Nearly 12 million Americans suffer from peripheral arterial disease (PAD). Many PAD patients are treated with titanium (Ti) coated Nitinol stents. However, these stents carry a high risk of in-stent restenosis and thrombosis. We recently showed that autologous late outgrowth endothelial progenitor cells (EPCs) harvested from peripheral blood can be seeded onto Ti tubes within minutes of implantation into pigs' blood vessels, and that they prevent thrombosis in vivo. However, clinical application of EPCs has been limited by the low yield from relatively large volumes of adult blood and EPCs' low proliferative potential in culture. Thus, our overall goal is to be able to isolate increased numbers of highly proliferative and functional EPCs from blood of PAD patients. We hypothesize that key growth factors and cytokines act in synergy with functionalized substrates to create a microenvironment that 1) stimulates EPC proliferation by upregulating integrins aVb3 and a5b1 and that 2) increases both the yield of EPCs from blood and their functionality when subjected to fluid shear stress on Ti surfaces by increasing expression of endothelial cell-specific genes.Aim I of our study is to define factors and substrates of an optimal microenvironment for EPC proliferation and to establish a crucial role for integrins aVb3 and a5b1 in EPC proliferation. We plan to utilize a high-throughput assay with an innovative adaptive search algorithm to identify synergistic effects of key growth factors, cytokines and functionalized substrates that increase EPC proliferation. To establish the role of integrins aVb3 and a5b1, we will quantify EPC integrin expression levels and the mitogen-activated protein kinase Erk1/2 activation. Lastly, we will use shRNA to knockdown integrins aV and a5 to assess the effect on EPC proliferation.In Aim II, we will use small volumes of blood from PAD patients to quantify EPC outgrowth and proliferation in our optimized microenvironment and to characterize resulting EPC phenotypes. We will then evaluate EPC functionality and gene expression under fluid shear stress on Ti. The knowledge gained will deepen our understanding of the mechanisms of EPC proliferation and function.If successful, we will have developed a technology to derive large numbers of highly proliferative and functional EPCs from small volumes of blood and characterized this cell population as a personalized tool to coat Ti implants for patients with PAD.
|Program type||Beginning Grant-in-Aid|
|Effective start/end date||07/01/2012 → 06/30/2014|