filariasis (LF) makes elimination a reality. However, successful elimination requires that NTD programs be sustainable-the ability to confirm that the disease has been eliminated and the capacity to ensure that it does not return. The WHO's guidelines on NTDs thoroughly detail how to reach elimination. Notwithstanding, comprehensive guidance regarding contextual and programmatic factors that influence sustainability is lacking. Moreover, a comprehensive NTD sustainability framework that includes these factors is nonexistent. This research aimed to develop a framework that identified the critical programmatic and contextual factors influencing sustainability of NTD elimination programs. The methodology included a literature review and a multiple case study. The literature review had two objectives: the first was to identify unique attributes of NTD elimination programs that should be considered when planning for sustainability and the second was to identify an existing sustainability framework that could be adapted to NTD elimination programs. The literature review resulted in a draft sustainability framework that was then tested, using a multi-case study methodology, on the Kenya National Program for Elimination of Lymphatic Filariasis. The result is the first comprehensive NTD sustainability framework that identifies key contextual and programmatic factors that influence NTD sustainability. This framework is an invaluable resource for practitioners and policy makers alike as many countries start to eliminate NTDs.

A 14-year-old girl was admitted to our institute with a history of intermittent bilateral ankle swelling, and moderate but progressively worsening pain which has lasted for 2 years.The patient's history was unremarkable. She did not take medications and was not involved in any sports activity. She reported no fever, gastrointestinal symptoms, fatigue, weight loss, travels abroad or previous infections. She reported moderate pain at night, associated with a sense of heaviness, tightness and general discomfort, and with no response to ibuprofen.Physical examination was remarkable only for bilateral ankle non-pitting oedema, more evident on the left leg, with a thickened skinfold at the base of the second toe, and without redness, swelling or skin warming.The patient had been previously examined, and her foot and ankle X-rays, ultrasound (US) and MRI were all negative. Blood tests (white cell count, C reactive protein, erythrocyte sedimentation rate, albumin, antinuclear antibodies, creatinine, transaminase, creatine kinase, lactate dehydrogenase, thyroid function and glucose) and urinalysis were in the normal range. Her ocular assessment and echocardiogram were also normal. QUESTION 1: Based on the clinical picture and laboratory tests, what is the most likely diagnosis?Deep venous thrombosis.Osteochondritis.Lymphoedema.Juvenile idiopathic arthritis. QUESTION 2: Based on what you see in figure 1, what is the underlying cause? Recurrent bacterial lymphangitis.Primary lymphoedema.Tumour.Filariasis.edpract;archdischild-2019-318096v1/F1F1F1figure 1Lymphoscintigraphy of the lower extremities showing insufficient deep lymphatic circulation in the left leg (red arrow, A) replaced by superficial drainage (B). QUESTION 3: Which is the best diagnostic test to confirm the diagnosis?US scan.MRI.Lymphoscintigraphy.Reassurance and clinical follow-up. QUESTION 4: What is the mainstay of management of this condition?Wait and see.Antibiotic course.Supportive therapy (i.e., physical activity, elevation of extremities, pneumatic compression).Surgical intervention. Answers can be found on page 2.

BACKGROUND: A ketogenic diet (KD) is an effective treatment for intractable epilepsy in children. Protein-losing enteropathy (PLE) is a rarely reported but serious complication of KDs. CASE PRESENTATION: A 3-month-old female patient presented with PLE while following a KD as treatment for intractable epilepsy. She also had genovariation of the STXBP1 gene. The patient suffered from general edema and hypoalbuminemia but no diarrhea. Esophagogastroduodenoscopy (EDG) revealed lymphatic ectasia in the lamina propria. We diagnosed her with intestinal lymphangiectasia, and after decreasing the KD ratio from 4:1 to 1.05:1, we successfully controlled her edema and hypoalbuminemia. As of now, the convulsions and hypsarrhythmia have disappeared, and the seizure-free state has lasted for 20 months. CONCLUSIONS: PLE may be managed by decreasing the ketogenic ratio rather than discontinuing a KD since for some patients, a KD is the only effective therapy available at present.

BACKGROUND: Noonan syndrome is an autosomal dominant, variably expressed multisystem disorder characterized by specific facial and cardiac defects, delayed growth, ectodermal abnormalities, and lymphatic dysplasias. Lymphedema and chylous pleural effusions are common in Noonan syndrome, but protein-losing enteropathy (PLE) has only rarely been described in the condition and little is known about its genetic associations. CASE PRESENTATION: We report the case of a 30-year-old Chinese woman who developed severe recurrent edema and hypoproteinemia. Gastroduodenoscopy showed a “snowflake” appearance of lymphangiectasia in the duodenum, and CT reconstruction of the small intestine showed segmental thickening of the intestinal wall with localized stenosis. Whole exome sequencing revealed that the patient harbored a pathogenic variant of PTPN11 (c.A922G p.N308D), which was unfortunately inherited by her 2.5-year-old daughter who had short stature and atrial septal defect but no hypoproteinemia. CONCLUSIONS: This case of Noonan syndrome with PLE was associated with a PTPN11 mutation. A comprehensive review of PLE in Noonan syndrome revealed that PLE often presents late in this context but there is no clear genotype-phenotype correlation. Genetic evaluation with next-generation sequencing can be useful for securing the diagnosis and planning early intervention and management.

BACKGROUND: The sensitivity of endolymphatic hydrops (EH) to the glycerol test varies in patients with Meniere's disease (MD). PURPOSE: To explore the features of EH and its glycerol-induced dynamics in MD. STUDY TYPE: Case-control study. POPULATION: Twenty patients with MD (24 affected ears) were included. FIELD STRENGTH/SEQUENCE: 3.0T 3D-FLAIR (fluid-attenuated inversion recovery) MRI and late gadolinium enhancement. ASSESSMENT: Intratympanic gadolinium-enhanced MRI was performed in the MD-affected ears before and after the glycerol test. The borders of the endolymphatic and total lymphatic space were contoured on the axial MRI slices to evaluate the volume of hydrops in both the cochlear and vestibular regions. STATISTICAL TESTS: Paired and unpaired t-tests, the Mann-Whitney U-test, linear discriminant analysis, Pearson's correlation, and linear regression. RESULTS: After glycerol ingestion, vestibular EH decreased in all patients, whereas cochlear EH significantly decreased only in patients with positive glycerol test results (all P < 0.01). At baseline, cochlear EH in the positive result group was greater than in the negative result group (P = 0.007). Unexpectedly, in the positive result group a drastic glycerol-induced dehydrating effect was observed in patients whose pretest cochlear EH ratio was >16% (P = 0.011). Moreover, the dehydrating role of glycerol was positively correlated with the baseline cochlear hydrops level (r = 0.7691, P < 0.001). DATA CONCLUSION: MRI provides evidence that glycerol administration improves the hearing threshold via dehydrating the EH. In the cochlear region, the baseline level of cochlear EH is a closely related factor for the validity of the glycerol test, whereas EH is consistently dehydrated in the vestibular component. LEVEL OF EVIDENCE: 2 TECHNICAL EFFICACY STAGE: 3.

Tumor-associated lymphangiogenesis and lymphatic invasion of tumor cells correlate with poor outcome in many tumor types, including breast cancer. Various explanations for this correlation have been suggested in the past, including the promotion of lymphatic metastasis and an immune-inhibitory function of lymphatic endothelial cells (LECs). However, the molecular features of tumor-associated lymphatic vessels and their implications for tumor progression have been poorly characterized. Here, we report the first transcriptional analysis of tumor-associated LECs directly isolated from the primary tumor in an orthotopic mouse model of triple negative breast cancer (4T1). Gene expression analysis showed a strong upregulation of inflammation-associated genes, including endothelial adhesion molecules such as VCAM-1, in comparison to LECs derived from control tissue. In vitro experiments demonstrated that VCAM-1 is not involved in the adhesion of tumor cells to LECs but unexpectedly promoted lymphatic permeability by weakening of lymphatic junctions, most likely through a mechanism triggered by interactions with integrin alpha4 which was also induced in tumor-associated LECs. In line with this, in vivo blockade of VCAM-1 reduced lymphatic invasion of 4T1 cells. Taken together, our findings suggest that disruption of lymphatic junctions and increased permeability via tumor-induced lymphatic VCAM-1 expression may represent a new target to block lymphatic invasion and metastasis.

BACKGROUND: Near-infrared (NIR) fluorescence imaging has recently been introduced to the sentinel lymph node (SLN) mapping because of the benefits of the SLN biopsy, such as providing real-time and high-resolution optical guidance. Methylene blue is available and less expensive as an SLN mapping tracer. Our study aims to identify SLN through the NIR fluorescence imaging system mediated by blue dye. METHODS: Early-stage breast cancer patients were prospectively enrolled. All participants received a subareolar or peritumoral injection of 1 mL methylene blue (MB) before surgery. The MB fluorescence system was set immediately after injection. SLNs were searched and removed under the guidance of fluorescence and blue dye. RESULTS: We identified SLN adequately with the help of real-time lymphography and blue dye. Symbolic lymphatic drainage patterns were also observed. CONCLUSION: NIR fluorescence imaging mediated by blue dye has benefits on the identification of lymph vessels, the location of SLN, and the patterns of breast lymphatic flow.

Arachidonic acid epoxides generated by cytochrome P450 (CYP) enzymes have been linked to increased tumor growth and metastasis, largely on the basis of overexpression studies and the application of exogenous epoxides. Here we studied tumor growth and metastasis in Cyp2c44(-/-) mice crossed onto the polyoma middle T oncogene (PyMT) background. The resulting PyMT(2c44) mice developed more primary tumors earlier than PyMT mice, with increased lymph and lung metastasis. Primary tumors from Cyp2c44-deficient mice contained higher numbers of tumor-associated macrophages, as well as more lymphatic endothelial cells than tumors from PyMT mice. While epoxide and diol levels were comparable in tumors from both genotypes, prostaglandin (PG) levels were higher in the PyMT(Delta2c44) tumors. This could be accounted for by the finding that Cyp2c44 metabolized the PG precursor, PGH2 to 12(S)-hydroxyheptadeca-5Z,8E,10E-trienoic acid (12-HHT), thus effectively reducing levels of effector PGs (including PGE2). Next, proteomic analyses revealed an up-regulation of WD repeating domain FYVE1 (WDFY1) in tumors from PyMT(Delta2c44) mice, a phenomenon that was reproduced in Cyp2c44-deficient macrophages as well as by PGE2 Mechanistically, WDFY1 was involved in Toll-like receptor signaling, and its down-regulation in human monocytes attenuated the LPS-induced phosphorylation of IFN regulatory factor 3 and nuclear factor-kappaB. Taken together, our results indicate that Cyp2c44 protects against tumor growth and metastasis by preventing the synthesis of PGE2 The latter eicosanoid influenced macrophages at least in part by enhancing Toll-like receptor signaling via the up-regulation of WDFY1.

BACKGROUND: Sentinel node navigation surgery (SNNS) has been frequently used in early cervical cancer. However, the incidence and potential reduction of lymphatic complications following the removal of the sentinel lymph node remain unknown. Thus, this study aimed to evaluate the occurrence of lymphatic complications post sentinel node navigation surgery in patients with early cervical cancer. METHODS: A total of 167 patients, including 70 and 97 patients who had undergone SNNS and pelvic lymphadenectomy (PLA), respectively, were enrolled in this study. We compared the lymphatic complications (lower extremity lymphedema and pelvic lymphocele) between the SNNS and PLA groups. RESULTS: The median number of sentinel lymph nodes removed was 2 (range: 1–14). Among the 70 patients in the SNNS group, there were 0 (0%) and 3 (4.3%) occurrences of lower extremity lymphedema and pelvic lymphocele, respectively. The occurrences of lower extremity lymphedema and pelvic lymphocele were significantly lower in the SNNS group than in the PLA group, despite circumflex iliac node removal. CONCLUSIONS: The occurrence of lymphatic complications (lower extremity lymphedema and pelvic lymphocele) was significantly lower in the SNNS group than in the PLA group. We found that SNNS, and not PLA, was the best treatment option for preventing the development of lower extremity lymphedema and pelvic lymphocele, despite circumflex iliac node preservation.

Lymph node metastasis is associated with tumor relapse and poor patient prognosis in bladder cancer. However, the mechanisms by which bladder carcinoma cells induce lymphangiogenesis and further promote metastasis in the lymphatic system remain unclear. Here, we show that the transcription factor GATA-binding factor 6 (GATA6) was substantially downregulated in bladder cancer via promoter hypermethylation. Low-level GATA6 expression significantly correlated with lymph node metastasis positivity and was able to predict earlier relapse and shorter survival of bladder cancer. Reconstitution of GATA6 inhibited lymphangiogenesis and lymph node metastasis in GATA6-low bladder cancer cells, while silencing of GATA6 rendered lymphatic metastasis in GATA6-high bladder cancer cells. Additionally, we demonstrated that GATA6 bound to the promoter of vascular endothelial growth factor (VEGF)-C, a lymphangiogenic factor, and acted as a transcriptional repressor. This GATA6/VEGF-C axis was essential for GATA6-mediated lymphatic metastasis. In bladder cancer patients, low GATA6 correlated with high VEGF-C and reduced overall survival. These findings indicate GATA6 as a pivotal regulator in the lymphatic dissemination of bladder cancer and suggest a new therapeutic target for the disease.

BACKGROUND: Local application of fluorouracil (Efudix, 5-FU) induces sclerosis in patients with sinonasal tumors and superficial basocellular skin carcinoma. As a ‘back against the wall’ treatment, we investigated the local effect of nasally applied 5-FU and whether this could decrease the burden of severe epistaxis in patients with hereditary hemorrhagic telangiectasia (HHT). METHODS: HHT patients with severe and frequent epistaxis, subsequent anemia and a necessity for blood and/or iron infusions were treated with a nasal tampon with 5-FU. This tampon was placed unilaterally in the nasal cavity on the side of the most severe epistaxis and replaced once weekly during 4 weeks. Outcome measures were safety and side effects, the aspect of the nasal mucosa measured with the mucosal HHT score, the epistaxis severity score (ESS), hemoglobin and ferritin plasma levels, and quality of life assessment pre-treatment, one and three months post-treatment. RESULTS: Six HHT patients participated. During treatment and follow-up, the nasal mucosa turned more pale and sclerotic and the number of telangiectases diminished. The mucosal HHT score improved and the ESS declined (p = 0.01). The decline of ESS persisted up to 3 months post-5-FU treatment. Moreover, mean hemoglobin levels increased from 6.0 pre-5-FU to 6.8 after one month post-5-FU. CONCLUSION: Unilateral application of 5-FU on a nasal tampon diminished the severity and frequency of epistaxis in all HHT patients. This effect sustained up to three months post-treatment, despite the fact that the contralateral side remained untreated. Subsequently, hemoglobin levels increased. Intranasal 5-FU is a promising entity for further research on epistaxis treatment in HHT patients.

BACKGROUND: Systemic sirolimus (rapamycin) has recently been found effective in treating complex vascular anomalies by reducing the size and associated complications. Many vascular anomalies have a cutaneous component, and thus, we sought to determine whether topical administration of sirolimus may be an effective therapy, as data on the use of topical sirolimus are limited. OBJECTIVE: We reviewed the efficacy and tolerability of topical formulations of sirolimus in the treatment of various simple and combined vascular malformations and tumors. METHODS: Eighteen patients with any vascular anomaly treated exclusively with topical sirolimus were retrospectively reviewed. RESULTS: Eleven patients had combined venous lymphatic malformations, three had tufted angiomas, two had a lymphatic malformation, one had a venous malformation, and one had a verrucous venous malformation. All (100%) patients reported some degree of improvement and 50% of patients reported marked improvement in one or more symptoms, most commonly blebs and lymphatic drainage, and bleeding. LIMITATIONS: The retrospective nature, small number of patients, and differences in topical preparations limit the broad application of the results. CONCLUSION: Topical sirolimus appears to be a safe and useful non-invasive therapy that is well-tolerated in the treatment of the cutaneous portion of a variety of vascular anomalies.

The lateral marginal vein is an anomalous clinical entity found in association with Klippel-Trenaunay and other PIK3CA-related overgrowth syndromes. Although it is reported to affect <20% of patients with Klippel-Trenaunay syndrome, this venous anomaly has been associated with significant morbidity and mortality attributable to venous hypertension and potentially lethal thromboembolic events. Limited literature exists on the diagnosis and management of this rare anomaly, with most of the reports focusing on retrospective clinical experience at a few centers of excellence. Despite these limitations, a systematic approach to diagnosis and treatment of this anomaly is warranted and expounded on herein. When plausible, clinical recommendations based on best available literature are made.

Infantile and congenital hemangiomas are difficult to distinguish in infants. The aim of this study was to compare the conventional ultrasonographic (US) and elastographic features of infantile and congenital hemangiomas. The US findings in 118 patients with congenital hemangioma (58 non-involuting, 36 rapidly involuting, 24 partially involuting) and 111 with 120 infantile hemangioma were retrospectively evaluated. On US imaging, 31.7% of infantile hemangiomas were hyperechoic, 31.7% hypoechoic and 36.6% mixed-echoic with hyperechoic and hypoechoic areas; 57.6% of congenital hemangiomas were mixed-echoic with a hypoechoic area and many vessels visible, 39.0% hypoechoic and 3.4% were mixed-echoic with hyperechoic and hypoechoic area. Calcifications were present in 6.8% and visible vessels involving muscle in 24.6% of congenital hemangiomas. All infantile hemangiomas and 82.2% of congenital hemangiomas were well-defined. All congenital hemangiomas were subcutaneous whereas 17.5% of the infantile hemangiomas were superficial. The maximum diameter and vascular density were greater in congenital hemangiomas. Elastography demonstrated that the congenital hemangiomas were softer than the infantile hemangiomas. The maximum diameter (including of visible vessels), thickness, vascular density, venous blood flow velocity and elasticity scores were greater for rapidly and partially involuting congenital hemangiomas than for non-involuting ones. The density of visible vessels in congenital hemangiomas decreased in the order of non-involuting, partially involuting and rapidly involuting. In conclusion, congenital hemangiomas have distinctive US imaging characteristics, including a greater maximum diameter, vascular density, number of visible vessels, visible vessels involving muscle, calcifications and elasticity score.

BACKGROUND: Intracranial arteriovenous malformation (AVM) is a common cause of primary intracerebral hemorrhage in young adults. Lesions typically are sporadic and contain somatic mutations in KRAS or BRAF. The purpose of this study was to identify somatic mutations in a cohort of participants with brain AVM and to determine if any genotype-phenotype associations exist. METHODS: Human brain AVM specimens (n = 16) were collected during a clinically-indicated procedure and subjected to multiplex targeted sequencing using molecular inversion probe (MIP-seq) for mutations in KRAS, BRAF, HRAS, NRAS, and MAP2K1. Endothelial cells (ECs) were separated from non-ECs by immune-affinity purification. Droplet digital PCR (ddPCR) was used to confirm mutations and to screen for mutations that may have been missed by MIP-seq. Patient and AVM characteristics were recorded. RESULTS: We detected somatic mutations in 10 of 16 specimens (63%). Eight had KRAS mutations [G12D (n = 5), G12V (n = 3)] and two had BRAF mutations [V600E (n = 1), Q636X (n = 1)]. We found no difference in age, sex, presenting symptom, AVM location, or AVM size between patients with a confirmed mutation and those without. Nor did we observe differences in these features between patients with KRAS or BRAF mutations. However, two patients with BRAF mutations presented at an older age than other study participants. CONCLUSIONS: Somatic mutations in KRAS and, less commonly in BRAF, are found in many but not all intracranial AVM samples. Currently, there are no obvious genotype-phenotype correlations that can be used to predict whether a somatic mutation will be detected and, if so, which gene will be mutated.

Brain and spinal arteriovenous malformations are congenital lesions causing intracranial haemorrhage or permanent disability especially in young people. We investigated whether the vast majority or all brain and spinal arteriovenous malformations are associated with detectable tumour-related somatic mutations. In a cohort of 31 patients (21 with brain and 10 with spinal arteriovenous malformations), tissue and paired blood samples were analysed with ultradeep next generation sequencing of a panel of 422 common tumour genes to identify the somatic mutations. We used droplet digital polymerase chain reaction to confirm the panel sequenced mutations and identify the additional low variant frequency mutations. The association of mutation variant frequencies and clinical features were analysed. The average sequencing depth was 1077 ± 298x. High prevalence (87.1%) of KRAS/BRAF somatic mutations was found in brain and spinal arteriovenous malformations with no other replicated tumour-related mutations. The prevalence of KRAS/BRAF mutation was 81.0% (17 of 21) in brain and 100% (10 of 10) in spinal arteriovenous malformations. We detected activating BRAF mutations and two novel mutations in KRAS (p.G12A and p.S65_A66insDS) in CNS arteriovenous malformations for the first time. The mutation variant frequencies were negatively correlated with nidus volumes of brain (P = 0.038) and spinal (P = 0.028) arteriovenous malformations but not ages. Our findings support a causative role of somatic tumour-related mutations of KRAS/BRAF in the overwhelming majority of brain and spinal arteriovenous malformations. This pathway homogeneity and high prevalence implies the development of targeted therapies with RAS/RAF pathway inhibitors without the necessity of tissue genetic diagnosis.10.1093/brain/awy307_video1awy307media15978667388001.

Increased risk of thromboembolism has been recognized in individuals with mosaic overgrowth disorders, Proteus syndrome (PS) and PIK3CA-related overgrowth spectrum (PROS), including Klippel-Trenaunay syndrome and CLOVES syndrome. PS and PROS have distinct, yet overlapping clinical findings and are caused by somatic pathogenic variants in the PI3K/AKT gene signaling pathway. PS is caused by a single somatic activating AKT1 c.49G > A p.E17K variant while PROS can be caused one of multiple variants in PIK3CA. The role of prothrombotic factors, endothelial cell adhesion molecules, and vascular malformations in both PS and PROS have not been previously investigated. A pilot study of prospective clinical and laboratory evaluations with the purposes of identifying potential risk factors for thrombosis was conducted. Doppler ultrasounds and magnetic resonance angiogram/venography (MRA/MRV) scans identified vascular malformations in PS and PROS that were not appreciated on physical examination. Abnormal D-dimers (0.60–2.0 mcg/ml) occurred in half of individuals, many having vascular malformations, but no thromboses. Soluble vascular endothelial markers, including thrombomodulin, soluble vascular adhesion molecule (sVCAM), soluble intercellular adhesion molecule (sICAM), E-selectin, and P-selectin were significantly higher in PS and PROS compared to controls. However, no single attribute was identified that explained the risk of thrombosis. Predisposition to thrombosis is likely multifactorial with risk factors including chronic stasis within vascular malformations, stasis from impaired mobility (e.g., following surgery), decreased anticoagulant proteins, and effects of AKT1 and PIK3CA variants on vascular endothelium. Based on our findings, we propose clinical recommendations for surveillance of thrombosis in PS and PROS.

Background and Objectives. Kaposiform hemangioendothelioma (KHE) is a vascular tumor of very low incidence, which occurs mostly in children and infants. The tumor is recognized for its locally aggressive, yet rarely metastatic behavior. It may cause consumptive coagulopathy known as Kasabach-Merritt phenomenon. We report a distinctive case, where an 11-year-old boy is presented with progressive thoracolumbar scoliosis without any symptom or neurological sign. Case Report. The patient underwent spinal deformity correction via posterior pedicle screw instrumentation and fusion, along with tumor biopsy. The pathology report confirmed KHE. The patient did not show a prominent progression of scoliosis after the surgery without any further treatments. Conclusions. Many of scoliotic patients do not have any apparent cause, thereby regarded as idiopathic scoliosis. The presented case is where kaposiform hemangioendothelioma is likely to be linked to the patient's scoliosis. We demonstrate the possibility of secondary scoliosis should always be kept in mind of orthopaedic doctors. We also conclude that secondary scoliosis does not show exacerbation after growth completion.

BACKGROUND: The indications for endoscopic dissection have been expanded to improve the quality of life of patients with early gastric cancer (EGC). This study aimed to develop a nomogram to predict the status of lymph node metastasis with the aim of avoiding unnecessary gastrectomies. METHODS: We reviewed the clinicopathological data of 10 579 patients who underwent curative resection for EGC. The nomogram was developed by multivariate analysis and was evaluated by external validation. Overall, disease-free and recurrence-free survival were compared between the gastrectomy group of 6641 patients and the endoscopic dissection group of 999 patients to show the efficacy of the nomogram. RESULTS: Multivariate analyses revealed that age, tumor size, lymphatic invasion, depth of invasion, and histologic differentiation were all significant prognostic factors for lymph node metastasis. The nomogram had good discriminatory performance, with a concordance index of 0.846. This was supported by the external validation point of 0.813. For patients with low risk of lymph node metastasis on the nomogram (</ = 3% of the provisional value in this study), the endoscopic dissection and gastrectomy groups had comparable rates of overall (P = 0.32), disease-free (P = 0.47), and recurrence-free (P = 0.09) survival. CONCLUSIONS: We developed and validated a nomogram that predicts the risk of lymph node metastasis in EGC based on a large database. This precision nomogram is useful to avoid unnecessary gastrectomy after endoscopic dissection, which may ultimately improve the quality of life of patients with EGC.

Since the discovery of propranolol in the treatment of infantile hemangioma (IH), there has been emergent investigation of beta-adrenergic receptor (beta-AR) signaling in IH and the mechanisms of action for which beta-AR blockers regulate hemangioma cell proliferation. However, beta-AR agonists and antagonists are known to act antithetically via the same intracellular beta-AR-driven proangiogenic pathways. We present the case of a patient with involuted IH treated with propranolol that showed a full and rapid regrowth during the intravenous administration of salbutamol, a selective beta2-adrenergic agonist, for an episode of severe obstructive bronchitis. This observation brings forward the clinical implication of beta-signaling effects in IH and raises awareness of the potential proliferative response of IH to beta-AR agonists such as salbutamol.

We present three African American infants with segmental, ulcerated infantile hemangiomas and concomitant, persistent hypertension. When treated with beta-blocker therapy, the hemangiomas decreased in size and the ulcerations resolved, but there was no impact on the elevated blood pressure in one of our patients. We failed to identify any associations between infantile hemangioma and hypertension in the literature.

OBJECTIVES: Infantile hemangiomas (IHs) are common; some cases require timely referral and treatment to prevent complications. We developed and validated a reliable instrument for timely and adequate referral of patients with IH to experts by nonexpert primary physicians. METHODS: In this multicenter, cross-sectional, observational study, we used a 3-stage process: (1) development of the Infantile Hemangioma Referral Score (IHReS) tool by IH experts who selected a representative set of 42 IH cases comprising images and a short clinical history, (2) definition of the gold standard for the 42 cases by a second independent committee of IH experts, and (3) IHReS validation by nonexpert primary physicians using the 42 gold standard cases. RESULTS: A total of 60 primary physicians from 7 different countries evaluated the 42 gold standard cases (without reference to the IHReS tool); 45 primary physicians evaluated these cases using the IHReS questionnaire, and 44 completed retesting using the instrument. IHReS had a sensitivity of 96.9% (95% confidence interval 96.1%–97.8%) and a specificity of 55.0% (95% confidence interval 51.0%–59.0%). The positive predictive value and negative predictive value were 40.5% and 98.3%, respectively. Validation by experts and primary physicians revealed substantial agreement for interrater reliability and intrarater repeatability. CONCLUSIONS: IHReS, a 2-part algorithm with a total of 12 questions, is an easy-to-use tool for primary physicians for the purpose of facilitating correct and timely referral of patients with IH. IHReS may help practitioners in their decision to refer patients to expert centers.

Infantile hemangioma (IH) is one of the most common soft-tissue neoplasms of infancy. Although clinical diagnosis for IH is well-established, the haematological parameters associated with IH are not well explored. In this short study, we observed significantly higher eosinophil (EO) numbers in IH patient blood compared to healthy controls. This contributed to the observed higher EO % in the peripheral blood of IH patients and was irrespective of age. This new haematological finding could carry a potential diagnostic/prognostic relevance for IH.

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.

BACKGROUND: Brain arteriovenous malformations (BAVMs) are vascular malformations composed of tangles of abnormally developed vasculature without capillaries. Abnormal shunting of arteries and veins is formed, resulting in high-pressure vascular channels, which potentially lead to rupture. BAVMs are generally considered a congenital disorder. But clinical evidence regarding involution, regrowth, and de novo formation argue against the static condition of this disease. Recently, the presence of the somatic activating KRAS mutations in more than half of BAVM cases was reported, suggesting the role of KRAS function in the pathogenesis. METHODS: KRAS mutation in codon35 (G—>A, G12D; G—>T, G12V) was examined by a digital polymerase chain reaction analysis using genome purified from paraffin-embedded slides of human BAVMs. We also examined protein expression of KRAS G12D in lesions to corroborate results from digital polymerase chain reaction analysis. RESULTS: We detected codon35 G—>A mutation in 15 (39.5%) among 38 samples and codon35 G—>T mutation in 10 (27.0%) among 37 samples we could assess mutations. There were no samples positive for both codon35 G—>A and G—>T mutation. The ratio of codon35 G—>A mutation ranged from 0.60% to 12.28% and that of G—>T was from 1.20% to 8.99%. We next examined protein expression of KRAS G12D in BAVM lesions in immunohistochemistry. A KRAS G12D mutant was detected mainly in endothelial cells of dilated vessels in lesions. CONCLUSIONS: KRAS mutations in codon35 were detected in about two thirds of specimens examined. KRAS function may actively contribute to the pathobiology of BAVM and can become a therapeutic target.

Background: Infantile hemangioma is the most common tumor of infancy. Currently, propranolol is a preferred drug for treating hemangioma. The exact mechanism of action of propranolol is not known. In this study, we attempted to assess whether propranolol has any effect on vascular endothelial growth factor (VEGF) and tissue inhibitor of metalloproteinase-2 (TIMP-2) over a period of time, and if it is there, how long it affects it. Materials and Methods: Propranolol was administered in the dosage of 2–3 mg/kg. The first serum sample was collected before starting the propranolol treatment. Thereafter, samples were collected at monthly intervals up to a total of six samples. The samples were assessed for TIMP-2 and VEGF using enzyme-linked immunosorbent assay kit. Results: The duration of this study was from June 2016 to November 2017. The total number of patients in this study was 15. Thirteen patients responded to treatment. The mean age of patients was 7.1 months. The mean value of baseline VEGF was 0.234 ± 0.059 and that of TIMP-2 was 1.338 ± 0.679. As compared to baseline value, the P value was statistically not significant in any of sequential values. In category-wise analysis, apart from statistically significant value in the 6(th) month in excellent category and good response category in the 1(st) month, all other values did not reveal any significant change in VEGF analysis. The analysis of TIMP-2 revealed a significant change in the levels from Sample 2 to Sample 6 in the excellent response group; however, the levels did not show a specific trend either increasing or decreasing. Conclusion: Despite its beneficial action in regression of hemangioma, the exact mechanism is yet to be identified. The exact duration of treatment needs further evaluation.

Introduction: The management of lymphatic malformations (LMs) continues to improve with advancement in molecular genetics, imaging, and treatment options. However, the management of tongue LMs remains a challenge due to the location, function involved, and long-term disabilities. We propose injection sclerotherapy with bleomycin in the management of spectrum of tongue LMs. Methods: Children with LMs involving the tongue were prospectively treated with bleomycin sclerotherapy. Outcome measured was the efficacy of sclerotherapy, complications, and functional outcome. Results: A total of 11 children underwent sclerotherapy with bleomycin for varying tongue lesions. Excellent outcome was seen in children with macroglossia. Eight children with isolated (focal) lesions had a resolution of symptoms with a clearance of lesions. Specific complications related to bleomycin toxicity were not encountered in our series during the follow-up of 4 years. Conclusion: In our series, children with macroglossia had an excellent outcome with normalization of tongue size and function. Children with focal tongue lesions also had good to excellent outcome. We recommend treatment of tongue LM with bleomycin sclerotherapy as the first line of management. Ease of treatment, early intervention, and excellent response makes it a favorable treatment option.

Patients with Sturge-Weber syndrome (SWS) are susceptible to ocular complications, and among them, glaucoma is one of the most frequent forms. In current study, we utilized multiplex human cytokine antibody array to simultaneously measure the concentration of 40 cytokines in aqueous humor (AH) of patients with SWS-induced glaucoma (SG), or from patients with senile cataract as controls. Compared with the control group, levels of interleukin (IL)-12p40, macrophage inflammatory protein (MIP)-1d, tumor necrosis factor-alpha (TNF-a), IL-5, IL-7, interleukin-6 receptor (IL-6R), and B lymphocyte chemoattractant (BLC) in AH were significantly higher in SG group. Samples from SG patients displayed significantly lower levels of MIP-1b, IL-6, MIP-1a, and monocyte chemoattractant protein (MCP)-1 than controls. Further analysis showed that IL-7, MIP-1a, TNF-a were positively correlated with intraocular pressure (IOP) in patients with early-onset SG. Moreover, IL-12p40 was negatively correlated with age in patients with SG. These cytokines may make contributions to the immunopathogenesis or progression of glaucoma in patients with SWS.

We report a case of Gorham-Stout disease (GSD) complicated by chylothorax and treated with a combination therapy with interferon and bisphosphonates. This treatment may be helpful in improving the usually unfavorable prognosis of GSD beginning with a chylothorax before 1 year of age, and in reducing bone lesions. Moreover, the use of bisphosphonates appears to be useful in treating pain.

Background and purpose—Gorham-Stout disease (GSD) is a rare mono- or polyostotic condition characterized by idiopathic intraosseous proliferation of angiomatous structures resulting in progressive destruction and resorption of bone. Little is known about the course of disease and no previous study has evaluated patients' quality of life (QoL).Patients and methods—This is a retrospective analysis of 7 consecutive patients (5 males) with a median age at diagnosis of 14 years and a median follow-up of 7 years who were diagnosed with GSD in our department between 1995 and 2018. Data regarding clinical, radiographic, and histopathological features, and treatment, as well as sequelae and their subsequent therapy, were obtained. QoL was assessed by Musculoskeletal Tumor Society Score (MSTS), Toronto Extremity Salvage Score (TESS), and Reintegration to Normal Living (RNL) Index.Results—3 patients had a monoostotic and 4 patients a polyostotic disease. Besides a diagnostic biopsy, 4 of the 7 patients had to undergo 8 surgeries to treat evolving sequelae. Using an off-label therapy with bisphosphonates in 6 patients, a stable disease state was achieved in 5 patients after a median of 20 months. The median MSTS, TESS, and RNL Index at last follow-up was between 87% and 79%.Interpretation—Due to its rare occurrence, diagnosis and treatment of GSD remain challenging. Off-label treatment with bisphosphonates appears to lead to a stable disease state in the majority of patients. QoL varies depending on the individual manifestations but good to excellent results can be achieved even in complex polyostotic cases with a history of possibly life-threatening sequelae.

BACKGROUND: The guide for monitoring and treatment of congenital hepatic hemangiomas (CHH) will depend on the subtype and the postnatal clinical behavior. Our aim is to present a series of CHH and characterize its clinical, histologic and genetic correlation, compared to cutaneous congenital hemangiomas (CCH). MATERIAL AND METHODS: A retrospective review of CHH patients diagnosed between 1991 and 2018 was performed. Clinical, morphological and histological data were analyzed and deep high-throughput sequencing was performed. MAIN RESULTS: Sixteen patients with CHH were included. Five patients were followed up with serial ultrasounds while pharmacological treatment (corticosteroids and propranolol) was decided in five. Surgical resection was performed in five owing to hemorrhage and suspicion of malignancy, and the last patient underwent embolization. Histologic analysis was available in 7 patients and confirmed CHH, showing two different histological patterns that could be associated with the presence of somatic pathogenic variants in GNAQ and/or PIK3CA detected in the genetic testing. Review of 7 samples of CCH revealed some histologic differences compared to CHH. CONCLUSION: CHH resemble its cutaneous homonym with similar clinical behavior. Histologic analysis can differentiate two subgroups while genetic testing can confirm mutations in GNAQ and in PIK3CA in a subset of CHH. TYPE OF STUDY: Treatment study. LEVEL OF EVIDENCE: IV.

IMPORTANCE: Capillary malformation-arteriovenous malformation (CM-AVM) syndrome is a recently described syndrome with distinctive cutaneous lesions. Very little is known about the histopathology of these lesions. OBJECTIVE: The purpose of the study was to evaluate the histopathological characteristics of the pink macules of the CM-AVM syndrome and to investigate if these pink macules could be classified as capillary malformations or arteriovenous malformations based on their histopathological features. DESIGN-SETTINGS-PARTICIPANTS: We conducted a retrospective multicenter study involving 8 hospitals in Spain. Fifteen biopsies from pink macules of the CM-AVM syndrome were analyzed, and compared with 5 biopsies of diverse capillary malformations and 3 stage I arteriovenous malformations. RESULTS: Pink macules' biopsies of the CM-AVM syndrome showed similar features including a high vascular density encompassing capillaries and numerous thick walled arterioles mainly located in the superficial dermis, a predominance of elongated over round vessels, scarce or absent erythrocytes within the lumina and discrete perivascular inflammation. CMs were characterized by an increased number of capillary-type vessels mostly rounded and located in the upper dermis. AVMs were composed by highly increased numbers of vessels with a branching pattern involving the full thickness of the dermis, without erythrocytes within the lumina, Wilms tumour 1 protein was positive in the endothelial cells both in pink macules of the CM-AVM and in arteriovenous malformations. CONCLUSIONS AND RELEVANCE: Pink macules of the CM-AVM syndrome seem to be different from capillary malformations. Our results suggest that histologically and immunohistochemically they are closer to incipient arteriovenous malformations than to capillary malformations. A deepened knowledge about the nature of these skin lesions will contribute to the better understanding of capillary malformation-arteriovenous malformation syndrome, and will open the possibility of new and more specific treatments in the future.

AIM: LncRNA MALAT1 is involved in regulation of angiogenesis, however, its expression and mechanism in infantile hemangioma (IH) are less reported. The study aimed to investigate MALAT1 in IH and to reveal the potential mechanism of MALAT1 acting on IH. METHODS: Isolated form IH tissue, human CD31(+) hemangioma endothelial cells (HemECs) were cultured and sorted by magnetic-activated cell sorting (MACS). Quantitative real-time (qRT)-PCR was performed to detect the expressions of MALAT1, miR-206 and VEGFA. The correlations among MALAT1, miR-206 and VEGFA were confirmed by bioinformatics analysis and dual-luciferase reporter assay. The effects of MALAT1, miR-206 and VEGFA on cell proliferation were detected by cell counting kit-8 (CCK-8) and cell colony formation assay. Flow cytometry, wound scratch, Transwell and Tube formation assay were performed to determine cell apoptosis, migration, invasion and vasoformation, respectively. Apoptosis-related proteins were determined by Western blot. RESULTS: The results showed that MALAT1 and VEGFA were high-expressed and miR-206 was low-expressed in IH tissues. SiMALAT1 negatively affected the cell proliferation, migration, invasion and vasoformation of HemECs and promoted apoptosis of HemECs. Moreover, Bcl-2 expression was significantly inhibited and the expressions of Bax and c cleaved-3 were greatly promoted. MALAT1 directly targeted and inhibited the expression of miR-206, and VEGFA was predicted to be the target gene for miR-206. SiMALAT1 suppressed the cell proliferation, migration, invasion and vasoformation of HemECs through modulating miR-206/VEGFA axis. CONCLUSION: Knock-down of MALAT1 inhibits the growth of HemECs through regulating miR-206/VEGFA axis, indicating that MALAT1 is a potential therapeutic mechanism for the treatment of IH.

Infantile hemangioma is one of the most common vascular tumors, which might result in morbidity and mortality without timely intervention. Propranolol is currently the first-line therapy for hemangiomas, but its potential side effects and high frequency of administration make it urgent to develop a suitable drug delivery system for propranolol. In the present study, we formulated a propranolol delivery system based on mesoporous silica nanoparticles (PRN@MSN) and investigated the interplay between autophagic activities mediated by nanoparticles and improved therapeutic efficacy of PRN@MSN. The results showed that PRN@MSN nanoparticles exhibited higher cytotoxicity compared with free propranolol in vitro and in vivo, which could induce excessive autophagosome accumulation through increased autophagosome formation and impaired autophagic degradation. Inhibition of autophagy in the early stage could attenuate the cytotoxicity of PRN@MSN. ROS generation was essential for nanoparticle-mediated autophagy and cytotoxicity, and PRN@MSN-induced autophagy dysfunction could enhance endoplasmic reticulum (ER) stress in hemangioma stem cells. Our study revealed a promising PRN delivery system based on a mesoporous silica nanoplatform that could induce autophagy dysfunction with excessive autophagosome accumulation to promote the therapeutic efficacy of PRN therapy. PRN@MSN drug delivery system combined with autophagy modulation may act as a promising treatment pattern in the treatment of hemangiomas.