Umbilical cord blood derived endothelial progenitor cells: Isolation, characterization, and adhesion potential in vitro and in vivo.

摘要

The number one cause of death in the industrialized world, atherosclerosis, can be treated through a variety of methods: angioplasty, stenting, vein graft bypass, synthetic grafts, and maybe one day tissue-engineered blood vessels (TEBVs). The long-term goal that motivated this research is to deliver umbilical cord blood-derived endothelial progenitor cells (CB-EPCs) to damaged arteries, and thereby reduce the rate of occlusion by re-establishing a healthy, functional, intact endothelium. The proposed research tested the following hypotheses: (1) Mild trypsinization methods produces strong endothelial cell (EC) adhesion strength, (2) CB-EPCs are functionally similar to native ECs (specifically human aortic endothelial cells (HAECs)) and exhibit similar antithrombotic and anti-inflammatory behavior compared to HAECs, (3) CB-EPCs are capable of adhering to smooth muscle cells (SMCs) and extracellular matrix (ECM) proteins under flow conditions, (4) CB-EPCs can be used to prevent thrombosis in mice that have undergone vein bypass grafts through re-endothelialization of the vessel, and (5) CB-EPCs are capable of proliferating under flow conditions. In order to produce supraphysiological adhesion strengths of HAECs or CB-EPCs, the cells must be detached using 0.025% trypsin for 5 minutes prior to adhesion to adsorbed ECM proteins or SMCs. CB-EPCs have a high proliferation rate and express similar levels of important anti-thrombotic genes and inflammatory proteins compared to HAECs. CB-EPCs and HAECs produce similar levels of nitric oxide and alignment in the direction of flow when exposed to laminar shear stress for at least 24 hours. CB-EPCs are capable of dynamic adhesion to many different substrates under flow conditions. The adhesion of CB-EPCs with response to shear stress appears to be biphasic and increases with shear stress up to 0.75 dyn/cm2 and then decreases above this value. CB-EPC adhesion is much greater than HAECs and EPCs isolated from peripheral blood (PB-EPCs) of healthy individuals, which can be related to their higher expression level of adhesion integrin alpha5beta1 and/or their smaller size. When seeded onto FN coated plastic, CB-EPCs proliferated under flow conditions and had a much shorter doubling time than PB-EPCs and HAECs. Proliferation of CB-EPCs and HAECs on SMCs was limited. Further, CB-EPCs formed network-like structures except when growth factors were removed and a shear stress of at least 5 dyne/cm2 was applied. To assess whether CP-EPCs could promote vessel repair in vivo, human CB-EPCs were injected into SCID mice that received a carotid interpositional vein grafts, resulting in 100% patency. In contrast, only 2 of the 8 saline injected mice had a patent vein graft 2 weeks post surgery. We found that CB-EPC injected mice had roughly 55% endothelialization compared to less than 20% for the patent saline controls, with CB-EPCs making up approximately 33% of this coverage. These results suggest that CB-EPCs could be used as a therapeutic method to prevent vessel occlusion in patients undergoing vein graft bypass surgery.