Update December 2014

The lymphatic vasculature plays a critical role in a number of disease conditions of increasing prevalence, such as autoimmune disorders, obesity, blood vascular diseases, and cancer metastases. Yet, unlike the blood vasculature, the tools available to interrogate the molecular basis of lymphatic dysfunction/disease have been lacking. More recently, investigators have reported that dysregulation of the PI3K pathway is involved in syndromic human diseases that involve abnormal lymphatic vasculatures, but there have been few compelling results that show the direct association of this molecular pathway with lymphatic dysfunction in humans. Using near-infrared fluorescence lymphatic imaging (NIRFLI) to phenotype and next generation sequencing (NGS) for unbiased genetic discovery in a family with non-syndromic lymphatic disease, we discovered a rare, novel mutation in INPPL1 that encodes the protein SHIP2, which is a negative regulator of the PI3K pathway, to be associated with lymphatic dysfunction in the family. In vitro interrogation shows that SHIP2 is directly associated with impairment of normal lymphatic endothelial cell (LEC) behavior and that SHIP2 associates with receptors that are associated in lymphedema, implicating its direct involvement in the lymphatic vasculature.

BACKGROUND: The interplay between Notch and Vegf signaling regulates angiogenesis in the embryo. Notch signaling limits the responsiveness of endothelial cells to Vegf to control sprouting. Despite the importance of this regulatory relationship, much remains to be understood about extrinsic factors that modulate the pathway. RESULTS: During a forward genetic screen for novel regulators of lymphangiogenesis, we isolated a mutant with reduced lymphatic vessel development. This mutant also exhibited hyperbranching arteries, reminiscent of Notch pathway mutants. Positional cloning identified a missense mutation in the carbamoyl-phosphate synthetase 2, aspartate transcarbamylase, and dihydroorotase (cad) gene. Cad is essential for UDP biosynthesis, which is necessary for protein glycosylation and de novo biosynthesis of pyrimidine-based nucleotides. Using a transgenic reporter of Notch activity, we demonstrate that Notch signaling is significantly reduced in cadhu10125 mutants. In this context, genetic epistasis showed that increased endothelial cell responsiveness to Vegfc/Vegfr3 signaling drives excessive artery branching. CONCLUSION: These findings suggest important post-translational modifications requiring Cad as an unappreciated mechanism that regulates Notch/Vegf signaling during angiogenesis. Developmental Dynamics, 2014. (c) 2014 Wiley Periodicals, Inc.

The cellular and physiologic mechanisms that regulate the resolution of inflammation remain poorly defined despite their widespread importance in improving inflammatory disease outcomes. We studied the resolution of two cardinal signs of inflammation-pain and swelling-by investigating molecular mechanisms that regulate neural and lymphatic vessel remodeling during the resolution of corneal inflammation. A mouse model of corneal inflammation and wound recovery was developed to study this process in vivo. Administration of nerve growth factor (NGF) increased pain sensation and inhibited neural remodeling and lymphatic vessel regression processes during wound recovery. A complementary in vivo approach, the corneal micropocket assay, revealed that NGF-laden pellets stimulated lymphangiogenesis and increased protein levels of VEGF-C. Adult human dermal lymphatic endothelial cells did not express canonical NGF receptors TrkA and p75NTR or activate downstream MAPK- or Akt-pathway effectors in the presence of NGF, although NGF treatment increased their migratory and tubulogenesis capacities in vitro. Blockade of the VEGF-R2/R3 signaling pathway ablated NGF-mediated lymphangiogenesis in vivo. These findings suggest a hierarchical relationship with NGF functioning upstream of the VEGF family members, particularly VEGF-C, to stimulate lymphangiogenesis. Taken together, these studies show that NGF stimulates lymphangiogenesis and that NGF may act as a pathogenic factor that negatively regulates the normal neural and lymphatic vascular remodeling events that accompany wound recovery.

Lymphangiogenesis is significantly involved in the pathogenesis of diseases, including graft rejection, cancer metastasis and various inflammatory conditions. The inhibition of lymphangiogenesis has become a new therapeutic target for the treatment of these diseases. Here, we explored the anti-lymphangiogenic effects of doxycycline in inflammation-induced lymphangiogenesis (ILA) in the cornea and the underlying mechanisms. In the present study, mice with ILA of the cornea were treated with topical doxycycline (0.1%) or vehicle control. Lymphangiogenesis was quantified using corneal immunostaining of lymphatic vessel endothelial hyaluronan receptor-1 (LYVE-1). Human dermal lymphatic endothelial cells (HDLECs) and a murine macrophage cell line (RAW264.7) were used to further explore the underlying mechanisms of doxycycline-mediated anti-lymphangiogenesis in vitro. Our results showed that doxycycline treatment dramatically inhibited ILA in the mouse cornea (p<0.001), with a significant decrease in vascular endothelial growth factor (VEGF)-C/VEGF receptor 3 signalling, macrophage infiltration and inflammatory cytokine expression. Doxycycline also significantly inhibited VEGF-C-induced HDLEC proliferation in vitro by modulating the PI3K/Akt/endothelial nitric oxide (NO) synthase (eNOS) pathway and significantly suppressed interleukin-1beta (IL-1beta), TNF-alpha and VEGF-C production in the RAW264.7 cell line by modulating the PI3K/Akt/nuclear factor-kappaB (NF-kappaB) pathway. Additionally, doxycycline treatment dramatically reduced the phosphorylation of NF-kappaBp65, Akt and eNOS in ILA and significantly inhibited matrix metalloproteinases (MMPs) activity in vitro and in ILA. In conclusion, doxycycline inhibited ILA, possibly through suppression of VEGF-C signalling, macrophage function and MMPs activity. This observation suggests that doxycycline is a potential therapeutic agent for lymphangiogenesis-related diseases.

During human ocular development, expression of proteins varies in different maturation stages. This study aims to characterize structures in human fetal eyes stained by the lymphatic marker podoplanin (D2-40) with emphasis on the stage of maturation and the presence of intraocular lymphatic structures. Formalin-fixed paraffin-embedded eyes from 40 human fetuses between 10 and 38 weeks of gestation (WoG) were investigated. Immunohistochemical stains were performed for D2-40, LYVE-1 as a secondary lymphatic marker, and CD34 as a control for endothelial reactivity. A semiquantitative analysis of antigen expression in different segments of the eye was performed by light microscopy. The intensity of antigen expression was graded with a score ranging from 0 to 3. Podoplanin expression was found with a variable intensity in 97.5% of the eyes, in particular in lymphatic vessels of the conjunctiva (n=26), conjunctival and corneal epithelium (n=33), corneal endothelium (n=4), trabecular meshwork (n=28), and optic nerve sheaths (n=23). A slight, equivocal staining reaction was noted in the choroid (n=14). There was a correlation of antigen reactivity and the gestational age for corneal endothelial reactivity in earlier gestational stages (p=0.003) and trabecular meshwork in older eyes (p=0.031). D2-40 positive Muller cells were detected in two eyes>/=32 WoG. Thus, aside from conjunctival lymphatic vessels, podoplanin was expressed in several structures of the human fetal eye and the ocular adnexae at different gestational stages. Podoplanin positive structures were also found in the choroid and the chamber angle. However, lymphatic vessels or its progenitors could not be unequivocally identified in intraocular structures during 10–38 weeks of gestation. There is no evidence from our data that transient intraocular lymphactics develop in the fetal eye between 10 and 38 weeks of gestation.

Mutations in SOX18, VEGFC and Vascular Endothelial Growth Factor 3 underlie the hereditary lymphatic disorders hypotrichosis-lymphedema-telangiectasia (HLT), Milroy-like lymphedema and Milroy disease, respectively. Genes responsible for hereditary lymphedema are key regulators of lymphatic vascular development in the embryo. To identify novel modulators of lymphangiogenesis, we used a mouse model of HLT (Ragged Opossum) and performed gene expression profiling of aberrant dermal lymphatic vessels. Expression studies and functional analysis in zebrafish and mice revealed one candidate, ArfGAP with RhoGAP domain, Ankyrin repeat and PH domain 3 (ARAP3), which is down-regulated in HLT mouse lymphatic vessels and necessary for lymphatic vascular development in mice and zebrafish. We position this known regulator of cell behaviour during migration as a mediator of the cellular response to Vegfc signalling in lymphatic endothelial cells in vitro and in vivo. Our data refine common mechanisms that are likely to contribute during both development and the pathogenesis of lymphatic vascular disorders.

BACKGROUND: Atherosclerosis is a chronic inflammatory disease of large- to medium-sized arteries and is the main underlying cause of death worldwide. The lymphatic vasculature is critical for processes that are intimately linked to atherogenesis such as the immune response and cholesterol metabolism. However, whether lymphatic vessels truly contribute to the pathogenesis of atherosclerosis is less clear despite increasing research efforts in this field. DESIGN: PubMed and Ovid MEDLINE databases were searched. In addition, key review articles were screened for relevant original publications. RESULTS: Current knowledge about lymphatic vessels in the arterial wall came from studies that examined the presence and location of such vessels in human atherosclerotic plaque specimens, as well as in a variety of arteries in animal models for atherosclerosis (e.g. rabbits, dogs, rats and mice). Generally, three experimental approaches have been used to investigate the functional role of plaque-associated lymphatic vessels; experimental lymphostasis was used to investigate lymphatic drainage of the arterial wall, and more recently, studies with genetic interventions and/or surgical transplantation have been performed. CONCLUSIONS: Lymphatic vessels seem to be mostly present in the adventitial layer of the arterial walls of animals and humans. They are involved in reverse cholesterol transport from atherosclerotic lesions, and arteries with a dense lymphatic network seem naturally protected against atherosclerosis. Lymphangiogenesis is a process that is an important part of the inflammatory loop in atherosclerosis. However, how augmenting or impeding the distribution of lymphatic vessels impacts disease progression remains to be investigated in future studies. This article is protected by copyright. All rights reserved.

Mesenchymal stem cells (MSCs) show great promise in blood vessel restoration and vascularization enhancement in many therapeutic situations. Typically, the co-implantation of MSCs with vascular endothelial cells (ECs) is effective for the induction of functional vascularization in vivo, indicating its potential applications in regenerative medicine. The effects of MSCs-ECs-induced vascularization can be modeled in vitro, providing simplified models for understanding their underlying communication. In this paper, a contact co-culture model in vitro and an RNA-seq approach were employed to reveal the active crosstalk between MSCs and ECs within a short time period at both morphological and transcriptional levels. The RNA-seq results suggested that angiogenic genes were significantly induced upon co-culture, and this pre-vascularization commitment might require the NF-kappaB signaling. NF-kappaB blocking and interleukin neutralization experiments demonstrated that MSCs potentially secreted interleukin factors including IL1beta and IL6 to modulate NF-kappaB signaling as well as downstream chemokines during co-culture. Conversely, RNA-seq results indicated that the MSCs were regulated by the co-culture environment to a smooth muscle commitment within this short period, which largely induced myocardin, the myogenic co-transcriptional factor. These findings demonstrate the mutual molecular mechanism of MSCs-ECs-induced pre-vascularization commitment in a quick response.

Lymphatic valves prevent the backflow of the lymph fluid and ensure proper lymphatic drainage throughout the body. Local accumulation of lymphatic fluid in tissues, a condition called lymphedema, is common in individuals with malformed lymphatic valves. The vascular endothelial growth factor receptor 3 (VEGFR3) is required for the development of lymphatic vascular system. The abundance of VEGFR3 in collecting lymphatic trunks is high before valve formation and, except at valve regions, decreases after valve formation. We found that in mesenteric lymphatics, the abundance of epsin 1 and 2, which are ubiquitin-binding adaptor proteins involved in endocytosis, was low at early stages of development. After lymphatic valve formation, the initiation of steady shear flow was associated with an increase in the abundance of epsin 1 and 2 in collecting lymphatic trunks, but not in valve regions. Epsin 1 and 2 bound to VEGFR3 and mediated the internalization and degradation of VEGFR3, resulting in termination of VEGFR3 signaling. Mice with lymphatic endothelial cell-specific deficiency of epsin 1 and 2 had dilated lymphatic capillaries, abnormally high VEGFR3 abundance in collecting lymphatics, immature lymphatic valves, and defective lymph drainage. Deletion of a single Vegfr3 allele or pharmacological suppression of VEGFR3 signaling restored normal lymphatic valve development and lymph drainage in epsin-deficient mice. Our findings establish a critical role for epsins in the temporal and spatial regulation of VEGFR3 abundance and signaling in collecting lymphatic trunks during lymphatic valve formation.

Capillary malformation-arteriovenous malformation (CM-AVM) is an autosomal dominant blood vascular (BV) disorder characterized by CM and fast flow BV lesions. Inactivating mutations of the RASA1 gene are the cause of CM-AVM in most cases. RASA1 is a GTPase-activating protein that acts as a negative regulator of the Ras small GTP-binding protein. In addition, RASA1 performs Ras-independent functions in intracellular signal transduction. Whether CM-AVM results from loss of an ability of RASA1 to regulate Ras or loss of a Ras-independent function of RASA1 is unknown. To address this, we generated Rasa1 knockin mice with an R780Q point mutation that abrogates RASA1 catalytic activity specifically. Homozygous Rasa1 R780Q/R780Q mice showed the same severe BV abnormalities as Rasa1-null mice and died midgestation. This finding indicates that BV abnormalities in CM-AVM develop as a result of loss of an ability of RASA1 to control Ras activation and not loss of a Ras-independent function of this molecule. More important, findings indicate that inhibition of Ras signaling is likely to represent an effective means of therapy for this disease.

BACKGROUND AND PURPOSE: The rupture of intracranial aneurysm (IA) causes subarachnoid hemorrhage associated with high morbidity and mortality. We compared gene expression profiles in aneurysmal domes between unruptured IAs and ruptured IAs (RIAs) to elucidate biological mechanisms predisposing to the rupture of IA. METHODS: We determined gene expression levels of 8 RIAs, 5 unruptured IAs, and 10 superficial temporal arteries with the Agilent microarrays. To explore biological heterogeneity of IAs, we classified the samples into subgroups showing similar gene expression patterns, using clustering methods. RESULTS: The clustering analysis identified 4 groups: superficial temporal arteries and unruptured IAs were aggregated into their own clusters, whereas RIAs segregated into 2 distinct subgroups (early and late RIAs). Comparing gene expression levels between early RIAs and unruptured IAs, we identified 430 upregulated and 617 downregulated genes in early RIAs. The upregulated genes were associated with inflammatory and immune responses and phagocytosis including S100/calgranulin genes (S100A8, S100A9, and S100A12). The downregulated genes suggest mechanical weakness of aneurysm walls. The expressions of Kruppel-like family of transcription factors (KLF2, KLF12, and KLF15), which were anti-inflammatory regulators, and CDKN2A, which was located on chromosome 9p21 that was the most consistently replicated locus in genome-wide association studies of IA, were also downregulated. CONCLUSIONS: We demonstrate that gene expression patterns of RIAs were different according to the age of patients. The results suggest that macrophage-mediated inflammation is a key biological pathway for IA rupture. The identified genes can be good candidates for molecular markers of rupture-prone IAs and therapeutic targets.

Lymphedema is a debilitating progressive condition that is frequently observed following cancer surgery and severely restricts quality of life. Although it is known that lymphatic dysfunction and obstruction underlie lymphedema, the pathogenic mechanism is poorly understood. Smooth muscle cells (SMCs) play pivotal roles in the pathogenesis of various vascular diseases, including atherosclerosis. We analyzed SMCs in lymphatic vessels from the lymphedematous legs of 29 patients. Expression of SM alpha-actin (SMalphaA) and smooth muscle myosin heavy chain (SM-MHC) isoforms SM1 and SM2 was investigated using immunohistochemistry. As compared to normal lymphatic vessels, all affected lymphatic vessels in chronic lymphedema showed marked wall thickening. In addition to increases in the numbers of rows of SMalphaA+ SM1+ SMCs in the tunica media, SMCs were also observed in the subendothelial region (tunica intima). While most intimal and medial cells were positive for SMalphaA and SM1, staining for SM1 and particularly SM2, a marker of mature SMCs, progressively declined in lymphatic vessels in increasingly severe lymphedema lesions. Consequently, the SM1+ and SM2+ cell fractions were significantly reduced in the tunica media and intima of lymphatic vessels. These observations indicate that the lymphatic tunica media and tunica intima consist mainly of phenotypically modulated SMCs, and that SMCs play a key role in the development of lymphedema. This article is protected by copyright. All rights reserved.

O-glycosylation of podoplanin (PDPN) on lymphatic endothelial cells (LECs) is critical for the separation of blood and lymphatic systems by interacting with platelet C-type lectin-like receptor 2 (CLEC-2) during development. However, how O-glycosylation controls endothelial PDPN function and expression remains unclear. Here we report that core 1 O-glycan-deficient or desialylated PDPN was highly susceptible to proteolytic degradation by various proteases including metalloproteinases MMP-2/9. We found that the lymph contained activated MMP-2/9 and incubation of the lymph reduced surface levels of PDPN on core 1 O-glycan-deficient endothelial cells (ECs), but not on WT ECs. The lymph from mice with sepsis induced by cecal ligation and puncture (CLP), which contained bacteria-derived sialidase, reduced PDPN levels on WT ECs. These reductions were rescued by metalloproteinase inhibitor GM6001. Additionally, GM6001 treatment rescued the reduction of PDPN level on LECs in mice lacking endothelial core 1 O-glycan or CLP-treated mice. Furthermore, core 1 O-glycan-deficient or desialylated PDPN impaired platelet interaction under physiological flow. These data indicate that sialylated O-glycans of PDPN are essential for platelet adhesion and prevent PDPN from proteolytic degradation primarily mediated by metalloproteinases in the lymph.

The interaction of CLEC-2 on platelets with Podoplanin on lymphatic endothelial cells initiates platelet signalling events that are necessary for prevention of blood-lymph mixing during development. In the present study, we show that CLEC-2 signalling via Src family and Syk tyrosine kinases promotes platelet adhesion to primary mouse lymphatic endothelial cells at low shear. Using supported lipid bilayers containing mobile Podoplanin, we further show that activation of Src and Syk in platelets promotes clustering of CLEC-2 and Podoplanin. Clusters of CLEC-2-bound Podoplanin migrate rapidly to the centre of the platelet to form a single structure. Fluorescence life-time imaging demonstrates that molecules within these clusters are within 10 nm of one another and that the clusters are disrupted by inhibition of Src and Syk family kinases. CLEC-2 clusters are also seen in platelets adhered to immobilised Podoplanin using direct stochastic optical reconstruction microscopy (dSTORM). These findings provide mechanistic insight by which CLEC-2 signalling promotes adhesion to Podoplanin and regulation of Podoplanin signalling thereby contributing to lymphatic vasculature development.

Lymphedema is a medically irreversible condition for which currently conservative and surgical therapies are either ineffective or impractical. The potential use of progenitor and stem cell-based therapies has offered a paradigm that may provide alternative treatment options for lymphatic disorders. Moreover, basic research, preclinical studies as well as clinical trials have evaluated the therapeutic potential of various cell therapies in the field of lymphatic regeneration medicine. Among the available cell approaches, MSCs seem to be the most promising candidate mainly due to its abundant sources and easy availability as well as evitable ethical and immunological issues confronted with ESCs and iPSCs. In this context, the purpose of this review is to summarize various cell-based therapies for lymphedema, together with strengths and weaknesses of these therapies in the clinical application for lymphedema treatment. Particularly, we will highlight the use of MSCs for lymphatic regeneration medicine. Additionally, the future perspectives of MSCs in the field of lymphatic regeneration will be discussed.

The mammalian lymphatic vasculature is important for returning fluids from the extracellular tissue milieu back to the blood circulation. We showed previously that Prox1 dosage is important for the development of the mammalian lymphatic vasculature. The lack of Prox1 activity results in the complete absence of lymphatic endothelial cells (LECs). In Prox1 heterozygous embryos, the number of LECs is reduced because of a decrease in the progenitor pool in the cardinal vein. This reduction is caused by some progenitor cells being unable to maintain Prox1 expression. In this study, we identified Vegfr3, the cognate receptor of the lymphangiogenic growth factor Vegfc, as a dosage-dependent, direct in vivo target of Prox1. Using various mouse models, we also determined that Vegfr3 regulates Prox1 by establishing a feedback loop necessary to maintain the identity of LEC progenitors and that Vegfc-mediated activation of Vegfr3 signaling is necessary to maintain Prox1 expression in LEC progenitors. We propose that this feedback loop is the main sensing mechanism controlling the number of LEC progenitors and, as a consequence, the number of budding LECs that will form the embryonic lymphatic vasculature.

Lymphatic metastasis of human malignant adenocarcinomas is a critical determinant of prognosis. Lymphangiogenesis, the growth of lymphatic vessels, is closely involved in lymphatic metastasis. However, the mechanisms of tumor lymphangiogenesis are not clearly understood. In a previous study, we showed that human gastric cancer MKN45 cells organize neighboring lymphatic vessels via recruitment of bone marrow-derived lymphatic endothelial progenitor cells in a nude mouse xenograft model. The present results also indicated that human colorectal cancer LS174T and breast cancer SK-BR-3 cells promoted lymphangiogenesis as well as the recruitment of lymphatic endothelial progenitor cells from bone marrow. Among growth factors, which are reported to be involved in lymphangiogenesis, only vascular endothelial growth factor (VEGF)-A was extensively secreted by these three types of adenocarcinoma cells in culture. The well-characterized lymphangiogenic factors VEGF-C and VEGF-D in the culture medium of these three types of adenocarcinoma cells were below the detectable levels in ELISA assay. Secretion of epidermal growth factor (EGF) and hepatocyte growth factor (HGF) was not detected. In in vitro culture assay, VEGF-A directly induced the differentiation of bone marrow mononuclear cells into LYVE-1-positive lymphatic endothelial lineage cells. These data collectively suggest the possibility that VEGF-A-rich human adenocarcinomas induce tumor lymphangiogenesis via recruitment of lymphangiogenic endothelial progenitor cells from bone marrow.

OBJECTIVE: This study is to establish the rhesus monkey model of lymphedema in the upper limbs, and assess the suitability of this model. METHODS: An animal model of lymphedema was established by the combined irradiation and surgical techniques in the upper limbs of these rhesus monkeys. Physical examination, high-resolution MR lymphangiography, bioelectrical impedance analysis (BIA), and immunohistochemical staining were performed to determine the severity of the edema in the upper limbs of the animal model. RESULTS: Our results from physical examination indicated that the rhesus monkey model present with typical appearance and features of lymphedema. MR lymphangiography further demonstrated pathologically modified lymphatic vessels in our rhesus monkey model. BIA revealed increased water content in the upper limb in these rhesus monkeys, which was in line with the pathology of lymphedema. Immunohistochemical staining showed the curvature of the lymphatic vessels in the rhesus monkey model, typical pathological changes in lymphedema. CONCLUSION: Rhesus monkey lymphedema model provides a more consistent background to elucidate the pathophysiology of the disease. This new model would help to increase our understanding of acquired upper limb lymphedema, and promote the development of new treatments for this intractable disorder.

Experimental autoimmune encephalomyelitis (EAE) is mediated by myelin-specific CD4+ T cells secreting Th1 and/or Th17 cytokines. Signal transducer and activator of transcription (STAT) family proteins have essential roles in transmitting Th1 and/or Th17 cytokine-mediated signals. However, most studies demonstrating the importance of the STAT signaling system in EAE have focused on distinct members of this family, often looking at their role specifically in the central nervous system, or in vitro. There is limited information available regarding the temporal and spatial expression patterns of each STAT protein and interplay between STAT proteins over the course of EAE development in critical lymphatic organs in vivo. In the present study, we demonstrate dramatic and progressive decrease of all six STAT family members (STAT1, STAT2, STAT3, STAT4, STAT5, STAT6) in the spleen and lymph nodes through the course of EAE development in SJL/J mice, in contrast with almost steady expression of thymic STAT proteins. Decreased splenic and lymphatic STAT expression was accompanied by significant enlargement of the spleen and lymph nodes, and histological proliferation of T cell areas with remodeling of the splenic microstructure in EAE mice. All STAT family members except STAT2 were mainly confined in T cell areas in spleen, whereas they were distributed in a protein specific manner in thymus. We present here a comprehensive analysis of all six members of the STAT family in spleen, lymph nodes and thymus through the development phase of EAE. Results suggest that EAE induced inflammatory T cells may develop distinct biological features different from normal splenic T cells due to altered STAT signaling.

Lymphatic vessels are intimately involved in the regulation of water and solute homeostasis by returning interstitial fluid back to the venous circulation and play an equally important role in immune responses by providing avenues for immune cell transport. Defects in the lymphatic vasculature result in a number of pathological conditions, including lymphedema and lymphangiectasia. Knowledge of molecular mechanisms underlying lymphatic development and maintenance is therefore critical for understanding, prevention and treatment of lymphatic circulation-related diseases. Research in the past two decades has uncovered several key transcriptional factors (Prox1, Sox18 and Coup-TFII) controlling lymphatic fate specification. Most recently, ERK signaling has emerged as a critical regulator of this transcriptional program. This review summarizes our current understanding of lymphatic fate determination and its transcriptional controls.

Murine bEnd.3 endothelioma cell line has been widely used in vascular research and here we report the novel finding that bEnd.3 cells express lymphatic vessel endothelial hyaluronan receptor-1 (LYVE-1) and vascular endothelial growth factor receptor-3 (VEGFR-3). Moreover, these cells express progenitor cell markers of Sca-1 and CD133. Upon stimulation with tumor necrosis factor-alpha (TNF-alpha), the bEnd.3 cells demonstrate enhanced formation of capillary-type tubes, which express LYVE-1. As the bEnd.3 cell line is derived from murine endothelioma, we further examined human tissues of endothelioma and identified lymphatic vessels in the tumor samples which express both LYVE-1 and podoplanin. Moreover, a significantly higher number of lymphatic vessels were detected in the endothelioma samples compared with normal control. Taken together, this study not only redefines bEnd.3 cells for vascular research, but also indicates a broader category of human diseases that are associated with lymphatics, such as endothelioma.

Whether primary or secondary, lymphedema is caused by failure to drain protein-rich interstitial fluid. Typically affecting a whole limb, it has become apparent that lymphedema can also affect localized regions of the skin, or it can be clinically silent but histologically evident, denoted by dilated lymphangiectases (latent lymphedema). Chronic lymph stasis has numerous consequences, including lipogenesis, fibrosis, inflammation, lymphangiogenesis, and immunosuppression. For example, lymphedema's disruption of immune cell trafficking leads to localized immune suppression, predisposing the area affected to chronic inflammation, infection (cellulitis and verrucosis), and malignancy (angiosarcoma and nonmelanoma skin cancer). The pathogenesis of lymphedema is reviewed and exemplified by describing how a combination of lymph stasis-promoting factors such as trauma, obesity, infection, and inflammatory disorders produces localized elephantiasis; furthermore, the finding of lymphangiectases is found to be common in numerous dermatologic disorders and argued to play a role in their pathogenesis. Lastly, it is discussed how antigen burden, which is controlled by lymphatic clearance, affects the immune response, resulting in immune tolerance, immunopathology, or normal adaptive immunity.

CONTEXT: Noonan syndrome (NS) is characterized by short stature and elevated risk of lymphedema. The mechanism underlying lymphedema may be mediated by vascular endothelial growth factors (VEGFs). OBJECTIVE: To assess the effect of growth hormone (GH) treatment on plasma insulin-like growth factor (IGF)-1, VEGF-A and VEGF-C levels in patients with NS as compared to short GH-sufficient children. DESIGN: Retrospective, comparative. SETTING: Endocrinology department of a tertiary pediatric medical center. PATIENTS AND METHODS: Plasma IGF-1, VEGF-A and VEGF-C levels were measured before and during GH treatment in 6 patients with NS and 18 age-matched short subjects (Turner, idiopathic short stature and small for gestational age). MAIN OUTCOME MEASURES: Changes in plasma VEGF and IGF-1 levels. RESULTS: Baseline IGF-1 SDS levels were slightly lower in NS patients compared with controls; IGF-1 response to GH therapy was markedly lower in NS patients compared with controls (p=0.017). Mean baseline VEGF-A levels were similar in NS patients and controls whilst mean baseline VEGF-C levels were significantly lower in the NS group as compared with controls (p=0.022). Plasma VEGF-A and VEGF-C levels did not significantly change during GH treatment in the study cohort. No correlation was found between VEGF-C levels and levels of IGF-1, VEGF-A and auxological parameters, either before or during GH administration. CONCLUSION: Children with NS have a distinct growth factor profile including low basal VEGF-C and flattened IGF-1 response to GH. Further studies are needed to confirm our findings and to elucidate the interaction between VEGF-C levels and lymphedema.

BACKGROUND: Angiogenesis is an early stage of psoriatic lesion development, but less is known about lymphagiogenesis and its role in the development of psoriasis. OBJECTIVE: To examine the expression of specific lymphatic markers and lymphatic growth factors in untreated psoriatic skin, in the unaffected skin of patients and skin of healthy volunteers, as well as their alteration after treatment with an anti-TNF agent. METHODS: Immunohistochemistry for the lymphatic markers D2-40 and LYVE-1, in addition to the VEGF-C and VEGF-D growth factors, was performed in the skin biopsies of psoriatic lesions and adjacent non-psoriatic skin of 19 patients before and after treatment with etanercept, as well as in the skin biopsies of 10 healthy volunteers. RESULTS: The expressions of D2-40, VEGF-C and VEGF-D on lymphatic vessels underwent statistically significant increases in untreated psoriatic skin compared with non-lesional skin, in contrast to LYVE-1, which did not involve significant increase in expression in psoriatic skin. VEGF-C expression on lymphatic vessels diminished after treatment with etanercept. Moreover VEGF-C and VEGF-D staining on fibroblasts presented with higher expression in lesional skin than in non-lesional adjacent skin. CONCLUSION: Remodeling of lymphatic vessels possibly occurs during psoriatic lesion development, parallel to blood vessel formation. The exact role of this alteration is not yet clear and more studies are necessary to confirm these results.

BACKGROUND/AIMS: We classified intestinal lymphangiectasia (IL) into two categories, the white and non-white villi types, and evaluated their clinical characteristics and therapeutic responses. METHODS: Of the 988 patients who underwent double-balloon enteroscopy, 14 consecutive patients (7 men and 7 women, median age at onset 34 years) were enrolled with immunohistochemically confirmed IL with protein-losing enteropathy. RESULTS: Enteroscopically the white villi type (n=8) showed white plaques and white-tipped villi were scattered in the small bowel, while non-white villi type (n=6) showed that apparently normal but under more detailed observation, low and round villi with a normal color were diffused. The serum albumin levels and fecal alpha1-antitrypsin clearance before treatment were significantly worse in the non-white villi type (p=0.017 and 0.039, respectively), whereas the serum immunoglobulin A and M levels were significantly lower in the white villi type (p=0.010 and 0.046, respectively). At gastroscopy, a non-cirrhotic snakeskin appearance was significantly observed in the non-white villi type (p=0.015). The corticosteroid response was better in the non-white villi type (p=0.015). CONCLUSION: Two distinct subgroups were found in IL. This classification was useful in pathophysiological clustering and in predicting the therapeutic response. (c) 2014 S. Karger AG, Basel.

OBJECTIVE: Cerebral arteriovenous malformations (AVMs) are vascular lesions whose pathogenesis, although not fully elucidated, is likely multifactorial. Recent research investigating vessel development suggests a potential hierarchical model in which capillary sprouts from higher-flow arteries give rise to lower-flow veins. It is possible that an embryologic structural vascular dysgenesis in this hierarchical development heavily contributes to the formation of AVMs. Subsequent genetic “second hits” may then allow development of a clinically significant cerebral AVM. We review this vascular developmental process and describe a novel proposal for the embryogenesis of AVMs and its implications in relation to recent research on polymorphisms and AVMs. METHODS: A comprehensive literature search was performed using PubMed for recent research relative to cerebral AVMs, embryologic vascular development, and polymorphisms involved in AVM pathology. RESULTS: It has recently been shown that both centrally, in the axial embryo, and peripherally, in the embryonic yolk sac, veins form via capillary sprouting from parent arteries. In developing intracranial vessels, a derangement in this embryonic process may lead to a primitive arteriovenous shunt. After this structural “first hit,” we suggest that single nucleotide polymorphisms (SNPs) are a major component in allowing AVM growth into symptomatic clinical lesions. CONCLUSIONS: This is a novel theory for the embryologic formation of cerebral AVMs. Hierarchical vessel development, where higher-flow parent arteries give rise to lower-flow veins, provides a potential mechanism for the formation of primitive arteriovenous shunts that, with the influence of polymorphisms, allows AVMs to develop.

BACKGROUND: In lower extremity lymphedema secondary to pelvic cancer treatments, lymphedema develops despite that the inguinal lymph nodes (LNs) are preserved. Obstruction of the efferent lymphatic vessels of the inguinal LNs causes lower extremity lymphedema, and it is considered a radical treatment to bypass the efferent lymphatic vessel. METHODS: Efferent lymphatic vessel anastomosis, supermicrosurgical efferent lymphatic vessel-to-venous anastomosis, was performed on 14 legs with subclinical lymphedema [leg dermal backflow (LDB) stage I]. Efferent lymphatic vessel anastomosis was performed under local anesthesia at the groin region, and an efferent lymphatic vessel of the inguinal LN is anastomosed to a recipient vein. Feasibility and postoperative results were evaluated. RESULTS: All 14 efferent lymphatic vessel anastomoses were successfully performed without perioperative complication. All legs could be free from lymphedematous symptoms without perioperative compression at postoperative 1 year. Postoperative LDB stage included LDB stage 0 (n=8) and LDB stage I (n=6), which was significantly downstaged compared with preoperative LDB stage (P<0.001). CONCLUSIONS: Efferent lymphatic vessel anastomosis allowed lymph flow bypass after filtration by the superficial inguinal LN through a skin incision along the inguinal crease, and was effective to prevent development of symptomatic lymphedema in subclinical lymphedema cases.

Lymphatic vessels (LVs) are involved in a number of physiological and pathophysiological processes such as fluid homoeostasis, immune surveillance, and resolution of inflammation and wound healing. Lymphangiogenesis, the outgrowth of existing LVs and the formation of new ones, has received increasing attention over the past decade on account of its prominence in organ physiology and pathology, which has been enabled by the development of specific tools to study lymph vessel functions. Several studies have been devoted to renal lymphatic vasculature and lymphangiogenesis in kidney diseases, such as chronic renal transplant dysfunction, primary renal fibrotic disorders, proteinuria, diabetic nephropathy and renal inflammation. This review describes the most recent findings on lymphangiogenesis, with a specific focus on renal lymphangiogenesis and its impact on renal diseases. We suggest renal lymphatics as a possible target for therapeutic interventions in renal medicine to dampen tubulointerstitial tissue remodelling and improve renal functioning.

Chronic lymphedema has a permissive effect with certain types of malignancies, particularly angiosarcomas, in what is known as Stewart-Treves syndrome. The presumed mechanism of this effect is an immunocompromised district of the affected area. Most other cutaneous malignancies have also been described in lymphedematous areas, including basal cell carcinoma, squamous cell carcinoma, melanoma, Kaposi sarcoma, Merkel cell carcinoma, and several cutaneous lymphomas. The occurrence of such malignancies suggests a more general immunosuppression within the skin. The formation of collateral lymphatic and vascular vessels in response to lymphedema produces an environment rich in growth factors, which may also play a role. In addition to infection and other general skin care issues, regions affected by lymphedema should be monitored for malignant changes not limited to angiosarcomas.