Abstract P241: Involvement of Mitochondrial Telomerase Reverse Transcriptase in the Restoration of Nitric Oxide Levels by Angiotensin-(1-7) in the Dysfunctional Diabetic CD34 ⁺ Cells

Abstract

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.