Hemodynamic regulation of perivalvular endothelial gene expression prevents deep venous thrombosis
John D. Welsh, Mark H. Hoofnagle, Sharika Bamezai, Michael Oxendine, Lillian Lim, Joshua D. Hall, Jisheng Yang, Susan Schultz, James Douglas Engel, Tsutomu Kume, Guillermo Oliver, Juan M. Jimenez, Mark L. Kahn
John D. Welsh, Mark H. Hoofnagle, Sharika Bamezai, Michael Oxendine, Lillian Lim, Joshua D. Hall, Jisheng Yang, Susan Schultz, James Douglas Engel, Tsutomu Kume, Guillermo Oliver, Juan M. Jimenez, Mark L. Kahn
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Research Article Vascular biology

Hemodynamic regulation of perivalvular endothelial gene expression prevents deep venous thrombosis

Abstract

Deep venous thrombosis (DVT) and secondary pulmonary embolism cause approximately 100,000 deaths per year in the United States. Physical immobility is the most significant risk factor for DVT, but a molecular and cellular basis for this link has not been defined. We found that the endothelial cells surrounding the venous valve, where DVTs originate, express high levels of FOXC2 and PROX1, transcription factors known to be activated by oscillatory shear stress. The perivalvular venous endothelial cells exhibited a powerful antithrombotic phenotype characterized by low levels of the prothrombotic proteins vWF, P-selectin, and ICAM1 and high levels of the antithrombotic proteins thrombomodulin (THBD), endothelial protein C receptor (EPCR), and tissue factor pathway inhibitor (TFPI). The perivalvular antithrombotic phenotype was lost following genetic deletion of FOXC2 or femoral artery ligation to reduce venous flow in mice, and at the site of origin of human DVT associated with fatal pulmonary embolism. Oscillatory blood flow was detected at perivalvular sites in human veins following muscular activity, but not in the immobile state or after activation of an intermittent compression device designed to prevent DVT. These findings support a mechanism of DVT pathogenesis in which loss of muscular activity results in loss of oscillatory shear–dependent transcriptional and antithrombotic phenotypes in perivalvular venous endothelial cells, and suggest that prevention of DVT and pulmonary embolism may be improved by mechanical devices specifically designed to restore perivalvular oscillatory flow.

Authors

John D. Welsh, Mark H. Hoofnagle, Sharika Bamezai, Michael Oxendine, Lillian Lim, Joshua D. Hall, Jisheng Yang, Susan Schultz, James Douglas Engel, Tsutomu Kume, Guillermo Oliver, Juan M. Jimenez, Mark L. Kahn

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Figure 1

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

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FOXC2- and PROX1-expressing endothelial cells around the venous valve ex...
(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.