Fsp1 cardiac embryonic expression delineates atrioventricular endocardial cushion, coronary venous and lymphatic valve development.

Fsp1 (a.k.a S100A4 or Metastatin) is an intracellular and secreted protein widely regarded as a fibroblast marker. Recent studies have nonetheless shown that Fsp1 is also expressed by other cell types, including small subsets of endothelial cells. Since no detailed and systematic description of Fsp1 spatio-temporal expression pattern in cardiac vascular cells is available in the literature, we have used a transgenic murine line (Fsp1-GFP) to study Fsp1 expression in the developing and postnatal cardiac vasculature and endocardium. Our work shows that Fsp1 is expressed in the endocardium and mesenchyme of atrioventricular valve primordia, as well as in some coronary venous and lymphatic endothelial cells. Fsp1 expression in cardiac venous and lymphatic endothelium is progressively restricted to the leaflets of cardiac venous and lymphatic valves. Our results suggest that Fsp1 could play a role in the development of atrioventricular valves and participate in the patterning and morphogenesis of cardiac venous and lymphatic vessel valves.

RASA1-driven cellular export of collagen IV is required for the development of lymphovenous and venous valves in mice.

RASA1, a negative regulator of Ras-MAPK signaling, is essential for the development and maintenance of lymphatic vessel valves. However, whether RASA1 is required for the development and maintenance of lymphovenous valves (LVV) and venous valves (VV) is unknown. In this study, we show that induced disruption of Rasa1 in mouse embryos did not affect initial specification of LVV or central VV, but did affect their continued development. Similarly, a switch to expression of a catalytically inactive form of RASA1 resulted in impaired LVV and VV development. Blocked development of LVV was associated with accumulation of the basement membrane protein, collagen IV, in LVV-forming endothelial cells (EC), and could be partially or completely rescued by MAPK inhibitors and drugs that promote collagen IV folding. Disruption of Rasa1 in adult mice resulted in venous hypertension and impaired VV function that was associated with loss of EC from VV leaflets. In conclusion, RASA1 functions as a negative regulator of Ras signaling in EC that is necessary for EC export of collagen IV, thus permitting the development of LVV and the development and maintenance of VV.

YAP and TAZ maintain PROX1 expression in the developing lymphatic and lymphovenous valves in response to VEGF-C signaling.

Lymphatic vasculature is an integral part of digestive, immune and circulatory systems. The homeobox transcription factor PROX1 is necessary for the development of lymphatic vessels, lymphatic valves (LVs) and lymphovenous valves (LVVs). We and others previously reported a feedback loop between PROX1 and vascular endothelial growth factor-C (VEGF-C) signaling. PROX1 promotes the expression of the VEGF-C receptor VEGFR3 in lymphatic endothelial cells (LECs). In turn, VEGF-C signaling maintains PROX1 expression in LECs. However, the mechanisms of PROX1/VEGF-C feedback loop remain poorly understood. Whether VEGF-C signaling is necessary for LV and LVV development is also unknown. Here, we report for the first time that VEGF-C signaling is necessary for valve morphogenesis. We have also discovered that the transcriptional co-activators YAP and TAZ are required to maintain PROX1 expression in LVs and LVVs in response to VEGF-C signaling. Deletion of Yap and Taz in the lymphatic vasculature of mouse embryos did not affect the formation of LVs or LVVs, but resulted in the degeneration of these structures. Our results have identified VEGF-C, YAP and TAZ as a crucial molecular pathway in valve development.

Antigen removal process preserves function of small diameter venous valved conduits, whereas SDS-decellularization results in significant valvular insufficiency.

Chronic venous disease (CVD) is the most common reported chronic condition in the United States, affecting more than 25 million Americans. Regardless of its high occurrence, current therapeutic options are far from ideal due to their palliative nature. For best treatment outcomes, challenging cases of chronic venous insufficiency (CVI) are treated by repair or replacement of venous valves. Regrettably, the success of venous valve transplant is dependent on the availability of autologous venous valves and hindered by the possibility of donor site complications and increased patient morbidity. Therefore, the use of alternative tissue sources to provide off-the-shelf venous valve replacements has potential to be extremely beneficial to the field of CVI. This manuscript demonstrates the capability of producing off-the-shelf fully functional venous valved extracellular matrix (ECM) scaffold conduits from bovine saphenous vein (SV), using an antigen removal (AR) method. AR ECM scaffolds maintained native SV structure-function relationships and associated venous valves function. Conversely, SDS decellularization caused significant changes to the collagen and elastin macromolecular structures, resulting in collagen fibril merging, elimination of fibril crimp, amalgaming collagen fibers and fragmentation of the inner elastic lamina. ECM changes induced by SDS decellularization resulted in significant venous valve dysfunction. Venous valved conduits generated using the AR approach have potential to serve as off-the-shelf venous valve replacements for CVI. STATEMENT OF SIGNIFICANCE: Retention of the structure and composition of extracellular matrix (ECM) proteins within xenogeneic scaffolds for tissue engineering is of crucial importance, due to the undeniable effect ECM proteins can impose on repopulating cells and function of the resultant biomaterial. This manuscript demonstrates that alteration or elimination of ECM proteins via commonly utilized decellularization approach results in complete disruption of venous valve function. Conversely, retention of the delicate ECM structure and composition of native venous tissue, using an antigen removal tissue processing method, results in preservation of native venous valve function.

Control of sinus venous valve and sinoatrial node development by endocardial NOTCH1.

AIMS: Sinus venous valve (SVV) and sinoatrial node (SAN) develop together at the sinoatrial junction during embryogenesis. SVV ensures unidirectional cardiac input and SAN generates sinus rhythmic contraction, respectively; both functions are essential for embryonic survival. We aim to reveal the potential role of endocardial NOTCH signalling in SVV and SAN formation. METHODS AND RESULTS: We specifically deleted Notch1 in the endocardium using an Nfatc1Cre line. This deletion resulted in underdeveloped SVV and SAN, associated with reduced expression of T-box transcription factors, Tbx5 andTbx18, which are essential for the formation of SVV and SAN. The deletion also led to decreased expression of Wnt2 in myocardium of SVV and SAN. WNT2 treatment was able to rescue the growth defect of SVV and SAN resulted from the Notch1 deletion in whole embryo cultures. Furthermore, the Notch1 deletion reduced the expression of Nrg1 in the SVV myocardium and supplement of NRG1 restored the growth of SVV in cultured Notch1 knockout embryos. CONCLUSION: Our findings support that endocardial NOTCH1 controls the development of SVV and SAN by coordinating myocardial WNT and NRG1 signalling functions.

Correlative Fluorescence and Scanning Electron Microscopy to Study Lymphovenous Valve Development.

Lymph collected from throughout the body is exclusively returned to blood circulation via two pairs of bilaterally located lymphovenous valves. Lymphovenous valves share numerous similarities with lymphatic and venous valves and are defective in multiple mouse models of lymphedema or lymphatic dysfunction. Here we describe a protocol that combines the strengths of fluorescence microscopy and scanning electron microscopy to precisely locate and analyze the topography of developing lymphovenous valves at high resolution.

Platelets Drive Thrombus Propagation in a Hematocrit and Glycoprotein VI-Dependent Manner in an In Vitro Venous Thrombosis Model.

OBJECTIVE: The objective of this study was to measure the role of platelets and red blood cells on thrombus propagation in an in vitro model of venous valvular stasis. APPROACH AND RESULTS: A microfluidic model with dimensional similarity to human venous valves consists of a sinus distal to a sudden expansion, where for sufficiently high Reynolds numbers, 2 countercurrent vortices arise because of flow separation. The primary vortex is defined by the points of flow separation and reattachment. A secondary vortex forms in the deepest recess of the valve pocket characterized by low shear rates. An initial fibrin gel formed within the secondary vortex of a tissue factor-coated valve sinus. Platelets accumulated at the interface of the fibrin gel and the primary vortex. Red blood cells at physiological hematocrits were necessary to provide an adequate flux of platelets to support thrombus growth out of the valve sinus. A subpopulation of platelets that adhered to fibrin expose phosphatidylserine. Platelet-dependent thrombus growth was attenuated by inhibition of glycoprotein VI with a blocking Fab fragment or D-dimer. CONCLUSIONS: A 3-step process regulated by hemodynamics was necessary for robust thrombus propagation: First, immobilized tissue factor initiates coagulation and fibrin deposition within a low flow niche defined by a secondary vortex in the pocket of a model venous valve. Second, a primary vortex delivers platelets to the fibrin interface in a red blood cell-dependent manner. Third, platelets adhere to fibrin, activate through glycoprotein VI, express phosphatidylserine, and subsequently promote thrombus growth beyond the valve sinus and into the bulk flow.

Smooth muscle cells of human veins show an increased response to injury at valve sites.

OBJECTIVE: Venous valves are essential but are prone to injury, thrombosis, and fibrosis. We compared the behavior and gene expression of smooth muscle cells (SMCs) in the valve sinus vs nonvalve sites to elucidate biologic differences associated with vein valves. METHODS: Tissue explants of fresh human saphenous veins were prepared, and the migration of SMCs from explants of valve sinus vs nonvalve sinus areas was measured. Proliferation and death of SMCs were determined by staining for Ki67 and terminal deoxynucleotidyl transferase dUTP nick end labeling. Proliferation and migration of passaged valve vs nonvalve SMCs were determined by cell counts and using microchemotaxis chambers. Global gene expression in valve vs nonvalve intima-media was determined by RNA sequencing. RESULTS: Valve SMCs demonstrated greater proliferation in tissue explants compared with nonvalve SMCs (19.3% ± 5.4% vs 6.8% ± 2.0% Ki67-positive nuclei at 4 days, respectively; mean ± standard error of the mean, five veins; P < .05). This was also true for migration (18.2 ± 2.7 vs 7.5 ± 3.0 migrated SMCs/explant at 6 days, respectively; 24 veins, 15 explants/vein; P < .0001). Cell death was not different (39.6% ± 16.1% vs 41.5% ± 16.0% terminal deoxynucleotidyl transferase dUTP nick end labeling-positive cells, respectively, at 4 days, five veins). Cultured valve SMCs also proliferated faster than nonvalve SMCs in response to platelet-derived growth factor subunit BB (2.9 ± 0.2-fold vs 2.1 ± 0.2-fold of control, respectively; P < .001; n = 5 pairs of cells). This was also true for migration (6.5 ± 1.2-fold vs 4.4 ± 0.8-fold of control, respectively; P < .001; n = 7 pairs of cells). Blockade of fibroblast growth factor 2 (FGF2) inhibited the increased responses of valve SMCs but had no effect on nonvalve SMCs. Exogenous FGF2 increased migration of valve but not of nonvalve SMCs. Unlike in the isolated, cultured cells, blockade of FGF2 in the tissue explants did not block migration of valve or nonvalve SMCs from the explants. Thirty-seven genes were differentially expressed by valve compared with nonvalve intimal-medial tissue (11 veins). Peptide-mediated inhibition of SEMA3A, one of the differentially expressed genes, increased the number of migrated SMCs of valve but not of nonvalve explants. CONCLUSIONS: Valve compared with nonvalve SMCs have greater rates of migration and proliferation, which may in part explain the propensity for pathologic lesion formation in valves. Whereas FGF2 mediates these effects in cultured SMCs, the mediators of these stimulatory effects in the valve wall tissue remain unclear but may be among the differentially expressed genes discovered in this study. One of these genes, SEMA3A, mediates a valve-specific inhibitory effect on the injury response of valve SMCs.

Human venous valve disease caused by mutations in FOXC2 and GJC2.

Venous valves (VVs) prevent venous hypertension and ulceration. We report that FOXC2 and GJC2 mutations are associated with reduced VV number and length. In mice, early VV formation is marked by elongation and reorientation ("organization") of Prox1hi endothelial cells by postnatal day 0. The expression of the transcription factors Foxc2 and Nfatc1 and the gap junction proteins Gjc2, Gja1, and Gja4 were temporospatially regulated during this process. Foxc2 and Nfatc1 were coexpressed at P0, and combined Foxc2 deletion with calcineurin-Nfat inhibition disrupted early Prox1hi endothelial organization, suggesting cooperative Foxc2-Nfatc1 patterning of these events. Genetic deletion of Gjc2, Gja4, or Gja1 also disrupted early VV Prox1hi endothelial organization at postnatal day 0, and this likely underlies the VV defects seen in patients with GJC2 mutations. Knockout of Gja4 or Gjc2 resulted in reduced proliferation of Prox1hi valve-forming cells. At later stages of blood flow, Foxc2 and calcineurin-Nfat signaling are each required for growth of the valve leaflets, whereas Foxc2 is not required for VV maintenance.

Segregated Foxc2, NFATc1 and Connexin expression at normal developing venous valves, and Connexin-specific differences in the valve phenotypes of Cx37, Cx43, and Cx47 knockout mice.

Venous valves (VVs) are critical for unidirectional blood flow from superficial and deep veins towards the heart. Congenital valve aplasia or agenesis may, in some cases, be a direct cause of vascular disease, motivating an understanding of the molecular mechanisms underlying the development and maintenance of VVs. Three gap junction proteins (Connexins), Cx37, Cx43, and Cx47, are specifically expressed at VVs in a highly polarized fashion. VVs are absent from adult mice lacking Cx37; however it is not known if Cx37 is required for the initial formation of valves. In addition, the requirement of Cx43 and Cx47 for VV development has not been studied. Here, we provide a detailed description of Cx37, Cx43, and Cx47 expression during mouse vein development and show by gene knockout that each Cx is necessary for normal valve development. The valve phenotypes in the knockout lines exhibit Cx-specific differences, however, including whether peripheral or central VVs are affected by gene inactivation. In addition, we show that a Cx47 null mutation impairs peripheral VV development but does not affect lymphatic valve formation, a finding of significance for understanding how some CX47 mutations cause inherited lymphedema in humans. Finally, we demonstrate a striking segregation of Foxc2 and NFATc1 transcription factor expression between the downstream and upstream faces, respectively, of developing VV leaflets and show that this segregation is closely associated with the highly polarized expression of Cx37, Cx43, and Cx47. The partition of Foxc2 and NFATc1 expression at VV leaflets makes it unlikely that these factors directly cooperate during the leaflet elongation stage of VV development.

P-selectin inhibition therapeutically promotes thrombus resolution and prevents vein wall fibrosis better than enoxaparin and an inhibitor to von Willebrand factor.

OBJECTIVE: Aptamers are oligonucleotides targeting protein-protein interactions with pharmacokinetic profiles and activity reversal options. Although P-selectin and von Willebrand factor (vWF) have been implicated in the development of venous thrombosis (VT), no studies have directly compared aptamer efficacy with standard of care in VT. In this study, ARC5692, an anti-P-selectin aptamer, and ARC15105, an anti-vWF aptamer, were compared with low-molecular-weight heparin, enoxaparin, to test the efficacy of P-selectin or vWF inhibition in promoting thrombus resolution and preventing vein wall fibrosis, in a baboon model of VT. APPROACH AND RESULTS: Groups were as follows: treatment arm: animals received P-selectin or vWF aptamer inhibitors or enoxaparin (n=3 per group). Controls received no treatment (n=3). Prophylactic arm: animals received P-selectin inhibitor (n=4) or vWF inhibitor (n=3). Treatment arm: P-selectin-inhibitor demonstrated a significant improvement in vein recanalization by magnetic resonance venography (73% at day 21), and significantly decreased vein wall collagen, compared with all groups. Anti-P-selectin equaled enoxaparin in maintaining valve competency by ultrasound. All control animals had compromised valve competency post thrombosis. Prophylactic arm: animals receiving P-selectin and vWF inhibitors demonstrated improved vein recanalization by magnetic resonance venography versus controls (80% and 85%, respectively, at day 21). Anti-P-selectin protected iliac valve function better than anti-vWF, and both improved valve function versus controls. No adverse bleeding events were observed. CONCLUSIONS: The P-selectin inhibitor aptamer promoted iliac vein recanalization, preserved valve competency, and decreased vein wall fibrosis. The results of this work suggest that P-selectin inhibition maybe an ideal target in the treatment and prophylaxis of deep VT, warranting clinical trials.

Integrin-α5ß1 is not required for mural cell functions during development of blood vessels but is required for lymphatic-blood vessel separation and lymphovenous valve formation.

Integrin α5ß1 is essential for vascular development but it remains unclear precisely where and how it functions. Here, we report that deletion of the gene encoding the integrin-α5 subunit (Itga5) using the Pdgfrb-Cre transgenic mouse line, leads to oedema, haemorrhage and increased levels of embryonic lethality. Unexpectedly, these defects were not caused by loss of α5 from Pdgfrb-Cre expressing mural cells (pericytes and vascular smooth muscle cells), which wrap around the endothelium and stabilise blood vessels, nor by defects in the heart or great vessels, but were due to abnormal development of the lymphatic vasculature. Reminiscent of the pathologies seen in the human lymphatic malformation, fetal cystic hygroma, α5 mutants display defects both in the separation of their blood and lymphatic vasculature and in the formation of the lymphovenous valves. As a consequence, α5-deficient mice develop dilated, blood-filled lymphatic vessels and lymphatic capillaries that are ectopically covered with smooth muscle cells. Analysis of the expression of Pdgfrb during lymphatic development suggests that these defects probably arise from loss of α5ß1 integrin in subsets of specialised Prox1(+)Pdgfrb(+) venous endothelial cells that are essential for the separation of the jugular lymph sac from the cardinal vein and formation of the lymphovenous valve leaflets.

Multifluorescence confocal microscopy: application for a quantitative analysis of hemostatic proteins in human venous valves.

Confocal laser scanning microscopy is commonly used to visualize and quantify protein expression. Visualization of the expression of multiple proteins in the same region via multifluorescence allows for the analysis of differential protein expression. The defining step of multifluorescence labeling is the selection of primary antibodies from different host species. In addition, species-appropriate secondary antibodies must also be conjugated to different fluorophores so that each protein can be visualized in separate channels. Quantitative analysis of proteins labeled via multifluorescence can be used to compare relative changes in protein expression. Multifluoresecence labeling and analysis of fluorescence intensity within and among human venous specimens, for example, allowed us to determine that the anticoagulant phenotype of the venous valve is defined not by increased anticoagulant expression, but instead by significantly decreased procoagulant protein expression (Blood 114:1276-1279, 2009 and Histochem Cell Biol 135:141-152, 2011).

Absence of venous valves in mice lacking Connexin37.

Venous valves play a crucial role in blood circulation, promoting the one-way movement of blood from superficial and deep veins towards the heart. By preventing retrograde flow, venous valves spare capillaries and venules from being subjected to damaging elevations in pressure, especially during skeletal muscle contraction. Pathologically, valvular incompetence or absence of valves are common features of venous disorders such as chronic venous insufficiency and varicose veins. The underlying causes of these conditions are not well understood, but congenital venous valve aplasia or agenesis may play a role in some cases. Despite progress in the study of cardiac and lymphatic valve morphogenesis, the molecular mechanisms controlling the development and maintenance of venous valves remain poorly understood. Here, we show that in valved veins of the mouse, three gap junction proteins (Connexins, Cxs), Cx37, Cx43, and Cx47, are expressed exclusively in the valves in a highly polarized fashion, with Cx43 on the upstream side of the valve leaflet and Cx37 on the downstream side. Surprisingly, Cx43 expression is strongly induced in the non-valve venous endothelium in superficial veins following wounding of the overlying skin. Moreover, we show that in Cx37-deficient mice, venous valves are entirely absent. Thus, Cx37, a protein involved in cell-cell communication, is one of only a few proteins identified so far as critical for the development or maintenance of venous valves. Because Cxs are necessary for the development of valves in lymphatic vessels as well, our results support the notion of common molecular pathways controlling valve development in veins and lymphatic vessels.

New insights into the mechanisms of venous thrombosis.

Venous thrombosis is a leading cause of morbidity and mortality in industrialized countries, especially in the elderly. Many risk factors have been identified for venous thrombosis that alter blood flow, activate the endothelium, and increase blood coagulation. However, the precise mechanisms that trigger clotting in large veins have not been fully elucidated. The most common site for initiation of the thrombus appears to be the valve pocket sinus, due to its tendency to become hypoxic. Activation of endothelial cells by hypoxia or possibly inflammatory stimuli would lead to surface expression of adhesion receptors that facilitate the binding of circulating leukocytes and microvesicles. Subsequent activation of the leukocytes induces expression of the potent procoagulant protein tissue factor that triggers thrombosis. Understanding the mechanisms of venous thrombosis may lead to the development of new treatments.

The endothelial microenvironment in the venous valvular sinus: thromboresistance trends and inter-individual variation.

The valve sinuses of the deep venous system are frequent sites of venous thrombus initiation. We previously reported that, in comparison with the non-valvular lumenal endothelium, the valve sinus endothelium had decreased expression of von Willebrand factor (vWF) and increased expression of endothelial protein C receptor (EPCR) and thrombomodulin (TM), suggesting alteration in the procoagulant/anticoagulant balance. We hypothesized that increased stasis in the deeper recesses of the venous valves would be associated with a gradient of increased thromboresistance. Expression of EPCR, TM, and vWF was analyzed via quantitative confocal immunofluorescence in residual saphenous veins collected following coronary artery bypass procedures. In agreement with our hypothesis, endothelial expression of vWF in the valve sinus decreased from the uppermost to the deepest region of the valve sinus. In contrast to our hypothesis, EPCR expression decreased from the uppermost to the deepest region of the valve sinus (p < 0.001) and TM expression remained unchanged throughout the valve sinus. Comparison of the non-valvular lumenal endothelium with the valve sinus endothelium demonstrated significantly decreased vWF expression (p < 0.001) in the valvular sinus consistent with our previous report; however, we did not observe statistically significant differences in EPCR or TM expression in this comparison. In addition, remarkable inter-individual variation in expression of these three proteins was also observed. These findings suggest that the genesis of these observations is more complex than predicted by our initial hypothesis, likely due, at least in part, to the complex rheology of the valvular sinus microenvironment.

Valves of the deep venous system: an overlooked risk factor.

Deep venous valves are frequent sites of deep venous thrombosis initiation. However, the possible contribution of the valvular sinus endothelium has received little attention in studies of thrombosis risk. We hypothesized that the endothelium of valve sinus differs from that of vein lumen with up-regulation of anticoagulant and down-regulation of procoagulant activities in response to the local environment. In pursuit of this hypothesis, we quantified endothelial protein C receptor (EPCR), thrombomodulin (TM), and von Willebrand factor (VWF) by immunofluorescence in great saphenous veins harvested at cardiac bypass surgery. We found significantly increased expression of EPCR and TM in the valvular sinus endothelium as opposed to the vein lumenal endothelium, and the opposite pattern with VWF (paired t test for TM and EPCR, each P < .001; for VWF, P = .01). These data support our hypothesis and suggest that variation in valvular sinus thromboresistance may be an important factor in venous thrombogenesis.