Diabetic Impairment of Nitric Oxide Generation in CD34 ⁺ Hematopoietic Stem/ Progenitor Cells is mediated by TGF‐β1/TSP‐1/CD47 pathway

Abstract

Circulating CD34+ hematopoietic stem progenitor cells (HSPCs) stimulate vasculogenesis and play an important role in the ischemic vascular repair. Long‐term diabetes is associated with impaired vasculogenic potential of HSPCs, which is at least in part due to decreased nitric oxide (NO) generation. Transforming growth factor‐ β1 (TGF‐β1) has pleiotropic functions in CD34+ HSPCs and is known to stimulate the expression of matrix protein thrombospondin‐1 (TSP‐1). Previous studies have shown that transient silencing of TGF‐β1 restores NO generation and improves migratory functions in diabetic CD34+ HSPCs. In this study, we tested the hypothesis that diabetic dysfunction in NO generation is mediated by activation of TSP‐1/CD47 receptor pathway. CD34+ HSPCs were isolated from peripheral blood mononuclear cells obtained from either male or female nondiabetic (ND, n=63) or diabetic (type 1 and type 2) (DB, n=51) individuals of age 38–85 years. TGF‐β1 expression was transiently blocked by using TGFβ1‐antisense delivered in the form of phosphorodiamidate morpholino oligomer (PMO‐TGFβ1). CD47 expression was blocked by siRNA approach. Migration and proliferation of cells were determined by chemotaxis assay and BrdU‐colorimetric ELISA, respectively. Activation of eNOS was evaluated by determining phosphorylation at Ser1177 and Thr495 by using fluorescent‐conjugated antibodies and flow cytometry. Expression of TGF‐β1 and TSP‐1 mRNA were higher in DB CD34+ cells compared to ND cells, which were decreased by PMO‐TGFβ1 (n=18). SDF‐induced migration and proliferation were impaired in DB cells compared to ND (P<0.05, n=5) that were reversed by PMO‐TGFβ1 (n=5). TSP‐1 decreased SDF‐induced migration and proliferation (P<0.05, n=6) that were reversed by knockdown of CD47 (n=6). CD47 siRNA restored SDF‐induced migration and proliferation in diabetic cells in the absence or in the presence of TSP‐1 (P<0.05, n=5). Diabetic cells showed decreased p‐eNOS‐Ser1177 and higher p‐eNOS‐Thr495 in response to SDF compared to ND cells (P<0.05, n=5). TSP‐1 decreased SDF‐induced changes in pSer1177 and pThr495 in ND cells (n=5) that were reversed by CD47 siRNA (n=5). In summary, diabetic impairment of eNOS activation and NO generation are mediated by TGF‐β1/TSP‐1/CD47 pathway.