Increased tissue transglutaminase activity contributes to central vascular stiffness in eNOS knockout mice.

Nitric oxide (NO) can modulate arterial stiffness by regulating both functional and structural changes in the arterial wall. Tissue transglutaminase (TG2) has been shown to contribute to increased central aortic stiffness by catalyzing the cross-linking of matrix proteins. NO S-nitrosylates and constrains TG2 to the cytosolic compartment and thereby holds its cross-linking function latent. In the present study, the role of endothelial NO synthase (eNOS)-derived NO in regulating TG2 function was studied using eNOS knockout mice. Matrix-associated TG2 and TG2 cross-linking function were higher, whereas TG2 S-nitrosylation was lower in the eNOS(-/-) compared with wild-type (WT) mice. Pulse-wave velocity (PWV) and blood pressure measured noninvasively were elevated in the eNOS(-/-) compared with WT mice. Intact aortas and decellularized aortic tissue scaffolds of eNOS(-/-) mice were significantly stiffer, as determined by tensile testing. The carotid arteries of the eNOS(-/-) mice were also stiffer, as determined by pressure-dimension analysis. Invasive methods to determine the PWV-mean arterial pressure relationship showed that PWV in eNOS(-/-) and WT diverge at higher mean arterial pressure. Thus eNOS-derived NO regulates TG2 localization and function and contributes to vascular stiffness.

Nitric oxide regulates tissue transglutaminase localization and function in the vasculature.

The multifunctional enzyme tissue transglutaminase (TG2) contributes to the development and progression of several cardiovascular diseases. Extracellular rather than intracellular TG2 is enzymatically active, however, the mechanism by which it is exported out of the cell remains unknown. Nitric oxide (NO) is shown to constrain TG2 externalization in endothelial and fibroblast cells. Here, we examined the role of both exogenous and endogenous (endothelial cell-derived) NO in regulating TG2 localization in vascular cells and tissue. NO synthase inhibition in endothelial cells (ECs) using N-nitro L-arginine methyl ester (L-NAME) led to a time-dependent decrease in S-nitrosation and increase in externalization of TG2. Laminar shear stress led to decreased extracellular TG2 in ECs. S-nitrosoglutathione treatment led to decreased activity and externalization of TG2 in human aortic smooth muscle and fibroblast (IMR90) cells. Co-culture of these cells with ECs resulted in increased S-nitrosation and decreased externalization and activity of TG2, which was reversed by L-NAME. Aged Fischer 344 rats had higher tissue scaffold-associated TG2 compared to young. NO regulates intracellular versus extracellular TG2 localization in vascular cells and tissue, likely via S-nitrosation. This in part, explains increased TG2 externalization and activity in aging aorta.

S-nitrosation of arginase 1 requires direct interaction with inducible nitric oxide synthase.

Arginase constrains endothelial nitric oxide synthase activity by competing for the common substrate, L -Arginine. We have recently shown that inducible nitric oxide synthase (NOS2) S-nitrosates and activates arginase 1 (Arg1) leading to age-associated vascular dysfunction. Here, we demonstrate that a direct interaction of Arg1 with NOS2 is necessary for its S-nitrosation. The specific domain of NOS2 that mediates this interaction is identified. Disruption of this interaction in human aortic endothelial cells prevents Arg1 S-nitrosation and activation. Thus, disruption of NOS2-Arg1 interaction may represent a therapeutic strategy to attenuate age related vascular endothelial dysfunction.