Extracellular Matrix Differentially Regulates Endothelial Nitric Oxide Synthase Production in HUVECs and Human Blood Outgrowth Endothelial Progenitor Cells

Abstract

Human outgrowth endothelial progenitor cells (BOECs), derived from peripheral blood mononuclear express endothelial protein profiles (i.e. CD31, 144, and vWF). Endothelial nitric oxide synthase (eNOS) is an important regulator of vascular tone and loss of eNOS activity is a hallmark of endothelial dysfunction. In this study, we examine the expression and activity of eNOS and elucidate the cause of differential regulation of eNOS in BOECs compared to mature endothelial cells. We found that BOECs express markedly lower eNOS protein (0.34 ± 0.13), mRNA (0.29 ± 0.17) as well as activity levels (0.49 ± 0.18) when compared to HUVECs or Human Aortic Endothelial Cells. When grown on fibronectin (FN), type I collagen (Col. I), and type IV collagen (Col. IV) we found significantly decreased eNOS protein, mRNA and mRNA stability in HUVECs compared to cells on polystyrene. The matrix mediated downregulation was blocked by β1 integrin siRNA and focal adhesion kinase siRNA transfection. In addition, rho‐associated protein kinase inhibitors including fasudil and Y27632 blocked the effect of ECM on eNOS downregulation in HUVECs. In contrast, in BOECs, eNOS protein expression was unchanged by cell‐ECM interactions. Interestingly, BOECs can highly deposit ECM molecules (Col. I, FN and Laminin) that assemble to an organized mesh‐like structure whereas HUVECs only express few ECM proteins which cannot form organized structures. Blocking Col. I synthesis with siRNA significantly enhanced eNOS expression in BOECs (1.77 ± 0.41 fold increase). Taken together, our results confirm a strong matrix mediated regulation of eNOS in endothelial cells and suggest that limited eNOS expression in BOECs results from higher ECM production.