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.

Usefulness of aliasing phenomenon for diagnosing venous valve stenosis of arteriovenous fistula in a hemodialysis patient.

We present venous valve stenosis, which is an uncommon cause of arteriovenous fistula (AVF) dysfunction. Owing to the thin structure in echography, venous valves are challenging to observe; however, we have found that the aliasing phenomenon is useful for diagnosing venous valve stenosis.

Surgical repair of a special category of arteriovenous fistula outflow stenosis caused by venous valve hyperplasia.

OBJECTIVE: This study evaluated a special category of arteriovenous fistula outflow stenosis caused by venous valve hyperplasia and explored the effectiveness of surgical repair in dealing with this kind of stenosis. STUDY DESIGN: This retrospective cohort study was conducted from February 2016 to January 2020 in our center. Patients with arteriovenous fistula dysfunction, including flow rate insufficiency, venous hypertension, thrombosis, and aneurysm dilation enlargement, were selected. Stenosis lesions presenting with venous valve hyperplasia were selected after ultrasound screening. All patients underwent surgical repair and were followed up every 6 months after surgery. RESULTS: Forty-three patients (median age, 54.5 ± 11.2 years; 65.1% men) were included. All procedures were technically successful. Based on intraoperative exploration, 56.5% were reconstructed via autologous vein patch, 17.4% of patients were reconstructed with end-to-end reconstruction after cutting the stenotic segment, 13.0% of cases simply had the valve resected, and 13.0% of cases involved a longitudinal incision and transverse suture. All patients returned to routine dialysis the following day and avoided catheter insertion. The mean follow-up time was 22.5 ± 14.0 (range, 1.3-49.8) months. The patency rates at 2 and 4 years were 92.2% and 79.0%, respectively. Valves harvested from patients were analyzed via Masson staining and immunohistochemical staining, indicating collagen fiber and myofibroblast hyperplasia in outflow venous valve hyperplasia (OVVH). CONCLUSIONS: Outflow venous valve hyperplasia can lead to fistula dysfunction. Ultrasound is the main method to diagnosis OVVH. Special surgical repair can preserve valuable vascular resources and relieve stenosis, is safe and effective, and has a high patency rate.

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.

"Quality of Life after Varicose Vein Surgery in Patients with High-ligation and Stripping, External Valvuloplasty and Sapheno-femoral Redo Surgery".

BACKGROUND: High-ligation and stripping (HL/S) and external valvuloplasty (eVP) with the implantation of an external device to restore the valve's function, are surgical methods to eliminate reflux at the saphenofemoral junction. Furthermore, redo-surgery (RedoS) can be performed in terms of same side groin recurrences. It is unclear, if there is a difference in quality of life (QoL) between these 3 surgical treatment options. Therefore, it was the aim of our study to elucidate QoL in patients before and after surgical treatment at the saphenofemoral junction by comparing HL/S, eVP, and RedoS. METHODS: A total of 303 participants (156 HL/S, 81eVP, 64 RedoS) were recruited during the daily clinical routine. QoL was measured at admission and 6 weeks after the surgical procedure by means of SF-12 (12 item short form health survey) and Aberdeen Varicose Vein Questionnaire. RESULTS: The mean value of Aberdeen Varicose Vein Questionnaire was 14.5 (SD 2.1) preoperatively and 4.9 (SD 3.3) postoperatively in the HL/S group, 16.4 (SD 1.4) preoperatively and 6.8 (SD 2.5) postoperatively in the eVP group and 15.5 (2.2) preoperatively and 5.8 (SD 4.2) postoperatively in the RedoS group, which was statistically significant (P< 0.05) in all groups. Postoperatively, the mean values were statistically significant within the groups. Concerning physical aspects of the SF-12 we found a significant improvement in the RedoS group, while mental aspects were significantly better in the HL/S and eVP group postoperatively. Nevertheless, the clinical relevance of these SF-12 differences is questionable under consideration of the minimal important difference. CONCLUSIONS: Varicose vein surgery leads to a significant improvement of QoL in all groups. The implantation of an external patch could have a negative influence in QoL.

Microengineered Human Vein-Chip Recreates Venous Valve Architecture and Its Contribution to Thrombosis.

Deep vein thrombosis (DVT) and its consequences are lethal, but current models cannot completely dissect its determinants-endothelium, flow, and blood constituents-together called Virchow's triad. Most models for studying DVT forego assessment of venous valves that serve as the primary sites of DVT formation. Therefore, the knowledge of DVT formed at the venous cusps has remained obscure due to lack of experimental models. Here, organ-on-chip methodology is leveraged to create a Vein-Chip platform integrating fully vascularized venous valves and its hemodynamic, as seen in vivo. These Vein-Chips reveal that vascular endothelium of valve cusps adapts to the locally disturbed microenvironment by expressing a different phenotype from the regions of uniform flow. This spatial adaptation of endothelial function recreated on the in vitro Vein-Chip platform is shown to protect the vein from thrombosis from disturbed flow in valves, but interestingly, cytokine stimulation reverses the effect and switches the valve endothelium to becoming prothrombotic. The platform eventually modulates the three factors of Virchow's triad and provides a systematic approach to investigate the determinants of fibrin and platelet dynamics of DVT. Therefore, this Vein-Chip offers a new preclinical approach to study venous pathophysiology and show effects of antithrombotic drug treatment.

Using two-dimensional ultrasound imaging to examine venous pressure.

Ultrasound imaging is being used increasingly to aid in the teaching of human physiology and anatomy. Here we describe how its use can be integrated into the teaching of concepts surrounding venous circulation, specifically 1) venous valves and the muscle pump, 2) the effects of hydrostatic pressure on venous pressure, and 3) central venous pressure. The imaging procedures described are relatively simple but add a dimension that helps deliver the teaching points clearly and is enjoyable for students. They also aid in the link of basic physiology to clinical aspects of venous circulatory physiology.

Morphology of the Vieussens valve and its imaging in cardiac multislice computed tomography.

INTRODUCTION: To deliver accurate morphological descriptions of the Vieussens valve (VV) and to investigate whether this structure could be visualized using standard contrast-enhanced electrocardiogram-gated multislice computed tomography (MSCT). METHODS: A total of 145 human autopsied hearts and 114 cardiac MSCT scans were examined. RESULTS: The VV was observed in both study groups, however, the detection rate was significantly worse in the MSCT examination (18.4% in MSCT vs 62.1% in cadavers, P < .0001). The VV height was larger in MSCT patients (2.8 ± 1.2 vs 5.4 ± 1.7 mm; P < .0001). No significant difference was found in the measured distance between the VV and the coronary sinus ostium between the two separate subgroups (27.3 ± 9.5 vs 24.4 ± 5.8 mm; P = .18). In autopsied material the most frequent valve location was the anterior wall of the coronary sinus (43.3%); the same was observed in MSCT scans (71.4%). CONCLUSION: The VV is a common heart structure, present in over 60% of humans, located mainly on the anterior and superior circuit of the coronary sinus, with relatively high morphological variability. Large VVs, which pose a significant obstacle in catheterization procedures, may be visualized using standard-protocol contrast-enhanced cardiac MSCT.

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.

Search for an Optimal Design of a Bioprosthetic Venous Valve: In silico and in vitro Studies.

OBJECTIVE/BACKGROUND: Valve incompetence is a progressive disease of the venous system that may eventually lead to venous hypertension, pain, and ulcers. There is a need for a venous valve prosthesis to replace incompetent valves. Computational and experimental investigations on venous valve design and associated haemodynamics will undoubtedly advance prosthesis design and treatments. Here, the objective is to investigate the effect of venous valve on the fluid and solid mechanics. The hypothesis is that there exists a valve geometry that maximises leaflet shear stress (LSS) but minimises leaflet intramural stress (LIS; i.e., minimise stress ratio = LIS/LSS). METHODS: To address the hypothesis, fully dynamic fluid-structure interaction (FSI) models were developed. The entire cycle of valve opening and closure was simulated. The flow validation experiments were conducted using a stented venous valve prosthesis and a pulse duplicator flow loop. RESULTS: Agreement between the output of FSI simulations and output of pulse duplicator was confirmed. The maximum flow rates were within 6% difference, and the total flow during the cycle was within 10% difference. The simulated high stress ratio region at the leaflet base (five times the leaflet average) predicted the disease location of the vast majority of explanted venous valves reported in clinical literature. The study found that the reduced valve height and leaflet dome shape resulted in optimal performance to provide the lowest stress ratio. CONCLUSION: This study proposes an effective design of venous prostheses and elaborates on the correlations of venous valve with clinical observations.

Prox1 dosage controls the number of lymphatic endothelial cell progenitors and the formation of the lymphovenous valves.

Arteries, veins, and lymphatic vessels are functionally linked, and their physical interaction is tightly regulated. The lymphatic vessels communicate with the blood vessels only at the junction of the jugular and subclavian veins. Here, we characterize the embryonic lymphovenous valves controlling this vital communication and show that they are formed by the intercalation of lymphatic endothelial cells (LECs) with a subpopulation of venous endothelial cells (ECs) at the junction of the jugular and subclavian veins. We found that unlike LEC progenitors, which move out from the veins and differentiate into mature LECs, these Prox1-expressing ECs remain in the veins and do not acquire LEC features. We demonstrate that the development of this Prox1-expressing venous EC population, and therefore of lymphovenous valves, requires two functional copies of Prox1, as the valves are absent in Prox1 heterozygous mice. We show that this is due to a defect in the maintenance of Prox1 expression in venous ECs and LEC progenitors promoted by a reduction in Coup-TFII/Prox1 complex formation. This is the first report describing the molecular mechanism controlling lymphovenous communication.

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.

A case report of cough headache with transient elevation of intracranial pressure and bilateral internal jugular vein valve incompetence: A primary or secondary headache?

Background Primary cough headache (PCH) is precipitated by coughing or the Valsalva manoeuver (VM), and its underlying pathophysiology remains unclear. Case report We report a case of recurrent cough headaches precipitated by VM with transient increase of intracranial pressure (ICP) diagnosed by measuring left sigmoid sinus pressure. Bilateral internal jugular vein valve incompetence (IJVVI) was also diagnosed by Doppler ultrasonography during a VM. Indomethacin was administered for over four months, and the headache had completely disappeared at the four-month follow-up. Conclusions Cough headache might be associated with a transient increase of ICP induced by IJVVI, which might partially explain the pathophysiology of VM-induced headache. Detecting the internal jugular vein during a VM might be used as diagnostic procedure for patients who have PCH during resting and VM. If it is necessary, monitoring the ICP could be considered.

The influence of age on valve disease in patients with varicose veins analysed by transmission electron microscopy and stereology.

BACKGROUND: The aim of this study was to investigate the influence of age on the ultrastructure of venous valve morphology in patients with C2 classified chronic venous disorders according to the CEAP classification. PATIENTS AND METHODS: The study population consisted of 16 consecutive patients with varicose veins (C2). The mean age was 49.8 years (30-66). The (pre-) terminal valve including the vessel wall was harvested within the proximal 2 centimetres of the great saphenous vein. The mean thickness (volume-to-surface ratio = V/S ratio) of elastin, collagen, endothelium and of the entire valve was determined. A blinded morphologist performed the examination by transmission electron microscopy and stereology. Analyses by Pearson's product moment correlation, Kendall's tau and Spearman's rank correlation were performed to investigate whether there is a correlation between age and the ultrastructural morphology. RESULTS: Stereological analysis of the valves demonstrated a mean V/S ratio (signifying a thickness estimation) for elastin of 0.87 µm3/µm2, for collagen of 18.0 µm3/µm2, for endothelium of 0.65 µm3/µm2, and for the entire valve of 25.2 µm³/µm². Statistical analyses showed no statistically significant correlation between age and the ultrastructural morphology in this patient group. CONCLUSIONS: The ultrastructural morphology of the venous valves in chronic venous disorders may not depend on age in patients presenting with C2 disease. This conclusion may or may not apply to all C classes as we investigated a homogenous group of patients with C2 limbs.

Human Femoral Vein Diameter and Topography of Valves and Tributaries: A Post Mortem Analysis.

The femoral vein (FV) is a clinically important vessel. Failure of its valves can lead to chronic venous insufficiency (CVI) with severe manifestations such as painful ulcers. Although they are crucial for identifying suitable implant sites for therapeutic valves, studies on the topography of FV tributaries and valves are rare. Moreover, the femoral vein diameter (FVD) must be known to assess the morphometric requirements for valve implants. To reassess the anatomical requirements for valve implants, 155 FVs from 82 human corpses were examined. FVDs and tributary and valve topographies were assessed using a laboratory straightedge. The FVD increased from 6 mm in the distal femoropopliteal vein to 11 mm in the iliofemoral vein proximal to the saphenofemoral junction (SFJ). Diameters were significantly bigger in males than females. Height correlated positively with FVD. Distal to the SFJ, within a distance of 38 cm, one to eight valves were present. Up to two valves were present within 10 cm proximal to the SFJ. Individual tributary and valve topography must be considered to ensure appropriate design and successful implantation of a venous valve for CVI therapy in the FV. A suitable implant site would be proximal to the SFJ via an infrainguinal transfemoral access. Clin. Anat. 31:1065-1076, 2018. © 2018 Wiley Periodicals, Inc.

[Functional assessment of primary lesion of the great saphenous vein when deciding upon stripping]. / Funktsional'naia otsenka pervichnogo porazheniia bol'shoi podkozhnoi veny pri reshenii voprosa o stripinge.

The purpose of the study was to examine a possibility of functional assessment of the great saphenous vein (GSV) with the help of a day orthostatic loading test for prognosis of restoration of the function of the GSV afterremoving its varicose tributaries in patients with primary varicose veins. Our prospective study included a total of sixty-five 29-to-53-year-old patients (15 men and 50 women, mean age - 36.7 years). The total number of the lower limb examined amounted to 87. All patients underwent ultrasonographic examination (duplex scanning) prior to operation, 1 and 12 months thereafter. The study was carried out using a day orthostatic loading test consisting in duplex scanning performed twice during 24 hours: in the evening after 18:00 hours and in the morning before 10:00 hours after a good night's rest. It was demonstrated that the day orthostatic loading test characterized the degree of preservation of the muscular-tonic properties of the GSV, making it possible to predict reversibility of reflux along it after removing the varicose tributaries, hence it may be used as a criterion for individualization of the choice of the scope of surgical intervention. A high orthostatic gradient prior to operation suggests preservation of the potential of the muscular-tonic function of the GSV; its decrease after surgery demonstrates reduction of the volemic loading on the GSV.

[Possibilities of local phlebectomy in correction of pelvioperineal venous reflux]. / Vozmozhnosti lokal'noi flebéktomii v korrektsii pel'vio-perineal'nogo venoznogo refliuksa.

The work was based on the results of examination and treatment of 43 female patients presenting with varicose transformation of pelvic, perineal, and lower-limb veins. The inclusion criteria were as follows: the presence of visually determined varicose transformation of the veins of the external genital organs, perineum, posterior surface of the thighs, as well as valvular insufficiency of the mentioned veins by the findings of ultrasonographic angioscanning (USAS). The following exclusion criteria were applied: the presence of pregnancy, symptoms of pelvic venous plethora (PVP), and varicothrombophlebitis. In 33 women correction of the pelvioperineal reflux (PPR) was performed with the help of local phlebectomy on the large pudendal lips and perineum, with the maximally possible mobilization of the vessel within the limits of the operative wound. Miniphlebectomy with the use of Varady phleboextractors was performed in 10 women presenting with isolated varicose transformation of the subcutaneous veins of the posterior femoral surface. The duration of follow up of patients amounted to 3 years. The criteria for efficiency of the carried out treatment were as follows: freedom from varicose syndrome both in the perineum and on the lower extremities, as well as no PVP symptoms during the whole term of follow up. Varicose syndrome of the external genital organs, perineum and posterior surface of the femurs was successfully eliminated in 100% of patients. Meticulous mobilization and removal of the veins of the labia majora, perineum and subcutaneous femoral veins is a reliable method of removing pathological reflux of blood from the intrapelvic to superficial veins of the perineum and lower limbs. 100% of our patients were found to be free from relapses of either vulvar or perineal varicosity, with no evidence of lower limb varicose veins. Local phlebectomy is an efficient method of elimination of varicose syndrome induced by PVP in patients with dilatation of intrapelvic, vulvar and perineal veins.

Multiple mouse models of primary lymphedema exhibit distinct defects in lymphovenous valve development.

Lymph is returned to the blood circulation exclusively via four lymphovenous valves (LVVs). Despite their vital importance, the architecture and development of LVVs is poorly understood. We analyzed the formation of LVVs at the molecular and ultrastructural levels during mouse embryogenesis and identified three critical steps. First, LVV-forming endothelial cells (LVV-ECs) differentiate from PROX1(+) progenitors and delaminate from the luminal side of the veins. Second, LVV-ECs aggregate, align perpendicular to the direction of lymph flow and establish lympho-venous connections. Finally, LVVs mature with the recruitment of mural cells. LVV morphogenesis is disrupted in four different mouse models of primary lymphedema and the severity of LVV defects correlate with that of lymphedema. In summary, we have provided the first and the most comprehensive analysis of LVV development. Furthermore, our work suggests that aberrant LVVs contribute to lymphedema.

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.