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

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.