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-AF-coated glass slides. Late outgrowth CB-EPCs expressed endothelial cell-specific 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 alpha(5)beta(1) and alpha(v)beta(3) integrins and antithrombotic genes compared to HAECs. There were some differences in mRNA levels of E-selectin 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-alpha 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.

FIG. 1.

Morphology of CB-EPCs compared to HAECs. (A) Phase contrast image of CB-EPCs seeded onto FN-coated Teflon-AF. (B) Phase contrast image of HAECs seeded onto FN-coated Teflon-AF. (C) CB-EPCs stained with VE-cadherin. (D) HAECs stained with VE-cadherin. CB-EPCs, umbilical cord blood–derived endothelial progenitor cells; HAEC, human aortic endothelial cells; FN, fibronectin.

FIG. 2.

CB-EPC and HAEC spreading. Spreading of CB-EPCs and HAECs on either FN-coated Teflon-AF or SMCs at 1 and 24 h. Cell area is greater at 24 h (*p < 0.001) and on FN-coated Teflon-AF compared to SMCs (p < 0.01), and, overall, HAECs are significantly larger than CB-EPCs (^#p < 0.05; n = 3, standard error based off of the n = 3). SMC, smooth muscle cell.

FIG. 3.

CB-EPC and HAEC strength of adhesion. CB-EPCs and HAECs were adhered to either FN-coated Teflon-AF or SMCs for 20 min before exposure to 2 min of shear stress, and the percentage of cells that remained adherent was determined (n = 4).

FIG. 4.

Integrin expression and blocking by CB-EPCs and HAECs. (A) Relative amount of adhesion integrins present on CB-EPCs and HAECs determined by flow cytometry. CB-EPCs express a significantly higher level of α₅β₁ compared to HAECs (*p < 0.05; n = 4). (B) Number of adherent cells after blocking α₅β₁, α_Vβ₃, or both α₅β₁ and α_Vβ₃ integrins with blocking antibodies. α₅β₁ significantly decreased adhesion compared to the control (no blocking antibodies) (*p < 0.05), both α₅β₁ and α_Vβ₃ significantly decreased adhesion compared to the control (*p < 0.01), and both α₅β₁ and α_Vβ₃ significantly decreased adhesion compared to α₅β₁ (^#p < 0.05).

FIG. 5.

CB-EPC and HAEC alignment under long-term flow. (A) Orientation and (B) elongation of CB-EPCs and HAECs on FN-coated Teflon-AF surfaces when exposed to 48 h of 15 dyn/cm² shear stress and under static conditions (0° is perfectly aligned with the direction of flow, 0 is a perfect line, and 1 is a perfect circle), *p < 0.001 compared to static conditions (n = 4).

FIG. 6.

CB-EPC and HAEC expression of adhesion molecules as measured by flow cytometry after exposure to static and/or flow conditions on both FN-coated Teflon-AF or SMCs and either unstimulated or stimulated with TNF-α for 4.5 h. (A) Expression of intracellular adhesion molecule 1. (B) Expression of E-Selectin. (C) Expression of vascular cell adhesion molecule: *p < 0.01 compared to non-TNF-α stimulated conditions, ^#p < 0.01 compared to all other conditions, and ^$p < 0.01 compared to static conditions (n = 4). TNF, tumor necrosis factor.

FIG. 6.

CB-EPC and HAEC expression of adhesion molecules as measured by flow cytometry after exposure to static and/or flow conditions on both FN-coated Teflon-AF or SMCs and either unstimulated or stimulated with TNF-α for 4.5 h. (A) Expression of intracellular adhesion molecule 1. (B) Expression of E-Selectin. (C) Expression of vascular cell adhesion molecule: *p < 0.01 compared to non-TNF-α stimulated conditions, ^#p < 0.01 compared to all other conditions, and ^$p < 0.01 compared to static conditions (n = 4). TNF, tumor necrosis factor.

FIG. 7.

CB-EPC and HAEC eNOS protein expression. eNOS protein expression was measured from CB-EPCs and HAECs on FN-coated Teflon-AF either exposed to 15 dyn/cm² for 24 h or under static conditions: *p < 0.05, compared to static conditions, and ^#p < 0.05, compared to CB-EPCs (n = 4). eNOS, endothelial nitric oxide synthase.

FIG. 8.

NO produced by CB-EPCs and HAECs. NO was measured, by total amount of nitrites and nitrates, in the supernatant of CB-EPCs and HAECs on FN-coated Teflon-AF or SMCs either exposed to 15 dyn/cm² of shear stress for 24 h or under static conditions, *p < 0.001, compared to static conditions (n = 4).