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

Abstract

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.

Keywords: S-nitrosylation; endothelial nitric oxide synthase; nitric oxide; pulse-wave velocity; tensile testing; tissue transglutaminase; vascular stiffness.

Fig. 1.

Effect of endothelial NO synthase (eNOS) knockdown on transglutaminase (TG2) in human aortic endothelial cells (HAEC). A: lentiviral delivery of eNOS short-hairpin RNA (shRNA) resulted in an ∼50% loss in eNOS protein expression in cultured HAEC. B: TG2 abundance in extracellular matrix (ECM) increased. C: TG2 S-nitrosylation decreased. D: TG2 cross-linking function increased with eNOS knockdown. Graphs show means ± SE (n = 6). *Significant difference by Student's t-test (P < 0.05).

Fig. 2.

TG2 localization and function in eNOS^−/− mice. A: TG cross-linking activity was higher. B: TG2 abundance in the decellularized aortic matrix was higher (n = 5). C: TG2 S-nitrosylation levels were lower in the eNOS^−/− mice compared with wild-type (WT) controls (n = 5). D: TG2 abundance was unaltered, whereas TG-specific cross-links were higher in the eNOS^−/− mouse (n = 8). *Significant difference by Student's t-test (P < 0.05).

Fig. 3.

Effect of TG inhibition on vascular compliance. TG inhibitor cystamine (40 mg·kg⁻¹·day⁻¹) was administered for 4 wk to WT and eNOS knockout mice using osmotic infusion pumps. Carotid artery compliance, measured by video-dimension analysis, was lower in eNOS^−/− mice compared with WT. Cystamine treatment restored carotid artery compliance toward WT controls. Values are means ± SE (n = 8). Significant difference by one-way ANOVA with Bonferroni post hoc analysis: *P < 0.05; **P < 0.01; ***P < 0.001.

Fig. 4.

Vascular stiffness measures in eNOS^−/− and WT mice. A: simultaneous pulsed-wave velocity (PWV) and mean arterial pressure (MAP) measurement in mice; a 1.2-Fr catheter was used to measure transit time in vivo. B: example waves/second derivative to determine transit time. C: example of beat-to-beat changes in arterial pressure. D: sample PWV and MAP relationship in a single animal. E: single-point, Doppler-based PWV measurement was higher in eNOS^−/− compared with WT. F: MAP was higher in eNOS^−/− mice. G: heart rate was similar in both groups. H: PWV-MAP correlation in eNOS^−/− mice diverged from WT mice at higher MAPs; dotted lines represent baseline pressure and PWV for eNOS^−/− (red) and WT (black) mice. I: intact aorta of eNOS^−/− mice were stiffer than WT. Inset: incremental elastic modulus of intact vessels at strain = 0.5. J: decellularized aortic segments of eNOS^−/− mice were stiffer than those from WT mice. Inset: incremental elastic modulus of decellularized vessels at strain = 0.8. Values are means ± SE (n = 8). Significant difference by Student's t-test for two groups or one-way ANOVA with Bonferroni post hoc analysis for more than two groups: *P < 0.05; **P < 0.01; ***P < 0.001.

Fig. 5.

Cell-matrix interactions and CSK stiffness of isolated vascular smooth muscle cells from eNOS^−/− and WT mice. A: proteins (50 μg) of aortic homogenates were immunoprecipitated using integrin β1 antibody; higher levels of TG2 co-immunoprecipitated with integrin β1 in eNOS^−/− mouse (n = 6); proteins from eNOS^−/− mice were immunoprecipitated with isotype IgG as control (right lane). *P < 0.05. B: CSK stiffness of freshly isolated vascular smooth muscle cells from the aorta of eNOS^−/− and WT mice was measured using magnetic twisting cytometry. Data are presented as geometric means ± SE (n = 248 cells for eNOS^−/−; n = 199 cells for WT).