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R2811 --> 703.3 --> Adult circulating CD34+ stem progenitor cells (HSPCs) participate in vascular repair by stimulating angiogenic functions of the endothelium via paracrine mechanisms. Aging and diabetes are associated with reduced number and impaired vasculogenic potential of CD34+cells. Previous studies showed that diabetic dysfunction was largely due to the paracrine pro‐inflammatory and anti‐angiogenic switch in the phenotype of CD34+ cells. Furthermore, diabetic cells have a higher potential to generate pro‐inflammatory monocyte‐macrophages. We have recently shown that older‐diabetic (DB) cells have increased expression of transforming growth factor β1 (TGF‐β1) compared to the age‐matched nondiabetic (ND) cells. Transient silencing of this pleiotropic regulator restored reparative functions such as migration, proliferation, and nitric oxide generation. The current study tested the hypothesis that TGF‐β1‐silencing reverses myelopoietic bias and paracrine pro‐inflammatory phenotype in CD34+ cells derived from diabetic older adults. Expression of alarmins and receptor for advanced glycation end‐products (RAGE) were evaluated. CD34+ cells were isolated from either male or female non‐diabetic (ND) (n=31) or diabetic (DB), both type 1 and type 2, (n=39) subjects. Phosphorodiamidate morpholino oligomers (PMO) were used for TGF‐β1‐silencing. Circulating CD34+cells and monocyte‐macrophages were enumerated by flow cytometry in both groups. Gene‐expression assays were carried out by qPCR. Myelopoiesis was determined by CFU‐GM assay followed by characterization of monocytes and macrophages by flow cytometry. Circulating CD34+ cells (p<0.05, n=31) and classical monocytes (anti‐inflammatory, CD14++CD16‐) were lower (p<0.05, n=16) whereas non‐classical (pro‐inflammatory, CD14lowCD16++) monocytes (P<0.05, n=16) were higher in DB compared to the ND group. However, no differences were observed in the intermediate monocyte and macrophage populations in circulation between the groups. DB‐CD34+ cells generated a higher number of colonies in the CFU‐GM assay than ND cells. Nonclassical monocytes were higher in DB‐CFUs (p<0.05 vs ND‐CFUs, n=4) with no difference in classical or intermediate monocytes. A higher number of pro‐inflammatory (CCR2+CD68+) (p<0.05, n=4) and lower number of anti‐inflammatory (CX3CR1+CD206+) macrophages (p<0.05, n=4) were detected in DB‐CFUs compared to that observed in ND‐CFUs. This imbalance was not observed in DB‐cells that were modified with TGF β1‐PMO. Gene expression of S100A8/S100A9, S100A10, S100A14, S100P, HMGb1 and RAGE were higher (p<0.05, n=5) in DB CD34+ cells compared to the ND. These changes were reversed by TGFβ1‐PMO. RAGE‐antagonist recapitulated the effects of TGFβ1‐PMO in assays for myelopoiesis. This study suggests that silencing of TGF‐β1 is a promising approach for enhancing the revascularization potential of CD34+cells from diabetic older adults by reversing the paracrine pro‐inflammatory functions that would negatively impact vascular repair. [ABSTRACT FROM AUTHOR]

R1748 --> 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. [ABSTRACT FROM AUTHOR]