Update October 2020

Lymphangiogenesis, the formation of lymphatic vessels from preexisting ones, is an important process in wound-healing physiology. Deregulation of lymphangiogenesis and lymphatic vascular remodeling have been implicated in a range of inflammatory conditions, such as lymphedema, lymphadenopathy, tumor growth, and cancer metastasis. Any attempt in understanding various parameters of the lymphangiogenic process and developing desirable therapeutic targets requires recapitulating these conditions in in vivo models. One pitfall with some experimental models is the absence of immune response, an important regulatory factor for lymphangiogenesis. We overcome this issue by using immune competent mice. In this chapter, by using Angiopoietin-2 (Ang2), a protein that belongs to the Ang/Tie signaling pathway, we describe the ear sponge assay with important adaptations, highlighting a reproducible and quantitative tool for assessment of in vivo lymphangiogenesis.

Epsins are a family of adaptor proteins involved in clathrin-dependent endocytosis. In the vasculature, epsins 1 and 2 are functionally redundant members of this family that are expressed in the endothelial cells of blood vessels and the lymphatic system throughout development and adulthood. These proteins contain a number of peptide motifs that allow them to interact with lipid moieties and a variety of proteins. These interactions facilitate the regulation of a wide range of cell signaling pathways. In this review, we focus on the involvement of epsins 1 and 2 in controlling vascular endothelial growth factor receptor signaling in angiogenesis and lymphangiogenesis. We also discuss the therapeutic implications of understanding the molecular mechanisms of epsin-mediated regulation in diseases such as atherosclerosis and diabetes.

The lymphatic system is essential for tissue regeneration and repair due to its pivotal role in resolving inflammation, immune cell surveillance, lipid transport, and maintaining tissue homeostasis. Loss of functional lymphatic vasculature is directly implicated in a variety of diseases, including lymphedema, obesity, and the progression of cardiovascular diseases. Strategies that stimulate the formation of new lymphatic vessels (lymphangiogenesis) could provide an appealing new approach to reverse the progression of these diseases. However, lymphangiogenesis is relatively understudied and stimulating therapeutic lymphangiogenesis faces challenges in precise control of lymphatic vessel formation. Biomaterial delivery systems could be used to unleash the therapeutic potential of lymphangiogenesis for a variety of tissue regenerative applications due to their ability to achieve precise spatial and temporal control of multiple therapeutics, direct tissue regeneration, and improve the survival of delivered cells. In this review, the authors begin by introducing therapeutic lymphangiogenesis as a target for tissue regeneration, then an overview of lymphatic vasculature will be presented followed by a description of the mechanisms responsible for promoting new lymphatic vessels. Importantly, this work will review and discuss current biomaterial applications for stimulating lymphangiogenesis. Finally, challenges and future directions for utilizing biomaterials for lymphangiogenic based treatments are considered.

The Alzheimer disease is a age related neurodegenerative disease. The factors causing alzheimer disease are numerous. Research on humans and rodent models predicted various causative factors involved in Alzheimer disease progression. Among them, neuroinflammation, oxidative stress and apoptosis play a major role because of accumulation of extracellular amyloid beta peptides. Here, the clearance of amyloid beta peptide plays a major role because of the imbalance in the production and clearance of the amyloid beta peptide. Additionally, neuroinflammation by microglia, astrocytes, cytokines, chemokines and the complement system also have a major role in Alzheimer disease. The physiological clearance pathways involved in amyloid beta peptide are glymphatic, vascular and immune pathways. Amyloid precursor protein, low density lipoprotein receptor-related protein 1, receptor for advanced glycation end product, apolipoprotein E, clusterin, aquaporin 4, auto-antibodies, complement system, cytokines and microglia are involved in amyloid beta peptide clearance pathways across the blood brain barrier. The plaque formation in the brain by alternative splicing of amyloid precursor protein and production of misfolded protein results in amyloid beta agglomeration. This insoluble amyloid beta leads to neurodegenerative cascade and neuronal cell death occurs. Studies had shown disturbed sleep may be a risk factor for dementia and cognitive decline. In this review, the therapeutic targets for alzheimer disease via focussing on pathways for amyloid beta clearance are discussed.

The lymphatic vasculature develops primarily from pre-existing veins. A pool of lymphatic endothelial cells (LECs) first sprouts from cardinal veins followed by migration and proliferation to colonise embryonic tissues. Although much is known about the molecular regulation of LEC fate and sprouting during early lymphangiogenesis, we know far less about the instructive and permissive signals that support LEC migration through the embryo. Using a forward genetic screen, we identified mbtps1 and sec23a, components of the COP-II protein secretory pathway, as essential for developmental lymphangiogenesis. In both mutants, LECs initially depart the cardinal vein but then fail in their ongoing migration. A key cargo that failed to be secreted in both mutants was a type II collagen (Col2a1). Col2a1 is normally secreted by notochord sheath cells, alongside which LECs migrate. col2a1a mutants displayed defects in the migratory behaviour of LECs and failed lymphangiogenesis. These studies thus identify Col2a1 as a key cargo secreted by notochord sheath cells and required for the migration of LECs. These findings combine with our current understanding to suggest that successive cell-to-cell and cell-matrix interactions regulate the migration of LECs through the embryonic environment during development.

Lymphatic vessels (LVs), lined by lymphatic endothelial cells (LECs), are indispensable for life(1). However, the role of metabolism in LECs has been incompletely elucidated. In the present study, it is reported that LEC-specific loss of OXCT1, a key enzyme of ketone body oxidation(2), reduces LEC proliferation, migration and vessel sprouting in vitro and impairs lymphangiogenesis in development and disease in Prox1(DeltaOXCT1) mice. Mechanistically, OXCT1 silencing lowers acetyl-CoA levels, tricarboxylic acid cycle metabolite pools, and nucleotide precursor and deoxynucleotide triphosphate levels required for LEC proliferation. Ketone body supplementation to LECs induces the opposite effects. Notably, elevation of lymph ketone body levels by a high-fat, low-carbohydrate ketogenic diet or by administration of the ketone body beta-hydroxybutyrate increases lymphangiogenesis after corneal injury and myocardial infarction. Intriguingly, in a mouse model of microsurgical ablation of LVs in the tail, which repeats features of acquired lymphoedema in humans, the ketogenic diet improves LV function and growth, reduces infiltration of anti-lymphangiogenic immune cells and decreases oedema, suggesting a novel dietary therapeutic opportunity.

The objective of this study was to obtain data on pathways of absorption of the synthetic pyrethroids deltamethrin (DLM) and cis-permethrin (CPM) following oral administration to rats. Adult male Sprague-Dawley rats with cannulated mesenteric lymph ducts and hepatic portal veins were given single doses of either 5 mg/kg DLM or 60 mg/kg CPM via the duodenum and lymph and portal blood samples collected for up to 300 min. The pyrethroid dosing vehicles (5 mL/kg body weight) were either corn oil or glycerol formal. Levels of DLM and CPM in lymph and portal blood samples were determined by high-performance liquid chromatography-mass spectrometry-mass spectrometry. Over the time period studied, levels of both DLM and CPM following administration in either corn oil or glycerol formal were greater in lymph than in portal blood. Lymphatic uptake of both DLM and CPM was enhanced following dosing in glycerol formal than in corn oil. The results of this study suggest that after oral administration to rats, these two pyrethroids are predominantly absorbed via the lymphatic system rather than via portal blood. The data obtained in this study thus support a recently developed physiologically-based pharmacokinetic (PBPK) model to evaluate age-related differences in pyrethroid pharmacokinetics in the rat, where it was assumed that absorption of pyrethroids was predominantly via lymphatic uptake.

Determining the abundance of leukocyte subtypes, including lymphocyte subpopulations, not only in blood but also in lymphatic tissues, is inevitable to assess the immune status of an organism for research purposes. However, nucleated thrombocytes and erythrocytes exacerbate many hematological techniques in avian species. In order to enable a rapid discrimination of leukocyte subsets from lymphatic tissues of chicken, we adapted existing flow cytometric methods for counting leukocytes in chicken blood. We established staining and gating strategies allowing the flow cytometric characterization and enumeration of total leukocytes, thrombocytes, monocytes/macrophages, CD8alpha(+) lymphocytes, CD4(+) T cells, gammadelta T cells, and B cells in chicken spleen and CD8alpha(+) lymphocytes, CD4(+) T cells, gammadelta T cells, and B cells among intraepithelial lymphocytes in chicken cecal tonsils. For this, we prepared single-cell suspensions of spleen and isolated intraepithelial lymphocytes from cecal tonsils without density centrifugation, and performed antibody staining of cells without subsequent washing steps to prevent cell loss and falsification of obtained cell counts. (c) 2020 The Authors. Cytometry Part A published by Wiley Periodicals LLC on behalf of International Society for Advancement of Cytometry.

Cardiovascular diseases are a leading cause of death worldwide. After an ischemic injury, the myocardium undergoes severe necrosis and apoptosis, leading to a dramatic degradation of function. Numerous studies have reported that cardiac fibroblasts (CFs) play a critical role in heart function even after injury. However, CFs present heterogeneous characteristics according to their development stage (i.e., fetal or adult), and the molecular mechanisms by which they maintain heart function are not fully understood. The aim of this study is to explore the hypothesis that a specific population of CFs can repair the injured myocardium in heart failure following ischemic infarction, and lead to a significant recovery of cardiac function. Flow cytometry analysis of CFs defined two subpopulations according to their relative expression of vascular cell adhesion molecule 1 (VCAM1). Whole-transcriptome analysis described distinct profiles for these groups, with a correlation between VCAM1 expression and lymphangiogenesis-related genes up-regulation. Vascular formation assays showed a significant stimulation of lymphatic cells network complexity by VCFs. Injection of human VCAM1-expressing CFs (VCFs) in postinfarct heart failure rat models (ligation of the left anterior descending artery) led to a significant restoration of the left ventricle contraction. Over the course of the experiment, left ventricular ejection fraction and fractional shortening increased by 16.65%+/-5.64% and 10.43%+/-6.02%, respectively, in VCF-treated rats. Histological examinations revealed that VCFs efficiently mobilized the lymphatic endothelial cells into the infarcted area. In conclusion, human CFs present heterogeneous expression of VCAM1 and lymphangiogenesis-promoting factors. VCFs restore the mechanical properties of ventricular walls by mobilizing lymphatic endothelial cells into the infarct when injected into a rat heart failure model. These results suggest a role of this specific population of CFs in the homeostasis of the lymphatic system in cardiac regeneration, providing new information for the study and therapy of cardiac diseases.

Pathologic lymphatic remodeling in lymphedema evolves during periods of tissue inflammation and hypoxia through poorly defined processes. In human and mouse lymphedema, there is a significant increase of hypoxia inducible factor (HIF)-1alpha, but a reduction of HIF-2alpha protein expression in lymphatic endothelial cells (LECs). We questioned whether dysregulated expression of these transcription factors contributes to disease pathogenesis and found that LEC-specific deletion of Hif-2alpha exacerbated lymphedema pathology. Even without lymphatic vascular injury, the loss of LEC-specific Hif-2alpha caused anatomic pathology and a functional decline in fetal and adult mice. These findings suggest that HIF-2alpha is an important mediator of lymphatic health. HIF-2alpha promoted protective phosphorylated TIE2 (p-TIE2) signaling in LECs, a process also replicated by upregulating TIE2 signaling through adenovirus-mediated angiopoietin-1 (Angpt1) gene therapy. Our study suggests that HIF-2alpha normally promotes healthy lymphatic homeostasis and raises the exciting possibility that restoring HIF-2alpha pathways in lymphedema could mitigate long-term pathology and disability.

Aim: Lymphangioleiomyomatosis is characterized by smooth muscle-like cells in the lungs that spread to other organs via lymphatic vessels. Oral rapamycin is restricted by low bioavailability approximately 15%. The aim of the present study is to systematically investigate the effect of inhaled rapamycin solid lipid nanoparticles (Rapa-SLN) surface charge on efficacy and penetration into the lymphatics. Materials & methods: Rapa-SLN formulations with different charge: neutral, positive and negative, were produced and assessed for their physicochemical particle characteristics and efficacy in vitro. Results: Negative Rapa-SLNs were significantly faster at entering the lymphatic endothelium and more potent at inhibiting lymphanigiogenesis compared with neutral and positive Rapa-SLNs. Conclusion: Negative Rapa-SLNs showed efficient lymphatic access and should therefore be investigated further as a treatment for targeting extrapulmonary lymphangioleiomyomatosis.

INTRODUCTION: Obesity results in lymphatic dysfunction, but the cellular mechanisms that mediate this effect remain largely unknown. Previous studies in obese mice have shown that inducible nitric oxide synthase-expressing (iNOS(+)) inflammatory cells accumulate around lymphatic vessels. In the current study, we therefore tested the hypothesis that increased expression of iNOS results in nitrosative stress and injury to the lymphatic endothelial cells (LECs). In addition, we tested the hypothesis that lymphatic injury, independent of obesity, can modulate glucose and lipid metabolism. METHODS: We compared the metabolic changes and lymphatic function of wild-type and iNOS knockout mice fed a normal chow or high-fat diet for 16 weeks. To corroborate our in vivo findings, we analyzed the effects of reactive nitrogen species on isolated LECs. Finally, using a genetically engineered mouse model that allows partial ablation of the lymphatic system, we studied the effects of acute lymphatic injury on glucose and lipid metabolism in lean mice. RESULTS: The mesenteric lymphatic vessels of obese wild-type animals were dilated, leaky, and surrounded by iNOS(+) inflammatory cells with resulting increased accumulation of reactive nitrogen species when compared with lean wild-type or obese iNOS knockout animals. These changes in obese wild-type mice were associated with systemic glucose and lipid abnormalities, as well as decreased mesenteric LEC expression of lymphatic-specific genes, including vascular endothelial growth factor receptor 3 (VEGFR-3) and antioxidant genes as compared with lean wild-type or obese iNOS knockout animals. In vitro experiments demonstrated that isolated LECs were more sensitive to reactive nitrogen species than blood endothelial cells, and that this sensitivity was ameliorated by antioxidant therapies. Finally, using mice in which the lymphatics were specifically ablated using diphtheria toxin, we found that the interaction between metabolic abnormalities caused by obesity and lymphatic dysfunction is bidirectional. Targeted partial ablation of mesenteric lymphatic channels of lean mice resulted in increased accumulation of iNOS(+) inflammatory cells and increased reactive nitrogen species. Lymphatic ablation also caused marked abnormalities in insulin sensitivity, serum glucose and insulin concentrations, expression of insulin-sensitive genes, lipid metabolism, and significantly increased systemic and mesenteric white adipose tissue (M-WAT) inflammatory responses. CONCLUSIONS: Our studies suggest that increased iNOS production in obese animals plays a key role in regulating lymphatic injury by increasing nitrosative stress. In addition, our studies suggest that obesity-induced lymphatic injury may amplify metabolic abnormalities by increasing systemic and local inflammatory responses and regulating insulin sensitivity. These findings suggest that manipulation of the lymphatic system may represent a novel means of treating metabolic abnormalities associated with obesity.

BACKGROUND: Soybean lecithin, a plant-based emulsifier widely used in food, is capable of modulating postprandial lipid metabolism. With arising concerns of sustainability, alternative sources of vegetal lecithin are urgently needed, and their metabolic effects must be characterized. OBJECTIVES: We evaluated the impact of increasing doses of rapeseed lecithin (RL), rich in essential alpha-linolenic acid (ALA), on postprandial lipid metabolism and ALA bioavailability in lymph-cannulated rats. METHODS: Male Wistar rats (8 weeks old) undergoing a mesenteric lymph duct cannulation were intragastrically administered 1 g of an oil mixture containing 4% ALA and 0, 1, 3, 10, or 30% RL (5 groups). Lymph fractions were collected for 6 h. Lymph lipids and chylomicrons (CMs) were characterized. The expression of genes implicated in intestinal lipid metabolism was determined in the duodenum at 6 h. Data was analyzed using either sigmoidal or linear mixed-effects models, or one-way ANOVA, where appropriate. RESULTS: RL dose-dependently increased the lymphatic recovery (AUC) of total lipids (1100 mug/mL.h per additional RL%; P = 0.010) and ALA (50 mug/mL.h per additional RL%; P = 0.0076). RL induced a faster appearance of ALA in lymph, as evidenced by the exponential decrease of the rate of appearance of ALA with RL (R2 = 0.26; P = 0.0064). Although the number of CMs was unaffected by RL, CM diameter was increased in the 30%-RL group, compared to the control group (0% RL), by 86% at 3–4 h (P = 0.065) and by 81% at 4–6 h (P = 0.0002) following administration. This increase was positively correlated with the duodenal mRNA expression of microsomal triglyceride transfer protein (Mttp; rho = 0.63; P = 0.0052). The expression of Mttp and secretion-associated, ras-related GTPase 1 gene homolog B (Sar1b, CM secretion), carnitine palmitoyltransferase IA (Cpt1a) and acyl-coenzyme A oxidase 1 (Acox1, beta-oxidation), and fatty acid desaturase 2 (Fads2, bioconversion of ALA into long-chain n-3 PUFAs) were, respectively, 49%, 29%, 74%, 48%, and 55% higher in the 30%-RL group vs. the control group (P < 0.05). CONCLUSIONS: In rats, RL enhanced lymphatic lipid output, as well as the rate of appearance of ALA, which may promote its subsequent bioavailability and metabolic fate.

BACKGROUND: Impaired lymphatic drainage of the arterial wall results in intimal lipid accumulation and atherosclerosis. However, the mechanisms regulating lymphangiogenesis in atherosclerotic arteries are not well understood. Our studies identified elevated levels of matrix protein R-Spondin 2 (RSPO2) in atherosclerotic arteries. In this study, we investigated the role of RSPO2 in lymphangiogenesis, arterial cholesterol efflux into lesion-draining lymph nodes and development of atherosclerosis. METHODS AND RESULTS: The effect of RSPO2 on lymphangiogenesis was investigated using human lymphatic endothelial cells in vitro and implanted Matrigel plugs in vivo. Cellular and molecular approaches, pharmacological agents, and siRNA silencing of RSPO2 receptor LGR4 were used to investigate RSPO2-mediated signaling in lymphatic endothelial cells. In vivo LDL tracking and perivascular blockade of RSPO2-LGR4 signaling using LGR4-ECD pluronic gel in hypercholesterolemic mice were utilized to investigate the role of RSPO2 in arterial reverse cholesterol transport and atherosclerosis. Immunoblotting and imaging experiments demonstrated increased RSPO2 expression in human and mouse atherosclerotic arteries compared to non-atherosclerotic controls. RSPO2 treatment inhibited lymphangiogenesis both in vitro and in vivo. LGR4 silencing and inhibition of RSPO2-LGR4 signaling abrogated RSPO2-induced inhibition of lymphangiogenesis. Mechanistically, we found that RSPO2 inhibits PI3K-AKT-eNOS signaling via LGR4 and inhibits activation of the canonical Wnt-beta-catenin pathway. ApoE-/- mice treated with LGR4-ECD developed significantly less atherosclerosis compared with control treatment. Finally, increased arterial lymphatic vessel density and improved lymphatic drainage of fluorescently-labeled LDL to deep cervical lymph nodes were observed in LGR4-ECD-treated mice. CONCLUSIONS: These findings demonstrate that RSPO2 inhibits lymphangiogenesis via LGR4 and downstream impairment of AKT-eNOS-NO signaling. These results may also inform new therapeutic strategies to promote lymphangiogenesis and improve cholesterol efflux from atherosclerotic arteries. TRANSLATIONAL PERSPECTIVE: Atherosclerotic cardiovascular disease is the leading cause of death worldwide. Thus, attenuation of atherosclerotic lesion formation and prevention of its cardiovascular complications is an urgent medical need. The findings of the present study that inhibition of LGR4-mediated signaling increases arterial lymphangiogenesis, improves lymphatic drainage from the vessel wall and attenuates atherosclerosis, provide a framework from which novel therapeutic strategies to augment lymphatic vessel density and reduce atherosclerotic lesion formation can be developed and used for the treatment of patients with atherosclerosis. This pathway may also have important implications in other pathological conditions associated with lymphatic dysfunction, such as lymphedema, obesity, hypertension, and impaired wound healing.

We have developed a macromolecular prodrug platform based on poly(l-lysine succinylated) (PLS) that targets scavenger receptor A1 (SR-A1), a receptor expressed by myeloid and endothelial cells. We demonstrate the selective uptake of PLS by murine macrophage, RAW 264.7 cells, which was eliminated upon cotreatment with the SR-A inhibitor polyinosinic acid (poly I). Further, we observed no uptake of PLS in an SR-A1-deficient RAW 264.7 cell line, even after 24 h incubation. In mice, PLS distributed to lymphatic organs following i.v. injection, as observed by ex vivo fluorescent imaging, and accumulated in lymph nodes following both i.v. and i.d. administrations, based on immunohistochemical analysis with high-resolution microscopy. As a proof-of-concept, the HIV antiviral emtricitabine (FTC) was conjugated to the polymer's succinyl groups via ester bonds, with a drug loading of 14.2% (wt/wt). The prodrug (PLS-FTC) demonstrated controlled release properties in vitro with a release half-life of 15 h in human plasma and 29 h in esterase-inhibited plasma, indicating that drug release occurs through both enzymatic and nonenzymatic mechanisms. Upon incubation of PLS-FTC with human peripheral blood mononuclear cells (PBMCs), the released drug was converted to the active metabolite FTC triphosphate. In a pharmacokinetic study in rats, the prodrug achieved approximately 7–19-fold higher concentrations in lymphatic tissues compared to those in FTC control, supporting lymphatic-targeted drug delivery. We believe that the SR-A1-targeted macromolecular PLS prodrug platform has extraordinary potential for the treatment of infectious diseases.

Lymphatic and blood vascular endothelial cells (ECs) share several molecular and developmental features. However, these two cell types possess distinct phenotypic signatures, reflecting their different biological functions. Despite significant advances in elucidating how the specification of lymphatic and blood vascular ECs is regulated at the transcriptional level during development, the key molecular mechanisms governing their lineage identity under physiological or pathological conditions remain poorly understood. To explore the epigenomic signatures in the maintenance of EC lineage specificity, we compared the transcriptomic landscapes, histone composition (H3K4me3 and H3K27me3) and DNA methylomes of cultured matched human primary dermal lymphatic and blood vascular ECs. Our findings reveal that blood vascular lineage genes manifest a more ‘repressed’ histone composition in lymphatic ECs, whereas DNA methylation at promoters is less linked to the differential transcriptomes of lymphatic versus blood vascular ECs. Meta-analyses identified two transcriptional regulators, BCL6 and MEF2C, which potentially govern endothelial lineage specificity. Notably, the blood vascular endothelial lineage markers CD34, ESAM and FLT1 and the lymphatic endothelial lineage markers PROX1, PDPN and FLT4 exhibited highly differential epigenetic profiles and responded in distinct manners to epigenetic drug treatments. The perturbation of histone and DNA methylation selectively promoted the expression of blood vascular endothelial markers in lymphatic endothelial cells, but not vice versa. Overall, our study reveals that the fine regulation of lymphatic and blood vascular endothelial transcriptomes is maintained via several epigenetic mechanisms, which are crucial to the maintenance of endothelial cell identity.

FOXC2 is a member of the human forkhead-box gene family and encodes a regulatory transcription factor. Mutations in FOXC2 have been associated with lymphedema distichiasis (LD), an autosomal dominant disorder that primarily affects the limbs. Most patients also show extra eyelashes, a condition known as distichiasis. We previously reported genetic and clinical findings in six unrelated families with LD. Half the patients showed missense mutations, two carried frameshift mutations and a stop mutation was identified in a last patient. Here we analyzed the subcellular localization and transactivation activity of the mutant proteins, showing that all but one (p.Y109*) localized to the nucleus. A significant reduction of transactivation activity was observed in four mutants (p.L80F, p.H199Pfs*264, p.I213Tfs*18, p.Y109*) compared with wild type FOXC2 protein, while only a partial loss of function was associated with p.V228M. The mutant p.I213V showed a very slight increase of transactivation activity. Finally, immunofluorescence analysis revealed that some mutants were sequestered into nuclear aggregates and caused a reduction of cell viability. This study offers new insights into the effect of FOXC2 mutations on protein function and shows the involvement of aberrant aggregation of FOXC2 proteins in cell death.

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

Background: While performing microsurgery, including lymphaticovenous anastomosis (LVA) for chronic limb lymphedema, it is a common procedure to identify the subcutaneous collecting lymph ducts with near-infrared fluorescence lymphangiography (NIR) using indocyanine green. However, due to limitations such as minimum observable depth, only a few lymphatic ducts can be identified with this procedure. Hence, we developed a new smaller-diameter “lymphatic wire” (LW) that could be inserted directly into lymphatic collecting ducts of the limbs, enabling accurate identification and localization. Methods and Results: First, used the LW on the hind limbs of 6 swine, and 36 porcine lymphatic collecting ducts were identified, the outer diameter of which varied from 0.3–0.7 mm (mean 0.41+/-0.11 mm). We could insert the LW after creating a side opening in 30 of these ducts. We encountered no difficulties during the procedure. In the pathological examination, adverse events such as valve dysfunction and perforation were not identified. Based on the results, a clinical evaluation of the LW was performed in two patients with lower extremity lymphedema, and the LW helped us identify lymphatic ducts in the subcutaneous layer, even at the sites where the NIR had proved ineffective. Conclusion: Based on our results, we suggest that the procedure for identifying lymphatic vessels using the newly developed LW is a useful technique that can be utilized before performing a LVA for lymphedema. However, further clinical study is required to develop this device and technique, for wider clinical application in the future.

Background: The aim was to compare edema and quality of life (QOL) after complex decongestive therapy (CDT) in two types of lymphedema: primary lower limb lymphedema (PLL) and secondary lower limb lymphedema (SLL). Methods and Results: Participants with PLL (n = 20) and SLL (n = 20) were recruited in this prospective single-blinded study. Patients in both groups were treated with CDT for 4 weeks 5 days a week. The amount of edema in their lower extremities was assessed by circumference measurement. The QOL for the patients was evaluated by a Lymphedema Functioning, Disability and Health Questionnaire for Lower Limb Lymphedema (Lymph-ICF-LL) before and immediately following the therapy. There was no significant difference in the volume reductions between the two groups (p > 0.05). Overall initial QOL was significantly lower in patients with PLL than in patients with SLL scores. Post-CDT differed significantly between PLL and SLL groups, QOL was significantly lower for patients with PLL than for patients with SLL scores (p < 0.05). When the changes in both groups were examined, it was found that their QOL increased after the treatment (p < 0.05). Conclusions: While there was no difference in the amount of edema in both groups, the results of patients with SLL were more positive than patients with PLL in terms of QOL. Lymphedema therapists should approach patients with different therapeutic considerations specific to each type of lymphedema before using CDT in clinical practice.

BACKGROUND: The physical activity (PA) of patients may change after microsurgical treatment for lower extremity lymphedema (LEL). We investigated whether PA changes perioperatively and whether it influences the treatment result. PATIENTS AND METHODS: Sixty patients with unilateral LEL (56 secondary and 4 primary) underwent lymphatic microsurgeries. Patients were divided into two groups based on improvement in International Physical Activity Questionnaire Short Form (IPAQ-SF) categories; the outcomes were compared. RESULTS: Fifty-three patients in whom linear pattern could be partially observed in indocyanine green lymphography or lymphoscintigraphy underwent lymphaticovenular anastomosis (LVA); seven patients in whom no linear pattern was observed underwent simultaneous LVA and vascularized lymph node transfer. No surgical complication was observed. The median IPAQ-SF score significantly improved from 990 (interquartile range: 231–2,376) to 1,386 (interquartile range: 940.5–4,158; p < .0001). The IPAQ-SF category improved in 22 patients (33.7%), who were categorized into the IPAQ-improved group. Improvement in excess limb volume was significantly larger in the IPAQ-improved group than that in the unimproved group (8.0+/-4.2 vs. 3.5+/-2.4%; p < .0001). CONCLUSION: The PA of patients may change after surgical treatment for unilateral LEL. Perioperative improvement in PA significantly correlated with the perioperative change in the excess limb volume. The change in PA is an important factor that might affect the outcome of surgical treatment for LEL. In evaluating the results of microsurgery for lymphedema, it may be necessary to consider changes in PA to avoid bias.

Objectives: This study aims to first measure patient doses during computed tomography (CT) chest, abdomen, and extremities procedures for evaluation lymphedema, and second to estimate the radiation dose-related risks during the procedures. Material and Methods: Radiation effective doses from CT lymphography procedures quantified using CT machines from different vendors. After the calibration of CT systems, the data collected for a total of 28 CT lymphography procedures. Effective and organ doses extrapolated using national radiological protection software based on Monte Carlo simulation. Results: The mean patient doses for chest and abdomen procedures in term of CTDIvol (mGy) and DLP (mGy.cm) are 10.0+/-3 and 425+/-222 and 24+/-12 and 1118+/-812 for CT 128 and CT 16 slice, respectively. The mean DLP (mGy.cm) for extremities was 320+/-140 and 424+/-212 for CT 128 and CT 16 slice, in that order. Conclusion: Patients’ dose showed significant differences due to variation in the scan length and clinical indication. Organs lay in the primary beam received high radiation doses especially in the chest region which increases the probability of radiation-induced cancer. The current patient's doses are higher compared to the previous studies.

BACKGROUND: Circulatory miRNAs are promising biomarkers. The feasibility of using miRNA from dried blood spots (DBS) was investigated using newborn screening cards from patients with cholestasis-lymphedema syndrome (Aagenaes syndrome) and controls. METHODS: Total amount of miRNA and specific miRNAs from DBS were analyzed. miRNA was also obtained from newborn screening cards in patients with cholestasis-lymphedema syndrome/Aagenaes syndrome and in healthy newborns. RESULTS: No differences in miRNA concentrations were found between multispotted samples and samples with one single drop of blood and between central and peripheral punches. Ten repeated freeze-thaw cycles did not significantly change miRNA levels from controls. miR-299 (1.73-fold change, p = 0.034) and miR-365 (1.46-fold change, p = 0.011) were upregulated and miR-30c (0.72-fold change, p = 0.0037), miR-652 (0.85-fold change, p = 0.025), and miR-744 (0.72-fold change, p = 0.0069) were downregulated in patients with Aagenaes syndrome at birth compared to controls. CONCLUSIONS: miRNAs were not affected by multispotting or punch location and were stable throughout repeated freeze-thaw cycles. miRNA in dried blood spots could be used to detect differential expression of miRNA in newborns with Aagenaes syndrome and healthy controls in newborn screening cards. Dried blood spots may be a useful source to explore circulating miRNA as biomarkers. IMPACT: Circulating miRNAs can be useful biomarkers.miRNAs from dried blood spots were not affected by multispotting or punch location and were stable throughout repeated freeze-thaw cycles.Discrimination between patients and controls are allowed even with few individuals.Early after birth, patients with cholestasis-lymphedema syndrome exhibit miRNA profiles associated with liver fibrosis.This study demonstrated that newborn screening cards may be a useful source for studying miRNA as the technical variability is smaller than biological variation. Figure 1RELATION BETWEEN THE AMOUNT OF MATERIAL AND YIELD OF MIRNA.: Comparison of material from three punches (black) and one single punch (gray) measured as a total miRNA detected using Qubit((R)) 2.0 Fluorometer with the Qubit((R)) microRNA Assay Kit; b miR-16; c miR-140, miR-210, and miR-4429 (not related to endogenous control); and d miR-140 (p = 0.97), miR-210 (p = 0.70), and miR-4429 (p = 0.50) (related to endogenous control miR-16). Data are given as mean and standard deviation (n = 8 for all assays). Two-tailed unpaired t test was used. **p < 0.0001. Figure 2EFFECT OF MULTISPOTTED SAMPLES AND LOCATION OF THE PUNCH.: No differences in punch location (central vs peripheral punch) or multispotting (one vs two drops of blood) were demonstrated with regard to a total miRNA levels (p = 0.33), b miR-16 (p = 0.95), or c miR-140 (p = 0.18), miR-210 (p = 0.56), and miR-4429 (p = 0.23) (related to endogenous control miR-16). Data are given as mean and standard deviation (n = 8). Comparisons were performed with one-way ANOVA. Figure 3RELATION BETWEEN MIRNA FROM DBS AND PLASMA.: Comparison of a miR-140, b miR-210, and c miR-4429 from dried blood spots (DBS) (black) and plasma (gray). miRNA are related to endogenous control (miR-16). Data are given as mean and standard deviation (n = 6 for all assays). Comparisons were performed with two-tailed unpaired t test. **p </ = 0.0001. Figure 4EFFECT OF REPEATED FREEZE-THAW CYCLES.: a Total miRNA in dried blood spots (DBS) (p = 0.93) and b miR-16 (p = 0.45), c miR-140 (p = 0.3), (d) miR-210 (p = 0.42), and e miR-4429 (p = 0.13) in DBS after repeated freeze-thaw cycles. miRNA was isolated from one 3.2-mm punch after 1 and 10 cycles. Data are given as mean and standard deviation (n = 6 for all groups). Comparisons were performed with unpaired t test.

Background: Filariasis is a tropical disease caused by infection with nematode parasites of the Filarioidea family. Filariasis is an endemic disease in parts of India, Sub-Saharan Africa, and Southeast Asia. Filariasis is a progressive disease predominantly affecting the lymphoreticular system, which can result in genitourinary complications (hydrocele, scrotal pain, and infertility), lymphedema, and elephantitis. Retroperitoneal fibrosis has a broad etiology, including secondary to chronic infection. Currently an estimated 25 million men are suffering from lymphatic filariasis with urogenital involvement worldwide. Case Presentation: We present a rare case of a 40-year-old man presenting with fever, groin lymphadenopathy, and a history of infertility. Imaging confirmed significant hydronephrosis and retroperitoneal fibrosis. Filariasis serology was positive. Prior bilateral testicular biopsy demonstrated chronic inflammation and atrophy. Disease course was not improved by empirical eradication and supportive retrograde ureteral stenting. The patient developed elephantitis and progressive retroperitoneal fibrosis leading to a solitary functioning right kidney with nephrostomy. Conclusion: Urologists should be aware of index presentations of filariasis and its associated urological complications, particularly in the travelling adult population in whom the etiology of renal impairment and infertility remains unclear.

PURPOSE: Lymphedema is progressive arm swelling from lymphatic dysfunction which can occur in 30% patients undergoing axillary dissection/radiation for breast cancer. Immediate lymphatic reconstruction (ILR) is performed in an attempt decrease the risk of lymphedema in patients undergoing axillary lymph node dissection (ALND). The purpose of this study was to assess the efficacy of ILR in preventing lymphedema rates in ALND patients. METHODS: An institutional review board-approved retrospective review was performed of all patients who underwent ILR from 2017 to 2019. Patient demographics, comorbidities, operative and pathologic findings, number of LVAs, limb measurements, complications, and follow-up were recorded and analyzed. Student's sample t-test, Fisher's exact test, and ANOVA were used to analyze data; significance was set at p < 0.05. RESULTS: Thirty-three patients were included in this analysis. Three patients (9.1%) developed persistent lymphedema, and two patients (6.1%) developed transient arm edema that resolved with compression and massage therapy. A significant effect was found for body mass index and the number of lymph nodes taken on the development of lymphedema (p < 0.01). CONCLUSIONS: The rate of lymphedema in this series was 9.1%, which is an improvement from historical rates of lymphedema. Our findings support ILR as a technique that potentially decreases the incidence of lymphedema after axillary lymphadenectomy. Obesity and number of lymph nodes removed were significant predictive variables for the development of lymphedema following LVA.

RATIONAL: Intestinal lymphangiectasia (IL) is a rare disease characterized by dilatation and rupture of intestinal lymphatic channels leading to protein-losing enteropathy. IL is classified as primary and secondary types. PATIENT CONCERNS: A 3-month-old girl born at term from vaginal delivery with an APGAR score of 10/10 and birth weight of 4.310 g (>97 degrees percentile) was admitted to our hospital because of increasing abdominal tenderness and diarrhea. At first examination, she presented an abdominal circumference of 60 cm, edema of the lower extremities and vulva, and facial dysmorphisms (hypertelorism, flat nasal bridge, flat mid-face). DIAGNOSIS: Once admitted, ultrasonography showed a large amount of ascites, while blood laboratory investigations revealed severe hypoproteinemia, hypoalbuminemia and hypogammaglobulinemia. Lymphoscintigraphy with 99m-Tc-nanocolloid demonstrated abnormal leakage of the tracer in the abdomen as evidence of IL. To detect a possible secondary, exams were performed and demonstrated positive antibody titres for CMV-IgM and IgG in blood and CMV-DNA positivity in blood, urine, saliva, maternal milk, and gastric and duodenal biopsies. Genetic investigations identified the genomic variant c.472C>T of the CCBE1 gene, coding for a protein variant (p.Arg158Cys), in homozygosity. INTERVENTIONS: Total parenteral nutrition was started and continued for a total of 18 days, then gradually bridged by enteral nutrition with a special formula. In addition, antiviral therapy for CMV infection was added first with intravenous ganciclovir for 14 days, resulting in the disappearance of blood viral load after 7 days of therapy and then with valganciclovir per os for another 30 days. OUTCOMES: The clinical course of the child gradually improved. A few days after starting treatments, lower extremities and vulvar edema disappeared, and abdominal circumference gradually decreased to a stable value of 38 cm, without any ultrasonographic signs of ascites left. Moreover, serum albumin and IgG rose to normal values after 3 months (4.3 g/dL and 501 mg/dL, respectively). LESSONS: This case suggests that in presence of IL both primary and secondary causes should be evaluated. On the other hand, genetic diagnosis is crucial not only for diagnosis but also for prognosis in HS. Life expectancy and quality could deeply vary among different gene mutations and protein variants of the same gene. Further studies and case reports are needed to better understand the clinical meaning of these genetic results and the role of CMV as trigger of IL.

Male genital lymphedema is a debilitating condition with significant physiologic and psychologic ramifications. Classical surgical treatments for male genital lymphedema include primarily ablative procedures through removal of excess soft tissue, which often have poor aesthetic and functional outcomes. Super microsurgical techniques (including lymphovenous bypass and lymph node transfers) are promising contemporary interventions. In this case report, we aim to share our experience of lymphovenous bypass with indocyanine green (ICG) lymphangiography in the management of penile and scrotal lymphedema. We performed ICG lymphography of the male genitalia and right thigh by injecting ICG at multiple sites followed by concomitant evaluation with a handheld fluorescent portable imager. Skin incisions were designed over the linear lymphatics upstream from the site of obstruction and dermal backflow. Four end-to-end and one end-to-side lymphovenous bypasses were performed. After completion, lymphovenous bypasses patency was confirmed by injecting ICG proximal to the incision and observing flow. At 10-month clinic follow-up, the patient showed marked improvement with improved skin tenting, softer tissues, improved sensation, visible dorsal penile vein, ability to retract foreskin for cleaning, and confidence to engage in sexual activities. This case report describes successful use of lymphovenous bypass in the treatment of penile and scrotal lymphedema using ICG lymphography intraoperatively to map functioning of superficial lymphatics. The full potential of this microsurgical approach is yet to be discovered, and future studies are needed to enhance the long-term outcomes for the treatment of penoscrotal lymphedema.

BACKGROUND/OBJECTIVES: Patients with obesity and lipedema commonly are misdiagnosed as having lymphedema. The conditions share phenotypic overlap and can influence each other. The purpose of this study was to delineate obesity-induced lymphedema, obesity without lymphedema, and lipedema in order to improve their diagnosis and treatment. SUBJECTS/METHODS: Our Lymphedema Center database of 700 patients was searched for patients with obesity-induced lymphedema (OIL), obesity without lymphedema (OWL), and lipedema. Patient age, sex, diagnosis, cellulitis history, body mass index (BMI), and treatment were recorded. Only subjects with lymphoscintigraphic documentation of their lymphatic function were included. RESULTS: Ninety-eight patients met inclusion criteria. Subjects with abnormal lymphatic function (n = 46) had a greater BMI (65+/-12) and cellulitis history (n = 30, 65%) compared to individuals with normal lymphatic function [(BMI 42+/-10); (cellulitis n = 8, 15%)] (p < 0.001). Seventeen patients had a history of lipedema and two exhibited abnormal lymphatic function (BMI 45, 54). The risk of having lower extremity lymphedema was predicted by BMI: BMI <40 (0%), 40–49 (17%), 50–59 (63%), 60–69 (86%), 70–79 (91%), >/ = 80 (100%). Five patients with OIL (11%) underwent resection of massive localized lymphedema (MLL) or suction-assisted lipectomy. Three individuals (18%) with lipedema were treated with suction-assisted lipectomy. CONCLUSIONS: The risk of lymphedema in patients with obesity and lipedema can be predicted by BMI; confirmation requires lymphoscintigraphy. Individuals with OIL are at risk for cellulitis and MLL. Patients with a BMI >40 are first managed with weight loss. Excisional procedures can further reduce extremity size once BMI has been lowered.

BACKGROUND: Obesity is a risk factor for the development of secondary lymphedema after axillary lymphadenectomy and radiation therapy. The purpose of this study was to determine whether obesity influences the morbidity of lymphedema in patients who have the condition. METHODS: Two cohorts of patients were compared: group 1, normal weight (body mass index </ = 25 kg/m); and group 2, obese (body mass index >/ = 30 kg/m). Inclusion criteria were patients aged 21 years or older with lymphedema confirmed by lymphoscintigraphy. Covariates included age, sex, lymphedema type (primary or secondary), location, comorbidities, lymph node dissection, radiation therapy, lymphoscintigram result, and disease duration. Outcome variables were infection, hospitalization, and degree of limb overgrowth. The cohorts were compared using the Mann-Whitney U test, Fisher's exact test, and multivariable logistic regression. RESULTS: Sixty-seven patients were included: group 1, n = 33; and group 2, n = 34. Disease duration did not differ between groups (p = 0.72). Group 2 was more likely to have an infection (59 percent), hospitalization (47 percent), and moderate or severe overgrowth (79 percent), compared to group 1 (18, 6, and 40 percent, respectively; p < 0.001). Multivariable logistic regression showed that obesity was an independent risk factor for infection (OR, 7.9; 95 percent CI, 2.5 to 26.3; p < 0.001), hospitalization (OR, 30.0; 95 percent CI, 3.6 to 150.8; p < 0.001), and moderate to severe limb overgrowth (OR, 6.7; 95 percent CI, 2.1 to 23.0; p = 0.003). CONCLUSIONS: Obesity negatively affects patients with established lymphedema. Obese individuals are more likely to have infections, hospitalizations, and larger extremities compared to subjects with a normal body mass index. Patients with lymphedema should be counseled about the negative effects of obesity on their condition. CLINICAL QUESTION/LEVEL OF EVIDENCE: Risk, II.

Guadagni, S., et al. (2020). “Contrast enhanced ultrasound for the diagnosis of bilateral renal lymphangiectasia: literature review and contrast enhanced ultrasound findings.” J Ultrasound, EPub 2020/09/05.

Renal lymphangiectasia (RLmp) is a rare benign lymphatic malformation which should be distinguished from other more common pathologies. Ultrasound (US) examination can define the first diagnostic suspicion, but the definitive diagnosis is usually reached with a second level imaging such as computed tomography (CT) or magnetic resonance imaging (MRI). We herein describe for the first time in literature, the use of the contrast enhanced ultrasound (CEUS) in disclosing the nature of peri-renal anechoic lesions in a 27-years old woman, that were initially confused with parenchymal cysts at B-Mode US. The diagnosis of RLmp may be particularly demanding due to its rarity and different clinical presentations. We suggest a possible role of CEUS as a real time, cost saving and easily accessible second level diagnostic tool, that can represent an appealing alternative in the diagnostic work up of suspected RLmp, respect to other imaging modalities.

BACKGROUND: Measurement of extremity volume is the most commonly used method for evaluation of lymphedema. However, volumetry would be inappropriate for comparing patients with different physiques, because body-type significantly affects extremity volume. Thus, we cannot evaluate using absolute values. To overcome this problem, we developed a simple index of proportion of the upper-extremity volume to total body volume (upper-extremity volume/total body volume ratio [UVR]) for body type-corrected volume evaluation of upper-extremity lymphedema. The purpose of this study was to compare upper-extremity volume and UVR in nonedematous upper extremities and to establish normative values of UVR in adult women. METHODS: Eighty-five normal female subjects were included in this study. The average age was 38+/-12 years, and the average body mass index (BMI) was 21.4+/-2.9. Volumetry of both upper extremities using water displacement method was tested in all subjects. Upper-extremity volume/total body volume ratio was calculated by dividing upper-extremity volume by total body volume. Total body volume was calculated by dividing body weight (g) by body density (g/mL). We used linear regression equation (body density = 1.0560–0.0005 x age) to calculate body density. RESULTS: UVR of 170 upper extremities averaged 2.580+/-0.202%. Although there was significant relationship between upper-extremity volume and BMI, there was no relationship between UVR and BMI. CONCLUSIONS: Although further studies are necessary to establish usefulness of UVR, UVR has a potential to allow better body type-corrected volume evaluation for upper-extremity lymphedema.

Neuroimmune interactions are involved in the development of endometriosis. Here, we examined the role of a neuropeptide, calcitonin gene-related peptide (CGRP), and its receptor, receptor activity-modifying protein (RAMP) 1, in growth of endometrial tissues and the formation of blood and lymphatic vessels in a mouse ectopic endometrial transplantation model. Endometrial fragments from donor wild-type (WT) mice transplanted into the peritoneal wall of recipient WT mice grew with increased density of blood and lymphatic vessels. When tissues from RAMP1-deficient (RAMP1(-/-)) mice were transplanted into RAMP1(-/-) mice, implant growth and angiogenesis/lymphangiogenesis were decreased. CGRP was up-regulated in dorsal root ganglia, and CGRP(+) nerve fibres were distributed into the implants from the peritoneum. RAMP1 was co-expressed with CD11b (macrophages) and S100A4 (fibroblasts), but did not co-localize with blood vessel endothelial cell marker CD31 or lymphatic vessel endothelial hyaluronan receptor (LYVE)-1. Cultured with CGRP, macrophages up-regulated vascular endothelial growth factor (VEGF)-A, VEGF-C and VEGF-D, whereas fibroblasts up-regulated VEGF-C, but not VEGF-A or VEGF-D, in a RAMP1-dependent manner. CGRP receptor antagonist CGRP8-37 inhibited growth of and angiogenesis/lymphangiogenesis within endometrial tissue implants. These results suggest that RAMP1 signalling is crucial for growth and angiogenesis/lymphangiogenesis in endometrial tissue. Blockade of RAMP1 is a potential tool for the treatment of endometriosis.

Angiosarcoma (AS) is a rare and aggressive tumor with high rates of local recurrence and distant metastasis. Stewart-Treves syndrome (STS) is defined as AS arising in the setting of chronic lymphedema, and is extremely uncommon in the lower abdominal wall. Eribulin mesylate (eribulin) is a non-taxane microtubule inhibitor that has been approved in Japan for treating soft tissue sarcoma. The current study reports the case of a 76 year-old woman with STS in the lower abdominal wall who exhibited an excellent response to eribulin. Having undergone surgery and postoperative radiation therapy (RT) for cervical cancer 12 years earlier, the patient presented with a mass in her left lower abdominal wall, where chronic lymphedema had developed. Contrast-enhanced computed tomography revealed multiple enhancing nodules in the left lower abdominal wall and edema of the subcutaneous tissues in the whole lower abdomen. A histologic analysis of the specimens revealed AS, and she was diagnosed as STS. A total of 3 cycles of combination chemotherapy with gemcitabine and docetaxel were administered, but the patient discontinued treatment owing to severe adverse events. RT was performed for the tumor, but multiple reddish nodules appeared in the whole lower abdominal wall 3 months later. At this point, eribulin administration was offered. After 4 cycles of treatment, there was a clear reduction in the size of the nodules. All lesions were stable, no new lesions had developed, and the side effects of treatment were minor over the course of 1 year. The results reveal that eribulin may serve as a potential therapeutic option for the treatment of STS.

Background: Secondary lymphedema (LE) can occur after breast cancer (BC) therapy with axillary lymph node surgery and/or radiotherapy. Reported incidence varies around 20%. The aim of this study was to see whether low-energy extracorporeal shockwave therapy (ESWT) is a therapeutic option in end-stage secondary upper limb fibro-LE. Methods and Results: A pilot study was performed on 10 adult patients who presented with an end-stage LE after BC treatment. They were all treated with usual physical therapy and all had lymphatic surgery before. Eight sessions of ESWT were applied, 2600 shocks at 0.1 mJ/mm(2), 2/week during 4 weeks. Upper limb volume decreased nonsignificantly, from 3086.4+/-539.47 to 2909.1+/-471.60 mL. Mean circumference of the upper limb was significantly decreased from 32.3+/-3.01 to 31.4+/-2.71 cm at the height of the upper arm, from 29.1+/-2.89 to 28.1+/-2.71 cm at the height of the elbow, and from 27.5+/-4.08 to 26.8+/-3.75 cm at the height of the forearm. Subjective measurements by visual analog scale showed significant decrease in both hardness from 57.3+/-15.84 to 24.4+/-21.89 mm and subjective feeling of edema from 44.2+/-16.90 to 23.2+/-21.16 mm. No adverse features were reported. Conclusion: We added some evidence that low-energy ESWT is well supported and has additional benefits also in longstanding fibro-lipo-LE on swelling of the arm leading to more subjective comfort for the patients.

There is growing evidence on an interconnection between the venous and lymphatic systems in venous leg ulceration, and the possible effects of prolonged oedema and lymphatic impairment in delayed wound healing. Compression therapy is a widely accepted treatment for venous and lymphatic disorders, as it decreases recurrence rates and prolongs the interval between recurrences. Compression bandages improve venous return, increase the volume and rate of venous flow, reduce oedema and stimulate anti-inflammatory processes. The pressure at the interface (IP) of the bandage and the skin is related to the elastic recoil of the product used and its resistance to expansion. The pressure difference between the IP in the supine and standing positions is called the static stiffness index (SSI). Elastic materials provide little resistance to muscle expansion during physical activity, resulting in small pressure differences between resting and activity, with an SSI <10mmHg. Stiff, inelastic materials with a stretch of <100% resist the increase of muscle volume during physical activity, producing higher peak pressures, an SSI of >10mmHg and a greater haemodynamic benefit than elastic systems. UrgoK2 is a novel dual-layer high-compression system consisting of an inelastic (short stretch) and elastic (long stretch) bandage, resulting in sustained tolerable resting pressure and elevated working pressures over extended wear times. It is indicated for the treatment of active venous leg ulcers and the reduction of chronic venous oedema. Each bandage layer has a visual aid to enable application at the correct pressure level. Published European studies have assessed this compression system, exploring its consistency of application, tolerability and efficacy. This article presents the first reports of health professionals’ clinical experience of using the compression system in the US, where it has been recently launched. Initial feedback is promising.

Primary lymphedema is caused by developmental and functional defects of the lymphatic vascular system that result in accumulation of protein-rich fluid in tissues, resulting in edema. The 28 currently known genes causing primary lymphedema can explain <30% of cases. Angiopoietin 1 (ANGPT1) and ANGPT2 function via the TIE1-TIE2 (tyrosine kinase with immunoglobulin-like and epidermal growth factor-like domains 1 and 2) receptor complex and alpha5beta1 integrin to form an endothelial cell signaling pathway that is critical for blood and lymphatic vessel formation and remodeling during embryonic development, as well as for homeostasis of the mature vasculature. By screening a cohort of 543 individuals affected by primary lymphedema, we identified one heterozygous de novo ANGPT2 whole-gene deletion and four heterozygous ANGPT2 missense mutations. Functional analyses revealed three missense mutations that resulted in decreased ANGPT2 secretion and inhibited the secretion of wild-type (WT)-ANGPT2, suggesting that they have a dominant-negative effect on ANGPT2 signaling. WT-ANGPT2 and soluble mutants T299M and N304K activated TIE1 and TIE2 in an autocrine assay in human lymphatic endothelial cells. Molecular modeling and biophysical studies showed that amino-terminally truncated ANGPT subunits formed asymmetrical homodimers that bound TIE2 in a 2:1 ratio. The T299M mutant, located in the dimerization interphase, showed reduced integrin alpha5 binding, and its expression in mouse skin promoted hyperplasia and dilation of cutaneous lymphatic vessels. These results demonstrate that primary lymphedema can be associated with ANGPT2 mutations and provide insights into TIE1 and TIE2 activation mechanisms.

BACKGROUND: Lymphatic filariasis is a neglected tropical disease that can cause permanent disability through disruption of the lymphatic system. This disease is caused by parasitic filarial worms that are transmitted by mosquitos. Mass drug administration (MDA) of antihelmintics is recommended by WHO to eliminate lymphatic filariasis as a public health problem. This study aims to produce the first geospatial estimates of the global prevalence of lymphatic filariasis infection over time, to quantify progress towards elimination, and to identify geographical variation in distribution of infection. METHODS: A global dataset of georeferenced surveyed locations was used to model annual 2000-18 lymphatic filariasis prevalence for 73 current or previously endemic countries. We applied Bayesian model-based geostatistics and time series methods to generate spatially continuous estimates of global all-age 2000-18 prevalence of lymphatic filariasis infection mapped at a resolution of 5 km(2) and aggregated to estimate total number of individuals infected. FINDINGS: We used 14 927 datapoints to fit the geospatial models. An estimated 199 million total individuals (95% uncertainty interval 174–234 million) worldwide were infected with lymphatic filariasis in 2000, with totals for WHO regions ranging from 3.1 million (1.6–5.7 million) in the region of the Americas to 107 million (91–134 million) in the South-East Asia region. By 2018, an estimated 51 million individuals (43–63 million) were infected. Broad declines in prevalence are observed globally, but focal areas in Africa and southeast Asia remain less likely to have attained infection prevalence thresholds proposed to achieve local elimination. INTERPRETATION: Although the prevalence of lymphatic filariasis infection has declined since 2000, MDA is still necessary across large populations in Africa and Asia. Our mapped estimates can be used to identify areas where the probability of meeting infection thresholds is low, and when coupled with large uncertainty in the predictions, indicate additional data collection or intervention might be warranted before MDA programmes cease. FUNDING: Bill & Melinda Gates Foundation.

BACKGROUND: Lymphedema is a debilitating chronic condition with no definitive curative treatment. There is a paucity of data on prevalence, mortality and burden of care in the United States. This study aimed to describe hospitalizations due to lymphedema and identify factors associated with poor outcomes. STUDY DESIGN: We performed a retrospective analysis of the Healthcare Cost and Utilization Project National Inpatient Sample for lymphedema-related hospitalizations from 2012 to 2017. All adults admitted with a primary diagnosis of lymphedema or a primary diagnosis of extremity cellulitis and secondary diagnosis of lymphedema were included. Weights were used to obtain national estimates and complex sampling structure was accounted for using survey methods. Chi-squared tests were performed to assess the association between patient demographics, hospital characteristics, and inpatient mortality. RESULTS: Between 2012 and 2017, there were an estimated 165,055 lymphedema admissions in the United States. The vast majority of hospitalizations were for cellulitis (92%), lower extremity disease (88%), and were admitted through the emergency room (77%). Median length of stay was 3.61 days (IQR 2.24–5.77) and inpatient mortality was 0.03% (n = 505). Age >80yrs (OR 3.83, 95% CI 1.72, 8.52) and Charlson comorbidity index >/ = 3 (OR 4.13, 95% CI 2.19, 7.79) were associated with increased risk of inpatient mortality. CONCLUSION: Although mortality is low, lymphedema-related hospitalizations are a significant burden to the US healthcare system. Counseling older lymphedema patients with comorbidities on early symptoms and signs of infection and prompt treatment of same may reduce mortality.

Emerging evidence shows that following injury or infection, the mesenteric lymph acts as a conduit for gut-derived toxic factors to enter the blood circulation, causing systemic inflammation and acute lung injury. Neither the cellular and molecular identity of lymph factors, nor their mechanisms of actions, have been well understood and thus become a timely topic of investigation. This review will first provide a summary of background knowledge on gut barrier and mesenteric lymphatics, followed by a discussion focusing on the current understanding of potential injurious factors in the lymph and their mechanistic contributions to lung injury. We also examine lymph factors with anti-inflammatory property as well as the bidirectional nature of the gut-lung axis in inflammation.

The role of the interstitial matrix in regulating exchanges and interactions at the level of the microvascular units, between the corpuscular component and the vascular and nerve structures, has long been known. Equally known are the objective and subjective clinical manifestations that these pathologies trigger in the patient, both in primary and secondary forms: embarrassment, asthenia, alterations of exteroceptive and proprioceptive sensitivity, pain, reduced capacity, and functional autonomy, affecting both the lower and the upper limbs. The authors studied 136 patients with I and II clinical stage lymphedema according to the International Society of Lymphology stadiation. Patients were treated with Lymdiaral, 20 drops three times a day for 90 consecutive days. The following parameters were examined in basal conditions and after treatment: ultrasound measurement of suprafascial thickness, short form healthy survey version 12 (SF12), body mass index, and limb circumferences in specific points of reference. The results demonstrate the effectiveness of the therapeutic principle both in the reduction of limb circumferences and in the improvement of the parameters related to the quality of life expressed by the SF12 items. There were no substantial differences in the results between primary and secondary forms.

Cholestatic liver disease affects millions of people worldwide and stems from a plethora of causes such as immune dysfunction, genetics, cancerous growths, and lifestyle choices. While not considered a classical lymphatic organ, the liver plays a vital role in the lymph system producing up to half of the body's lymph per day. The lymphatic system is critical to the health of an organism with its networks of vessels that provide drainage for lymphatic fluid and routes for surveilling immune cells. Cholestasis results in an increase of inflammatory cytokines, growth factors, and inflammatory infiltrate. Left unchecked, further disease progression will include collagen deposition which impedes both the hepatic and lymphatic ducts, eventually resulting in an increase in hepatic decompensation, increasing portal pressures, and accumulation of fluid within the abdominal cavity (ascites). Despite the documented interplay between these vital systems, little is known about the effect of liver disease on the lymph system and its biological response. This review looks at the current cholestatic literature from the perspective of the lymphatic system and summarizes what is known about the role of the lymph system in liver pathogenesis during hepatic injury and remodeling, immune-modulating events, or variations in interstitial pressures.

Background: ARAP3 is a small GTPase-activating protein regulator, which has important functions in lymphatic vessel organogenesis and modulation of cell adhesion and migration. Mutations in the ARAP3 gene are associated with impaired lymphatic vessel formation. Objective: The aim of our study was to determine the genotypes of lymphedema patients in relation to variants in the ARAP3 gene in order to explore its role in the development of lymphedema. Methods and Results: We applied next-generation sequencing to DNA samples of a cohort of 246 Italian patients with lymphatic malformations. When we tested probands for known lymphedema genes, 235 out of 246 were negative. Retrospectively, we tested the DNA of these 235 patients for new candidate lymphedema-associated genes, including ARAP3. Three out of 235 probands proved to carry rare missense heterozygous variants in ARAP3. In the case of two families, other family members were also tested and proved negative for the ARAP3 variant, besides being unaffected by lymphedema. According to in silico analysis, alterations due to these variants have a significant impact on the overall structure and stability of the resulting proteins. Conclusions: Based on our results, we propose that variants in ARAP3 could be included in genetic testing for lymphedema.

Although there is increasing importance placed on the use of mathematical models for the effective design and management of long-term parasite elimination, it is becoming clear that transmission models are most useful when they reflect the processes pertaining to local infection dynamics as opposed to generalized dynamics. Such localized models must also be developed even when the data required for characterizing local transmission processes are limited or incomplete, as is often the case for neglected tropical diseases, including the disease system studied in this work, viz. lymphatic filariasis (LF). Here, we draw on progress made in the field of computational knowledge discovery to present a reconstructive simulation framework that addresses these challenges by facilitating the discovery of both data and models concurrently in areas where we have insufficient observational data. Using available data from eight sites from Nigeria and elsewhere, we demonstrate that our data-model discovery system is able to estimate local transmission models and missing pre-control infection information using generalized knowledge of filarial transmission dynamics, monitoring survey data, and details of historical interventions. Forecasts of the impacts of interventions carried out in each site made by the models estimated using the reconstructed baseline data matched temporal infection observations and provided useful information regarding when transmission interruption is likely to have occurred. Assessments of elimination and resurgence probabilities based on the models also suggest a protective effect of vector control against the reemergence of LF transmission after stopping drug treatments. The reconstructive computational framework for model and data discovery developed here highlights how coupling models with available data can generate new knowledge about complex, data-limited systems, and support the effective management of disease programs in the face of critical data gaps.

Secondary lymphedema (SL)is a frequent and devastating complication of modern oncological therapy and filarial infections. A lack of a reliable preclinical model to investigate the underlying mechanism of clinical stage progression has limited the development of new therapeutic strategies. Current first line treatment has shown to be merely symptomatic and relies on lifetime use of compression garments and decongestive physiotherapy. In this study, we present the development of a secondary lymphedema model in 35 rats using pre- and intraoperative fluorescence-guided mapping of the lymphatics and microsurgical induction. In contrast to the few models reported so far, we decided to avoid the use of radiation for lymphedema induction. It turned out, that the model is nearly free of complications and capable of generating a statistically significant limb volume increase by water displacement measurements, sustained for at least 48 days. A translational, accurate lymphatic dysfunction was visualized by a novel VIS-NIR X-ray ICG-Clearance-Capacity imaging technology. For the first-time SL stage progression was validated by characteristic histological alterations, such as subdermal mast cell infiltration, adipose tissue deposition, and fibrosis by increased skin collagen content. Immunofluorescence confocal microscopy analysis suggested that stage progression is related to the presence of a characteristic alpha SMA+/HSP-47+/vimentin+ fibroblast subpopulation phenotype. These findings demonstrate that the in-vivo model is a reliable and clinically relevant SL model for the development of further secondary lymphedema therapeutic strategies and the analysis of the veiled molecular mechanisms of lymphatic dysfunction.

The aim of this study was to compare the incidence of infection and verrucous hyperkeratosis in patients who underwent surgery for advanced lymphedema according to the algorithm designed by the senior author, and were treated concurrently with/without toe treatment. A case series (Between 2004–2015) of 46 patients with unilateral advanced lower limb lymphedema was reviewed. Lymphoscintigraphy was used for evaluation of lymphedema severity. The ICG lymphography was used for staging. Fibrosis and skin induration were reflected by the tonicity. They were divided into two groups: (1) patients who underwent further treatment of toes according to the algorithm, and (2) patients who did not have toe-related treatment. Infection episodes and verrucous hyperkeratosis were recorded. There were 21 and 25 patients in Groups 1 and 2, respectively. All lymphoscintigrams showed severe dermal backflow with severe stagnation by 2.5h after injection of Tc-99 colloid. All patients were stage IV or V. Tonicity values of skin were <60. Group 1 was reduced to an average of 0.6 episodes per year in the past year of follow-up, and Group 2 was reduced to an average of 1.5 episodes per year in the past year of follow-up (p < 0.001). The average frequency of preoperative cellulitis was 3.6 episodes per year. The occurrence of verrucous hyperkeratosis was observed in 14.3% and 32% of Groups 1 and 2, respectively. The incidences of cellulitis and verrucous hyperkeratosis were significantly lower in Group 1 than in Group 2 (p < 0.001). To achieve successful control of infection, they should be treated carefully according to the strategy described above.

Background: To examine the correlation between lymphedema severity on lymphoscintigraphy and indocyanine green (ICG) lymphography in patients with secondary lower extremity lymphedema. Methods and Results: The maximal circumference difference (MCD) between the two legs was recorded. Lymphoscintigraphy and ICG lymphography images were classified into type I to V according to dermal backflow (DB) stage and MD Anderson Cancer Center (MDACC) stage based on lymphatic flow preservation and how DB was extended. Correlation between the scales was analyzed. Forty-four patients attended our hospital for evaluation of secondary lower extremity lymphedema. The most common cause of lymphedema was a postoperative complication of a malignant tumor (32 patients; 72.5%). Correlation analysis showed that lymphoscintigraphy and ICG DB (anterior) stage (r = 0.92), lymphoscintigraphy and ICG DB (posterior) stage (r = 0.94), and lymphoscintigraphy and MDACC stage (r = 0.93) exhibited very strong positive correlations. Intrarater agreement between lymphoscintigraphy and ICG DB (posterior) stage was substantial (kappa = 0.65), and moderate between lymphoscintigraphy and ICG DB (anterior) stage (kappa = 0.59) and lymphoscintigraphy and MDACC stage (kappa = 0.52). Lymphedema severity stages and MCDs exhibited moderate positive correlations. Conclusion: Lymphoscintigraphy and ICG lymphography stage were strongly and positively correlated. These studies can work synergistically as complementary studies of lymphedema severity.

BACKGROUND: Ulcerative colitis (UC) was a type of inflammatory bowel diseases, which was difficult to cure and even would malignant turn into colon cancer. The specific etiology and molecular mechanism of UC were unclear to date. The purpose of this study was to search for new targets for the diagnosis and treatment of UC. METHODS: Firstly, we downloaded the gene expression data of UC from the gene expression omnibus database database (GSE107499), and used multiple bioinformatics methods to find differently expressed genes (DEGs) in UC. Subsequently, we evaluated the lymphocyte infiltration in UC inflamed colon tissue by using the cell type identification by estimating relative subset of known RNA transcripts method. RESULTS: We obtained 1175 DEGs and 8 hub genes (IL6, TNF, PTPRC, CXCL8, FN1, CD44, IL1B, and MMP9) in this study. Among them, 903 DEGs were up-regulated and 272 DEGs were down-regulated. Compared with non-inflamed colon tissues, the inflamed colon tissues had higher levels of memory B cells, activated memory CD4 T cells, follicular helper T cells, M1 macrophages, resting dendritic cells, activated dendritic cells, activated mast cells, and neutrophils, whereas the proportions of plasma cells, resting memory CD4 T cells, gamma delta T cells, activated NK cells, M2 macrophages and resting mast cells were relatively lower. CONCLUSIONS: The DEGs, hub genes and different lymphatic infiltration conditions can provide new targets for diagnosis and treatment of UC. However, these were just predictions through some theoretical methods, and more basic experiments will be needed to prove in the future.

Breast-cancer-acquired lymphedema is routinely diagnosed from the appearance of irreversible swelling that occurs as a result of lymphatic dysfunction. Yet in head and neck cancer survivors, lymphatic dysfunction may not always result in clinically overt swelling, but instead contribute to debilitating functional outcomes. In this review, we describe how cancer metastasis, lymph node dissection, and radiation therapy alter lymphatic function, as visualized by near-infrared fluorescence lymphatic imaging. Using custom gallium arsenide (GaAs)-intensified systems capable of detecting trace amounts of indocyanine green administered repeatedly as lymphatic contrast for longitudinal clinical imaging, we show that lymphatic dysfunction occurs with cancer progression and treatment and is an early, sub-clinical indicator of cancer-acquired lymphedema. We show that early treatment of lymphedema can restore lymphatic function in breast cancer and head and neck cancer patients and survivors. The compilation of these studies provides insights to the critical role that the lymphatics and the immune system play in the etiology of lymphedema and associated co-morbidities.

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

Lymphedema is a complex disease caused by the accumulation of fluid in the tissues resulting from a dysfunctional or damaged lymphatic vasculature. In developed countries, lymphedema most commonly occurs as a result of cancer treatment. Initially, impaired lymph flow causes edema, but over time this results in inflammation, fibrotic and fatty tissue deposition, limited mobility, and bacterial infections that can lead to sepsis. While chronically impaired lymph flow is generally believed to be the instigating factor, little is known about what pathophysiological changes occur in the lymphatic vessels to inhibit lymph flow. Lymphatic vessels not only regulate lymph flow through a variety of physiologic mechanisms, but also respond to lymph flow itself. One of the fascinating ways that lymphatic vessels respond to flow is by growing bicuspid valves that close to prevent the backward movement of lymph. However, lymphatic valves have not been investigated in cancer-related lymphedema patients, even though the mutations that cause congenital lymphedema regulate genes involved in valve development. Here, we review current knowledge of the regulation of lymphatic function and development by lymph flow, including newly identified genetic regulators of lymphatic valves, and provide evidence for lymphatic valve involvement in cancer-related lymphedema.

BACKGROUND: This study aimed to examine the prevalence, location, and severity of chronic internal, external, and combined head and neck lymphedema (HNL) in patients with head and neck (HNC) who were treated with definitive chemoradiotherapy (CRT) or postoperative radiotherapy (PORT). METHODS: Sixty-two participants between 1 and 3 years post-treatment were recruited. Internal HNL was rated with Patterson's Scale. External HNL was graded with the MD Anderson Cancer Center Lymphedema Rating Scale. RESULTS: Ninety-eight percent of participants presented with some form of chronic HNL. Sixty-one percent had internal HNL only, 35% had combined HNL, and 2% had external HNL only. Participants treated with PORT were more likely to experience combined HNL (69% vs 24%, P = .001), whereas those treated with CRT were more likely to have internal HNL only (74% vs 25%, P = .001). CONCLUSIONS: Chronic HNL is highly prevalent following multimodal treatment, and differences in HNL presentations exist between treatment modalities.

Clinical response to immunotherapy is closely associated with the immunosuppressive tumour microenvironment (TME), and influenced by the dynamic interaction between tumour cells and lymphatic endothelial cells (LECs). Here, we show that high levels of miR-142-5p positively correlate with indoleamine 2,3-dioxygenase (IDO) expression in tumour-associated lymphatic vessels in advanced cervical squamous cell carcinoma (CSCC). The miR-142-5p is transferred by CSCC-secreted exosomes into LECs to exhaust CD8(+) T cells via the up-regulation of lymphatic IDO expression, which was abrogated by an IDO inhibitor. Mechanistically, miR-142-5p directly down-regulates lymphatic AT-rich interactive domain-containing protein 2 (ARID2) expression, inhibits DNA methyltransferase 1 (DNMT1) recruitment to interferon (IFN)-gamma promoter, and enhances IFN-gamma transcription by suppressing promoter methylation, thereby leading to elevated IDO activity. Furthermore, increased serum exosomal miR-142-5p levels and the consequent IDO activity positively correlate with CSCC progression. In conclusion, exosomes secreted by CSCC cells deliver miR-142-5p to LECs and induce IDO expression via ARID2-DNMT1-IFN-gamma signalling to suppress and exhaust CD8(+) T cells. Our study suggests that LECs act as an integral component of the immune checkpoint(s) in the TME and may serve as a potential new target for CSCC diagnosis and treatment.

Proteus syndrome is a mosaic genetic overgrowth disorder caused by a postzygotic, mosaic activating mutation in AKT1. Rare prenatal presentations include segmental tissue overgrowth, and skeletal and CNS anomalies. We present the first report of prenatally diagnosed and molecularly confirmed Proteus syndrome. Prenatal imaging identified megalencephaly, brain and eye malformations, focal soft tissue enlargement, and ambiguous genitalia. Exome sequencing performed on cultured amniocytes demonstrated an AKT1 pathogenic variant consistent with Proteus syndrome, and postnatal examination confirmed the diagnosis. Postnatal Sanger sequencing could not identify the AKT1 pathogenic variant. This case underscores the importance of prenatal exome sequencing on cultured amniocytes for mosaic overgrowth disorders, as well as provides additional information on the prenatal phenotype of Proteus syndrome, and highlights the impact of prenatal diagnosis on postnatal management.

Hereditary hemorrhagic telangiectasia (HHT, Osler-Weber-Rendu disease) is a rare multisystem vascular disorder causing chronic gastrointestinal bleeding, epistaxis, and severe anemia. Bevacizumab, an anti-vascular endothelial growth factor antibody, may be effective to treat bleeding in HHT. This international, multicenter, retrospective study evaluated the use of systemic bevacizumab to treat HHT-associated bleeding and anemia at 12 HHT treatment centers. Hemoglobin, epistaxis severity score, red cell units transfused, and intravenous iron infusions before and after treatment were evaluated using paired means testing and mixed-effects linear models. 238 HHT patients received bevacizumab for a median of 12 (range, 1–96) months. Compared with pretreatment, bevacizumab increased mean hemoglobin by 3.2 g/dL (95% CI, 2.9–3.5 g/dL) [mean hemoglobin 8.6 (8.5, 8.8) g/dL versus 11.8 (11.5, 12.1) g/dL, p < 0.0001)] and decreased the epistaxis severity score (ESS) by 3.4 (3.2–3.7) points [mean ESS 6.8 (6.6–7.1) versus 3.4 (3.2–3.7), P < 0.0001] during the first year of treatment. Compared with 6 months pretreatment, RBC units transfused decreased by 82% [median of 6.0 (IQR 0.0–13.0) units versus 0 (IQR, 0.0–1.0) units, P < 0.0001] and iron infusions decreased by 70% [median of 6.0 (1.0–18.0) infusions versus 1.0 (0.0–4.0) infusions, P < 0.0001] during the first 6 months of bevacizumab treatment. Outcomes were similar regardless of underlying pathogenic mutation. Following initial induction infusions, continuous/scheduled bevacizumab maintenance achieved higher hemoglobin and lower ESS than intermittent/as needed maintenance but with more drug exposure. Bevacizumab was well tolerated: hypertension, fatigue, and proteinuria were the most common adverse events. Venous thromboembolism occurred in 2% of patients. In conclusion, systemic bevacizumab was safe and effective to manage chronic bleeding and anemia in HHT.

BACKGROUND: Hereditary Hemorrhagic Telangiectasia (HHT) is a rare inherited disorder characterized by development of mucocutaneous telangiectases and visceral organ arteriovenous malformations, which can lead to recurrent, spontaneous bleeding and development of iron deficiency anemia. The primary objective of this study was to ascertain the relationship between epistaxis severity scores (ESS), laboratory values, genotype, and phenotype in HHT. Our secondary objective was to assess efficacy of systemic antifibrinolytic therapy in reducing ESS in HHT. METHODOLOGY: We conducted a retrospective review of patients seen at the UNC HHT Center from January 1, 2009 to February 28, 2015. ESS, demographics, and results of genetic testing were abstracted from the medical record. Response to antifibrinolytic therapy was evaluated by comparing pre-post ESS. RESULTS: One hundred and forty nine patients were eligible with 116 having genetic testing and 33 without. Age, hemoglobin and ferritin levels were predictive of ESS. Of the 116 patients that underwent genetic testing: 63 had an ACVRL1 mutation, 40 had an ENG mutation, 2 had a SMAD4 mutation, and 11 patients had no pathologic HHT genetic variation detected. Compared to patients without a detectable HHT-associated genetic variation, patients with a HHT-associated genetic variation had higher ESS scores (p < 0.05). Neither ESS nor genotype was predictive of pulmonary or brain AVMs. Twenty-four HHT patients with ESS >4 were started on antifibrinolytic therapy (tranexamic acid or aminocaproic acid) and had a post-treatment ESS recorded. All patients had a decrease in ESS of >0.71 (minimal meaningful difference), but patients taking antifibrinolytics displayed larger decreases. No patients on antifibrinolytics experienced a VTE with median follow up of 13 months. CONCLUSIONS: We demonstrate that the ESS correlates with age, hemoglobin and ferritin. Additionally, we demonstrate that HHT patients with genetic mutations have higher ESS scores. Our data demonstrate that antifibrinolytics are effective in decreasing epistaxis severity and safe with long-term use in HHT patients.

BACKGROUND: We determined the frequency of cerebrovascular malformations in a pediatric cohort with hereditary hemorrhagic telangiectasia. METHODS: Retrospective cohort study of 54 children diagnosed with hereditary hemorrhagic telangiectasia at a tertiary care center. All neuroimaging was reviewed to assess for number and types of cerebrovascular malformations and for intracerebral hemorrhage and arterial ischemic stroke. Clinical charts were reviewed for clinical manifestations, genetic mutation, and clinically evident intracerebral hemorrhages and arterial ischemic strokes. RESULTS: Among 54 children with hereditary hemorrhagic telangiectasia with a median age of 3.5 years (interquartile range 0.4 to 7.9 years) at diagnosis, neuroimaging was performed in 52 (96.3%) at a median age of 5.2 years (interquartile range 1.8 to 9 years). Fourteen of 52 imaged children (26.9%) had cerebrovascular malformations. Cerebrovascular malformations included arteriovenous malformations, arteriovenous fistulas, vein of Galen malformations, and developmental venous anomalies. Six of the 14 children with cerebrovascular malformations (42.9%) had multiple malformations. Three children developed new cerebral arteriovenous malformations over time. Six children (11.1%) had clinically evident intracerebral hemorrhage, arterial ischemic stroke, or transient ischemic attack. The three children with intracerebral hemorrhage presented at young ages (4.3 to 7.7 years). CONCLUSIONS: More than a quarter of children with hereditary hemorrhagic telangiectasia who were imaged had cerebrovascular malformations, and overt stroke occurred in more than 10%. Intracerebral hemorrhages can occur in pediatric hereditary hemorrhagic telangiectasia patients at young ages, and new cerebral arteriovenous malformations may develop over time. Early screening with neuroimaging including neurovascular imaging as well as repeat neuroimaging may be warranted in children with hereditary hemorrhagic telangiectasia.

A 2-month-old boy developed a protuberant, blue nodule inferomedial to the left medial canthus. It was unresponsive to oral and intramuscular antibiotics. After developing difficulty breathing, he was admitted, with the diagnosis of a dacryocele, and, after an inconclusive ultrasound, underwent probing and irrigation with nasal endoscopy. Intraoperatively, the lesion appeared discontinuous with the nasolacrimal system and could not be decompressed. Postoperative magnetic resonance imaging suggested a hemangioma or possible collapsed dacryocele. Doppler ultrasound confirmed a perinasolacrimal duct hemangioma. Systemic propranolol treatment was initiated.

BACKGROUND: Significant advances have been reported recently in the genetic and mechanistic characterization of extracranial venous malformations. However, intracranial purely venous malformations (icVM) analogous to those outside the CNS have not been systematically described. PURPOSE: We sought to ascertain whether such an entity as icVM could in fact be identified, distinct from previously described CNS venous anomalies and analogous to extracranial venous malformations. METHODS: Our prospectively collected pediatric cerebrovascular database was reviewed to identify patients with icVM; 1458 consecutive angiograms and/or angiographic interventions performed on 706 children at our institution from October, 2006 through May, 2019 were evaluated, in addition to outside imaging studies on 192 additional patients sent to our Vascular Anomalies Center for cerebrovascular review during the same time period. Thus, the cohort consisted of 898 children. RESULTS: Nineteen of 898 patients (2.1%) were found to harbor icVM, including 9 (47.3%) with sinus pericranii, 15 (78.9%) with associated large, complex extracranial venous malformations, and 3 (15.7%) with neurocognitive delay. There was no intracranial hemorrhage or venous hypertension seen in the cohort. Asymptomatic venous thrombosis in the superior sagittal sinus was seen in three patients. CONCLUSION: Venous malformations, both extracranial and icVM, share many characteristics that are distinct from developmental venous anomalies. icVM were not associated with venous hypertension. The underlying genetic mutations involved in the development of icVM, germ-line or somatic, remain to be elucidated, but may very well involve shared mechanisms and pathways with extracranial venous malformations.

Kaposiform lymphangiomatosis (KLA) is a rare, life-threatening congenital lymphatic malformation. Diagnosis is often delayed due to complex indistinct symptoms. Blood angiopoietin-2 (ANG2) levels are elevated in KLA and may be useful as a biomarker to monitor disease status. We report a 7-year-old male child with easy bruising, inguinal swelling, and consumptive coagulopathy, diagnosed with KLA. A multimodal treatment regimen of prednisone, sirolimus, vincristine, and adjunctive zoledronate was used. Plasma ANG2 levels were highly elevated at diagnosis but decreased during treatment. The patient showed significant clinical improvement over a 38-month period and normalization of ANG2 levels correlated with resolution of the coagulopathy.

Intussusception in lymphatic vessels has received less attention than in blood vessels. In tumors and pseudotumors of blood vessels with intravascular papillary structures, including sinusoidal hemangioma and intravascular papillary endothelial hyperplasia, we observed exuberant intussusceptive angiogenesis, as well as the similarity between papillae (term used by pathologists) and pillars/folds (hallmarks of intussusceptive angiogenesis). A similar response could be expected in lymphangiomas (lymphatic malformations and reactive processes rather than tumors) with papillae. The aim of this work is to assess whether papillae/pillars/folds and associated structures (vessel loops and septa) are present in lymphangiomas, and to establish the characteristics and formation of these structures. For this purpose, we selected lymphangiomas with intraluminal papillae (n = 18), including cystic, cavernous, circumscriptum, and progressive types, of which two cases of each type with a greater number of papillae were used for serial histologic sections and immunohistochemistry. The studies showed a) dilated lymphatic spaces giving rise to lymphatic-lymphatic vascular loops, which dissected and encircled perilymphatic structures (interstitial tissue structures/ITSs and pillars/posts), b) ITSs and pillars, surrounded by anti-podoplanin-positive endothelial cells, protruding into the lymphatic spaces (papillary aspect), and c) splitting, remodeling, linear arrangement, and fusion of papillae/pillars/folds, forming papillary networks and septa. In conclusion, as occurs in blood vessel diseases, the development of lymphatic vessel loops, papillae/pillars/folds, and septa (segmentation) supports intussusceptive lymphangiogenesis and suggests a piecemeal form of intussusception. This intussusceptive lymphangiogenesis in lymphatic diseases can provide a basis for further studies of lymphatic intussusception in other conditions, with clinical and therapeutic implications. Anat Rec, 302:2003–2013, 2019. (c) 2019 American Association for Anatomy.

Infantile haemangiomas are very common benign tumours in the first months of life. They are mostly cutaneous; however, extracutaneous lesions are possible, and occur in very rare cases in the central nervous system. A European multicentre observational retrospective study was conducted in the last 5 years. Seven patients with intracranial or intraspinal infantile haemangiomas were selected and treated with oral propranolol. Propranolol was interrupted after complete or almost complete resolution of infantile haemangiomas. All patients tolerated the treatment well without side-effects. Central nervous system infantile haemangiomas are probably underestimated due to the frequent absence of symptoms and their spontaneous involution. However, they should be investigated in case of segmental cutaneous infantile haemangiomas, particularly on the head, neck, upper trunk, lumbar or sacral area in order to diagnosis intra-central nervous system involvement at an early stage.

PHACE syndrome is a rare disorder with posterior fossa brain malformations, segmental infantile haemangiomas, arterial anomalies, cardiac defects and eye anomalies. Cerebral and cervical arterial abnormalities occur commonly in these patients, predisposing subjects with PHACE syndrome to neurovascular complications including migraine-like headaches, moyamoya vasculopathy, arterial dissection and arterial ischaemia stroke. We leveraged institutional MRI protocols developed for adult neurovascular disease to better elucidate the pathogenesis of the arterial alternations observed in PHACE. Using high-resolution vessel wall and 4D flow MRI, we demonstrated enhancement, focal dissection and altered blood flow in a 7-year-old girl with PHACE syndrome. This is the first-time vessel wall imaging has been used to detail the known arterial changes in PHACE, and these findings may indicate that progressive vascular narrowing and vessel wall changes/inflammation are a factor in chronic headaches and other arterial complications seen in subjects with PHACE syndrome.

Background We report two cases of RASA1-related capillary malformation-arteriovenous malformation (CM-AVM1) syndrome mimicking hereditary haemorrhagic telangiectasia (HHT).Methods and results A 28-year-old man, previously embolised for cerebral arteriovenous malformations (AVMs), presented with epistaxis and typical nasal telangiectasias of HHT. CT scan revealed a large portocaval shunt. The second patient was a 9-year-old girl presenting with cyanosis and several mucocutaneous telangiectasias, similar to those observed in typical cases of HHT. CT scan revealed a huge and complex pulmonary AVM of the right lower lobe and a hepatic AVM within the left lobe. HHT diagnosis was considered possible according to the Curacao criteria for the two patients, with at least two criteria for each. Genetic tests did not find any mutation in the three classic genes (Endoglin, Activin receptor-like kinase 1 or Mothers against decapentaplegic homolog 4), but identified in both cases an RASA1 mutation, known to cause CM-AVM1 syndrome.Conclusions Pulmonary AVM and portocaval shunt, usually encountered in HHT, have not yet been described in the CM-AVM1 syndrome. RASA1 screening may be considered in case of HHT suspicion, particularly when mutations are not found in the usually affected genes.

Parkes Weber syndrome is associated with autosomal dominant inheritance, caused by germline heterozygous inactivating changes in the RASA1 gene, characterized by multiple micro arteriovenous fistulas and segmental overgrowth of soft tissue and skeletal components. The focal nature and variable expressivity associated with this disease has led to the hypothesis that somatic “second hit” inactivating changes in RASA1 are necessary for disease development. We report a 2-yr-old male with extensive capillary malformation and segmental overgrowth of his lower left extremity. Ultrasound showed subcutaneous phlebectasia draining the capillary malformation; magnetic resonance imaging showed overgrowth of the extremity with prominence of fatty tissues, fatty infiltration, and enlargement of all the major muscle groups. Germline RASA1 testing was normal. Later somatic testing from affected tissue showed two pathogenic variants in RASA1 consistent with the c.934_938del, p.(Glu312Argfs*14) and the c.2925del, p.(Asn976Metfs*20) with variant allele fractions of 3.6% and 4.2%, respectively. The intrafamilial variability of Parkes Weber syndrome involving segmental overgrowth of soft tissue, endothelium, and bone is strongly suggestive of a somatic second-hit model. There are at least two reports of confirmed second somatic hits in RASA1 To our knowledge, this is the first report of an individual with two somatic pathogenic variants in the RASA1 gene in DNA from a vascular lesion.

Lymphatic malformation (LM) can occur in the head and neck regions and cause cosmetic problems in adults. Sclerotherapy and surgical resection have been frequently applied; however, both are far from being minimally invasive in terms of aesthetic satisfaction, including the aesthetic downtime. We performed a less-invasive treatment using the venous anastomosis technique, named the lymphatic malformation-venous anastomosis (LMVA), mainly in pediatric patients with intractable microcystic lesions, in whom general anesthesia was required because the pediatric patients could not remain still. Here, we report the case of a 35-year-old man with a cystic submandibular LM successfully treated with LMVA under local anesthesia. He presented with a gradually enlarging LM on the neck. For improving aesthetics, LMVA was planned under local anesthesia. Lymphography by injecting indocyanine green revealed no inflow or outflow connection to the malformation; thus, we created an outflow bypass using the sidewall of the LMVA technique. The patient was discharged on the following day of the operation without any postoperative complications. A volumetric analysis 6 months later showed a 43.5% reduction of the malformation, with the patient being completely satisfied with the result. To the best of our knowledge, there has been no previous report on performing LMVA under local anesthesia in an adult. LMVA can be a novel treatment of choice when other options are less feasible.

PURPOSE: To determine if the size and location of facial port-wine stain (PWS) can predict glaucoma risk for neonates. DESIGN: Retrospective cohort study METHODS: Children with facial PWS who had undergone ophthalmologic examination within four weeks of their birth were included. Clinical information including facial photograph, intraocular pressure, corneal diameter, optic-disc cup-to-disc ratio, and Sturge-Weber syndrome (SWS) diagnosis were collected. Based on facial photographs, PWS distribution, eyelid involvement, and PWS scores according to degree of involvement in each embryonic facial vasculature distribution (segment [S]1, S2 and S3) were evaluated. RESULTS: Among the 34 patients, 7 (21%) had bilateral PWS lesions. Eighteen (53%) were diagnosed as glaucoma. The proportion of eyes showing PWS involving both S1 and S2 was the highest (n = 15, 37%) and the frequency of glaucoma diagnosis (n = 9, 60%) was also the greatest. In eyelid involvement analysis, among the 7 eyes with lower-eyelid lesion only, 5 (83%) had glaucoma. Among the 11 eyes having upper-eyelid lesion only, however, 2 (18%) were diagnosed with glaucoma. A logistic regression model showed that the significant factors associated with glaucoma risk were greater PWS score in S2 (OR 3.604; 95% CIs 1.078–12.050; P = 0.037) or lower-eyelid involvement (OR 12.816; 95% CIs 1.698–96.744; P = 0.013). CONCLUSIONS: Among the newborns with facial PWS, (1) greater extent of birthmarks involving the S2 area, and (2) lesions including the lower eyelid were associated with higher risk of glaucoma development within the neonatal period.

AIM: We assessed the utilization of the National Institutes of Health Quality of Life in Neurological Disorders (Neuro-QoL) in pediatric patients with Sturge-Weber syndrome, a rare neurovascular disorder which frequently results in seizures, brain atrophy, calcification, and a range of neurological impairments. METHODS: Subjects were seen clinically and consented for research. All 22 patients filled out the Pediatric Neuro-QoL. The Neuro-QoL subscores were converted to T-scores to compare with the referenced control population. Twenty-one participants also filled out the Brain Vascular Malformation Consortium Database Questionnaire containing data pertaining to Sturge-Weber syndrome-related medical history, medications, comorbidities, and family history. All data were analyzed with a significance threshold of P < 0.05. RESULTS: Cognitive function quality of life was significantly lower (P < 0.001) in pediatric patients with Sturge-Weber syndrome compared with referenced control subjects. Male gender (P = 0.02) was associated with lower cognitive function Neuro-QoL. The extent of skin (R = -0.46, P = 0.04), total eyelid port-wine birthmark (R = -0.56, P = 0.007), eye (R = -0.58, P = 0.005), and total Sturge-Weber syndrome involvement (R = -0.63, P = 0.002) were negatively correlated with cognitive function Neuro-QoL. A younger age at seizure onset was associated with lower cognitive function Neuro-QoL (hazard ratio = 0.90, P = 0.004) even after controlling for extent of brain, skin, or eye involvement. Antidepressant use was associated with lower cognitive function Neuro-QoL (P = 0.005), and cognitive function Neuro-QoL was negatively correlated with depression Neuro-QoL; however, after adjusting for depression this relationship was no longer significant. CONCLUSIONS: The results suggest targeting cognitive function Neuro-QoL in treatment trials and reiterate the prognostic value of early seizure onset. In addition, sex-related differences were noted, which should be further studied.

Cerebral cavernous malformations (CCMs) form following loss of the CCM protein complex in brain endothelial cells due to increased endothelial MEKK3 signaling and KLF2/4 transcription factor expression, but the downstream events that drive lesion formation remain undefined. Recent studies have revealed that CCM lesions expand by incorporating neighboring wild-type endothelial cells, indicative of a cell nonautonomous mechanism. Here we find that endothelial loss of ADAMTS5 reduced CCM formation in the neonatal mouse model. Conversely, endothelial gain of ADAMTS5 conferred early lesion genesis in the absence of increased KLF2/4 expression and synergized with KRIT1 loss of function to create large malformations. Lowering versican expression reduced CCM burden, indicating that versican is the relevant ADAMTS5 substrate and that lesion formation requires proteolysis but not loss of this extracellular matrix protein. These findings identify endothelial secretion of ADAMTS5 and cleavage of versican as downstream mechanisms of CCM pathogenesis and provide a basis for the participation of wild-type endothelial cells in lesion formation.

Cystic lymphatic malformations result from an abnormal embryological development of the lymphatic structures. Here we report on a case of a preterm female baby, born at 34 weeks of gestation, with a voluminous cervicofacial cystic lymphatic malformation responsible for an airway obstruction. An mTOR inhibitor, sirolimus, was started from the first day of life, and was combined with iterative sclerotherapy procedures. This case illustrates a safe and successful early administration of sirolimus in a preterm neonate.

BACKGROUND: Infantile hemangiomas (IH) are the most common benign tumor of infancy. Although oral propranolol is currently first-line therapy, optimal dosing for treatment of IH remains debated. We sought to identify hemangioma characteristics associated with poor response to standard dosing (2 mg/kg/d) and to assess the therapeutic benefit of higher dosing. METHODS: Retrospective chart review was conducted of 559 patients with IH seen at Johns Hopkins between 2008 and 2018, of whom 245 (44%) were treated with propranolol. Baseline characteristics were compared between patients who received increased propranolol dosing (>/ = 2.5 mg/kg/d) and those who remained on standard dose (2 mg/kg/d). Changes in the Hemangioma Activity Score (HAS) during the increased dosage period were scored by two trained, blinded pediatric dermatologists. RESULTS: Of 245 patients, 204 (83%) received standard 2 mg/kg/d propranolol dosing while 41 (17%) received a higher dose of >/ = 2.5 mg/kg/d. The most common location of IH in both groups was the face. In the increased dosage group, 85.4% of IH were of mixed or deep morphology with a mean greatest diameter of 4.6 cm. IH requiring increased dosing received longer courses of propranolol (mean of 389 vs. 282 days, P < .001) and underwent higher rates of excision by plastic surgery (26.8% vs. 5.9%, P < .001). Mean change in HAS over the period with dosage >/ = 2.5 mg/kg/d was minimal (-0.70; P < .001). CONCLUSIONS: Most recalcitrant IH were located on the face, larger in diameter, and of mixed or deep morphology. Patients had little improvement in HAS score with increased propranolol dosing implemented late in the treatment course with over one-fourth ultimately receiving surgical excision.

Hereditary hemorrhagic telangiectasia (HHT), a rare autosomal dominant disease mostly caused by mutations in three known genes (ENG, ACVRL1, and SMAD4), is characterized by the development of vascular malformations (VMs). Patients with HHT may present with mucocutaneous telangiectasia, as well as organ arteriovenous malformations (AVMs) of the central nervous system, lungs, and liver. Genotype-phenotype correlations have been well described in adults with HHT. We aimed to investigate genotype-phenotype correlations among pediatric HHT patients. Demographic, clinical, and genetic data were collected and analyzed in 205 children enrolled in the multicenter Brain Vascular Malformation Consortium HHT Project. A chi-square test was used to determine the association between phenotypic presentations and genotype. Among 205 patients (age range: 0–18 years; mean: 11 years), ENG mutation was associated with the presence of pulmonary AVMs (p < 0.001) and brain VM (p < 0.001). The presence of a combined phenotype-defined as both pulmonary AVMs and brain VMs-was also associated with ENG mutation. Gastrointestinal bleeding was rare (4.4%), but was associated with SMAD4 genotype (p < 0.001). We conclude that genotype-phenotype correlations among pediatric HHT patients are similar to those described among adults. Specifically, pediatric patients with ENG mutation have a greater prevalence of pulmonary AVMs, brain VMs, and a combined phenotype.

Rationale: Hereditary hemorrhagic telangiectasia (HHT) is a genetic disease caused by mutations in ENG, ALK1, or SMAD4. Since proteins from all three HHT genes are components of signal transduction of TGF-beta family members, it has been hypothesized that HHT is a disease caused by defects in the ENG-ALK1-SMAD4 linear signaling. However, in vivo evidence supporting this hypothesis is scarce. Objective: We tested this hypothesis and investigated the therapeutic effects and potential risks of induced-ALK1 or -ENG overexpression for HHT. Methods and Results: We generated a novel mouse allele (ROSA26(Alk1)) in which HA-tagged ALK1 and bicistronic eGFP expression are induced by Cre activity. We examined whether ALK1-overexpression (OE) using the ROSA26(Alk1) allele could suppress the development of AVMs in wounded adult skin and developing retinas of Alk1- and Eng-inducible knockout (iKO) mice. We also used a similar approach to investigate whether ENG-OE could rescue AVMs. Biochemical and immunofluorescence analyses confirmed the Cre-dependent overexpression of the ALK1-HA transgene. We could not detect any pathological signs in ALK1-OE mice up to 3 months after induction. ALK1-OE prevented the development of retinal AVMs and wound-induced skin AVMs in Eng-iKO as well as Alk1-iKO mice. ALK1-OE normalized expression of SMAD and NOTCH target genes in ENG-deficient endothelial cells (ECs) and restored the effect of BMP9 on suppression of phosphor-AKT levels in these ECs. On the other hand, ENG-OE could not inhibit the AVM development in Alk1-iKO models. Conclusions: These data support the notion that ENG and ALK1 form a linear signaling pathway for the formation of a proper arteriovenous network during angiogenesis. We suggest that ALK1 overexpression or activation can be an effective therapeutic strategy for HHT1 and HHT2 in Alk1- and Eng-inducible knockout (iKO) mice. Further research is required to study whether this therapy could be translated into treatment for humans.

Anomalous marginal veins of the trunk or extremities are congenitally incompetent entities found in association with phosphatidylinositol 4,5-bisphosphate 3-kinase catalytic subunit alpha (PIK3CA)-related overgrowth syndromes, such as Klippel-Trenaunay syndrome and congenital lipomatous overgrowth, vascular malformations, epidermal nevi, and skeletal deformities (CLOVES) syndrome. When present, they can be a major source of venous hypertension-related morbidity and potentially lethal thromboembolic events. Herein, we describe a rare case of an upper extremity marginal vein in a patient with CLOVES syndrome. Through a multimodal therapeutic approach, we identified a somatic PIK3CA mutation in the excised anomalous vein. This finding questions the validity of commonly employed terminology, such as persistent embryonic vein, in reference to these anomalous entities.

Hereditary hemorrhagic telangiectasia (HHT) can be clinically diagnosed, but children often lack characteristic features. We report a family with homozygous growth differentiation factor 2 (GDF2)-related HHT diagnosed by genetic testing. A boy aged 5 years and 2 months presented with isolated hypoxemia. He was the product of a consanguineous marriage; his parents were second cousins. Physical examination revealed cyanosis of nail beds and clubbed fingers. Pulse oxygen saturation was 84% to 89%. Lung function, contrast-enhanced lung computed tomography, and noncontrast echocardiography were normal. A pulmonary perfusion scan revealed radioactivity in the brain and bilateral kidney, suggesting the existence of a intrapulmonary shunt. Whole-exome sequencing revealed a homozygous variant [c.1060_1062delinsAG (p.Tyr354ArgfsTer15)] in GDF2, which was found to be inherited from his heterozygous parents. At the age of 8 years, he developed epistaxis, and an angiogram revealed diffuse pulmonary arteriovenous malformations. At the age of 9 years, he was treated with sirolimus, and his condition improved significantly. However, his now 7-year-old sister with the same homozygous variant currently has no symptoms. Physical examinations revealed 1 pinpoint-sized telangiectasia on the chest of his mother and a vascular lesion on the forehead of his sister. Additionally, the patient's father and great-uncle had a history of mild to moderate epistaxis. Mutation in GDF2 is a rare cause of HHT. Ours is the first report of homozygous GDF2-related HHT; in addition, this variant has not been reported previously. In our report, we also confirm variable expressivity, even with the same pathogenic variant in GDF2-related HHT.

BACKGROUND: Hemangioma (Hem) is a benign tumor commonly seen in infancy with a relative high morbidity. Human umbilical vein endothelial cell (HUVEC)-derived extracellular vesicles (EVs) are actively participated in Hem. Therefore, this study is designed to figure out the underlying mechanism of HUVEC-derived EVs in Hem. METHODS: Initially, EVs were separated from HUVECs and identified. HUVEC-derived EVs in normoxia or hypoxia were then cultivated with Hem endothelial cells (HemECs) to test the proliferation, apoptosis, and migration of HemECs. Microarray analysis was performed to select microRNAs (miRs) with differential expression. miR-210 in hypoxia-induced HUVECs was silenced, and the relevant EVs were extracted and then co-cultured with HemECs to perform biological effect experiments. Then, the target relation between miR-210 and homeobox A9 (HOXA9) was identified by the dual luciferase reporter gene assay and RNA immunoprecipitation assay. Moreover, xenograft transplantation was also applied to confirm the in vitro experiments. RESULTS: Hypoxia-induced HUVECs promoted release of EVs, which were absorbed by HemECs. Hypoxia-induced HUVEC-EVs promoted HemEC proliferation and migration and inhibited apoptosis. miR-210 from the hypoxia-induced HUVEC-EVs was highly expressed and promoted HemEC growth. Silencing miR-210 expression in the hypoxia-induced HUVEC-EVs suppresses Hem development in vivo. In addition, miR-210 targeted HOXA9. CONCLUSION: Silencing miR-210 in HUVEC-derived EVs could suppress Hem by targeting HOXA9. This investigation may provide novel insights for Hem treatment.

OBJECTIVE: To study the clinical application of high-frequency ultrasound in the treatment of infantile hemangiomas. METHODS: Ultrasonographic data before and after treatment for 1 month, 3 months and 5 months were recorded from 80 patients with infantile hemangioma. The hemangioma was monitored dynamically with relevant indexes before and after treatment, and the treatment results were divided into 2 groups: the treatment-effective group of 70 cases and the treatment-ineffective group of 10 cases. The differences of tumor size, internal echo and hemodynamic parameters (peak arterial systolic flow velocity, resistance index and venous velocity) were compared before and after treatment, and reliable ultrasound observation indexes were summarized. RESULTS: After treatment, in the treatment-effective group, the thickness of tumor was reduced in all cases, the blood flow signals decreased in 55 cases, the peak systolic velocity decreased in 55 cases, resistance index increased in 55 cases, and venous flow velocity decreased in 53 cases; the differences were statistically significant compared with those before treatment (P < 0.05). The thickness of tumors was significantly decreased after 5 months of treatment compared with 1 month and 2 month treatment (P < 0.05). There was no significant difference in the longitudinal and transverse diameter of tumors after treatment (P > 0.05). In the treatment-ineffective group, there was no significant difference in the above indexes before and after treatment (P > 0.05). CONCLUSION: High-frequency ultrasound had primary application value in the treatment of infantile hemangiomas, where the change of tumor thickness provided the most important observational indicator.

Proliferative disorders of lymphatic origin in animals are mostly congenital or occur within the first few months of life. Involvement of internal organs is extremely infrequent. A seven-year-old entire female mixed-breed dog was presented with apathy and poor appetite. Abdominal ultrasonography revealed a focally enlarged spleen with an anechoic round lesion. Splenectomy was performed and pathological examination demonstrated a sponge-like, compressible tumour composed of endothelium-lined vascular cystic spaces filled with eosinophilic proteinaceous material lacking erythrocytes. Immunohistochemical stains showed that cyst-lining cells were strongly positive for CD31 and factor VIII and focally positive for lymphatic vessel endothelial hyaluronan receptor 1 (LYVE-1). Based on these findings, the lesion was identified as splenic cystic lymphangioma. To the best of our knowledge, this is the first description of solitary lymphangioma of the spleen in animals.

Hereditary hemorrhagic telangiectasia (HHT) is an autosomal dominant, vascular disorder that presents with telangiectases and arteriovenous malformations. HHT is a genetically heterogeneous disorder, involving mutations in endoglin (ENG; HHT1) and activin receptor-like kinase 1 (ACVRL1/ALK1; HHT2) genes that account for over 85% of all HHT patients. The current diagnosis of HHT patients remains at the clinical level, but many suspected patients do not have a clear HHT diagnosis or do not show pathogenic mutations in HHT genes. This situation has prompted the search for biomarkers to help in the early diagnosis of the disease. We have analyzed the plasma levels in HHT patients of selected micro-RNAs (miRNAs), small single-stranded RNAs that regulate gene expression at the transcriptional level by interacting with specific RNA targets. A total of 16 HHT1 and 17 HHT2 plasma samples from clinically confirmed patients and 16 controls were analyzed in this study. Total RNA was purified from plasma, and three selected miRNAs (miRNA-10a, miRNA-214, and miRNA-370), related to the pathobiology of cardiovascular diseases and potentially targeting ENG or ALK1, were measured by quantitative polymerase chain reaction. Compared with controls, levels of miRNA-370, whose putative target is ENG, were significantly downregulated in HHT1, but not in HHT2, whereas the levels of miRNA-10a, whose putative target is ALK1, were significantly upregulated in HHT2, but not in HHT1. In addition, the levels of miRNA-214, potentially targeting ENG and ALK1, did not change in either HHT1 or HHT2 patients versus control samples. While further studies are warranted, these results suggest that dysregulated plasma levels of miRNA-370 or miRNA-10a could help to identify undiagnosed HHT1 or HHT2 patients, respectively.

Fetal lymphangioma, also known as cystic hygroma, is a hamartomatous congenital tumor which involves the fetal skin and the subcutaneous tissue. The most common site of appearance is in the neck region. Location of the lesion in the axilla as well as in other anathomical sites is very rare.Prenatal diagnosis can be made on ultrasound examination. Other structural or chromosomal anomalies are frequently associated with this diagnosis. The antepartum management and type of delivery should be set up by a multidisciplinary team.We present a rare case diagnosed with axillary lymphangioma during the second trimester morphological ultrasound 2D/4D scan.

Extracranial arteriovenous malformation (AVM) is most commonly caused by a somatic mutation in MAP2K1. We report two patients with vascular anomalies that had an unclear clinical diagnosis most consistent with either an AVM or congenital hemangioma. Lesions were cutaneous, reddish-purple with telangiectasias, present at birth, and had defined borders. Histopathology indicated AVM and both lesions contained somatic KRAS mutations. A rare AVM phenotype exists that shares clinical features with congenital hemangioma.

Klippel-Trenaunay syndrome (KTS) is a rare disorder characterized by triad of vascular malformations, varicose veins, and bony or soft tissue hypertrophy involving an extremity. Port wine stain is the most common vascular malformation associated with this syndrome. Various other rare vascular malformations described are angiokeratomas, angiokeratoma circumscriptum naeviformae, angiodermatitis, etc. This is the first instance, to the best of our knowledge, describing the capillary, venous, and lymphatic malformations in a same case of KTS.

Oral propranolol (OP) demonstrated high efficacy and safety profile for treatment of critical infantile hemangiomas (IHs). Our aim was to assess the morphologic changes of IHs with standard and high-resolution video dermoscopy (HRVD) from baseline to 18 months either in presence or absence of OP therapy; to investigate if extended anamnestic perinatal data and clinical-dermoscopic characteristics of the IHs can correlate with therapeutic outcome. We enrolled 94 patients (112 IHs): 58 were treated with OP, 35 (42 IHs) for 6 months (group 1), and 23 (25 IHs) for 12-months (group 2); 36 (45 IHs) were followed-up. Clinical-dermoscopic examinations were performed every 3 months during therapy and follow-up. Among 67 treated IHs, superficial and deep IHs with homogenous clinical-dermoscopic aspect developed after the 2 weeks of life achieved the better outcome, stable at 9-month follow-up, independently form treatment duration. Under HRVD, glomerular vessels were prevalent at baseline; corckscrew, comma, and linear-irregular vessels were the prevalent pattern at 1, 3, and 6 months of therapy, respectively. At 12-month follow-up, adequate healing was achieved by 96% of IHs in group 2 and by 78% in group 1, showing dotted vessels. Persistent IHs displayed a reticulated aspect and linear irregular vessels, while arborizing vessels characterized relapsed IHs. A 12-month OP therapy can be considered for newborns presenting with nonhomogenous mixed IHs >3 cm on the perineal area/lower extremities. In conclusion, HRVD allows a real time monitoring of vascular changes in IHs treated with OP and can support physicians in identifying relapses before they become clinically evident.

PURPOSE: Tissue disaggregation and the cell sorting technique by surface markers has played an important role in isolating lymphatic endothelial cells (LECs) from lymphatic malformation (LM). However, this technique may have the drawback of impurities or result in isolation failure because it is dependent on surface marker expressions, the heterogeneity of which has been found in the lymphatic system. We developed a novel method for isolating LM-LECs without using whole tissue disaggregation. METHODS: Seven LM surgical specimens were collected from seven patients with LMs. LM-LECs were detached from the LM cyst wall by “lumen digestion” and irrigating the cystic cavity with trypsin, and maintained in culture. RESULTS: The cells formed a monolayer with a cobblestone-like appearance. Immunohistochemistry and quantitative RT-PCR of these cells revealed high expression of lymphatic-specific genes, confirming their identity as LM-LECs. The whole-exome sequencing and PIK3CA sequencing of these cells revealed somatic mutations in PIK3CA in all cases. CONCLUSIONS: We established a novel technique for isolating LM-LECs from LM tissue by “lumen digestion” without whole-tissue disaggregation. The limited incorporation of non-LM LECs in the isolate in our method could make it an important tool for investigating the heterogeneity of gene expression as well as mutations in LM-LECs.

INTRODUCTION: Capillary malformation-arteriovenous malformation (CM-AVM) syndrome is a rare syndrome that associates characteristic skin lesions with fast-flow vascular malformations (FFVM) in one-third of patients. Few case series have been described, and none in Spain. METHODS: We conducted an observational study of patients with CM-AVM syndrome diagnosed in 15 Spanish hospitals over 3 years. The main clinical, radiological, genetic findings and associated diseases were analyzed. RESULTS: Sixty-four patients have been collected. In 26.5% of the cases, the diagnosis was incidental. In 75% of patients, one macule significantly larger was identified, which we have called herald patch. FFVMs have been detected in 34% of the patients, distributed in 30% on the skin, in 7.8% on the brain, and in 1.5% at the spinal level. 65% had a positive family history. 42 out of 57 patients tested (73%) were positive for the RASA1 mutations, and all 4 patients tested for EPHB4 mutations were positive. No tumor lesions were detected in the series, except for 5 infantile hemangiomas. CONCLUSIONS: The data that we have obtained regarding clinical lesions, associated FFVM, family history, and genetics are similar to those previously published in the literature. An extensive data analysis failed to demonstrate any statistically significant association between the presence of a FFVM and any clinical, familiar, or genetic parameter that could predict its onset, although a link between the presence of a herald patch on the midline face and a brain FFVM could be observed. No correlation between genotype and phenotype could be detected neither.

OBJECTIVE: The objective of this study was to characterize the role of NOTCH signaling cascade activation in the angiogenic drive of infantile hemangioma (IH) tissue. METHODS: Both IH tissue and normal skin were collected from 55 surgical patients. Of these, 14 were proliferating hemangiomas, 14 were stationary, 14 were involuted, and 13 had been treated with propranolol. Protein and RNA were extracted from all tissues and subjected to Western blotting and reverse transcription-polymerase chain reaction, respectively, for components of the NOTCH signaling pathway. RESULTS: Compared with normal skin from the same patients, proliferating IHs contained higher levels of messenger RNA and protein for the majority of NOTCH receptors and ligands as well as the downstream coactivator MAML1. Expression of NOTCH receptor ligand messenger RNA and protein was significantly lower in involuting and propranolol-treated IHs. CONCLUSIONS: Perturbations in NOTCH signaling follow the natural course and treatment outcome of IHs, suggesting a critical role in their pathogenesis.

* Klippel-Trenaunay syndrome increases the risk for thromboembolic events. * The individual risk for thromboembolic events is difficult to predict. The presence of specific aberrations in venous anatomy increases the risk for thromboembolic events. This knowledge provides a guide to identify high-risk patients eligible for prophylactic treatment.