Influence of the oxygen microenvironment on the proangiogenic potential of human endothelial colony forming cells

Abstract

Therapeutic angiogenesis is a promising strategy to promote the formation of new or collateral vessels for tissue regeneration and repair. Since changes in tissue oxygen concentrations are known to stimulate numerous cell functions, these studies have focused on the oxygen microenvironment and its role on the angiogenic potential of endothelial cells. We analyzed the proangiogenic potential of human endothelial colony-forming cells (hECFCs), a highly proliferative population of circulating endothelial progenitor cells, and compared outcomes to human dermal microvascular cells (HMVECs) under oxygen tensions ranging from 1% to 21% O2, representative of ischemic or healthy tissues and standard culture conditions. Compared to HMVECs, hECFCs (1) exhibited significantly greater proliferation in both ischemic conditions and ambient air; (2) demonstrated increased migration compared to HMVECs when exposed to chemotactic gradients in reduced oxygen; and (3) exhibited comparable or superior proangiogenic potential in reduced oxygen conditions when assessed using a vessel-forming assay. These data demonstrate that the angiogenic potential of both endothelial populations is influenced by the local oxygen microenvironment. However, hECFCs exhibit a robust angiogenic potential in oxygen conditions representative of physiologic, ischemic, or ambient air conditions, and these findings suggest that hECFCs may be a superior cell source for use in cell-based approaches for the neovascularization of ischemic or engineered tissues.

Fig. 1

a hECFC and HMVEC proliferation on collagen I. *P < 0.05 versus same cell type cultured in the absence of collagen. Data are mean ± SE (n = 3). b Proliferation of hECFCs and HMVECs at varying oxygen tensions. *P < 0.001 versus HMVECs at corresponding oxygen tension. Data are mean ± SE (n = 6)

Fig. 2

Fold increase in cell migration when exposed to a chemotactic gradient at varying oxygen tensions. Data are mean ± SE (n = 4). *P < 0.05 versus HMVECs at same O₂ tension

Fig. 3

Fluorescence microscopy images of transwell undersides reveal calcein-stained hECFCs (a, b) and HMVECs (c, d) that crossed the barrier with (a, c) or without (b, d) a chemotactic gradient in 1% oxygen. Images are representative of four independent experiments. Scale bars represent 200 μm

Fig. 4

Tubulogenesis with hECFCs (a–c) and HMVECs (d–f) cultured at 1, 5, or 21% oxygen. Images are representative of four independent experiments. Scale bars represent 200 μm

Fig. 5

Apoptotic response of hECFCs and HMVECs under varying oxygen tensions. *P < 0.05 versus same cell type at 5 or 21% O₂. Data are mean ± SE (n = 4). **P < 0.05 versus HMVECs at 21% O₂

Fig. 6

Acetylated LDL uptake by hECFCs (a–c) and HMVECs (d–f) at 1, 5, and 21% oxygen. Images are representative of five independent experiments. Scale bars represent 200 μm

Fig. 7

Expression of HIF-1α in hECFCs and HMVECs under varying oxygen tensions after 4 h. Bands represent protein expression at 1, 5, and 21% oxygen, or in the presence of CoCl₂ (left to right). Lower band represents α-tubulin expression