Nitric Oxide Induces Angiogenesis and Upregulates αvβ3 Integrin Expression on Endothelial Cells

Abstract

Nitric oxide (NO) has been implicated as a mediator of angiogenesis. However, its precise role in angiogenesis and its mechanism of action have not been established. We performed in vivo and in vitro angiogenesis assays using NO donor S-nitroso-N-acetylpenicillamine (SNAP) and NO synthase inhibitor N-iminoethyl-l-ornithine (L-NIO). SNAP significantly increased and L-NIO significantly suppressed capillary ingrowth into subcutaneously implanted Matrigel plugs in mice. For the in vitro angiogenesis assay, human umbilical vein endothelial cells (HUVECs) (4 × 104 cells/well) were treated with placebo, SNAP (100 μM), or L-NIO (100 μM) and cultured on Matrigel for 18 h. The typical capillary networks formed on Matrigel by HUVECs as a result of cell migration and differentiation were quantified by computer-assisted image analysis as a measure of angiogenesis. Treatment of HUVECs with SNAP significantly increased the capillary network area compared with control, 8701 ± 693 vs 6258 ± 622 area units (P < 0.05), whereas L-NIO significantly decreased the capillary area (4540 ± 342, P < 0.05). Furthermore, we have shown with a blocking monoclonal antibody that formation of capillary networks on Matrigel is mediated by the functional expression of the αvβ3 integrin, which plays a role in facilitating endothelial cell adhesion to basement membrane matrix and endothelial cell migration. After an 18-h culture, flow cytometry revealed that SNAP significantly upregulated and L-NIO significantly downregulated in a concentration-dependent manner αvβ3 integrin expression on endothelial cells. In conclusion, NO induces angiogenesis in vivo and in vitro by promoting endothelial cell migration and differentiation into capillaries. One possible mechanism might involve the upregulation of αvβ3 integrin on endothelial cells, a critical mediator of cell–matrix adhesion and migration.