Update December 2013

Despite the key roles of lymphatic vessels in homeostasis and disease, the cellular sources of signals that direct lymphatic vascular growth and patterning remain unknown. Using high-resolution imaging in two and three dimensions, we demonstrated that postnatal mouse mammary gland lymphatic vessels share an intimate spatial association with epithelial ducts and large blood vessels. We further demonstrated that the lymphatic vasculature is remodeled together with the mammary epithelial tree and blood vasculature during postnatal mouse mammary gland morphogenesis. Neither estrogen receptor alpha nor progesterone receptor were detected in lymphatic endothelial cells in the mouse mammary gland, suggesting that mammary gland lymphangiogenesis is not likely regulated directly by these steroid hormones. Epithelial cells, especially myoepithelial cells, were determined to be a rich source of prolymphangiogenic stimuli including VEGF-C and VEGF-D with temporally regulated expression levels during mammary gland morphogenesis. Blockade of VEGFR-3 signaling using a small-molecule inhibitor inhibited the proliferation of primary lymphatic endothelial cells promoted by mammary gland conditioned medium, suggesting that lymphangiogenesis in the mammary gland is likely driven by myoepithelial-derived VEGF-C and/or VEGF-D. These findings provide new insight into the architecture of the lymphatic vasculature in the mouse mammary gland and, by uncovering the proximity of lymphatic vessels to the epithelial tree, suggest a potential mechanism by which metastatic tumor cells access the lymphatic vasculature.

Mutations in gene RASA1 have been historically associated with capillary malformation-arteriovenous malformation, but sporadic reports of lymphatic involvement have yet to be investigated in detail. To investigate the impact of RASA1 mutations in the lymphatic system, we performed investigational near-infrared fluorescence lymphatic imaging and confirmatory radiographic lymphangiography in a Parkes-Weber syndrome (PKWS) patient with suspected RASA1 mutations and correlated the lymphatic abnormalities against that imaged in an inducible Rasa1 knockout mouse. Whole-exome sequencing (WES) analysis and validation by Sanger sequencing of DNA from the patient and unaffected biological parents enabled us to identify an early-frameshift deletion in RASA1 that was shared with the father, who possessed a capillary stain but otherwise no overt disease phenotype. Abnormal lymphatic vasculature was imaged in both affected and unaffected legs of the PKWS subject that transported injected indocyanine green dye to the inguinal lymph node and drained atypically into the abdomen and into dermal lymphocele-like vesicles on the groin. Dermal lymphatic hyperplasia and dilated vessels were observed in Rasa1-deficient mice, with subsequent development of chylous ascites. WES analyses did not identify potential gene modifiers that could explain the variability of penetrance between father and son. Nonetheless, we conclude that the RASA1 mutation is responsible for the aberrant lymphatic architecture and functional abnormalities, as visualized in the PKWS subject and in the animal model. Our unique method to combine investigatory near-infrared fluorescence lymphatic imaging and WES for accurate phenoptyping and unbiased genotyping allows the study of molecular mechanisms of lymphatic involvement of hemovascular disorders.

Maintenance of tissue homeostasis and immune surveillance are important functions of the lymphatic vascular system. Lymphatic vessels are lined by lymphatic endothelial cells (LECs). By gene micro-array expression studies we recently compared human lymphangioma-derived LECs with umbilical vein endothelial cells (HUVECs). Here, we followed up on these studies. Besides well-known LEC markers, we observed regulation of molecules involved in immune regulation, acetylcholine degradation and platelet regulation. Moreover we identified differentially expressed WNT pathway components, which play important roles in the morphogenesis of various organs, including the blood vascular system. WNT signaling has not yet been addressed in lymphangiogenesis. We found high expression of FZD3, FZD5 and DKK2 mRNA in HUVECs, and WNT5A in LECs. The latter was verified in normal skin-derived LECs. With immunohistological methods we detected WNT5A in LECs, as well as ROR1, ROR2 and RYK in both LECs and HUVECs. In the human, mutations of WNT5A or its receptor ROR2 cause the Robinow syndrome. These patients show multiple developmental defects including the cardio-vascular system. We studied Wnt5a-knockout (ko) mouse embryos at day 18.5. We show that the number of dermal lymphatic capillaries is significantly lower in Wnt5a-null-mice. However, the mean size of individual lymphatics and the LEC number per vessel are greater. In sum, the total area covered by lymphatics and the total number of LECs are not significantly altered. The reduced number of lymphatic capillaries indicates a sprouting defect rather than a proliferation defect in the dermis of Wnt5a-ko-mice, and identifies Wnt5a as a regulator of lymphangiogenesis.

OBJECTIVE: Lymphangiogenesis is regulated by transcription factors and by growth factor pathways, but their interplay has not been extensively studied so far. We addressed this issue in zebrafish. APPROACH AND RESULTS: Mutations in the transcription factor-coding gene SOX18 and in VEGFR3 cause lymphedema, and the VEGFR3/Flt4 ligand VEGFC plays an evolutionarily conserved role in lymphangiogenesis. Here, we report a strong genetic interaction between Sox18 and VegfC in the early phases of lymphatic development in zebrafish. Knockdown of sox18 selectively impaired lymphatic sprouting from the cardinal vein and resulted in defective lymphatic thoracic duct formation. Sox18 and the related protein Sox7 play redundant roles in arteriovenous differentiation. We used a novel transgenic line that enables inducible expression of a dominant-negative mutant form of mouse Sox18 protein. Our data led us to conclude that Sox18 is crucially involved in lymphangiogenesis after arteriovenous differentiation. Combined partial knockdown of sox18 and vegfc, using subcritical doses of specific morpholinos, revealed a synergistic interaction in both venous and lymphatic sprouting from the cardinal vein and greatly impaired thoracic duct formation. CONCLUSIONS: This interaction suggests a previously unappreciated crosstalk between the growth factor and transcription factor pathways that regulate lymphangiogenesis in development and disease.

The low stiffness of reconstituted collagen hydrogels has limited their use as scaffolds for engineering implantable tissues. Although chemical crosslinking has been used to stiffen collagen and protect it against enzymatic degradation in vivo, it remains unclear how crosslinking alters the vascularization of collagen hydrogels. In this study, we examine how the crosslinking agents genipin and 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) alter vascular stability and function in microfluidic type I collagen gels in vitro. Under moderate perfusion (10 dyn/cm2 shear stress), tubes of blood endothelial cells exhibited indistinguishable stability and barrier function in untreated and crosslinked scaffolds. Surprisingly, under low perfusion (5 dyn/cm2 shear stress) or nearly zero transmural pressure, microvessels in crosslinked scaffolds remained stable, while those in untreated gels rapidly delaminated and became poorly perfused. Similarly, tubes of lymphatic endothelial cells under intermittent flow were more stable in crosslinked gels than in untreated ones. These effects correlated well with the degree of mechanical stiffening, as predicted by analysis of fracture energies at the cell-scaffold interface. This work demonstrates that crosslinking of collagen scaffolds does not hinder normal endothelial cell physiology; instead, crosslinked scaffolds promote vascular stability. Thus, routine crosslinking of scaffolds may assist in vascularization of engineered tissues.

Vascular endothelial growth factor receptor 2 (VEGFR2) is highly expressed by lymphatic endothelial cells and has been shown to stimulate lymphangiogenesis in adult mice. However, the role VEGFR2 serves in the development of the lymphatic vascular system has not been defined. Here we use the Cre-lox system to show that the proper development of the lymphatic vasculature requires VEGFR2 expression by lymphatic endothelium. We show that Lyve-1(wt/Cre);Vegfr2(flox/flox) mice possess significantly fewer dermal lymphatic vessels than Vegfr2(flox/flox) mice. Although Lyve-1(wt/Cre);Vegfr2(flox/flox) mice exhibit lymphatic hypoplasia, the lymphatic network is functional and contains all of the key features of a normal lymphatic network (initial lymphatic vessels and valved collecting vessels surrounded by smooth muscle cells (SMCs)). We also show that Lyve-1(Cre) mice display robust Cre activity in macrophages and in blood vessels in the yolk sac, liver and lung. This activity dramatically impairs the development of blood vessels in these tissues in Lyve-1(wt/Cre);Vegfr2(flox/flox) embryos, most of which die after embryonic day14.5. Lastly, we show that inactivation of Vegfr2 in the myeloid lineage does not affect the development of the lymphatic vasculature. Therefore, the abnormal lymphatic phenotype of Lyve-1(wt/Cre);Vegfr2(flox/flox) mice is due to the deletion of Vegfr2 in the lymphatic vasculature not macrophages. Together, this work demonstrates that VEGFR2 directly promotes the expansion of the lymphatic network and further defines the molecular mechanisms controlling the development of the lymphatic vascular system.

Rationale: The emergence of lymphatic endothelial cells (LECs) appears to be highly regulated during development. While several factors that promote the differentiation of LECs in embryonic development have been identified, those that negatively regulate this process are largely unknown. Objective: To delineate the role of BMP2 signaling on lymphatic development. Methods and Results: BMP2 signaling negatively regulates the formation of LECs. Developing LECs lack any detectable BMP signaling activity in both zebrafish and mouse embryos, and excess BMP2 signaling in zebrafish embryos and mouse embryonic stem (ES) cell-derived embryoid bodies (EBs) substantially decrease the emergence of LECs. Mechanistically, BMP2 signaling induces expression of miR-31 and miR-181a in a SMAD-dependent mechanism, which in turn, result attenuated expression of PROX1 during development. Conclusions: Our data identify BMP2 as a key negative regulator for the emergence of the lymphatic lineage during vertebrate development.

We have previously demonstrated a principal role for nitric oxide (NO) in the endothelium/shear-dependent regulation of contractility in rat thoracic duct (TD). In this study we tested the hypothesis that cGMP/PKG (cyclic guanosine monophosphate/cGMP-dependent protein kinase) is central to the intrinsic and extrinsic flow-dependent modulation of lymphatic contractility. Lymphatic diameters and indices of pumping in isolated, cannulated and pressurized segments of rat TD were measured. The influences of increased transmural pressure (1 to 5 cm H2O) and imposed flow (1 to 5 cm H2O transaxial pressure gradients) on lymphatic function were studied before and after: 1. inhibition of guanylate cyclase (GC) with and without a NO donor, 2. an application of stable cGMP analog and 3. An inhibition of the cGMP activation of PKG. Additionally, western blotting and immunofluorescent tissue staining were used to analyze the PKG isoforms expressed in TD. We found that the GC inhibitor ODQ induced changes in TD contractility similar to NO synthase blockade and prevented the relaxation induced by the NO donor SNAP. The cGMP analog - 8pCPTcGMP mimicked the extrinsic flow-induced relaxation in a dose-dependent manner, whereas treatment with the cGMP/PKG inhibitor - Rp-8-Br-PET-cGMPS eliminated the intrinsic flow-dependent relaxation, and largely inhibited the extrinsic flow-dependent relaxation. Western blotting demonstrated that both PKG-Ialpha and Ibeta isoforms are found in TD, with ∼10-times greater expression of the PKG-Ialpha protein in TD compared with aorta and vena cava. The PKG-Ibeta isoform expressed equally in TD and vena cava, both being ∼2 times higher than that in the aorta. Immunofluorescent labeling of PKG-Ialpha protein in the wall of rat thoracic duct confirmed it's localization inside the TD muscle cells. These findings demonstrate that cGMP is critical to the flow-dependent regulation of TD contractility; they also indicate an important involvement of PKG, especially PKG-Ialpha in these processes and identifies PKG protein as a potential therapeutic target.

Abstract We have previously demonstrated a principal role for nitric oxide (NO) in the endothelium/shear-dependent regulation of contractility in rat thoracic duct (TD). In this study we tested the hypothesis that cyclic guanosine monophosphate (cGMP) and the dependent protein kinase (PKG) are central to the intrinsic and extrinsic flow-dependent modulation of lymphatic contractility. Lymphatic diameters and indices of pumping in isolated, cannulated and pressurized segments of rat TD were measured. The influences of increased transmural pressure (1–5 cmH2O) and imposed flow (1–5 cm H2O transaxial pressure gradients) on lymphatic function were studied before and after: (1) inhibition of guanylate cyclase (GC) with and without a NO donor; (2) application of stable cGMP analogue; and (3) inhibition of the cGMP activation of PKG. Additionally, Western blotting and immunofluorescent tissue staining were used to analyse the PKG isoforms expressed in TD. We found that the GC inhibitor ODQ induced changes in TD contractility similar to NO synthase blockade and prevented the relaxation induced by the NO donor S-nitroso-N-acetylpenicillamine. The cGMP analogue, 8-(4-Chlorophenylthio)-guanosine 3,5-cyclic monophosphate sodium salt (8pCPTcGMP), mimicked the extrinsic flow-induced relaxation in a dose-dependent manner, whereas treatment with the cGMP/PKG inhibitor, guanosine 3,5-cyclic monophosphorothioate, 8-(4-chlorophenylthio)-, Rp-isomer, triethylammonium salt (Rp-8-Br-PETcGMPS), eliminated intrinsic flow-dependent relaxation, and largely inhibited extrinsic flow-dependent relaxation. Western blotting demonstrated that both PKG-Ialpha and -Ibeta isoforms are found in TD, with approximately 10 times greater expression of the PKG-Ialpha protein in TD compared with the aorta and vena cava. The PKG-Ibeta isoform expressed equally in TD and vena cava, both being approximately 2 times higher than that in the aorta. Immunofluorescent labelling of PKG-Ialpha protein in the wall of rat thoracic duct confirmed its localization inside TD muscle cells. These findings demonstrate that cGMP is critical to the flow-dependent regulation of TD contractility; they also indicate an important involvement of PKG, especially PKG-Ialpha in these processes and identifies PKG protein as a potential therapeutic target.

PURPOSE: To investigate the specific targeting property of lymphatic vessel endothelial hyaluronan receptor-1 binding polyethylene glycol-coated ultrasmall superparamagnetic iron oxide (LYVE-1-PEG-USPIO) nanoparticles to mouse lymphatic endothelial cells (MLECs). METHODS: A ligand specific target to lymphatic vessels was selected by immunohistochemical staining on the sections of a Lewis subcutaneous transplanted tumor. The z-average hydrodynamic diameter (HD), zeta potential, and the relaxivity of PEG-USPIO and LYVE-1-PEG-USPIO nanoparticles were determined with a laser particle analyzer and magnetic resonance T2 spin echo sequence, respectively. Prussian blue staining and transmission electron microscopy (TEM) of nanoparticle labeled cells were performed to determine the nanoparticles' binding form. Magnetic resonance imaging (MRI) was performed in vitro to evaluate the signal enhancement on the T2 spin echo sequence of the nanoparticle labeled cells. The iron content of the labeled cells after the Prussian blue staining and MRI scanning was determined by atomic absorption spectroscopy (AAS). RESULTS: The anti-LYVE-1 antibody was used as the specific ligand to synthesize the target probe to the MLECs. The mean z-average HDs of the LYVE-1-PEG-USPIO and PEG-USPIO nanoparticles were 57.42 +/− 0.31 nm and 47.91 +/− 0.73 nm, respectively, and the mean zeta potentials of the LYVE-1-PEG-USPIO and PEG-USPIO nanoparticles were 12.38 +/− 4.87 mV and 2.57 +/− 0.83 m V, respectively. The relaxivities of the LYVE-1-PEG-USPIO and PEG-USPIO nanoparticles were 185.48 mM(−1)s(−1) and 608.32 mM(−1)s(−1). Cells binding nanoparticles were visualized as blue granules in the Prussian blue staining. The TEM results of the labeled cells showed the specific localization of nanoparticles. The AAS results of labeled cells after the Prussian blue staining and MRI scanning showed that the LYVE-1-PEG-USPIO nanoparticles had good binding selectivity for MLECs. MRI results indicated that the PEG-USPIO and LYVE-1-PEG-USPIO nanoparticles could generate contrast on T2-weighted imaging, and the correlation between R2 and the iron content of the labeled cells was significantly positive. CONCLUSION: This study demonstrated that LYVE-1-PEG-USPIO nanoparticles might potentially be used as an MRI contrast agent for targeting MLECs, and the magnetic properties of LYVE-1-PEG-USPIO nanoparticles were suitable for MRI.

All vertebrate animals share in common the production of lymph through net capillary filtration from their closed circulatory system into their tissues. The balance of forces responsible for net capillary filtration and lymph formation is described by the Starling equation, but additional factors such as vascular and interstitial compliance, which vary markedly among vertebrates, also have a significant impact on rates of lymph formation. Why vertebrates show extreme variability in rates of lymph formation, and how non-mammalian vertebrates maintain plasma volume homeostasis is unclear. This gap hampers our understanding of the evolution of the lymphatic system and its interaction with the cardiovascular system. The evolutionary origin of the vertebrate lymphatic system is not clear, but recent advances suggest common developmental factors for lymphangiogenesis in teleost fishes, amphibians and mammals with some significant changes in the water-land transition. The lymphatic system of anuran amphibians is characterized by large lymphatic sacs and two pairs of lymph hearts that return lymph into the venous circulation, but no lymph vessels per se. The lymphatic systems of reptiles and some birds have lymph hearts, and both groups have extensive lymph vessels, but their functional role in both lymph movement and plasma volume homeostasis is almost completely unknown. The purpose of this review is to present an evolutionary perspective in how different vertebrates have solved the common problem of the inevitable formation of lymph from their closed circulatory systems, and to point out the many gaps in our knowledge of this evolutionary progression.

Purpose: The influence of autologous serum eye drops on the corneal vasculature is undefined. Therefore, we analyzed the corneal vascular effects of serum eye drops in comparison and in combination with Bevacizumab. Methods: In vitro, blood and lymphatic endothelial cells were treated with serum, Bevacizumab or a combination of both and cell proliferation was measured. In vivo, inflammatory corneal neovascularization was induced by suture placement. Subsequently, corneal blood and lymphatic vessel progression and regression were analyzed after treatment with serum or Bevacizumab eye drops or a combination of both. Hem- and lymphangiogenesis was quantified in wholemounts using CD31 and LYVE-1; inflammatory cell infiltration was analyzed using CD11b. Furthermore, corneal expression levels of IL-1beta, TNFalpha, VEGF-A, VEGF-C and VEGF-D were analyzed by realtime PCR. Results: In vitro, addition of serum increased whereas Bevacizumab reduced endothelial cell proliferation. In vivo, serum eye drops had no significant influence on corneal vessel progression or regression. Bevacizumab eye drops reduced blood and lymphatic vessel progression and promoted blood and lymphatic vessel regression. The combination of serum and Bevacizumab attenuated the anti(lymph)angiogenic effects of Bevacizumab. Inflammatory corneal cell counts were not significantly altered by serum or Bevacizumab. Serum changed the proinflammatory and pro(lymph)angiogenic status of the cornea. Bevacizumab significantly reduced proinflammatory and pro(lymph)angiogenic factor expression. Higher doses of Bevacizumab could not restore its anti(lymph)angiogenic effects when used in combination with serum. Conclusions: The counteracting influences of serum eye drops and Bevacizumab on the corneal vasculature should be taken into account when combinatory therapeutic regimens are considered.

The molecular basis of the lymphatic development remains largely unknown. Using zebrafish as a model, we discovered a novel role for the Ras guanine-releasing protein 1 (RasGRP1), a protein involved in Ras activation in lymphangiogenesis. Secondary lymphatic sprouts from the posterior cardinal vein give rise to thoracic duct which is the first lymphatic vessel in zebrafish. Knockdown of rasgrp1 by injecting morpholino in zebrafish embryos impaired formation of thoracic duct accompanied by pericardial and truck edema, whereas blood vessel development of the embryos was largely unaffected. In rasgrp1-knockdown embryos, the number of sprouts producing the string of parachordal lymphangioblast cells was reduced. Meanwhile the total number of the secondary sprouts was not changed. As a result, the number of venous intersegmental vessels was increased, whereas the number of lymphatic vessel was reduced at a later stage. The lymphatic developmental defects caused by rasgrp1 knockdown could be rescued by ectopic expression of a constitutively active HRas. Further analysis revealed that RasGRP1 knockdown could synergize with flt4/vegfr3 knockdown to induce defects in lymphangiogenesis. Taken together, this finding demonstrates a critical role for RasGRP1 in lymphatic development in zebrafish.

The cytokine Interleukin-7 (IL-7) exerts essential roles in lymph node (LN) organogenesis and lymphocyte development and homeostasis. Recent studies have identified lymphatic endothelial cells (LECs) as a major source of IL-7 in LNs. Here, we report that LECs not only produce IL-7 but also express the IL-7 receptor chains IL-7Ralpha and CD132. Stimulation with recombinant IL-7 enhanced LEC in vitro activity and induced lymphangiogenesis in the cornea of wild-type (WT) mice. While in IL-7Ralpha-/- mice dermal lymphatic vessels (LVs) were abnormally organized and lymphatic drainage was compromised, transgenic overexpression of IL-7 in mice resulted in an expanded dermal LV network with increased drainage function. Moreover, systemic treatment with recombinant IL-7 enhanced lymphatic drainage in the skin of WT mice and of mice devoid of lymphocytes. Experiments in IL-7Ralpha-/- bone marrow (BM) chimeras demonstrated that the drainage-enhancing activity of IL-7 was exclusively dependent on IL-7Ralpha expression in stromal but not in hematopoietic cells. Finally, near-infrared in vivo imaging performed in IL-7Ralpha-/- mice revealed that the pumping activity of collecting vessels was normal but fluid uptake into lymphatic capillaries was defective. Overall, our data point towards an unexpected new role for IL-7 as a potential autocrine mediator of lymphatic drainage.

One of the key mechanisms linking cell signaling and control of gene expression is reversible phosphorylation of transcription factors. FOXC2 is a forkhead transcription factor that is mutated in human vascular disease lymphedema-distichiasis and plays an essential role in lymphatic vascular development. However, the mechanisms regulating FOXC2 transcriptional activity are not well understood. Here we report that FOXC2 is phosphorylated on eight evolutionarily conserved proline-directed serine/threonine residues. Loss of phosphorylation at these sites triggers substantial changes in the FOXC2 transcriptional program. Through genome-wide location analysis in lymphatic endothelial cells, we demonstrate that the changes are due to selective inhibition of FOXC2 recruitment to chromatin. The extent of the inhibition varied between individual binding sites, suggesting a novel rheostat-like mechanism by which expression of specific genes can be differentially regulated by FOXC2 phosphorylation. Furthermore, unlike the wild type protein, the phosphorylation-deficient mutant of FOXC2 failed to induce vascular remodeling in vivo. Collectively, our results point to the pivotal role of phosphorylation in the regulation of FOXC2-mediated transcription in lymphatic endothelial cells and underscore the importance of FOXC2 phosphorylation in vascular development.

Dermal lymphatic endothelial cells (LECs) emerge from the dorsolateral region of the cardinal veins within the anterior trunk to form an intricate, branched network of lymphatic vessels during embryogenesis. Multiple growth factors and receptors are required for specification and maintenance of LECs, but the mechanisms coordinating LEC movements and morphogenesis to develop three-dimensional lymphatic network architecture are not well understood. Here, we demonstrate in mice that precise LEC sprouting is a key process leading to stereotypical lymphatic network coverage throughout the developing skin, and that transforming growth factor beta (TGFbeta) signaling is required for LEC sprouting and proper lymphatic network patterning in vivo. We utilized a series of conditional mutants to ablate the TGFbeta receptors Tgfbr1 (Alk5) and Tgfbr2 in LECs. To analyze lymphatic defects, we developed a novel, whole-mount embryonic skin imaging technique to visualize sprouting lymphangiogenesis and patterning at the lymphatic network level. Loss of TGFbeta signaling in LECs leads to a severe reduction in local lymphangiogenic sprouting, resulting in a significant decrease in global lymphatic network branching complexity within the skin. Our results also demonstrate that TGFbeta signaling negatively regulates LEC proliferation during lymphatic network formation. These data suggest a dual role for TGFbeta signaling during lymphatic network morphogenesis in the skin, such that it enhances LEC sprouting and branching complexity while attenuating LEC proliferation.

To unveil organotypic role and vulnerability of lymphatic vessels, we generated a LYVE-1-Cre/iDTR double-transgenic mouse and ablated LYVE-1-expressing lymphatic vessels in adult mice in a diphtheria toxin-inducible manner based on selective expression of LYVE-1 in most lymphatic vessels. Strikingly, lymphatic vessels in the small intestine and lymph nodes were rapidly ablated, but lymphatic vessels in the other organs were relatively intact at 24 h after diphtheria toxin administration. Unexpectedly, LYVE-1-Cre/iDTR mice died from sepsis without visible edema at 24 and 60 h after diphtheria toxin administration. The cause of death appeared to be related to acute failure of immune surveillance systems in the small intestine and draining lymph nodes. Of note, acute loss of lymphatic lacteals in intestinal villi appeared to trigger distortion of blood capillaries and whole architecture of the villi whereas acute loss of lymphatic vessels in lymph nodes caused dysfunction of lymph drainage and abnormal distribution of dendritic cells and macrophages. Thus, intact lymphatic vessels are required for structural and functional maintenance of surrounding tissues in an organotypic manner, at least in the intestine and lymph nodes.

Tissue inflammation is characterized by increased trafficking of antigen-loaded dendritic cells (DC) from the periphery via afferent lymphatics to draining lymph nodes, with resulting stimulation of ongoing immune responses. Transmigration across lymphatic endothelium constitutes the first step in this process and is known to involve the chemokine CCL21 and its receptor CCR7. However, the precise details of DC transit remain obscure and it is likely that additional chemokine-receptor pairs have roles in lymphatic vessel entry. Here, we report that the transmembrane chemokine CX3CL1 (fractalkine) is induced in inflamed lymphatic endothelium, both in vitro in TNF-alpha-treated human dermal lymphatic endothelial cells (HDLEC) and in vivo in a mouse model of skin hypersensitivity. However, unlike blood endothelial cells, which express predominantly transmembrane CX3CL1 as a leukocyte adhesion molecule, HDLEC shed virtually all CX3CL1 at their basolateral surface via matrix metalloproteinases. We show for the first time that both recombinant soluble CX3CL1 and endogenous secreted CX3CL1 promote basolateral-to-luminal migration of DC across HDLEC monolayers in vitro. Furthermore, we show in vivo that neutralizing antibodies against CX3CL1 dramatically reduce allergen-induced trafficking of cutaneous DC to draining lymph nodes as assessed by FITC skin painting in mice. Finally, we show that deletion of CX3CL1 receptor in CX3CR1-/- DC results in markedly delayed lymphatic trafficking in vivo and impaired translymphatic migration in vitro, thus establishing a previously unrecognized role for this atypical chemokine in regulating DC trafficking through the lymphatics.

A number of genetic diseases in man have been described in which abnormalities in the development and function of the lymphatic vascular (LV) system are prominent features. The genes that are mutated in these diseases are varied and include genes that encode lymphatic endothelial cell (LEC) growth factor receptors and their ligands and transcription factors that control LEC fate and function. In addition, an increasing number of genes have been identified that encode components of the Ras signal transduction pathway that conveys signals from cell surface receptors to regulate cell growth, proliferation, and differentiation. Gene targeting studies performed in mice have confirmed that the LV system is particularly susceptible to perturbations in the Ras pathway.

BACKGROUND: Obesity is linked with an increased risk of lymphedema, which is a serious clinical problem. Adiponectin is a circulating adipokine that is down-regulated in obese states. We investigated the effects of adiponectin on lymphatic vessel formation in a model of lymphedema and dissected its mechanisms. METHODS AND RESULTS: A mouse model of lymphedema was created via ablation of tail surface lymphatic network. Adiponectin-knockout mice showed the greater diameter of the injured tail compared with wild-type mice, which was associated with lower numbers of lymphatic endothelial cells (LECs). Systemic delivery of adiponectin reduced the thickness of the injured tail and enhanced LEC formation in wild-type and adiponectin-knockout mice. Adiponectin administration also improved the edema of injured tails in obese KKAy mice. Treatment with adiponectin protein stimulated the differentiation of human LECs into tubelike structures and increased LEC viability. Adiponectin treatment promoted the phosphorylation of AMP-activated protein kinase (AMPK), Akt, and endothelial nitric oxide synthase n LECs. Blockade of AMPK or Akt activity abolished adiponectin-stimulated increase in LEC differentiation and viability and endothelial nitric oxide synthase phosphorylation. Inhibition of AMPK activation also suppressed adiponectin-induced Akt phosphorylation in LECs. In contrast, inactivation of Akt signaling had no effects on adiponectin-mediated AMPK phosphorylation in LECs. Furthermore, adiponectin administration did not affect the thickening of the damaged tail in endothelial nitric oxide synthase-knockout mice. CONCLUSIONS: Adiponectin can promote lymphatic vessel formation via activation of AMPK/Akt/endothelial nitric oxide synthase signaling within LECs, thereby leading to amelioration of lymphedema.

The circle of Willis (cW) is a major arterial collateral structure interconnecting hemispheric circulation within the brain, and in humans, anatomical variation of the cW is linked to stroke risk. Our prior studies on adult mice deficient in vascular smooth muscle cell (vSMC) Notch signaling revealed altered cerebroarterial maturation and patterning, including an anatomically incompetent cW similar to human variants. However, a developmental dependency on Notch signaling for cW formation in this model remained uncharacterized. Through temporospatial embryonic analyses, we now demonstrate that cW assembly is a pre-natal process highly sensitive to vSMC Notch signals, whose absence results in delayed nascent vascular plexus formation and under-development of the cW including the key anterior communicating artery (AComA) interconnecting anterior forebrain circulation. Mutant embryos additionally feature reduced vSMC coverage, non-uniform calibers and asymmetric branching at bifurcations of the major proximal cerebral arteries. At the cellular level, a notable reduction in vascular endothelial cell proliferation exists in the region of AComA assembly despite the presence of Vegfa. Furthermore, Notch signaling-deficient vSMCs in developing cerebral vessels feature reduced Pdgfrbeta and Jagged1 levels and impaired proliferation. These collective findings in the embryonic brain support studies in adult animals demonstrating a reliance on intact vSMC Notch signaling for optimal neovascular responses to angiogenic stimuli. Importantly, the new data provide unique insights into the native formation of the cW and underscore a pioneering developmental role for vSMC Notch signaling in regulating temporospatial assembly of the clinically relevant cW.

The lymphedema-lymphangiectasia-intellectual disability (Hennekam) syndrome (HS) is characterised by a widespread congenital lymph vessel dysplasia manifesting as congenital lymphedema of the limbs and intestinal lymphangiectasia, accompanied by unusual facial morphology, variable intellectual disabilities and infrequently malformations. The syndrome is heterogeneous as mutations in the gene CCBE1 have been found responsible for the syndrome in only a subset of patients. We investigated whether it would be possible to predict the presence of a CCBE1 mutation based on phenotype by collecting clinical data of patients diagnosed with HS, with or without a CCBE1 mutation. We report here the results of 13 CCBE1 positive patients, 16 CCBE1 negative patients, who were clinically found to have classical HS, and 8 patients in whom the diagnosis was considered possible, but not certain, and in whom no CCBE1 mutation was identified. We found no statistically significant phenotypic differences between the 2 groups with the clinical HS phenotype, although the degree of lymphatic dysplasia tended to be more pronounced in the mutation positive group. We also screened 158 patients with less widespread and less pronounced forms of lymphatic dysplasia for CCBE1 mutations, and no mutation was detected in this group. Our results suggest that (1) CCBE1 mutations are present only in patients with a likely clinical diagnosis of HS, and not in patients with less marked forms of lymphatic dysplasia, and (2) that there are no major phenotypic differences between HS patients with or without CCBE1 mutations.

Mutations in SOX18, VEGFC and VEGFR3 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 hypotrichosis-lymphedema-telangiectasia (Ragged Opossum) and performed gene expression profiling of aberrant dermal lymphatic vessels. Expression studies and functional analysis in zebrafish and mice revealed one candidate, ARAP3 (ArfGAP, RhoGAP, ankyrin repeats and PH domains 3), 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.

Lymphedema is caused by dysfunction of lymphatic vessels, leading to disabling swelling that occurs mostly on the extremities. Lymphedema can be either primary (congenital) or secondary (acquired). Familial primary lymphedema commonly segregates in an autosomal dominant or recessive manner. It can also occur in combination with other clinical features. Nine mutated genes have been identified in different isolated or syndromic forms of lymphedema. However, the prevalence of primary lymphedema that can be explained by these genetic alterations is unknown. In this study, we investigated 7 of these putative genes. We screened 78 index patients from families with inherited lymphedema for mutations in FLT4, GJC2, FOXC2, SOX18, GATA2, CCBE1, and PTPN14. Altogether, we discovered 28 mutations explaining 36% of the cases. Additionally, 149 patients with sporadic primary lymphedema were screened for FLT4, FOXC2, SOX18, CCBE1, and PTPN14. Twelve mutations were found that explain 8% of the cases. Still unidentified is the genetic cause of primary lymphedema in 64% of patients with a family history and 92% of sporadic cases. Identification of those genes is important for understanding of etiopathogenesis, stratification of treatments and generation of disease models. Interestingly, most of the proteins that are encoded by the genes mutated in primary lymphedema seem to act in a single functional pathway involving VEGFR3 signaling. This underscores the important role this pathway plays in lymphatic development and function and suggests that the unknown genes also have a role.

Propranolol, a beta 1 (ADBR1) and beta 2 (ADBR2) adrenergic receptor blocker, accelerates regression of proliferating infantile hemangiomas (IH-P) while not affecting non-involuting congenital hemangiomas (NICH) and nonproliferating IH (IH-NP). To determine the expression of ADBRs in vascular tumors, immunofluorescent staining and confocal microscopy were employed to determine the in situ cellular distribution of ADBRs in formalin-fixed paraffin-embedded tissue sections of IH-P, IH-NP, and NICH. In situ cellular proliferation, indexed by Ki-67 expression, distinguished IH-P (n=3) from both IH-NP (n=3) and NICH (n=2). In IH-P, IH-NP, and NICH tumor sections, both ADBR1 and ADBR2 were co-localized in both endothelial cells (ECs; GLUT1+ in IH; CD31+ in NICH) and pericytes (smooth muscle actin). We tentatively conclude that either EC and/or pericytes in IH-P could be target(s) of propranolol. Cell proliferation, but not absence of either class of ADBR, distinguished the propranolol responsive IH-P from the nonresponsive IH-NP and NICH.

Capillary malformation-arteriovenous malformation (CM-AVM) is an autosomal dominant disorder, caused by heterozygous RASA1 mutations, and manifesting multifocal capillary malformations and high risk for fast-flow lesions. A limited number of patients has been reported, raising the question of the phenotypic borders. We identified new patients with a clinical diagnosis of CM-AVM, and patients with overlapping phenotypes. RASA1 was screened in 261 index patients with: CM-AVM (n=100), common capillary malformation(s) (port-wine stain; n=100), Sturge-Weber syndrome (n=37), or isolated arteriovenous malformation(s) (n=24). Fifty-eight distinct RASA1 mutations (43 novel) were identified in 68 index patients with CM-AVM and none in patients with other phenotypes. A novel clinical feature was identified: cutaneous zones of numerous small white pale halos with a central red spot. An additional question addressed in this study was the “second-hit” hypothesis as a pathophysiological mechanism for CM-AVM. One tissue from a patient with a germline RASA1 mutation was available. The analysis of the tissue showed loss of the wild-type RASA1 allele. In conclusion, mutations in RASA1 underscore the specific CM-AVM phenotype and the clinical diagnosis is based on identifying the characteristic capillary malformations. The high incidence of fast-flow lesions warrants careful clinical and radiologic examination, and regular follow-up. This article is protected by copyright. All rights reserved.

Hereditary hemorrhagic telangiectasia (HHT), the most common inherited vascular disorder, is caused by mutations in genes involved in the transforming growth factor beta (TGF-beta) signaling pathway (ENG, ACVRL1, and SMAD4). Yet, approximately 15% of individuals with clinical features of HHT do not have mutations in these genes, suggesting that there are undiscovered mutations in other genes for HHT and possibly vascular disorders with overlapping phenotypes. The genetic etiology for 191 unrelated individuals clinically suspected to have HHT was investigated with the use of exome and Sanger sequencing; these individuals had no mutations in ENG, ACVRL1, and SMAD4. Mutations in BMP9 (also known as GDF2) were identified in three unrelated probands. These three individuals had epistaxis and dermal lesions that were described as telangiectases but whose location and appearance resembled lesions described in some individuals with RASA1-related disorders (capillary malformation-arteriovenous malformation syndrome). Analyses of the variant proteins suggested that mutations negatively affect protein processing and/or function, and a bmp9-deficient zebrafish model demonstrated that BMP9 is involved in angiogenesis. These data confirm a genetic cause of a vascular-anomaly syndrome that has phenotypic overlap with HHT.