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. 2011 Aug;121(8):2984-92.
doi: 10.1172/JCI58050. Epub 2011 Jul 18.

Genes Regulating Lymphangiogenesis Control Venous Valve Formation and Maintenance in Mice

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Free PMC article

Genes Regulating Lymphangiogenesis Control Venous Valve Formation and Maintenance in Mice

Eleni Bazigou et al. J Clin Invest. .
Free PMC article

Abstract

Chronic venous disease and venous hypertension are common consequences of valve insufficiency, yet the molecular mechanisms regulating the formation and maintenance of venous valves have not been studied. Here, we provide what we believe to be the first description of venous valve morphogenesis and identify signaling pathways required for the process. The initial stages of valve development were found to involve induction of ephrin-B2, a key marker of arterial identity, by venous endothelial cells. Intriguingly, developing and mature venous valves also expressed a repertoire of proteins, including prospero-related homeobox 1 (Prox1), Vegfr3, and integrin-α9, previously characterized as specific and critical regulators of lymphangiogenesis. Using global and venous valve-selective knockout mice, we further demonstrate the requirement of ephrin-B2 and integrin-α9 signaling for the development and maintenance of venous valves. Our findings therefore identified molecular regulators of venous valve development and maintenance and highlighted the involvement of common morphogenetic processes and signaling pathways in controlling valve formation in veins and lymphatic vessels. Unexpectedly, we found that venous valve endothelial cells closely resemble lymphatic (valve) endothelia at the molecular level, suggesting plasticity in the ability of a terminally differentiated endothelial cell to take on a different phenotypic identity.

Figures

Figure 1
Figure 1. Characterization of venous valves in mice and humans.
(AC) Stereomicroscopic visualization of murine valves in the iliac veins. The boxed region in A was photographed after X-gal staining in a Tie2lacZ reporter mouse (B) to visualize the valve leaflets (C). Arrow, vein; arrowhead, artery. (DJ) SEM images of the lumen of mouse (D, F, H, and J; n = 24) and human (E, G, and I; n = 4) veins, showing the overall valve structure (D and E), commissure (F and G), valve leaflet (H and I), and vessel wall upstream of the valve (J). (H, I, and F) Arrowheads denote a round endothelial cell on the leaflet (H and I) or downstream of the valve (F); arrows denote fusiform cells forming the free edge of the leaflet. The arrow in J shows the direction of flow. Scale bars: 1 mm (A, B, and E); 250 μm (C); 125 μm (D); 10 μm (F and HJ); 250 μm (G).
Figure 2
Figure 2. Development of venous valves.
(A) Visualization of valve development by X-gal staining in Tie2lacZ reporter mice and SEM imaging of veins that were opened with a longitudinal incision. 5 developmental stages were discerned: stage 0, rounding up of endothelial cells at the sites of developing valves; stage 1, valve endothelial cell alignment perpendicular to the longitudinal axis of the vessel; stage 2, formation of a circular shelf; stage 3, formation of the first commissure; stage 4, formation of the second commissure. Venous valve formation through these stages are shown in schematic form below, with the developing leaflets shown in blue. For stage 4 valve, only 1 of the leaflets is clearly visible. (B) Distribution of stage 0–stage 4 valves in mice of different ages (E18 to P42, n = 89, 143 valves; >P42, n = 12). Boxes denote interquartile range; lines within boxes denote median; whiskers denote range. Extreme outliers were excluded. (C) Percent stage 0–stage 4 valves at the indicated ages. (D) SEM images of resin cast of a stage 3 valve. Shown are 2 images from opposite sides of the same valve to demonstrate the single commissure stage in an adult mouse. Asterisks mark the entrance of a tributary. (E) SEM image of resin cast of a stage 4 valve (representative of 10 valves at various anatomical positions). Scale bars: 250 μm (A, photographs); 50 μm (A, SEM images); 200 μm (D and E).
Figure 3
Figure 3. Unique molecular identity of venous valve endothelial cells.
(AL) Immunofluorescence staining of murine venous valves using antibodies against integrin-α9 (A, B, D, E, and G), Prox1 (D, F, J, and K), and Fn-EIIIA (G and H) at the indicated stages. Visualization of ephrin-B2 and Vegfr3 expression was achieved using reporter mice expressing nuclear GFP (Efnb2GFP; A, C, J, and K) or β-gal (Vegfr3lacZ; I and L), respectively. (K) Higher-magnification view of the boxed region in J. Note high expression of Prox1 in ephrin-B2–negative cells on the free edges of valve leaflets (asterisk). (I and L) Vegfr3lacZ reporter activity was present in developing (I) but not in adult (L; asterisk) valves. Arrows denote an adjacent lymphatic vessel; dotted lines outline the iliac vein. (M and N) Immunostaining of adult human venous valve using antibodies against PROX1. (N) Higher-magnification view of the boxed region in M. Arrow denotes a Prox1-positive (dark blue) endothelial cell on the free edge of the valve leaflet; arrowhead denotes a cell on the wall of the vein. Scale bars: 50 μm (AJ and L); 20 μm (K and N); 100 μm (M).
Figure 4
Figure 4. Prox1 expression in developing and mature venous valves allows genetic targeting of valve endothelial cells in Prox1-CreERT2 mice.
(A and B) Immunofluorescence of ear skin from 3-week-old R26-mTmG;Prox1-CreERT2 (Prox1::GFP) mouse administered 4-OHT at P1. Arrow, lymphatic vessel (GFP+PECAM-1+); arrowhead, blood vessel (GFPPECAM-1+). (C) Expression of GFP in venous valve of P7 R26-mTmG;Prox1-CreERT2 mouse administered 4-OHT at P1. (DI) Immunofluorescence staining of iliac veins of R26-mTmG;Prox1-CreERT2 (D and G) and Efnb2GFP (E, F, H, and I) mice at the indicated times using Prox1 antibodies (red). Note GFP expression in the developing valve (arrow) and in vessel wall (asterisk) in R26-mTmG;Prox1-CreERT2 mice after 4-OHT treatment at P0 (D), but more restricted expression in the valve after treatment at P2 (G, arrow). (E, F, H, and I) Arrows, developing venous valves; arrowheads, adjacent lymphatic vessels. (J and K) Immunofluorescence of iliac vein (J) and ear skin (K) from a 4-week-old R26-mTmG;Prox1-CreERT2 mouse fed tamoxifen-containing diet for 2 weeks. (J) Arrows, GFP+ cells on the free edges of valve leaflets; asterisk, recombination in some individual endothelial cells on the vein wall. (K) Arrow, lymphatic vessel (GFP+PECAM-1+); arrowhead, blood vessel (GFPPECAM-1+). Scale bars: 50 μm (AI and K); 300 μm (J).
Figure 5
Figure 5. Integrin-α9 and ephrin-B2 are required for the development of venous valves.
(A and B) SEM images of venous valves (arrows) in P6 WT (A) and Itga9–/– (B; 5 valves) mice. (CF) SEM images of venous valves in control (C; 10 valves), Itga9lx/lx;Prox1-CreERT2 (D; 7 valves), Fn-EIIIA–/– (E; 8 valves), and Efnb2lx/lx;Prox1-CreERT2 (F; 10 valves) mice at P11. Arrows, valves in external iliac veins; arrowheads, valves in internal iliac veins. Note the rudimentary valves in the external iliac vein and absent valves (asterisks) in the internal iliac vein in the mutant mice. (GL) Immunofluorescence staining of iliac veins of R26-mTmG;Prox1-CreERT2 (G and H), Itga9lx/lx; Prox1-CreERT2 (I and J; n = 2), and Efnb2lx/lx;R26-mTmG;Prox1-CreERT2 (K and L; n = 3) mice at P7, after administration of 4-OHT at P2, using an antibody against Prox1 (red). PECAM-1 antibodies were used to visualize endothelial cells in I. Note the endothelial cells on the free ends of the valve leaflets (arrows) showed colocalization of GFP and strong Prox1 immunolabeling (G and H), reduced number of Prox1-positive cells in Itga9 mutants (I and J), and loss of Prox1-positive cells in Efnb2 mutants (K and L). Asterisks in K and L denote a lymphatic vessel. Scale bars: 100 μm.
Figure 6
Figure 6. Differential requirement of integrin-α9 and ephrin-B2 for the maintenance of venous and lymphatic valves.
(AF) SEM images of venous valves of adult control and Itga9 and Efnb2 mutant mice (3 valves per genotype) after oral administration of tamoxifen for 2 weeks. (B, D, and F) Higher-magnification views of the boxed regions in A, C, and E, respectively. Arrows denote valve leaflets. (GL) Immunofluorescence of ears of control Prox1-CreERT2 (G and H), Itga9lx/lx;Prox1-CreERT2 (I and J) and Efnb2lx/lx;Prox1-CreERT2 (K and L) adult mice (n = 3 per genotype) using antibodies against PECAM-1 (green) and podoplanin (red). Arrows denote valves. (H, J, and L) Higher-magnification views of boxed regions in G, I, and K, respectively. (M and N) Immunofluorescence of ears of control Prox1-CreERT2 (M) and Itga9lx/lx;Prox1-CreERT2 (N) adult mice after oral administration of tamoxifen for 2 weeks, using antibodies against PECAM-1 (red) and integrin-α9 (green). Note the reduced expression of integrin-α9 in the Itga9 mutants. (O) Number of lymphatic valves in the ear skin of adult control and Itga9 and Efnb2 mutant mice. Data represent mean ± SD valves in 0.5-mm2 dermal area (n = 3 per genotype, 2 random areas per mouse). **P = 0.005, ***P < 0.001, t test. Scale bars: 500 μm (A, C, and E); 50 μm (B, D, F, G, I, and K); 20 μm (H, J, L, M, and N).

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