Abstract 35: Integrin alpha-5 beta-1 and Fibronectin Receptor-Ligand Pair Determines Reparative and Neovascularization Potential of Endothelial Progenitor Cells
Endothelial Progenitor Cell (EPC)-based therapy may be useful for the treatment of microvascular disorders associated with ischemic diseases and for therapeutic neovascularization. Here, we addressed whether mouse bone marrow derived EPCs (mEPCs) express integrins, synthesize and deposit fibronectin, requirements for EPC engraftment in injured microvessels. We used anti-CD34, anti-Flk-1, and anti-VE-cadherin antibodies as mEPC markers. Ex vivo expanded mEPCs were analyzed by fluorescent microscopy and Western blotting for expression of integrins, fibronectin, and von Willebrand Factor (ligands for integrins alpha-5 beta-1 and alpha-v beta-3). Fibronectin and vWF expression of mEPCs was compared with adult mouse lung endothelial cells (mECs). Fluorescent microscopy showed markedly increased fibronectin and vWF staining and Western blotting showed >20-fold increase in fibronectin and vWF expression in mEPCs compared with mECs. The appearance of polymerized fibronectin matrix suggested that mEPCs synthesized, deposited, and assembled fibronectin. Next, ex vivo expanded mEPCs were subjected to shRNA-mediated silencing of alpha-5 integrin, fibronectin and vWF genes. Silencing of alpha-5 integrin and fibronectin genes reduced the proliferative potential of mEPCs whereas vWF silencing had no effect. To evaluate the effectiveness of mEPCs treatment, we irradiated C57BL/6J mice (5.0 Gy) and challenged the mice i.p. with bacterial lipopolysaccharide (LPS) to induce endothelial barrier injury and pulmonary edema. Infusion of mEPCs induced greater survival (~50% at 7.5 mg of LPS/kg BW, n=6) and prevented pulmonary edema resulting from microvessel injury; in contrast, mECs infusion had no protective effect. Thus, EPC expression of alpha-5 beta-1 integrin and secretion of fibronectin matrix may be crucial determinants of engraftment of EPCs at sites of vascular injury, and their ability to promote repair of injured microvessels and neovascularization.