Abstract P526: MMP-2 Activation By S-glutathiolation Decreases Calponin-1 During Vascular Remodeling In Hypertension

Abstract

Aim: Antioxidant tempol may decrease MMP-2 activation by S-glutathiolation as it reduces oxidative stress, and then prevents calponin-1 cleavage and aortic remodeling in hypertension. Methods: Male Wistar rats were submitted to the two kidney-one clip (2K-1C) model of hypertension or sham surgery and were treated with tempol (18 mg/kg/day) or water by gavage for one week. Systolic blood pressure (SBP) was accessed by tail-cuff plethysmography. Aortas were used to perform in situ zymography, immunofluorescence for dihydroethidium and calponin-1, immunohistochemistry for nitrotyrosine, co-immunoprecipitation for glutathione followed by Western blot for MMP-2 and histomorphology. Two-way ANOVA followed by Bonferroni test was done (Ethics Committee approval number: 0015/2017). Results: SBP increased in 2K-1C rats compared to Sham group (156±2.9 vs. 128.6±4.1, p<0.05) and tempol did not reduce it (147.3±4.7, p>0.05). Morphological analysis showed increased that 2K-1C aortas had increased media to lumen ratio (M/L) (8.6±0.7 vs. 6.5±0.2) and cross-sectional area (CSA) (1.4x10 6 ±1.5x10 5 vs. 9.7x10 5 ±4.2x10 4 ) (p<0.05 vs. Sham), but tempol did not decrease them (M/L: 7.9±0.8; CSA: 1.3x10 6 ±1.3x10 5 , p>0.05 vs. 2K-1C). However, tempol decreased 2K-1C-induced increased nitrotyrosine levels (5.9x10 5 ±1.3x10 4 vs. 4.0x10 5 ±6.1x10 4 ), dihydroethidium (16.9±1.7 vs. 8.9±1.1) and MMP-2 activity (57.5±6.4 vs. 39.4±2.6; p<0.05). All four groups presented S-glutathiolation of MMP-2, however, the levels of calponin-1 were only reduced in aortas from 2K-1C rats (17.2±1.5 vs. 28.5±1.2, p<0.05 vs. Sham groups) and tempol reverted such loss (23.6±1.6, p<0.05 vs. 2K-1C). As calponin-1 is a potential intracellular target of MMP-2 in VSMC, these results suggest that the intracellular activation of MMP-2 may be more pronounced in hypertension. Different concentrations of tempol (0.5, 1 or 1.5 mM) did not directly inhibit MMP-2 activity in gelatin zymography (respectively: 4.6x10 6 ±7.4x10 4 , 4.9x10 6 ±1.2x10 5 and 4.8x10 6 ±1.5x10 5 vs. 4.6x10 6 ±2.2x10 5 ; p>0.05). Conclusion: Increased oxidative stress may contribute to activate aortic MMP-2 by S-glutathiolation in early hypertension, thus resulting in calponin-1 loss and maladaptive remodeling.