Adult hematopoietic stem/progenitor cells (HSPCs) participate in the cardiovascular repair by stimulating regeneration and angiogenic functions of endothelium. Long‐term diabetes is associated with impaired vasoprotective functions of HSPCs. Transforming growth factor β (TGFβ1) is pleiotropic regulator of HSPC functions. Previous studies have shown that transient silencing of TGFβ1 expression improves in vivo migratory functions of diabetic HSPCs partly by restoring nitric oxide (NO) generation. This study tested the hypothesis that restoration of NO by TGFβ1‐silencing is mediated by thrombospondin‐1 (TSP1)/CD47 pathway. TSP1 is known to attenuate NO generation via inhibition of endothelial nitric oxide synthase (eNOS). HSPCs were isolated from peripheral blood samples obtained from either male or female healthy (n=25) or diabetic (both type 1 and type 2) (n=27) individuals (50–80 years of age) by immunomagnetic enrichment. TGFβ1 expression was transiently blocked by using TGFβ1‐antisense delivered in the form of phosphorodiamidate morpholino oligomer (PMO‐TGFβ1). TGFβ1 and TSP1 gene expressions were determined in HSPCs treated with either PMO‐control or PMO‐TGFβ1. NO generation induced by stromal‐derived factor‐1α (SDF) was determined by DAF‐FM flow cytometry. Diabetic cells have higher expression of TGFβ1 or TSP1 compared to that observed in cells derived from healthy individuals (n=10). Treatment with TGFβ1‐PMO decreased the expression of TGFβ1, which was associated with decreased TSP1 expression. CD47 expression is similar in healthy or diabetic individuals. NO generation by SDF is attenuated in diabetic compared to healthy cells (P<0.01, n=5). PMO‐TGFβ1‐treated diabetic cells showed increased generation of NO in response to SDF compared to PMO‐control‐treatment (P<0.01, n=5). Future studies will determine the effect of TSP1 on SDF/NO generation in cells with or without CD47‐siRNA, to further support the hypothesis. Collectively, these studies indicate the involvement of TGFβ1/TSP1/CD47/eNOS pathway in diabetic impairment of NO generation however involvement of other pathways cannot be ruled out. Support or Funding Information This study is partly supported by funding from NIH National Institute of Aging (AG056681).

Telomerase reverse transcriptase (TERT) has been shown to translocate to mitochondria (mtTERT), and decrease the generation of mitochondrial reactive oxygen species (mtROS). Angiotensin (Ang)-(1-7) stimulates vasoreparative functions in diabetic CD34 + stem/progenitor cells in part by decreasing ROS levels and increasing nitric oxide (NO) bioavailability. This study tested the involvement of mtTERT in mediating the effect of Ang-(1-7) on NO levels in diabetic CD34 + cells. CD34 + cells were isolated from the peripheral blood mononuclear cells (MNCs) of nondiabetic subjects (ND), and type 1 or type 2 diabetic (DB) patients (male or female, age 48-76 years) (DB: HbA1C 6.5-11.2). Cells were treated with Ang-(1-7) (100nM) or Stromal derived factor-1α (SDF) and evaluated for NO, and mtROS levels by flow cytometry by using DAF-FAM and mitoSOX, respectively. Number of cells used was ≥5x10 4 per treatment. The Mean Fluorescence Intensity is expressed in arbitrary fluorescence units (AFIx10 5 ). Inhibitors of Mas receptor and TERT, A779 and BIBR-1532, respectively, and decoy peptides that inhibit mitochondrial translocation or nuclear transport of TERT, mtXTERT and nucXTERT, respectively, were used. SDF (100nM) failed to stimulate NO generation in DB-cells (1.2±0.07 vs basal 0.8±0.05, n=10) compared to ND-cells (3.5±0.2 vs basal 1.6±0.2) (n=10, P<0.0001). Ang-(1-7) induced NO levels in ND- (2.9±0.2) and restored NO generation in DB-cells (2.2±0.1) (n=10). DB-cells have increased mitoROS (3.6±0.4) compared to ND-cells (1.7±0.1, P<0.001, n=7) and Ang-(1-7) normalized the mitoROS in DB-cells (1.8±0.3, n=7). The effects of Ang-(1-7) on NO and mitoROS levels were reversed by A-779 or BIBR-1532 (n=5). Decoy peptide mtXTERT not nucXTERT increased mitoROS in ND- (4.4±0.7 vs basal 1.8±0.2, P<0.01) and in DB-cells (12±1 vs basal 6±0.4, P<0.001) (n=5). The decreasing effect of Ang-(1-7) on mitoROS (1.6±0.3) was reversed by mtXTERT (10±0.9, P<0.001, n=5) but not by nucXTERT. Along similar lines, restoration of NO levels by Ang-(1-7) in DB-cells was reversed by mtXTERT but not by nucXTERT. These observations provide compelling evidence for the involvement of TERT in stimulating the vasoreparative functions of diabetic stem/progenitor cells by Ang-(1-7)/Mas pathway.