Figure 1. FOXC2- and PROX1-expressing endothelial cells around the venous valve exhibit a strong antithrombotic phenotype.

(A) Schematic illustration of mouse venous valve sections defining the regions of luminal endothelium (Lumen endo), valve leaflet endothelium (Valve endo), and valve sinus endothelium (Sinus endo). (B) FOXC2 and PROX1 were measured using anti-FOXC2 and anti-GFP immunostaining of wild-type and PROX1-GFP transgenic mice. Cell nuclei were detected with DAPI staining. White dashed lines indicate luminal venous endothelial cells; green dashed lines indicate perivalvular endothelial cells. Arrows indicate the direction of venous blood flow. Images are representative of 8 separate experiments using different animals. (C–F) Mouse saphenous veins were immunostained to detect expression of vWF (n = 8 valves), THBD (n = 9), EPCR (n = 8), and TFPI (n = 13). Relative quantitation of staining in luminal (L), valvular (V), and sinus (S) endothelial cells is shown at right for each protein. (G and H) Mouse saphenous veins were immunostained to detect expression of the adhesion proteins ICAM1 (n = 7) and P-selectin (n = 7). Relative quantitation of protein levels is shown at right for each protein. (I) P-selectin is not expressed on the surface of perivalvular endothelial cells. Surface P-selectin was detected by i.v. injection of Alexa Fluor 647–labeled anti–P-selectin antibodies into PROX1-GFP transgenic animals. Images are representative of 6 separate experiments in 4 mice. White dashed lines indicate luminal venous endothelial cells, and green dashed lines indicate perivalvular endothelial cells. Arrows indicate the direction of venous blood flow. For each graph the mean is shown as the bar with dots representing each data point, and error bars indicate SD. Significance was determined by ratio paired t test and corrected for multiple comparisons. *P < 0.025; **P < 0.01; ***P < 0.001; ****P < 0.0001.