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Objective: To investigate the role of matrix metalloproteinase 2 (MMP2) in pancreatic beta cell injury induced by oxidative stress., Methods: Rat pancreatic beta cell line INS-1 cells were treated with advanced glycation end-products (AGE) to induce intracellular oxidative stress. Intracellular MMP2 expression and activity were determined by quantitative reverse transcription polymerase chain reaction (RT-PCR), Western blotting, and zymography, respectively. MMP2 expression and activity were manipulated by over-expression with recombinant MMP2 plasmids or knockdown with either MMP2 specific siRNA or inhibitors, and effects on apoptosis and insulin-secretion were measured by flow cytometry and ELISA., Results: AGE treatment induced intracellular oxidative stress in INS-1 cells, as indicated by elevated ROS levels, apoptotic cell death, and suppressed insulin secretion. This was accompanied by increased MMP2 expression and activity. However, Antioxidant N-acetylcysteine (NAC) treatment inhibited MMP2 expression and activity, and partially reversed cell apoptosis and insulin secretion dysfunction induced by AGE. Forced expression of MMP2 mimicked the effects of AGE treatment while inhibition of MMP2 either by a specific MMP2 inhibitor or MMP2 siRNA protected oxidative stress induced by AGE., Conclusion: MMP2 expression and intracellular activity are increased by oxidative stress, contributing to cellular dysfunction and apoptosis in INS-1 cells after AGE challenge.

Abstract: Volume overload (VO) caused by aortocaval fistula (ACF) is associated with oxidative/inflammatory stress. The resulting inflammation, matrix metalloproteinase (MMP) activation, and collagen degradation is thought to play a pivotal role in left ventricular (LV) dilatation and failure. Since mitochondria are also targets for inflammation and oxidative stress, we hypothesized that there would be bioenergetic dysfunction with acute VO. In Sprague-Dawley rats subjected to 24hrs of ACF, there was a two-fold increase in LV pressure-volume area in vivo, consistent with increased LV myocardial oxygen usage and increased bioenergetic demand in cardiomyocytes. Isolated cardiomyocytes from ACF LVs demonstrated increased hydrogen peroxide and superoxide formation and increased MMP activity. Subsarcolemmal mitochondria (SSM) showed a 40% decrease in state 3 respiration and proteomic analysis of SSM demonstrated decreased levels of complexes I-V in ACF. Immunohistochemical analysis revealed disruption of the subsarcolemmal location of the SSM network in ACF. To test for a potential link between SSM dysfunction and loss of interstitial collagen, rats were treated with the MMP-inhibitor PD166793 prior to ACF. MMP-inhibitor preserved interstitial collagen, integrin-α5 and the SSM structural arrangement. In addition, the decrease in state 3 mitochondrial respiration with ACF was prevented by PD166793. These studies established an important interaction between degradation of interstitial collagen in acute VO and the disruption of SSM structure and function which could contribute to progression to heart failure. [ABSTRACT FROM AUTHOR]

Abstract: Xanthine oxidoreductase (XOR) is increased in the left ventricle (LV) of humans with volume overload (VO), and mitochondrial inhibition of the respiratory chain occurs in animal models of VO. Because mitochondria are both a source and a target of reactive oxygen and nitrogen species, we hypothesized that activation of XOR and mitochondrial dysfunction are interdependent. To test this we used the aortocaval fistula (ACF) rat model of VO and a simulation of the stretch response in isolated adult cardiomyocytes with and without the inhibitor of XOR, allopurinol, or the mitochondrially targeted antioxidant MitoQ. Xanthine oxidase (XO) activity was increased in cardiomyocytes from ACF vs sham rats (24h) without an increase in XO protein. A twofold increase in LV end-diastolic pressure/wall stress and a decrease in LV systolic elastance with ACF were improved when allopurinol treatment (100mg/kg) was started at ACF induction. Subsarcolemmal State 3 mitochondrial respiration was significantly decreased in ACF and normalized by allopurinol. Cardiomyocytes subjected to 3h cyclical stretch resulted in an increase in XO activity and mitochondrial swelling, which was prevented by allopurinol or MitoQ pretreatment. These studies establish an early interplay between cardiomyocyte XO activation and bioenergetic dysfunction that may provide a new target that prevents progression to heart failure in VO. [Copyright &y& Elsevier]

Prolyl endopeptidase (PREP) is a serine endopeptidase widely distributed in the body, and accumulated evidence suggests that PREP participates in inflammation and oxidative stress. Here, we explored the effect of PREP gene disruption on hepatic inflammation and oxidative stress status in a methionine-choline-deficient (MCD)-induced nonalcoholic steatohepatitis (NASH) model. PREP gene disruption (PREPgt) mice and wild-type (WT) littermates were placed on a control or an MCD diet for 4 weeks, respectively. The liver histopathological analysis and the number of inflammatory cells were determined by hematoxylin-eosin (HE) and immunohistochemical staining. Inflammation-associated genes and cytokine levels in liver tissue were evaluated by quantitative PCR and ELISA. The levels of P53, Sesn2, Nrf2, HO-1, and oxidative stress indicators in mice and the palmitic acid (PA)-treated human hepatocellular carcinoma cells (HepG2) were examined by immunoblotting and commercially available kits, respectively. We found that PREP expression was upregulated in the MCD-induced NASH model. In addition, PREP disruption alleviated MCD-induced hepatic inflammation accompanied by diminished infiltration of inflammatory cells and secretion of inflammatory mediators. More importantly, the results of this study indicate that targeting PREP can improve oxidative stress status in the liver of MCD-diet mice and PA-exposed HepG2 cells. The effect is most likely mediated by the activation of P53 and its downstream signaling pathways (Sesn2/Nrf2/HO-1). Our results showed that PREP disruption (or inhibition) could decrease oxidative stress and inflammation and improve liver function, indicating that targeting PREP might be a new potential therapeutic option for NAFLD/NASH. [ABSTRACT FROM AUTHOR]