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Diabetes increases the risk for ischemic vascular diseases, which is further elevated in older adults. Bone marrow-derived hematopoietic CD34+ stem/progenitor cells have the potential of revascularization; however, diabetes attenuates vasoreparative functions. Angiotensin-converting enzyme 2 (ACE2) is the vasoprotective enzyme of renin-angiotensin system in contrast with the canonical angiotensin-converting enzyme (ACE). The present study tested the hypothesis that diabetic dysfunction is associated with ACE2/ACE imbalance in hematopoietic stem/progenitor cells (HSPCs) and that increasing ACE2 expression would restore reparative functions. Blood samples from male and female diabetic (n=71) or nondiabetic (n=62) individuals were obtained and CD34+ cells were enumerated by flow cytometry. ACE and ACE2 enzyme activities were determined in cell lysates. Lentiviral (LV) approach was used to increase the expression of soluble ACE2 protein. Cells from diabetic older adults (DB) or nondiabetic individuals (Control) were evaluated for their ability to stimulate revascularization in a mouse model of hindlimb ischemia (HLI). DB cells attenuated the recovery of blood flow to ischemic areas in nondiabetic mice compared with that observed with Control cells. Administration of DB cells modified with LV-ACE2 resulted in complete restoration of blood flow. HLI in diabetic mice resulted in poor recovery with amputations, which was not reversed by either Control or DB cells. LV-ACE2 modification of Control or DB cells resulted in blood flow recovery in diabetic mice. In vitro treatment with Ang-(1-7) modified paracrine profile in diabetic CD34+ cells. The present study suggests that vasoreparative dysfunction in CD34+ cells from diabetic older adults is associated with ACE2/ACE imbalance and that increased ACE2 expression enhances the revascularization potential. [ABSTRACT FROM AUTHOR]

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]

Aging with diabetes is associated with impaired vasoprotective functions and decreased nitric oxide (NO) generation in CD34+ cells. Transforming growth factor- β1 (TGF-β1) is known to regulate hematopoietic functions. This study tested the hypothesis that transforming growth factor- β1 (TGF-β1) is upregulated in diabetic CD34+ cells and impairs NO generation via thrombospondin-1 (TSP-1)/CD47/NO pathway. CD34+ cells from nondiabetic (ND) (n=58) or diabetic older adults (DB) (both type 1 and type 2) (n=62) were isolated from peripheral blood. TGF-β1 was silenced by using an antisense delivered as phosphorodiamidate morpholino oligomer (PMO-TGF-β1). Migration and proliferation in response to stromal-derived factor-1α (SDF-1α) were evaluated. NO generation and eNOS phosphorylation were determined by flow cytometry. CD34+ cells from older, but not younger, diabetics have higher expression of TGF-β1 compared to that observed in cells derived from healthy individuals (P<0.05, n=14). TSP-1 expression was higher (n=11) in DB compared to ND cells. TGFβ1-PMO decreased the secretion of TGF-β1, which was accompanied with decreased TSP-1 expression. Impaired proliferation, migration and NO generation in response to SDF-1α in DB cells were reversed by TGF-β1-PMO (n=6). TSP-1 inhibited migration and proliferation of nondiabetic CD34+ cells that was reversed by CD47-siRNA, which also restored these responses in diabetic CD34+ cells. TSP-1 opposed SDF-1α-induced eNOS phosphorylation at Ser1177 that was reversed by CD47-siRNA. These results infer that increased TGF-β1 expression in CD34+ cells induces dysfunction in CD34+ cells from diabetic older adults via TSP-1/CD47-dependent inhibition of NO generation. [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]