Endothelial Precursor Cells (EPCs) of Diabetic and Nondiabetic Origin are Differentally Modulated by Hypoxia Inducible Factors Present in Serum

Abstract

Purpose: : EPCs are circulating bone marrow–derived cells that are exquistely sensitive to hypoxia. The aim of this study was to compare the migratory response and receptor profile of EPCs of diabetic vs. nondiabetic origin.

Methods: : CD 34⁺ EPCs were isolated from individuals with proliferative diabetic retinopathy (PDR) and age–matched nondiabetic controls. The hypoxia–regulated factors tested included VEGF, IGF–1, stromal derived growth factor (SDF–1), IGF binding protein– 3 (IGFBP–3) and the adenosine analogue, 5'–N–ethylcarboxamido–adenosine (NECA). Each factor was assessed using the modified Boyden chamber migration assay and was tested from a concentration of 1 nM to 10 µM. VEGF receptor (VEGFR)–1, VEGFR–2 and adenosine receptor surface expression was determined by FACS and by real time PCR. EPC differentiation was determined by loss of CD133.

Results: : Diabetic EPCs demonstrated defective migration in response to VEGF, IGF–1 and SDF–1 compared to nondiabetic controls. However, both diabetic and nondiabetic EPCs migrated in a dose–dependent manner to NECA [p<0.05] with diabetic EPCs showing a much greater response than nondiabetic EPCs [p<0.001]. EPCs from patients with PDR demonstrated increased mRNA and protein levels of the adenosine A_2b receptor, known to modulate pathological neovascularization, compared to nondiabetic controls that showed increased A_2a receptor, the receptor known to modulate the beneficial vasodilator effects of adenosine. Exposure to SDF–1, IGF–1 or NECA resulted in a dose– and time–dependent increase in VEGFR–1 and VEGFR–2 with the increase VEGFR–2 being greater than VEGFR–1 in both diabetic and control EPCs. Exposure of EPCs of diabetic as well as nondiabetic origin to IGFBP–3, the major IGFBP found in serum and a hypoxia regulated protein, resulted in loss of CD133 and differentiation into endothelial cells.

Conclusions: : In diabetic and nondiabetic EPCs, SDF–1, IGF and NECA play a critical role in regulating VEGF receptor expression and IGFBP–3 promotes EPC differentiation. The adenosine receptor profile on EPCs of diabetic patients promotes an angiogenic phenotype. Diabetic EPCs retain their ability to migrate in response to adenosine, unlike the other factors tested. In diabetic EPCs signaling mechanisms responsible for cytoskeletal changes leading to migration are defective in response to growth factors but are intact in response to nucleotides.

Keywords: growth factors/growth factor receptors • diabetes • differentiation