The regulation of endothelial nitric oxide synthase by extracellular matrix in human late outgrowth endothelial progenitor cells

摘要

Introduction: There is a need for a readily available source of functional autologous endothelial cells to cover blood contacting surfaces in order to induce and maintain homeostatic environment (reviewed in). A subpopulation of circulating, bone-marrow-derived cells, termed endothelial progenitor cells (EPCs), can potentially contribute to neovascularization and home to sites of vascular trauma participating in re-endothelialization of damaged or denuded surfaces, If maintained in culture these EPCs undergo a transition to cells with homologous gene expression (e.g. CD31, CD144, CD146, vWF and KDR) to mature endothelial cells and display similar phenotype (proliferative, cobblestone morphology, and contact inhibited growth). These blood derived endothelial cells have been termed late outgrowth EPCs (LEPCs). Endothelial nitric oxide synthase (eNOS) is an important factor that regulates homeostasis at the blood contacting surface and loss of eNOS activity is a hallmark of endothelial dysfunction. Here, we examined the expression and activity of eNOS and elucidated the cause of differential regulation of eNOS in LEPCs compared to mature endothelial cells. Methods and Results: We found that LEPCs express markedly lower eNOS protein (0.34 ±0.13, Western blot), mRNA (0.29 ±0.17, qRT-PCR) as well as activity levels (0.49 ±0.18, Nitrite analysis) when compared to Human Umbilical Vein Endothelial cells (HUVECs) or Human Aortic Endothelial Cells (HAECs). When grown on fibronectin (FN), type Ⅰ collagen (Col. Ⅰ), and laminin (LAMA) we found significantly decreased eNOS protein in HUVECs (0.52 ± 0.08 fold change on FN, 0.49 ± 0.07 on Col. Ⅰ and 0.53 ± 0.07 on LAMA) compared to cells on polystyrene, these findings were associated with decreased eNOS mRNA. The matrix mediated downregulation was blocked by β1 integrin siRNA and focal adhesion kinase siRNA transfection. In addition, rho-associated protein kinase (ROCK) inhibitors including fasudil and Y27632 blocked the effect of ECM on eNOS downregulation in HUVECs. Interestingly, when cultured on polystyrene, LEPCs express significantly more focal adhesion sites, extracellular matrix (ECM) proteins especially Col. Ⅰ and lower MMP-2 activity compared to HUVECs and HAECs. Blocking Col. Ⅰ synthesis (siRNA) or treatment with Y27632 were found significantly enhanced eNOS expression in LEPCs (1.77 ± 0.41 and 1.39 ± 0.11 fold increase for Col. Ⅰ siRNA and Y27632, respectively). Increased eNOS level further improved cell survival in LEPCs after serum deprivation. Conclusions: Taken together, our results suggest LEPCs contain lower eNOS level compared to mature endothelial cells due to their higher ECM (e.g. Col. 1) deposition and demonstrate that blockage of ECM synthesis or cell-ECM interactions could help to develop functional endothelialized surfaces.