Update December 2019

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

The above manuscript, “ARAF recurrent mutation causes central conducting lymphatic anomaly treatable with a MEK inhibitor,” by Li et al., is one of a number of emerging reports of medical therapies derived from genetic testing of tissue specimens from patients with vascular anomalies, and provides evidence for effective targeted medical therapy for these disorders. Many vascular anomalies are caused by somatic mutations (mostly in the PI3K/AKT/mTOR pathway and/or the RAS/MAPK/ERK pathway), which are present in the affected tissue, often at low allele frequencies. While the mutant genes are typically proto-oncogenes, tumorigenesis is not a key feature in patients with vascular anomalies (1–4). The patient in Li's paper, who had a central conducting lymphatic anomaly did not respond to the mTor inhibitor Sirolimus, which has been effective for many patient with predominantly lymphatic malformations (5). A mutation in ARAF, serine/threonine kinase proto-oncogene involved in the MAP kinase signaling pathway, was detected. Mutations in the RAS/MAPK pathway have been described primarily in high-flow vascular malformations (6–8). Greenberger et al. identified a somatic mutation in NRAS in Generalized lymphatic anomaly (GLA) and performed further studies on pharmacologic inhibition of the disease causing pathway (9). Although only 2 patients were studied in the highlighted publication by Li, the presented data is convincing. An accompanying commentary on this paper, by Koltowska, is also included (10).

The glycocalyx is a dense layer of carbohydrate chains involved in numerous and fundamental biological processes, such as cellular and tissue homeostasis, inflammation and disease development. Composed of membrane-bound glycoproteins, sulfated proteoglycans and glycosaminoglycan side-chains, this structure is particularly essential for blood vascular barrier functions and leukocyte diapedesis. Interestingly, whilst the glycocalyx of blood vascular endothelium has been extensively studied, little is known about the composition and function of this glycan layer present on tissue-associated lymphatic vessels (LVs). Here, we applied confocal microscopy to characterize the composition of endothelial glycocalyx of initial lymphatic capillaries in murine cremaster muscles during homeostatic and inflamed conditions using an anti-heparan sulfate (HS) antibody and a panel of lectins recognizing different glycan moieties of the glycocalyx. Our data show the presence of HS, alpha-D-galactosyl moieties, alpha2,3-linked sialic acids and, to a lesser extent, N-Acetylglucosamine moieties. A similar expression profile was also observed for LVs of mouse and human skins. Interestingly, inflammation of mouse cremaster tissues or ear skin as induced by TNF-stimulation induced a rapid (within 16 h) remodeling of the LV glycocalyx, as observed by reduced expression of HS and galactosyl moieties, whilst levels of alpha2,3-linked sialic acids remains unchanged. Furthermore, whilst this response was associated with neutrophil recruitment from the blood circulation and their migration into tissue-associated LVs, specific neutrophil depletion did not impact LV glycocalyx remodeling. Mechanistically, treatment with a non-anticoagulant heparanase inhibitor suppressed LV HS degradation without impacting neutrophil migration into LVs. Interestingly however, inhibition of glycocalyx degradation reduced the capacity of initial LVs to drain interstitial fluid during acute inflammation. Collectively, our data suggest that rapid remodeling of endothelial glycocalyx of tissue-associated LVs supports drainage of fluid and macromolecules but has no role in regulating neutrophil trafficking out of inflamed tissues via initial LVs.

OBJECTIVE: Emerging areas of vascular biology focus on lymphatic/blood vessel mispatterning and the regulation of endothelial cell identity. However, a fundamental question remains unanswered: can lymphatic vessels become blood vessels in adult tissues? Leveraging a novel tissue culture model, the objective of this study was to track lymphatic endothelial cell fate over the time-course of adult microvascular network remodeling. METHODS: Cultured adult Wistar rat mesenteric tissues were labeled with BSI-lectin and time-lapse images were captured over five days of serum stimulated remodeling. Additionally, rat mesenteric tissues on day 0 and day 3 and 5 post-culture were labelled for PECAM + LYVE-1 or PECAM + podoplanin. RESULTS: Cultured networks were characterized by increases in blood capillary sprouting, lymphatic sprouting and the number of lymphatic/blood vessel connections. Comparison of images from the same network regions identified incorporation of lymphatic vessels into blood vessels. Mosaic lymphatic/blood vessels contained lymphatic marker positive and negative endothelial cells. CONCLUSIONS: Our results reveal the ability for lymphatic vessels to transition into blood vessels in adult microvascular networks and discovers a new paradigm for investigating lymphatic/blood endothelial cell dynamics during microvascular remodeling.

Changes in the intestinal lymphatic vascular system, such as lymphatic obstruction, are characteristic features of inflammatory bowel diseases. The lymphatic vasculature forms a conduit to enable resolution of inflammation; this process is driven by specialized endogenous proresolving mediators (SPMs). To evaluate contributions of lymphatic obstruction to intestinal inflammation and to study profiles of SPMs, we generated a novel animal model of lymphatic obstruction using African green monkeys. Follow-up studies were performed at 7, 21, and 61 days. Inflammation was determined by histology. Luminex assays were performed to evaluate chemokine and cytokine levels. In addition, lipid mediator metabololipidomic profiling was performed to identify SPMs. After 7 days, lymphatic obstruction resulted in a localized inflammatory state, paralleled by an increase in inflammatory chemokines and cytokines, which were found to be up-regulated after 7 days but returned to baseline after 21 and 61 days. At the same time, a distinct pattern of SPMs was profiled, with an increase for D-series resolvins, protectins, maresins, and lipoxins at 61 days. These results indicate that intestinal lymphatic obstruction can lead to an acute inflammatory state, accompanied by an increase in proinflammatory mediators, followed by a phase of resolution, paralleled by an increase and decrease of respective SPMs.

BACKGROUND: Vascularized lymph node transfer (VLNT) is an effective surgery for extremity lymphedema. This study evaluated a lymphatic drainage device (LDD) for the drainage of accumulated fluid into the venous system. METHODS: Micropore filtering membranes with pore sizes of 5, 0.65, and 0.22 mum polyvinylidene difluoride, and 0.8 mum Nylon Net Filter were evaluated to determine the in vitro efficiency of drainage flow of an LDD. The two superior membranes were further used for the evaluation of the inflow and outflow of the LDD in vivo using 5% albumin. RESULTS: At 5 minutes, the volumes drained with 5, 0.65, and 0.22 mum polyvinylidene difluoride and 0.8 mum nylon membranes were 15.2, 2.77, 2.37, and 0.59 mL, respectively (P < .01). At 10 minutes, the collected volumes of 5 and 0.65 mum polyvinylidene difluoride were 1788 and 1051 muL (P = .3). The indocyanine green fluorescence was detected at 50 seconds for the 5 mum polyvinylidene difluoride membrane but not for the 0.65 mum membrane. CONCLUSIONS: The study successfully demonstrated the proof-of-concept of the LDD prototype that mimicked VLNT with drainage of 5% albumin into the venous system in a rat model.

Lymphatic filariasis (LF), a morbid disease caused by the tissue-invasive nematodes Wuchereria bancrofti, Brugia malayi, and Brugia timori, affects millions of people worldwide. Global eradication efforts have significantly reduced worldwide prevalence, but complete elimination has been hampered by limitations of current anti-filarial drugs and the lack of a vaccine. The goal of this study was to evaluate B. malayi intestinal UDP-glucuronosyltransferase (Bm-UGT) as a potential therapeutic target. To evaluate whether Bm-UGT is essential for adult filarial worms, we inhibited its expression using siRNA. This resulted in a 75% knockdown of Bm-ugt mRNA for 6 days and almost complete suppression of detectable Bm-UGT by immunoblot. Reduction in Bm-UGT expression resulted in decreased worm motility for 6 days, 70% reduction in microfilaria release from adult worms, and significant reduction in adult worm metabolism as detected by MTT assays. Because prior allergic-sensitization to a filarial antigen would be a contraindication for its use as a vaccine candidate, we tested plasma from infected and endemic normal populations for Bm-UGT-specific IgE using a luciferase immunoprecipitation assay. All samples (n = 35) tested negative. We then tested two commercially available medicines known to be broad inhibitors of UGTs, sulfinpyrazone and probenecid, for in vitro activity against B. malayi. There were marked macrofilaricidal effects at concentrations achievable in humans and very little effect on microfilariae. In addition, we observed that probenecid and sulfinpyrazone exhibit a synergistic macrofilaricidal effect when used in combination with albendazole. The results of this study demonstrate that Bm-UGT is an essential protein for adult worm survival. Lack of prior IgE sensitization in infected and endemic populations suggest it may be a feasible vaccine candidate. The finding that sulfinpyrazone and probenecid have in vitro effects against adult B. malayi worms suggests that these medications have promise as potential macrofilaricides in humans.

Head and neck squamous cell carcinoma (HNSCC) cells bind to lymphocytes via L-selectin in a shear-dependent manner. This interaction takes place exclusively under low shear stress conditions, such as those found within the lymph node parenchyma. This represents a novel functional role for L-selectin/selectin ligand interactions. Our previous work has characterized as of yet unidentified L-selectin ligands expressed by HNSCC cells that are specifically active under conditions of low shear stress consistent with lymph flow. Using an affinity purification approach, we now show that nucleolin expressed on the cell surface of HNSCC cells is an active ligand for L-selectin. Parallel plate chamber flow based experiments and atomic force microscopy (AFM) experiments show that nucleolin is the main functional ligand in these low force conditions. Furthermore, AFM shows a clear relationship between work of de-adhesion and physiological loading rates. Our results reveal nucleolin as the first major ligand reported for L-selectin that operates in low shear stress conditions.

Despite the recent description of meningeal lymphatic vessels draining solutes from the brain interstitium and cerebrospinal fluid (CSF), the physiological factors governing cranial lymphatic efflux remain largely unexplored. In agreement with recent findings, cervical lymphatic drainage of 70 kD and 2000 kD fluorescent tracers injected into the adult mouse cortex was significantly impaired in the anesthetized compared to waking animals (tracer distribution across 2.1 +/− 4.5% and 23.7 +/− 15.8% of deep cervical lymph nodes, respectively); however, free-breathing anesthetized mice were markedly hypercapnic and acidemic (paCO2 = 64 +/− 8 mmHg; pH = 7.22 +/− 0.05). Mechanical ventilation normalized arterial blood gases in anesthetized animals, and rescued lymphatic efflux of interstitial solutes in anesthetized mice. Experimental hypercapnia blocked cervical lymphatic efflux of intraparenchymal tracers. When tracers were injected into the subarachnoid CSF compartment, glymphatic influx into brain tissue was virtually abolished by hypercapnia, while lymphatic drainage was not appreciably altered. These findings demonstrate that cervical lymphatic drainage of interstitial solutes is, in part, regulated by upstream changes in glymphatic CSF-interstitial fluid exchange. Further, they suggest that maintaining physiological blood gas values in studies of glymphatic exchange and meningeal lymphatic drainage may be critical to defining the physiological regulation of these processes.

Endothelial cells line blood and lymphatic vessels and form intercellular junctions, which preserve vessel structure and integrity. The vascular endothelial cadherin, VE-cadherin, mediates endothelial adhesion and is indispensible for blood vessel development and permeability regulation. However, its requirement for lymphatic vessels has not been addressed. During development, VE-cadherin deletion in lymphatic endothelial cells resulted in abortive lymphangiogenesis, edema, and prenatal death. Unexpectedly, inducible postnatal or adult deletion elicited vessel bed-specific responses. Mature dermal lymph vessels resisted VE-cadherin loss and maintained button junctions, which was associated with an upregulation of junctional molecules. Very different, mesenteric lymphatic collectors deteriorated and formed a strongly hyperplastic layer of lymphatic endothelial cells on the mesothelium. This massive hyperproliferation may have been favored by high mesenteric VEGF-C expression and was associated with VEGFR-3 phosphorylation and upregulation of the transcriptional activator TAZ Finally, intestinal lacteals fragmented into cysts or became highly distended possibly as a consequence of the mesenteric defects. Taken together, we demonstrate here the importance of VE-cadherin for lymphatic vessel development and maintenance, which is however remarkably vessel bed-specific.

Cranial lymphatic vessels (LVs) are involved in the transport of fluids, macromolecules and central nervous system (CNS) immune responses. Little information about spinal LVs is available, because these delicate structures are embedded within vertebral tissues and difficult to visualize using traditional histology. Here we show an extended vertebral column LV network using three-dimensional imaging of decalcified iDISCO(+)-clarified spine segments. Vertebral LVs connect to peripheral sensory and sympathetic ganglia and form metameric vertebral circuits connecting to lymph nodes and the thoracic duct. They drain the epidural space and the dura mater around the spinal cord and associate with leukocytes. Vertebral LVs remodel extensively after spinal cord injury and VEGF-C-induced vertebral lymphangiogenesis exacerbates the inflammatory responses, T cell infiltration and demyelination following focal spinal cord lesion. Therefore, vertebral LVs add to skull meningeal LVs as gatekeepers of CNS immunity and may be potential targets to improve the maintenance and repair of spinal tissues.

Podoplanin (PDPN)/T1alpha/Aggrus, a small mucin-type transmembrane glycoprotein, has been shown to be expressed on lymphatic endothelial cells and epithelial cells of many organs. PDPN is also upregulated in many cancers, and is involved in cancer metastasis and malignant progression. Human PDPN possesses three platelet aggregation-stimulating (PLAG) domains and the PLAG-like domain, which bind to C-type lectin-like receptor-2 (CLEC-2). Previously, we reported a novel antipig PDPN (pPDPN) monoclonal antibody (PMab-210) using Cell-Based Immunization and Screening (CBIS) method. PMab-210 specifically detected pPDPN-overexpressed Chinese hamster ovary (CHO)-K1 cells by flow cytometry and Western blot analysis. Immunohistochemical analyses demonstrated that PMab-210 stained pulmonary type I alveolar cells strongly and renal corpuscles weakly in pig or microminipig. However, the specific binding epitope of PMab-210 for pPDPN could not be determined by enzyme-linked immunosorbent assay using a series of pPDPN peptides. In this study, deletion mutants or point mutants of pPDPN were produced for analyzing the PMab-210 epitope using flow cytometry. The analysis of deletion mutants showed that N-terminus of PMab-210 epitope exists between 45th amino acid (aa) and 50th aa of pPDPN. In addition, the analysis of point mutants demonstrated that the critical epitope of PMab-210 could include Glu47, Asp48, Tyr49, Thr50, and Val51 of pPDPN, indicating that PMab-210 epitope is located in PLAG3 domain of pPDPN.

Background: Knowledge of functional parameters that can be used for evaluation of upper extremity lymphatic function is limited. This study aims to evaluate near-infrared fluorescence (NIRF) imaging for evaluation of lymphatic function in the human arm. Methods and Results: Ten healthy male volunteers (age 25.7 +/− 1.3 years) were included and examined at two occasions. The lymphatic functional frequency and velocity were examined at baseline, during hyperthermia and after exercise. In addition, the maximum pressure generated by contraction of the lymphatic vessels was estimated. The mean contraction frequency was found to be 0.9 +/− 0.4/min, and the mean velocity of the propulsions was 1.1 +/− 0.3 cm/s. The average maximal pressure generated by the contraction of the lymphatic vessels was 59 +/− 12 mmHg. No significant difference in frequency, velocity, or pumping pressure was found between the two visits (p > 0.05). Local hyperthermia increased contraction frequency significantly, whereas exercise decreased frequency and increased propulsion velocity. Conclusions: The functional lymphatic parameters evaluated by NIRF imaging showed good repeatability with no significant difference between visits. Future examinations should be conducted with standardization of temperature and exercise, as these parameters were shown to alter lymphatic function.

OBJECTIVE: Determine the effect of bradykinin on solute permeability and cellular junctional proteins in human dermis microvascular endothelial cells. METHODS: Cells were characterized by immunofluorescence and fluorescence-activated cell sorting. Macromolecular transport of dextran and albumin was monitored. Junctional protein expression and phosphorylation were determined by immunoblot analyses. Intracellular calcium and cAMP levels were evaluated. Target gene expression at mRNA and protein levels was determined. RESULTS: Human dermis microvascular endothelial cells comprised 97% lymphatic endothelial cells. Bradykinin increased the permeability to dextran in a concentration-dependent manner, while reduced the permeability to albumin. Bradykinin treatment down-regulated VE-cadherin expression and affected its phosphorylation status at Tyr731. It also down-regulated claudin-5 expression at the transcriptional level through bradykinin-2-receptor signaling. An increase in the intracellular calcium levels and a reduction in the cAMP concentration were associated effects. Finally, bradykinin induced the up-regulation of vascular endothelial growth factor-C protein which was found increased in BK-induced human dermis microvascular endothelial cells culture supernates. CONCLUSIONS: Human dermis microvascular endothelial cells represent a model of lymphatic endothelial cells, in which bradykinin-2-receptor is expressed. Bradykinin-induced bradykinin-2-receptor signaling through intracellular calcium mobilization and reduction in cAMP levels, triggered changes in solute permeability and cellular junction expression. It further up-regulated vascular endothelial growth factors-C protein expression, which is a key modulator of lymphatic vessels function and lymphangiogenesis.

Lymphedema is a chronic condition caused by disruption of lymphatic vessels, which often occurs after invasive surgery. Calcitonin gene-related peptide (CGRP) is a 37-amino acid peptide produced by alternative splicing of the primary transcript of the calcitonin/CGRP gene. CGRP was initially identified as a neuropeptide released primarily from sensory nerves and involved in regulating pathophysiological nociceptive pain. However, recent studies have shown CGRP is also released from a variety of other cells and possesses multiple functions. In this study, CGRP knockout (−/−) mice were used to show the actions of endogenous CGRP in postoperative lymphedema. After generating a mouse postoperative tail lymphedema model, the edema was observed to be more severe in CGRP(−/−) mice than in wild-type mice. Numbers of LYVE-1-positive lymphatic capillaries were decreased and lymphatic capillary formation-related factors were down-regulated in CGRP(−/−) mice. In addition, accumulation of M2 but not M1 macrophages was selectively reduced in the edematous tissue of CGRP(−/−) mice. Selective depletion of M2 macrophages decreased lymphatic capillary formation and worsened lymphedema in wild-type mice but not CGRP(−/−) mice, where numbers of M2 macrophages were already diminished. These findings suggest that endogenous CGRP acts to ameliorate postoperative lymphedema by enhancing lymphatic capillary formation and that M2 macrophages play critical roles. CGRP may be a useful therapeutic target for the treatment of postoperative lymphedema.

INTRODUCTION: The glial-lymphatic pathway is a fluid-clearance pathway consisting of a para-arterial route for the flow of cerebrospinal fluid along perivascular spaces and subsequently toward the brain interstitium. In this case series we aim to investigate an empirical demonstration of glymphatic clearance of extravasated iodine following perforation incurred during endovascular therapy on serial CT. METHODS AND RESULTS: Six consecutive cases of endovascular perforation during thrombectomy performed between 2005 and 2018 were retrospectively collected by searching our internal database of total 446 thrombectomies. Two cases were excluded because care was withdrawn shortly following the procedure and no follow-up imaging was available. One case was excluded because a ventricular drain was placed. Three cases were hence included in this analysis. All three cases demonstrated progressive absorption of contrast by the brain parenchyma with eventual contrast disappearance. CONCLUSION: We described a likely in vivo CT correlate of the glymphatic system in a cohort of patients who sustained intraprocedural extravasation during thrombectomy for acute ischemic stroke.

The progress in microneedle research is evidenced by the transition from simple ‘poke and patch’ solid microneedles fabricated from silicon and stainless steel to the development of bioresponsive systems such as hydrogel-forming and dissolving microneedles. In this review, we provide an outline on various microneedle fabrication techniques which are currently employed. As a range of factors, including materials, geometry and design of the microneedles, affect the performance, it is important to understand the relationships between them and the resulting delivery of therapeutics. Accordingly, there is a need for appropriate methodologies and techniques for characterization and evaluation of microneedle performance, which will also be discussed. As the research expands, it has been observed that therapeutics delivered via microneedles has gained expedited access to the lymphatics, which makes them a favorable delivery method for targeting the lymphatic system. Such opportunity is valuable in the area of vaccination and treatment of lymphatic disorders, which is the final focus of the review.

Interstitial, e.g., subcutaneous (SC) or intradermal (ID), administration of monoclonal antibodies (mAb) is less invasive than intravenous administration and leads to mAb uptake into both lymphatic and blood capillaries draining the injection site. Interstitial administration, however, is hindered by the presence of hyaluronan (HA), a glycosaminoglycan that is a major fluid barrier in the interstitial space. The transient removal of HA with recombinant human hyaluronidase (rHuPH20) helps facilitate the interstitial administration of often high therapeutic doses of mAb in the clinic. rHuPH20's impact on the systemic pharmacokinetics of several mAbs has been previously described, however effects on route of absorption (lymph vs blood) are unknown. The current study has therefore explored the lymphatic transport and bioavailability of cetuximab and trastuzumab after SC and ID coadministration in the presence and absence of rHuPH20 in rats. After SC administration cetuximab absolute bioavailability increased from 67% to 80% in the presence of rHuPH20. Cetuximab recovery in the lymphatics also increased after SC (35.8% to 49.4%) and ID (26.7% to 58.8%) administration in the presence of rHuPH20. When the injection volume (and therefore dose) was increased 10-fold in the presence of rHuPH20 cetuximab plasma exposure increased approximately linearly (12- and 8.9-fold respectively after SC and ID administration), although the proportional contribution of cetuximab lymphatic transport reduced slightly (6.2-fold increase for both administration routes). In contrast co-administration with rHuPH20 did not lead to increases in plasma exposure for trastuzumab after SC or ID administration, most likely reflecting the fact that the reported absolute bioavailability of trastuzumab (in the absence of rHuPH20) is high (approximately 77–99%). However, lymphatic transport of trastuzumab did increase when coadministered ID with rHuPH20 in spite of the lack of change to overall bioavailability. The data suggest that co-administration with rHuPH20 is able to increase the volume of mAb that can be administered interstitially, and in some instances can increase the amount absorbed into both the blood and the lymph. In the current studies the ability of rHuPH20 to enhance interstitial bioavailability was higher for cetuximab where intrinsic interstitial bioavailability was low, when compared to trastuzumab where interstitial bioavailability was high.

BACKGROUND: Lymphatic vessel formation (lymphangiogenesis) plays important roles in cancer metastasis, organ rejection, and lymphedema, but the underlying molecular events remain unclear. Furthermore, despite significant overlap in the molecular families involved in angiogenesis and lymphangiogenesis, little is known about the crosstalk between these processes. The ex vivo aortic ring assay and lymphatic ring assay have enabled detailed studies of vessel sprouting, but harvesting and imaging clear thoracic duct samples remain challenging. Here we present a modified ex vivo dual aortic ring and thoracic duct assay using tissues from dual fluorescence reporter Prox1-GFP/Flt1-DsRed (PGFD) mice, which permit simultaneous visualization of blood and lymphatic endothelial cells. OBJECTIVE: To characterize the concurrent sprouting of intrinsically fluorescent blood and lymphatic vessels from harvested aorta and thoracic duct samples. METHODS: Dual aorta and thoracic duct specimens were harvested from PGFD mice, grown in six types of endothelial cell growth media (one control, five that each lack a specific growth factor), and visualized by confocal fluorescence microscopy. Linear mixed models were used to compare the extent of vessel growth and sprouting over a 28-day period. RESULTS: Angiogenesis occurred prior to lymphangiogenesis in our assay. The control medium generally induced superior growth of both vessel types compared with the different modified media formulations. The greatest decrease in lymphangiogenesis was observed in vascular endothelial growth factor-C (VEGF-C)-devoid medium, suggesting the importance of VEGF-C in lymphangiogenesis. CONCLUSION: The modified ex vivo dual aortic ring and thoracic duct assay represents a powerful tool for studying angiogenesis and lymphangiogenesis in concert.

Impairment of cardiac lymphatic vessels leads to cardiac lymphedema. Recent studies have suggested that stimulation of lymphangiogenesis may reduce cardiac lymphedema. However, effects of lymphatic endothelial progenitor cells (LEPCs) on cardiac lymphangiogenesis are poorly understood. Therefore, this study investigated effectiveness of LEPC transplantation and VEGF-C release with self-assembling peptide (SAP) on cardiac lymphangiogenesis after myocardial infarction (MI). CD34(+)VEGFR-3(+) EPCs isolated from rat bone marrow differentiated into lymphatic endothelial cells after VEGF-C induction. VEGF-C also stimulated the cells to incorporate into the lymphatic capillary-like structures. The functionalized SAP could adhere with the cells and released VEGF-C sustainedly. In the condition of hypoxia and serum deprivation or abdominal pouch assay, the SAP hydrogel protected the cells from apoptosis and necrosis. At 4 weeks after intramyocardial transplantation of the cells and VEGF-C loaded with SAP hydrogel in rat MI models, cardiac lymphangiogenesis was increased, cardiac edema and reverse remodeling were reduced, and cardiac function was improved significantly. Delivery with SAP hydrogel favored survival of the engrafted cells. VEGF-C released from the hydrogel promoted differentiation and incorporation of the cells as well as growth of pre-existed lymphatic vessels. Cardiac lymphangiogenesis was beneficial for elimination of the inflammatory cells in the infarcted myocardium. Moreover, angiogenesis and myocardial regeneration were enhanced after reduction of lymphedema. These results demonstrate that the combined delivery of LEPCs and VEGF-C with the functionalized SAP promotes cardiac lymphangiogenesis and repair of the infarcted myocardium effectively. This study represents a novel therapy for relieving myocardial edema in cardiovascular diseases.

Hennekam lymphangiectasia-lymphedema syndrome is an autosomal recessive disorder characterized by congenital lymphedema, intestinal lymphangiectasia, facial dysmorphism, and variable intellectual disability. Known disease genes include CCBE1, FAT4, and ADAMTS3. In a patient with clinically diagnosed Hennekam syndrome but without mutations or copy-number changes in the three known disease genes, we identified a homozygous single-exon deletion affecting FBXL7. Specifically, exon 3, which encodes the F-box domain and several leucine-rich repeats of FBXL7, is eliminated. Our analyses of databases representing >100,000 control individuals failed to identify biallelic loss-of-function variants in FBXL7. Published studies in Drosophila indicate Fbxl7 interacts with Fat, of which human FAT4 is an ortholog, and mutation of either gene yields similar morphological consequences. These data suggest that FBXL7 may be the fourth gene for Hennekam syndrome, acting via a shared pathway with FAT4.

Everolimus is a mammalian target of rapamycin (mTOR) inhibitor, which is used in immunosuppressive treatment regimens in solid-organ transplant recipients. Although mTOR inhibitors are usually well tolerated, their adverse effects have been reported: sirolimus treatment in transplant patients has been rarely reported to be associated with lymphedema of the skin and subcutaneous tissues, whereas the use of everolimus seemed to be less burdened by this type of adverse effect. We report the case of a 58-year-old man with a history of end-stage renal disease of unknown etiology who had undergone right renal transplantation 11 years before. After the transplant, the patient soon developed bilateral progressive swelling involving feet and legs. The symptoms of the left limb improved markedly after discontinuing everolimus. This condition has been classified as everolimus-induced lymphedema. The patient was referred to our department for the execution of a noncontrast magnetic resonance lymphangiography, that is, a noninvasive magnetic resonance imaging technique that has recently proposed for the study of lymphedema. Noncontrast magnetic resonance lymphangiography showed asymmetry between the lower extremities with signs of advanced lymphedema located in the right lower limb and dilated peripheral lymphatic vessels.Drug withdrawal is currently the only effective solution for treating this type of secondary lymphedema; however, with the prolonged use of the drug, lymphedema tends to persist even after mTOR inhibitor suspension, with only partial clinical improvement, as in this case. This case report describes the imaging characteristics of such condition at noncontrast magnetic resonance lymphangiography and discusses the rare adverse effects of everolimus. Immediate suspension of the drug is the only effective strategy to avoid the persistence of this disorder.

BACKGROUND: Supermicrosurgical lymphaticovenous anastomosis (LVA) alleviates lymphedema by draining stagnant lymph from the lymphatic vessels into the venous system. Nevertheless, LVA is believed to be unsuitable for treating moderate-to-severe lymphedema presenting diffuse-pattern dermal backflow (DB). DB is considered to be the sign of superficial lymphatic functional failure that renders LVA ineffective. Based on a current algorithm, a more invasive vascularized lymph node flap transfer is recommended instead of LVA. This retrospective study aimed to further investigate and possibly challenge this concept. METHODS: One-hundred patients with unilateral lymphedematous lower limbs who underwent LVA were included. Patients were divided into Group I (10 patients with mild lymphedema) and Group II (90 patients with moderate-to-severe lymphedema). Demographic data and intraoperative findings were recorded. The post-LVA volume reductions by magnetic resonance volumetry were recorded and analyzed. RESULTS: Preoperatively, significant differences were found in body mass index (20.6 vs. 26.0, p = 0.004) and the volume gained in the lymphedematous limb (396.8 mL vs. 1056.8 mL, p = 0.005) between Groups I and II. Postoperatively, a significant median post-LVA volume reduction (−282.0 mL vs. −763.5 mL, p = 0.022) was found in Group II. However, there were no differences in the percentage of post-LVA volume reduction (−43.8% vs. −36.4%, p = 0.793) in Groups I and II. CONCLUSION: The use of supermicrosurgical LVA is as effective at treating moderate-to-severe lymphedema as milder lymphedema. The indication for LVA should be broadened to include such cases.

BACKGROUND: Although lipedema is often clinically distinguished from lymphedema, there is considerable overlap between the two entities. The purpose of this study was to evaluate lymphoscintigraphic findings in patients with lipedema to better characterize lymphatic flow in this patient population. METHODS: Patients with lipedema receiving lymphoscintigraphy between January 2015 and October 2017 were included. Patient demographics, clinical characteristics, and lymphoscintigraphic findings were extracted. Klienhan's transport index (TI) was utilized to assess lymphatic flow in patient's lower extremities (LEs).Scores ranged from 0 to 45, with values >10 denoting pathologic lymphatic transport. RESULTS: A total of 19 total patients with lipedema underwent lymphoscintigraphic evaluation. Mean age was 54.8 years and mean body mass index was 35.9 kg/m(2). Severity of lipedema was classified as stage 1 in five patients (26.3%), stage 2 in four patients (21.1%), stage 3 in four patients (21.1%), and stage 4 in six patients (31.6%). The mean TI for all extremities was 12.5; 24 (63.2%) LEs had a pathologic TI, including 7 LEs with stage 1 (29.2%), 3 LEs with stage 2 (12.5%), 6 LEs with stage 3 (25.0%), and 8 LEs with stage 4 lipedema (33.3%). The mean TI was significantly greater for extremities with severe (stage 3/4) lipedema than those with mild or moderate (stage 1/2) lipedema (15.1 vs. 9.7, p = 0.049). Mean difference in TI scores between each LE for individual patients was 6.43 (standard deviation +7.96). CONCLUSION: Our results suggest that patients with lipedema have impaired lymphatic transport, and more severe lipedema may be associated with greater lymphatic transport abnormalities.

RATIONALE: Chylothorax remains a poorly understood phenomenon, and no optimal treatment or guidelines have been established. This is the first report of treating congenital chylothorax and lymphedema in a low-birth-weight infant by lymphovenous anastomosis (LVA). PATIENT CONCERNS: We report a case of successful LVA for persistent congenital chylothorax and lymphedema resistant to other conservative therapies. DIAGNOSIS: The diagnosis of chylothorax was confirmed by the predominance of lymphocytes in the pleural fluid draining from the chest tube. In addition, the infant developed oliguria and generalized lymphedema. INTERVENTIONS: LVA under local anesthesia combined with light sedation was performed at his medial thighs and left upper arm. OUTCOMES: Although his subcutaneous edema markedly improved, the decrease in chest tube drainage was gradual. No additional treatment was required. LESSONS: LVA is of considerable value as a surgical treatment option in the setting of persistent congenital chylothorax and lymphedema, because LVA is a less invasive procedure.

BACKGROUND: Investigating correlations between clinical, instrumental, and genetic features of primary lymphedema (PL) with the aim to facilitate the diagnosis, the staging, and the management of this subgroup of patients. METHODS: A prospective observational study was conducted from September 2016 to May 2018, including patients with diagnosis of nonsyndromic PL. All patients underwent a lymphoscintigraphic rest-stress test, an indocyanine green (ICG) lymphographic test, and a genetic test from sputum sample. RESULTS: A total of 20 patients were enrolled in the study and 44 limbs were examined. The totality of clinically affected limbs (32/44) showed lymphographic and lymphoscintigraphic abnormalities. Concerning clinically healthy limbs (12/44), an abnormal pattern was demonstrated in 33.3% of ICG lymphographic test and 75% of lymphoscintigraphy. Regarding lymphography findings, the most frequent pattern was the distal dermal backflow (DDB). We distinguished four grades of DDB, which correlates with clinical and lymphoscintigraphic features. Furthermore, we found a new lymphographic alteration consisting of fluorescence appearing distally to the injection site of ICG, including fingers/toes and palmar/plantar surface of the hand and of the foot. This alteration, called “print sign," seems to be typical of DDB pattern PL. Genetic test did not help us make any etiological diagnosis. CONCLUSION: To our knowledge, this is the first study about PL comparing clinical, ICG lymphographic, lymphoscintigraphic, and genetic findings. As expected, all clinically affected limbs showed abnormalities in ICG lymphographic and lymphoscintigraphic tests. In opposition to what has previously been reported, also clinically healthy limbs showed lymphographic and lymphoscintigraphic alterations and this could suggest the existence of a subclinical form of PL. We proposed a grading of the DDB pattern, which correlates with clinical and lymphoscintigraphic features. Furthermore, we have described a new lymphographic alteration that seems to be typical of DDB pattern PL, the “print sign."

General lymphatic anomaly (GLA) is a very rare disorder, characterised by multifocal lymphatic malformations into various tissues that is due to congenital abnormalities of lymphatic development. No treatment has ever proved its efficiency.We report a 22-year-old man with recurrent bronchial casts due to thoracic involvement of GLA. After a 6-month treatment with sildenafil (20 mg three times a day), a phosphodiesterase 5 inhibitor, chest CT scan showed a complete regression of ground-glass opacities and lung function test results improved substantially and remained stable for 1 year. The treatment was well tolerated.This observation suggests that sildenafil may be a therapeutic approach to be tested in thoracic involvement of GLA.

Conventional oral therapy of lymphatic filariasis drugs is only effective to kill microfilariae in the bloodstream, but is often ineffective to kill adult filarial (macrofilariae) in the complex anatomy of the lymphatic system. The encapsulation of drugs into lipid-based nanoparticles with sizes of <100 nm, and administration intradermally, could be used to enhance lymphatic uptake. Therefore, we developed an innovative approach, using solid lipid nanoparticles (SLNs) and dissolving microneedles (MNs) to deliver antifilariasis drugs, namely doxycycline, diethylcarbamazine and albendazole, intradermally. The SLNs were prepared from Geleol(R) and Tween(R)80 as a lipid matrix and stabilizer, respectively. The formulations were optimized using a central composite design, producing SLNs with sizes of <100 nm. Drug release was sustained over 48 h from SLNs, compared to pure drugs. The SLNs were then incorporated into a polymeric hydrogel which was casted to form SLNs-loaded MNs. SLNs-loaded MNs exhibited sufficient mechanical and insertion properties. Importantly, dermatokinetic studies showed that >40% of drugs were retained in the dermis of excised neonatal porcine skin up to 24 h post-MN application, indicating the high possibility of the SLNs to be taken by the lymphatic system. In in vivo studies, the maximal lymph concentrations of the three drugs in rat, achieved following intradermal delivery, ranged between 4- and 7-fold higher than that recorded after oral administration. Additionally, compared to oral administration, despite the lower plasma Cmax and organ-distribution, the AUC and relative bioavailability of the three drugs in rat plasma was also higher using our delivery approach. Accordingly, this delivery approach could maximize the drugs concentrations in the lymph system without essentially increasing their plasma concentrations. This could potentially deliver the drugs efficiently to the bloodstream, where the microfilariae reside, while also targeting drug to the lymph nodes, where filarial nematodes reside in infected patients, leading to an effective therapy for lymphatic filariasis.

The lymphatic vasculature has traditionally been considered important for removal of excessive fluid from the interstitial space, absorption of fat from the intestine and the immune system. Advances in molecular medicine and imaging have provided us with new tools to study the lymphatics. This has revealed that the vessels are actively involved in regulation of immune cell trafficking and inflammation. We now know much about how new lymphatic vessels are created (lymphangiogenesis) and that this is important in, for example, wound healing and tissue repair. The best characterised pathway for lymphangiogenesis is the vascular endothelial growth factor C (VEGF-C)/VEGFR3 pathway. Over recent years, there has been an increasing interest in the role of the lymphatics in cardiovascular medicine. Preclinical studies have shown that lymphangiogenesis and immune cell trafficking play a role in cardiovascular conditions such as atherosclerosis, recovery after myocardial infarction and rejection of cardiac allografts. Targeting the VEGF-C/VEGFR3 pathway can be beneficial in these conditions. The clinical spectrum of lymphatic abnormalities and lymphoedema is wide and overlaps with congenital heart disease. Important long-term complications to the Fontan circulation involves the lymphatics. New and improved imaging modalities has improved our understanding and management of these patients. Lymphatic leaks and flow abnormalities can be successfully treated, minimally invasively, with percutaneous embolisation. Future research will prove if the preclinical findings that point to a role of the lymphatics in several cardiovascular conditions will result in new treatment options.

Background: Delayed onset muscle soreness (DOMS) in runners is classified as a leg muscle strain injury and presents with tenderness or stiffness to palpation and movement limitation. Most attention is directed at muscles but not at the mass of other limb soft tissues, including their lymphatic vasculature, although they undergo mechanical stress and bruises, edema, nail destruction, and pains contributing to symptoms. Methods: The study was done on lower limbs of long-distance runners suffering from DOMS complaints. There were 16 runners, 11 males and 5 females, age 22–28, practicing long-distance running over the last 5 years, with body mass index (BMI) 23 +/− 4. Inclusion criteria: three to five marathon runs per year and daily 3–5 km slow runs. Last long distance run 3 to 7 days before the investigation. Controls were six subjects initiating running, of the same age group and BMI. Testing of blood and lymph flow was done before and after standard ergometer 300 W 30 minutes cycling. The measurement methods were leg and big toe venous plethysmography, big toe capillary Doppler, tonometry of skin and deep tissues, lymphoscintigraphy, and indocyanine green (ICG) fluorescent lymphography. Results: (a) Strain gauge plethysmography of the calf and big toe revealed a two- to three-times higher venous capacity in runners than in controls, (b) the increased toe venous capacity was confirmed by point Doppler recordings showing two- to three-times higher blood capillary flow compared to controls, (c) lymphoscintigraphy revealed retention of tracer in feet, dilated superficial and deep lymphatics, and enlarged popliteal and inguinal lymph nodes, and (d) ICG lymphograms showed confluents of accumulated fluid in foot and calf subcutaneous tissue with fluorescence level reaching 40%–50% compared to 20% in controls. Conclusion: Our results show that, 3–5 days after run, not only muscles but also skin and subcutaneous tissue reveal major tissue fluid accumulation, an overload bringing about functional lymphatic transport insufficiency. This may be an additional factor responsible for DOMS symptoms.

Tongue squamous cell carcinoma (TSCC) has a poor prognosis due to its early metastasis through blood and lymphatic vessels. We undertook a systematic review to investigate the prognostic significance of blood microvessel density (MVD) and lymphatic vessel density (LVD) in TSCC patients. We carried out a systematic search in Ovid Medline, Scopus, and Cochrane libraries. All studies that evaluated the prognostic significance of MVD/LVD markers in TSCC were systematically retrieved. Our results showed that MVD/LVD markers, CD31, CD34, CD105, factor VIII, lymphatic vessel endothelial hyaluronan receptor-1, and D2–40 were evaluated in TSCC patients until 28 June 2018. Six out of 13 studies reported markers that were associated with poor prognosis in TSCC. Two out of three studies suggested that a high number of D2–40(+) vessels predicated low overall survival (OS); the third study reported that the ratio of D2–40(+) over factor VIII(+) vessels is associated with low OS. Most of the other markers had controversial results for prognostication. We found higher expression of MVD/LVD markers were commonly, but not always, associated with shorter survival in TSCC patients. It is therefore not currently possible to recommend implementation of these markers as reliable prognosticators in clinical practice. More studies (especially for D2–40) with larger patient cohorts are needed.

Non-coding RNAs (ncRNAs), which do not encode proteins, have pivotal roles in manipulating gene expression in development, physiology, and pathology. Emerging data have shown that ncRNAs can regulate lymphangiogenesis, which refers to lymphatics deriving from preexisting vessels, becomes established during embryogenesis, and has a close relationship with pathological conditions such as lymphatic developmental diseases, inflammation, and cancer. This review summarizes the molecular mechanisms of lymphangiogenesis in lymphatic development, inflammation and cancer metastasis, and discusses ncRNAs' regulatory effects on them. Therapeutic targets with regard to lymphangiogenesis are also discussed.

AIMS: Infantile hemangioma (IH) is the most common vascular neoplasm in infant and young children. Long non-coding RNAs (lncRNAs) are known to be associated with IH. This study aims to investigate the role and underlying mechanism of lncRNA-MALAT1 in IH. MAIN METHODS: qRT-PCR was used to quantify the expressions of MALAT1, miR-424, and MEKK3 in IH tissues. The cell proliferation, apoptosis, migration, invasion, and tube formation ability were assessed by MTT assay, colony formation assay, flow cytometric analysis, transwell assay and tube formation assay, respectively. The interaction among MALAT1, miR-424 and MEKK3 was evaluated by luciferase reporter assay. Immunohistochemistry (IHC) and Western blotting were utilized to evaluate the expression levels of MEKK3, Ki-67 and NF-kappaB pathway-related proteins both in vitro and in vivo. KEY FINDINGS: In IH tissues, MALAT1 and MEKK3 were overexpressed while miR-424 was down-regulated. Silencing MALAT1 or overexpression of miR-424 significantly inhibited the IH cell proliferation, migration and tube formation, but promoted the cell apoptosis. Knockdown of MALAT1 suppressed the expression of MEKK3 and inactivated the IKK/NF-kappaB pathway by sponging miR-424. Overexpression of MEKK3 in HemEcs reversed the impact of knockdown of MALAT1 and overexpression of miR-424 on the cell proliferation, apoptosis, migration, invasion and tube formation rate. The tumor xenografts experiments demonstrated that silencing MALAT1 significantly inhibited the tumor growth in vivo and Ki-67 in the tumor tissues was also significantly suppressed. SIGNIFICANCE: MALAT1 promoted the IH progression through inhibiting miR-424 to activate MEKK3-mediated IKK/NF-kappaB pathway, suggesting that MALAT1, miR-424 and MEKK3 could be used as potential targets to improve IH treatment efficiency.

OBJECTIVE: To determine the effectiveness of Kinesio taping compared to compression garments during maintenance phase of complex decongestive therapy for breast cancer-related lymphedema. DESIGN: Randomized, cross-over, controlled trial. SETTING: Outpatient tertiary-level hospital rehabilitation setting. SUBJECTS: Randomized sample of 30 women with breast cancer-related lymphedema. INTERVENTIONS: Participants received two interventions, Kinesio taping and compression garment, both lasting four weeks, whose order was randomized by blocks. A four-week washout period was established prior to the interventions and between them. MEASUREMENTS: The main outcome was the lymphedema Relative Volume Change. Secondary outcomes were range of motion of arm joints, self-perception of comfort, and lymphedema-related symptoms (pain, tightness, heaviness, and hardness). RESULTS: The decrease in the Relative Volume Change was greater in the Kinesio taping intervention (−5.7%, SD = 2.0) compared to that observed using compression garments (−3.4%, SD = 2.9) (P < 0.001). The range of motion of five upper-limb movements increased after applying taping (between 5.8 degrees and 16.7 degrees) (P < 0.05), but not after compression (P > 0.05). In addition, taping was perceived as more comfortable by patients (between 2.4 and 3 points better than compression in four questions with a 5-point scale (P < 0.001)) and further reduced lymphedema-related symptoms compared to compression (between 0.96 and 1.40 points better in four questions with a 6-point scale (P < 0.05)). CONCLUSION: Kinesio taping was more effective than compression garments for reducing the lymphedema volume, with less severe lymphedema-related symptoms, better improvement of upper-limb mobility, and more comfort.

BACKGROUND: Acquired lymphangiectasias represent cystic dilatations of the cutaneous lymphatic vessels resulting from damage and/or obstruction of previously normal lymphatics, usually secondary to surgery, radiotherapy, and/or infections. MATERIALS AND METHODS: The clinical, dermoscopic, and confocal microscopy features of three cases of acquired lymphangiectasias occurring after breast surgery are described along with histopathological correlations. RESULTS: Polarized dermoscopy revealed in all lesions the presence of well-circumscribed, white-yellowish lacunae surrounded by pale septa. In addition, some lesions showed scattered reddish areas and red lacunae. Confocal microscopy showed in the upper dermis numerous, roundish dark cavities separated by thin septa. These features corresponded histopathologically to saccular dilations and ectatic lymphatic vessels lined by a single layer of endothelial cells in the papillary/reticular dermis. CONCLUSION: The use of non-invasive diagnostic techniques may be addressed to an enhanced non-invasive diagnosis of acquired lymphangiectasias by showing peculiar features, thus avoiding the need for skin biopsy.

BACKGROUND & AIMS: The microenvironment regulates hepatoma stem cell behavior. However, the contributions of lymphatic endothelial cells to the hepatoma stem cell niche remain largely unknown; we aimed to analyze this contribution and elucidate the mechanisms behind it. METHODS: Associations between lymphatic endothelial cells and CD133(+) hepatoma stem cells were analyzed by immunofluorescence and adhesion assays; with the effects of their association on IL-17A expression examined using western blot, quantitative reverse transcription PCR and luciferase reporter assay. The effects of IL-17A on the self-renewal and tumorigenesis of hepatoma stem cells were examined using sphere and tumor formation assays. The role of IL-17A in immune escape by hepatoma stem cells was examined using flow cytometry. The expression of IL-17A in hepatoma tissues was examined using immunohistochemistry. RESULTS: CD133(+) hepatoma stem cells preferentially interact with lymphatic endothelial cells. The interaction between the mannose receptor and high-mannose type N-glycans mediates the interaction between CD133(+) hepatoma stem cells and lymphatic endothelial cells. This interaction activates cytokine IL-17A expression in lymphatic endothelial cells. IL-17A promotes the self-renewal of hepatoma stem cells. It also promotes their immune escape, partly through upregulation of PD-L1. CONCLUSION: Interactions between lymphatic endothelial cells and hepatoma stem cells promote the self-renewal and immune escape of hepatoma stem cells, by activating IL-17A signaling. Thus, inhibiting IL-17A signaling may be a promising approach for hepatoma treatment. LAY SUMMARY: The microenvironment is crucial for the self-renewal and development of hepatoma stem cells, which lead to the development of liver cancer. Lymphatic endothelial cells are an important component of this niche microenvironment, helping hepatoma stem cells to self-renew and escape immune attack, by upregulating IL-17A signaling. Thus, targeting IL-17A signaling is a potential strategy for the treatment of hepatoma.

PURPOSE: The Outcome measures for vascular malformation (OVAMA) group reached consensus on the core outcome domains for the core outcome set (COS) for peripheral vascular malformations (venous, lymphatic and arteriovenous malformations). However, it is unclear which instruments should be used to measure these domains. Therefore, our aims were to identify all outcome measurement instruments available for vascular malformations, and to evaluate their measurement properties. METHODS: With the first literature search, we identified outcomes and instruments previously used in prospective studies on vascular malformations. A second search yielded studies on measurement properties of patient- and physician-reported instruments that were either developed for vascular malformations, or used in prospective studies. If the latter instruments were not specifically validated for vascular malformations, we performed a third search for studies on measurement properties in clinically similar diseases (vascular or lymphatic diseases and benign tumors). We assessed the methodological quality of these studies following the Consensus-based Standards for the selection of health Measurement Instruments methodology, and evaluated the quality of the measurement properties. RESULTS: The first search yielded 27 studies, none using disease-specific instruments. The second and third search included 22 development and/or validation studies, concerning six instruments. Only the Lymphatic Malformation Function Instrument was developed specifically for vascular malformations. Other instruments were generic QoL instruments developed and/or partly validated for clinically similar diseases. CONCLUSIONS: Additional research on measurement properties is needed to assess which instruments may be included in the COS. This review informs the instrument selection and/or the development of new instruments. SYSTEMATIC REVIEW REGISTRATION: PROSPERO, 42017056242.

OBJECTIVE: Complicated lymphatic anomalies (CLAs) are chronic, progressive, and debilitating conditions that share clinical features, yet key elements for optimal evaluation and management have not been established. We aimed to formulate expert opinion consensus-based guidelines for comprehensive evaluation of CLAs. STUDY DESIGN: Patient support groups dedicated to CLAs organized an international conference for vascular anomaly experts from 16 specialties to address the objective. Participants received a set of questions before the meeting and reviewed the literature. Data extracted from international lymphatic anomaly registries were presented and the group separated for panel discussions during the conference. The recommendations achieving consensus within the panel were presented to the entire audience. Open debate occurred until majority approval was achieved. RESULTS: The expert group was composed of 52 physicians who defined the clinical elements required to evaluate and diagnose a CLA. The radiology panel established the preferred anatomical and functional imaging methods for diagnosis and the elements required to be described during interpretation. Two medical panels compiled the metabolic and hematologic tests at diagnosis and also recommended functional studies. The surgical group recommended precautions for biopsy and the pathology panel provided biopsy specimen processing guidelines. CONCLUSIONS: Patients with CLAs require a comprehensive and targeted diagnostic plan for appropriate management, prevention of complications, and conservation of resources. As this population is managed by diverse medical and surgical specialties, we offer an expert multidisciplinary consensus-based opinion on the current literature and on data extracted from international lymphatic anomaly registries.

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

The hedgehog (Hh) pathway is highly conserved and required for embryonic patterning and determination. Mutations in the Hh pathway are observed in sporadic tumors as well as under syndromic conditions. Common to these syndromes are the findings of polydactyly/syndactyly and brain overgrowth. The latter is also a finding most commonly observed in the cases of mutations in the PI3K/AKT/mTOR pathway. We have identified novel Hh pathway mutations and structural copy number variations in individuals with somatic overgrowth, macrocephaly, dysmorphic facial features, and developmental delay, which phenotypically closely resemble patients with phosphatase and tensin homolog (PTEN) mutations. We hypothesized that brain overgrowth and phenotypic overlap with syndromic overgrowth syndromes in these cases may be due to crosstalk between the Hh and PI3K/AKT/mTOR pathways. To test this, we modeled disease-associated variants by generating PTCH1 and Suppressor of Fused (SUFU) heterozygote cell lines using the CRISPR/Cas9 system. These cells demonstrate activation of PI3K signaling and increased phosphorylation of its downstream target p4EBP1 as well as a distinct cellular phenotype. To further investigate the mechanism underlying this crosstalk, we treated human neural stem cells with sonic hedgehog (SHH) ligand and performed transcriptional analysis of components of the mTOR pathway. These studies identified decreased expression of a set of mTOR negative regulators, leading to its activation. We conclude that there is a significant crosstalk between the SHH and PI3K/AKT/mTOR. We propose that this crosstalk is responsible for why mutations in PTCH1 and SUFU lead to macrocephaly phenotypes similar to those observed in PTEN hamartoma and other overgrowth syndromes associated with mutations in PI3K/AKT/mTOR pathway genes.

BACKGROUND: A somatic mutation of GNAQ (c.548G>A, p.Arg183Gln) plays a key role in capillary malformation development. The present study aimed to evaluate clinical manifestations of port-win stain (PWS) associated with this genetic mutation. METHODS: Skin tissue was obtained from 70 patients with capillary malformation who had been treated with excision for lesions. Droplet digital polymerase chain reaction was used to quantify the abundance of cells with the GNAQ mutation. RESULTS: The GNAQ mutation was found in 50 patients. Patients with lesions involving upper facial region, which included forehead, eyebrow, and upper eyelid, showed a significantly higher rate of positive GNAQ mutation than those not involving it. Cases with facial PWS involving all three facial regions (upper, middle, and lower) showed significantly higher positive rate of GNAQ mutation compared to those involving one or two. CONCLUSIONS: Presence of the somatic mutation GNAQ p.Arg183Gln might be associated with clinical manifestations of PWS.

Hepatic hemangioma (HH) is a common asymptomatic, self-limiting benign vascular tumor of the liver in neonates. Although complicated HHs are rare, they have significant risks of morbidity and mortality, especially during the perinatal period. Because of the high risks of complications from surgical interventions, there is an unmet need for effective medical therapy. We report 2 neonates with life-threatening HH who were evaluated for a liver transplant before being treated successfully with combined medical therapy, which included sirolimus, corticosteroids, and propranolol.

BACKGROUND: Infantile hemangioma (IH) is the most common childhood benign vascular tumor. Recently, propranolol has been found to be an effective therapy for IH, but its mechanism of action is not yet understood. Hemangioma stem cells (HemSCs) have a mesenchymal morphology, robust proliferation, and multilineage differentiation (into adipocytes). Therefore, we hypothesized that propranolol could accelerate the transdifferentiation of HemSCs and prevent the growth of proliferating IH. In this study, the fibrofatty tissue of IH that received therapy with propranolol appeared much earlier than without the treatment. METHODS: We isolated HemSCs with CD133-tagged immunomagnetic beads, and then we used flow cytometry technology to analyze the HemSC phenotypes and determine whether propranolol induced HemSC death. The proliferation and adipogenesis abilities of propranolol-treated HemSCs were analyzed by 3-(4,5)-dimethylthiahiazo(-z-y1)-3,5-diphenytetrazoliumromide assay, Oil red O staining, and Western blotting. RESULTS: We observed that the HemSC morphological traits gradually became spindle shaped, like fibroblasts, and the average extraction yield of HemSCs was about 0.25%. The HemSCs had high rates of expression for CD90 (98.8%) and CD105 (97.8%) but did not significantly express CD31 (0.7%). We also found a 100 muM concentration of propranolol cutoff point. Propranolol did not affect HemSC survival significantly at low concentrations (6.25, 12.5, 25.0, and 50.0 muM). However, propranolol resulted in a sharp and significant variation in cell morphology and survival rates at high concentrations (100, 200, and 400 muM). The results suggest that treatment with propranolol inhibited HemSC proliferation and induced cell death and apoptosis in a concentration-dependent manner. Oil droplets determined by Oil red O staining showed that propranolol increased the transdifferentiation rate of HemSCs into adipocytes. Furthermore, the expressions of phosphorylated AKT and peroxisome proliferator-activated receptor gamma (PPARgamma) were increased with a 100 muM concentration of propranolol in HemSC culture. CONCLUSIONS: Our study found that propranolol inhibited proliferation, induced apoptosis and necrosis, and promoted differentiation of HemSCs. Propranolol may upregulate PPARgamma via PI3K pathways, thereby accelerating lipogenesis and enhancing IH HemSC adipogenesis.

Disorders of somatic mosaicism (DoSM) are a diverse group of syndromic and non-syndromic conditions caused by mosaic variants in genes that regulate cell survival and proliferation. Despite overlap in gene space and technical requirements, few clinical labs specialize in DoSM compared to oncology. We adapted a high-sensitivity next-generation sequencing cancer assay for DoSM in 2014. Some 343 individuals have been tested over the past 5 years, 58% of which had pathogenic and likely pathogenic (P/LP) findings, for a total of 206 P/LP variants in 22 genes. Parameters associated with the high diagnostic yield were: (1) deep sequencing (approximately 2,000x coverage), (2) a broad gene set, and (3) testing affected tissues. Fresh and formalin-fixed paraffin embedded tissues performed equivalently for identification of P/LP variants (62% and 71% of individuals, respectively). Comparing cultured fibroblasts to skin biopsies suggested that culturing might boost the allelic fraction of variants that confer a growth advantage, specifically gain-of-function variants in PIK3CA. Buccal swabs showed high diagnostic sensitivity in case subjects where disease phenotypes manifested in the head or brain. Peripheral blood was useful as an unaffected comparator tissue to determine somatic versus constitutional origin but had poor diagnostic sensitivity. Descriptions of all tested individuals, specimens, and P/LP variants included in this cohort are available to further the study of the DoSM population.

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

Vascular anomalies (VAs) may be associated with significant morbidity and mortality. The aim of this study was to evaluate the efficacy and safety of sirolimus (rapamycin) in the treatment of children and young adults with complicated VAs. A retrospective chart was created that included 19 patients treated with sirolimus for complicated VAs. Concurrently, a search of the PubMed database for VA cases treated with sirolimus was conducted. Descriptive analysis was performed and the efficiency rate of sirolimus was calculated. This retrospective study included 19 patients, 17 of whom were treated with oral sirolimus and 2 with topical sirolimus. Clinical improvement occurred in 15 patients (79%). One patient experienced near-complete resolution. Only 2 patients showed poor response and discontinued treatment. The literature review analysed 150 cases of VA treated with sirolimus. Sirolimus was efficient in 85% of cases, including 5 cases of complete resolution. Sirolimus appears to be an effective and safe treatment for children and young adults with complicated VAs.

Infantile hemangiomas (IH) are the most common vascular tumor of infancy (1). Most hemangiomas follow a predictable pattern with proliferation followed by slow involution and many do not require medical or surgical intervention. However, some hemangiomas cause complications, including disfigurement, visual obstruction and/or functional impairment, which require therapeutic intervention. (2) Since the discovery of beta blockers as effective topical and oral therapy for IH, hemangioma specialists suggest early therapy and referral for IH to prevent permanent sequalae (3). Yet, little is known about utilization patterns for hemangiomas in the United States.

Hereditary hemorrhagic telangiectasia (HHT) is a Mendelian disease characterized by vascular malformations (VMs) including visceral arteriovenous malformations and mucosal telangiectasia. HHT is caused by loss-of-function (LoF) mutations in one of three genes, ENG, ACVRL1, or SMAD4, and is inherited as an autosomal-dominant condition. Intriguingly, the constitutional mutation causing HHT is present throughout the body, yet the multiple VMs in individuals with HHT occur focally, rather than manifesting as a systemic vascular defect. This disconnect between genotype and phenotype suggests that a local event is necessary for the development of VMs. We investigated the hypothesis that local somatic mutations seed the formation HHT-related telangiectasia in a genetic two-hit mechanism. We identified low-frequency somatic mutations in 9/19 telangiectasia through the use of next-generation sequencing. We established phase for seven of nine samples, which confirms that the germline and somatic mutations in all seven samples exist in trans configuration; this is consistent with a genetic two-hit mechanism. These combined data suggest that bi-allelic loss of ENG or ACVRL1 may be a required event in the development of telangiectasia, and that rather than haploinsufficiency, VMs in HHT are caused by a Knudsonian two-hit mechanism.

BACKGROUND/OBJECTIVE: The pathogenesis of infantile hemangiomas (IH), PHACE, and LUMBAR syndromes remains unknown. We aim to describe histopathologic features of midline anomalies associated with IH, including patients with PHACE and LUMBAR syndromes. METHODS: A multicenter retrospective chart review was performed to identify patients with IH, PHACE, and LUMBAR syndrome with histopathologic specimens from sternal or midline anomalies. A total of 18 midline lesions from 13 patients were included. Out of 18, 14 midline lesions underwent both histopathologic and clinical review. Three hamartoma-like chin plaques and one supraumbilical raphe underwent only clinical review. RESULTS: All 13 patients had midline lesions and IH. Histopathologic diagnoses were as follows: rhabdomyomatous mesenchymal hamartoma (3), folliculosebaceous cystic hamartoma (1), fibroepithelial polyp (1), verrucous epidermal hyperplasia with vascular proliferation and fibroplasia (1), congenital midline cervical cleft (1), pericardium with fibrosis (1), fibrous components with increased collagen (1), atrophic skin/membrane (3), angiolipomatous mass with neural components (1), and lipomatous mass (1). Due to the retrospective nature of this study, it was not possible to obtain pathology slides for all midline lesions that had previously been biopsied or resected. We show clinically and histopathologically a new association between PHACE syndrome and rhabdomyomatous mesenchymal hamartoma (RMH), in addition to demonstrating the association between PHACE syndrome and chin hamartomas. We also display histopathologic findings seen in midline lesions resected from LUMBAR patients. CONCLUSION: Rhabdomyomatous mesenchymal hamartoma is thought to be related to aberrations of mesenchymal cells during development; therefore, this may provide clues to the pathogenesis of IH and related syndromes.

BACKGROUND/OBJECTIVES: Congenital hemangiomas (CH) are a group of benign vascular tumors that are present at birth and exhibit variable involution during infancy. Congenital hemangiomas that do not involute are typically solitary patch or plaque-type tumors that grow proportionally with somatic growth. We report a case series of 9 patients with persistent CH, which exhibited uncommon features including segmental involvement, recurrent or severe pain, or growth via volumetric increase in size or apparent increased extent of anatomic involvement over time. METHODS: Via retrospective chart review, we included patients with persistent CH and atypical presentations. Available data regarding clinical characteristics, natural history, histopathology, imaging, and genetic tests were collected. RESULTS: Data on 9 patients were collected, including 7 noninvoluting CH and 2 partially involuting CH. Three of the 9 cases had segmental distribution, 6 had apparent growth or clinical evolution, and 4 were symptomatic with pain. One also had marked localized intravascular coagulopathy. CONCLUSIONS: Ongoing or recurrent pain and large extent of anatomic involvement can be features of CH, albeit uncommon ones, and can pose both diagnostic and management challenges. Tissue genomic studies can offer a novel tool for CH diagnosis.

We present a 7-year-old boy with tuberous sclerosis and congenital segmental lymphedema (CSL) of the left leg, as well as two aortic aneurysms. He was treated with everolimus (EVE) since the age of 14 months. His CSL regressed under treatment with EVE. His first aneurysms required operative intervention at age of 17 months. Four months afterward a new aortic aneurysm had been detected above the Dracon graft, but this one remained stable since that time. The patient didn't experience severe side effects. EVE has been well tolerated without disturbance of somatic growth or serious adverse effect.

Lymphatic malformations (LMs) are congenital, nonneoplastic vascular malformations associated with postzygotic activating PIK3CA mutations. The mutation spectrum within LMs is narrow, with the majority having 1 of 3 hotspot mutations. Despite this relative genetic homogeneity, clinical presentations differ dramatically. We used molecular inversion probes and droplet digital polymerase chain reaction to perform deep, targeted sequencing of PIK3CA in 271 affected and unaffected tissue samples from 81 individuals with isolated LMs and retrospectively collected clinical data. Pathogenic PIK3CA mutations were identified in affected LM tissue in 64 individuals (79%) with isolated LMs, with variant allele fractions (VAFs) ranging from 0.1% to 13%. Initial analyses revealed no correlation between VAF and phenotype variables. Recognizing that different mutations activate PI3K to varying degrees, we developed a metric, the genotype-adjusted VAF (GVAF), to account for differences in mutation strength, and found significantly higher GVAFs in LMs with more severe clinical characteristics including orofacial location or microcystic structure. In addition to providing insight into LM pathogenesis, we believe GVAF may have broad applicability for genotype-phenotype analyses in mosaic disorders.