Abstract P141: Downregulation Of Deletion Variants Of Telomerase Reverse Transcriptase (TERT) By Transforming Growth Factor-β1-silencing Decreases Mitochondrial ROS In Diabetic CD34 + Cells

Abstract

Telomerase reverse transcriptase (TERT) modulates mitochondrial levels of reactive oxygen species (mitoROS). Deletion variants, α-, β- and αβ, are negative regulators of TERT. Increased ROS levels in diabetic CD34 + cells impair vasoreparative functions. Our recent studies showed that silencing of transforming growth factor-β1 (TGF-β1) decreased ROS levels and stimulated vasoreparative functions. This study tested the hypothesis that prevalence of TERT deletion variants is higher in diabetic CD34 + cells and that TGF-β1-silencing decreases mitoROS by downregulation of the variants. CD34 + cells derived from either male or female nondiabetic (ND) (n=38) or diabetic (DB), both type 1 and type 2, (n=43) subjects were studied. Phosphorodiamidate morpholino oligomers (PMO) were used for TGF-β1-silencing. TERT variants were characterized by qPCR and agarose gel electrophoresis. MitoSOX-flow cytometry and ELISA were used for determining mitoROS levels and telomerase activity, respectively. TGF-β1-silencing decreased mitoROS levels in DB-CD34+ cells (P<0.01 vs ND-cells, n=6). This effect was reversed by TERT inhibitor, BIBR1532 (1μM) (n=6) or mito-X-TERT, a decoy peptide that prevents mitochondrial translocation of TERT (n=5). TERT expression was higher in DB compared to ND cells (P<0.05, n=18) but telomerase activity was lower (P<0.05 vs ND, n=10). Prevalence of TERT deletion variants was higher among DB cells compared to ND (DB - 8/10 vs ND - 2/10 subjects have one or more of α, β, or αβ variants). TGF-β1-silencing downregulated the expression of TERT variants and increased full-length TERT that was accompanied by increased telomerase activity in DB-CD34+ cells (P<0.05 vs untreated, n=5). This study suggests that diabetes is associated with higher prevalence of TERT variants that would impede mitochondrial functions of TERT. TGF-β1-silencing decreases mitoROS levels at least in part by downregulation of TERT variants.