Characterization of Umbilical Cord Blood–Derived Late Outgrowth Endothelial Progenitor Cells Exposed to Laminar Shear Stress

Abstract

Endothelial progenitor cells isolated from umbilical cord blood (CB-EPCs) represent a promising source of endothelial cells for synthetic vascular grafts and tissue-engineered blood vessels since they are readily attainable, can be easily isolated, and possess a high proliferation potential. The objective of this study was to compare the functional behavior of late outgrowth CB-EPCs with human aortic endothelial cells (HAECs). CB-EPCs and HAECs were cultured on either smooth muscle cells in a coculture model of a tissue-engineered blood vessels or fibronectin adsorbed to Teflon-AFÔ-coated glass slides. Late outgrowth CB-EPCs expressed endothelial cellspecific markers and were negative for the monocytic marker CD14. CB-EPCs have higher proliferation rates than HAECs, but are slightly smaller in size. CB-EPCs remained adherent under supraphysiological shear stresses, oriented and elongated in the direction of flow, and expressed similar numbers of a 5 b 1 and a v b 3 integrins and antithrombotic genes compared to HAECs. There were some differences in mRNA levels of Eselectin and vascular cell adhesion molecule 1 between CB-EPCs and HAECs; however, protein levels were similar on the two cell types, and CB-EPCs did not support adhesion of monocytes in the absence of tumor necrosis factor-a stimulation. Although CB-EPCs expressed significantly less endothelial nitric oxide synthase protein after exposure to flow than HAECs, nitric oxide levels induced by flow were not significantly different. These results suggest that late outgrowth CB-EPCs are functionally similar to HAECs under flow conditions and are a promising cell source for cardiovascular therapies.

Endothelial Outgrowth Cells: Function and Performance in Vascular Grafts

“…As with native ECs, EOCs significantly up-regulate these adhesion proteins compared with their basal state in response to inflammatory cytokines or aberrant flow conditions. The inflammatory response of EOCs can be induced by treating the cells with the cytokine tumor necrosis factor-a (TNF-a), which results in an increase in both ICAM-1 expression 58 and prothrombotic tissue factor expression. 59 In addition, oscillatory fluid shear has a pro-inflammatory effect on EOCs, resulting in increased monocyte adhesion.…”

“…Commonly, eNOS gene or protein expression levels are used as an indicator of NO production. Alternatively, NO can be measured in an in vitro assay quantifying NO metabolites, 58,61 a fluorescent NO indicator probe, 17 or a functionbased NO assay that measures smooth muscle cell relaxation after controlled activation of eNOS. 27 The majority of groups 17,26,35,46,58 though some groups have found no difference between cell types.…”

“…Alternatively, NO can be measured in an in vitro assay quantifying NO metabolites, 58,61 a fluorescent NO indicator probe, 17 or a functionbased NO assay that measures smooth muscle cell relaxation after controlled activation of eNOS. 27 The majority of groups 17,26,35,46,58 though some groups have found no difference between cell types. 36 However, eNOS expression does not directly correlate with NO production, as evidenced by studies demonstrating that EOCs have lower eNOS expression but similar NO production as aortic ECs when stimulated by calcium 17 or fluid flow.…”

“…36 However, eNOS expression does not directly correlate with NO production, as evidenced by studies demonstrating that EOCs have lower eNOS expression but similar NO production as aortic ECs when stimulated by calcium 17 or fluid flow. 58 Intimal hyperplasia after vascular injury can also be reduced by activated Protein C. 62,63 Activated Protein C acts to inhibit inflammation-induced EC apoptosis and permeability, thereby maintaining functional EC regulation of underlying smooth muscle cells. 64 Similar to ECs, EOCs can activate Protein C. 46,65 To enhance Protein C activation, EOCs were engineered to overexpress thrombomodulin via adenoviral transfection.…”

“…46,60 Steady shear causes EOCs to increase eNOS gene expression 37,42,60 and NO production, suggesting an improved capacity to limit intimal hyperplasia. 37,58,61,75 Models that incorporate both fluid shear stress and inflammatory cytokines enable a better characterization of how these factors interact to influence EOC behavior. After 24 h of 6 dyn/cm 2 shear stress preconditioning, TNF-ainduced tissue factor activity of EOCs was reduced compared with non-preconditioned EOCs.…”