Diabetic impairments in NO-mediated endothelial progenitor cell mobilization and homing are reversed by hyperoxia and SDF-1α
Katherine A. Gallagher, … , Stephen R. Thom, Omaida C. Velazquez
Katherine A. Gallagher, … , Stephen R. Thom, Omaida C. Velazquez
Published May 1, 2007
Citation Information: J Clin Invest. 2007;117(5):1249-1259. https://doi.org/10.1172/JCI29710.
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Research Article

Diabetic impairments in NO-mediated endothelial progenitor cell mobilization and homing are reversed by hyperoxia and SDF-1α

Abstract

Endothelial progenitor cells (EPCs) are essential in vasculogenesis and wound healing, but their circulating and wound level numbers are decreased in diabetes. This study aimed to determine mechanisms responsible for the diabetic defect in circulating and wound EPCs. Since mobilization of BM EPCs occurs via eNOS activation, we hypothesized that eNOS activation is impaired in diabetes, which results in reduced EPC mobilization. Since hyperoxia activates NOS in other tissues, we investigated whether hyperoxia restores EPC mobilization in diabetic mice through BM NOS activation. Additionally, we studied the hypothesis that impaired EPC homing in diabetes is due to decreased wound level stromal cell–derived factor–1α (SDF-1α), a chemokine that mediates EPC recruitment in ischemia. Diabetic mice showed impaired phosphorylation of BM eNOS, decreased circulating EPCs, and diminished SDF-1α expression in cutaneous wounds. Hyperoxia increased BM NO and circulating EPCs, effects inhibited by the NOS inhibitor N-nitro-l-arginine-methyl ester. Administration of SDF-1α into wounds reversed the EPC homing impairment and, with hyperoxia, synergistically enhanced EPC mobilization, homing, and wound healing. Thus, hyperoxia reversed the diabetic defect in EPC mobilization, and SDF-1α reversed the diabetic defect in EPC homing. The targets identified, which we believe to be novel, can significantly advance the field of diabetic wound healing.

Authors

Katherine A. Gallagher, Zhao-Jun Liu, Min Xiao, Haiying Chen, Lee J. Goldstein, Donald G. Buerk, April Nedeau, Stephen R. Thom, Omaida C. Velazquez

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Figure 1

Impaired phosphorylation of BM eNOS with attenuation of HBO-induced NO levels results in decreased circulating EPCs in diabetic mice.

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Impaired phosphorylation of BM eNOS with attenuation of HBO-induced NO l...
(A) Representative Western blot analysis for BM eNOS. Diabetic mice demonstrated decreased phosphorylated eNOS compared with nondiabetic controls. Insulin failed to restore impaired eNOS phosphorylation. Quantification of phospho-eNOS (p-eNOS). Results are based on 4 experiments and show the amount of phospho-eNOS relative to total eNOS and β-actin. Nondiabetic controls are used as the standard (value set at 100). *P < 0.01. (B) Changes in cell composition in BM of diabetic mice. EPC (VEGFR2/CXCR4) and HSC (CD34/CD45) populations are unchanged, while mesenchymal stromal (CD73/CD44) and inflammatory cell (SSC/CD3/CD45RA) populations are slightly decreased in diabetic mice compared with nondiabetic mice. Percentages indicate positive cells in total BM cells counted. (C) Quantification of the number of Tie2+/VEGFR2+ EPCs/μl of peripheral blood in Tie2-GFP mice by flow cytometry at 7 days following STZ treatment. A substantial reduction in circulating EPCs was found in diabetic compared with nondiabetic mice. (D and E) NO production in the BM cavity of diabetic and nondiabetic mice during 10 minutes of HBO treatment. Baseline NO levels were obtained 5 minutes prior to onset of pressurization at 100% O2 (gray bar). Solid lines represent mean values, with surrounding gray or black shading representing SEM. (D) Hyperoxia increases BM NO levels significantly, but the NO response is attenuated in diabetic mice compared with nondiabetic animals (P < 0.05). Insulin did not reverse the impairment of NO production. (E) Total iNOS and nNOS proteins were upregulated in diabetic mice. –, nondiabetic mice; +, diabetic mice. (F) Complete inhibition of BM NO production in diabetic and nondiabetic mice undergoing HBO treatment after pretreatment with l-NAME.