Abstract 1708: Decreased S-Nitrosylation of Tissue Transglutaminase Contributes to Age-Related Increases in Vascular Stiffness

Abstract

Vascular aging is marked by an increase in vascular stiffness. Tissue-type transglutaminase (tTG) enzymatically forms crosslinks between extracellular matrix proteins and may contribute to this pathobiology. Recently, it has been demonstrated that s-nitrosylation of cysteine residues within the enzyme leads to its inhibition. We hypothesize that tTG is more active in the aging vasculature due to decreased bioavailability of nitric oxide (NO) and impaired S-nitrosylation. tTG activity in aorta measured by the incorporation of biotin labeled pentylamine, a tTG substrate, into crosslinks was markedly increased in old (O) animals compared to young (Y) (3.2 fold increase, n = 6, p = 0.0018). S-nitrosylation of tTG, as determined by the biotin switch assay, was significantly reduced in O vs. Y rat aorta. tTG specific (ϵ-(γ-glutamyl)lysine) crosslinks measured by immunofluorescent staining was significantly increased in O vs Y rat aortic rings. Denitrosylation of tTG in Y rats with DTT increased tTG activity by 41% as did the NOS-inhibitor L-NAME. Treatment of O rats with cystamine (tTG inhibitor) lead to a decrease in vascular stiffness as measured by pulse wave velocity (5.55 ± 0.11 m/s to 4.99 ± 0.22 m/s, n = 6, p = 0.038). Given that tTG nitrosylation is likely NOS-3 dependent, we measured tTG activity and function in NOS-3 −/− mice. tTG activity was significantly increased in NOS-3 −/− vs. WT mice (18.94 ± 0.70 RU vs. 31.63 ± 2.06 RU, n = 4, p = 0.015). Treatment of NOS-3 mice with cystamine decreased aortic stiffness and increased carotid artery compliance. Finally, tTG activity in old human aortic tissue (collected post-mortem) is increased by 30% compared to young despite unchanged protein abundance. These data suggest that NOS-3 dependent NO constrains the activity of tTG by S-nitrosylation of the enzyme. Furthermore decreased NO bioavailability contributes to tTG mediated increases in vascular stiffness. Finally tTG promises to be a critical target in age-related vascular stiffness.