Type I collagen proteolysis by matrix metalloproteinase-2 contributes to focal adhesion kinase activation and vascular smooth muscle cell proliferation in the aorta in early hypertension.

INTRODUCTION: Increased matrix metalloproteinase (MMP)-2 activity contributes to increase vascular smooth muscle cell (VSMC) proliferation in the aorta in early hypertension by cleaving many proteins of the extracellular matrix. Cleaved products from type I collagen may activate focal adhesion kinases (FAK) that trigger migration and proliferation signals in VSMC. We therefore hypothesized that increased activity of MMP-2 proteolyzes type I collagen in aortas of hypertensive rats, and thereby, induces FAK activation, thus leading to increased VSMC proliferation and hypertrophic remodeling in early hypertension. METHODS: Male Sprague-Dawley rats were submitted to renovascular hypertension by the two kidney-one clip (2K1C) model and treated with doxycycline (30 mg/kg/day) by gavage from the third to seventh-day post-surgery. Controls were submitted to sham surgery. Systolic blood pressure (SBP) was measured daily by tail-cuff plethysmography and the aortas were processed for zymography and Western blot for MMP-2, pFAK/FAK, integrins and type I collagen. Mass spectrometry, morphological analysis and Ki67 immunofluorescence were also done to identify collagen changes and VSMC proliferation. A7r5 cells were stimulated with collagen and treated with the MMP inhibitors (doxycycline or ARP-100), and with the FAK inhibitor PND1186 for 24 h. Cells were lysed and evaluated by Western blot for pFAK/FAK. RESULTS: 2K1C rats developed elevated SBP in the first week as well as increased expression and activity of MMP-2 in the aorta (p < 0.05 vs. Sham). Treatment with doxycycline reduced both MMP activity and type I collagen proteolysis in aortas of 2K1C rats (p < 0.05). Increased pFAK/FAK and increased VSMC proliferation (p < 0.05 vs. Sham groups) were also seen in the aortas of 2K1C and doxycycline decreased both parameters (p < 0.05). Higher proliferation of VSMC contributed to hypertrophic remodeling as seen by increased media/lumen ratio and cross sectional area (p < 0.05 vs Sham groups). In cell culture, MMP-2 cleaves collagen, an effect reversed by MMP inhibitors (p < 0.05). Increased levels of pFAK/FAK were observed when collagen was added in the culture medium (p < 0.05 vs control) and MMP and FAK inhibitors reduced this effect. CONCLUSIONS: Increase in MMP-2 activity proteolyzes type I collagen in the aortas of 2K1C rats and contributes to activate FAK and induces VSMC proliferation during the initial phase of hypertension.

Quercetin decreases cardiac hypertrophic mediators and maladaptive coronary arterial remodeling in renovascular hypertensive rats without improving cardiac function.

Oxidative stress and MMP activity are found in the hearts and arteries in hypertension and contribute to the resulting hypertrophy and dysfunction. Quercetin is a flavonoid that reduces MMP-2 activity and ameliorates hypertrophic vascular remodeling of hypertension. The hypothesis is that treatment of hypertensive rats with quercetin ameliorates coronary maladaptive remodeling and decreases hypertrophic cardiac dysfunction by decreasing oxidative stress and MMP activity. Male Sprague-Dawley two-kidney, one-clip (2K1C) and Sham rats were treated with quercetin (10 mg/kg/day) or its vehicle for 8 weeks by gavage. Rats were analyzed at 10 weeks of hypertension. Systolic blood pressure (SBP) was examined by tail-cuff plethysmography. Cardiac left ventricles were used to determine MMP activity by in situ zymography and oxidative stress by dihydroethidium. Immunofluorescence was performed to detect transforming growth factor (TGF)-ß and nuclear factor kappa B (NFkB). Morphological analyses of heart and coronary arteries were done by H&E and picrosirius red, and cardiac function was measured by Langendorff. SBP was increased in 2K1C rats, and quercetin did not reduce it. However, quercetin decreased both oxidative stress and TGF-ß in the left ventricles of 2K1C rats. Quercetin also decreased the accentuated MMP activity in left ventricles and coronary arteries of 2K1C rats. Quercetin ameliorated hypertension-induced coronary arterial hypertrophic remodeling, although it did not reduce cardiac hypertrophic remodeling and dysfunction. Quercetin decreases cardiac oxidative stress and TGF-ß and MMP activity in addition to improving coronary remodeling, yet does not ameliorate cardiac dysfunction in 2K1C rats.

Omeprazole induces vascular remodeling by mechanisms involving xanthine oxidoreductase and matrix metalloproteinase activation.

Proton pump inhibitors (PPI) are commonly used drugs that may increase the cardiovascular risk by mechanisms not entirely known. We examined whether the PPI omeprazole promotes vascular oxidative stress mediated by xanthine oxidoreductase (XOR) leading to activation of matrix metalloproteinases (MMPs) and vascular remodeling. We studied Wistar rats treated with omeprazole (or vehicle) combined with the XOR inhibitor allopurinol (or vehicle) for four weeks. Systolic blood pressure (SBP) measured by tail-cuff plethysmography was not affected by treatments. Omeprazole treatment increased the aortic cross-sectional area and media/lumen ratio by 25% (P < 0.05). Omeprazole treatment decreased gastric pH and induced vascular remodeling accompanied by impaired endothelium-dependent aortic responses (assessed with isolated aortic ring preparation) to acetylcholine (P < 0.05). Omeprazole increased vascular active MMP-2 expression and activity assessed by gel zymography and in situ zymography, respectively (P < 0.05). Moreover, omeprazole enhanced vascular oxidative stress assessed in situ with the fluorescent dye DHE and with the lucigenin chemiluminescence assay (both P < 0.05). All these biochemical changes caused by omeprazole were associated with increased vascular XOR activity (but not XOR expression assessed by Western blot) and treatment with allopurinol fully prevented them (all P < 0.05). Importantly, treatment with allopurinol prevented the vascular dysfunction and remodeling caused by omeprazole. Our results suggest that the long-term use of omeprazole induces vascular dysfunction and remodeling by promoting XOR-derived reactive oxygen species formation and MMP activation. These findings provide evidence of a new mechanism that may underlie the unfavorable cardiovascular outcomes observed with PPI therapy. Clinical studies are warranted to validate our findings.

Nebivolol Prevents Up-Regulation of Nox2/NADPH Oxidase and Lipoperoxidation in the Early Stages of Ethanol-Induced Cardiac Toxicity.

Changes in redox state are described in the early stages of ethanol-induced cardiac toxicity. Here, we evaluated whether nebivolol would abrogate ethanol-induced redox imbalance in the heart. Male Wistar rats were treated with a solution of ethanol (20% v/v) for 3 weeks. Treatment with nebivolol (10 mg/kg/day; p.o. gavage) prevented the increase of both superoxide (O2•-) and thiobarbituric acid reactive substances (TBARS) in the left ventricle of rats chronically treated with ethanol. Neither ethanol nor nebivolol affected the expression of Nox4, p47phox, or Rac-1. Nebivolol prevented ethanol-induced increase of Nox2 expression in the left ventricle. Superoxide dismutase (SOD) activity as well as the concentration of reduced glutathione (GSH) was not altered by ethanol or nebivolol. Augmented catalase activity was detected in the left ventricle of both ethanol- and nebivolol-treated rats. Treatment with nebivolol, but not ethanol increased eNOS expression in the left ventricle. No changes in the activity of matrix metalloproteinase (MMP)2 or in the expressions of MMP2, MMP9, and tissue inhibitor metalloproteinase (TIMP)1 were detected after treatment with ethanol or nebivolol. However, ethanol increased the expression of TIMP2, and this response was prevented by nebivolol. Our results provided novel insights into the mechanisms underlying the early stages of the cardiac injury induced by ethanol consumption. We demonstrated that Nox2/NADPH oxidase-derived ROS play a role in ethanol-induced lipoperoxidation and that this response was prevented by nebivolol. In addition, we provided evidence that MMPs are not activated in the early stages of ethanol-induced cardiac toxicity.

Verapamil decreases calpain-1 and matrix metalloproteinase-2 activities and improves hypertension-induced hypertrophic cardiac remodeling in rats.

AIMS: Increased activity of calpain-1 and matrix metalloproteinase (MMP)-2 was observed in different models of arterial hypertension and contribute to thicken the left ventricle (LV) walls and to hypertrophy cardiac myocytes. MMP-2 activity may be regulated by calpain-1 via bioactive molecules activation such as transforming growth factor (TGF)-ß in cardiovascular diseases. This study analyzed whether calpain-1 causes cardiac hypertrophy and dysfunction by modulating the expression and activity of MMP-2 in renovascular hypertension. MAIN METHODS: Male Wistar rats were submitted to two kidneys, one clip (2K1C) model of hypertension or sham surgery and were treated with verapamil (VRP, 8 mg/kg/bid) by gavage from the second to tenth week post-surgery. Systolic blood pressure (SBP) was weekly assessed by tail-cuff plethysmography and morphological and functional parameters of LV were analyzed by echocardiography. MMP-2 activity was analyzed by in situ and gelatin zymography, while calpain-1 activity by caseinolytic assay. MMP-2, calpain-1, TGF-ß and MMP-14/TIMP-2 levels were identified in the LV by western blots. Fluorescence assays were performed to evaluate oxidative stress, MMP-2 and calpain-1 levels. KEY FINDINGS: SBP increased in 2K1C rats and was unaltered by VRP. However, VRP notably ameliorated hypertension-induced increase in LV thickness. VRP decreased hypertension-induced enhances in calpain-1 and MMP-2 activities, oxidative stress and mature TGF-ß levels. Treatment with VRP also decreased the accentuated MMP-14/TIMP-2 levels in 2K1C. SIGNIFICANCE: Treatment with VRP decreases calpain-1 and MMP-2 activities and also reduces TGF-ß and MMP-14/TIMP-2 levels in the LV of hypertensive rats, thus contributing to ameliorate cardiac hypertrophy.

Matrix metalloproteinase (MMP)-2 activation by oxidative stress decreases aortic calponin-1 levels during hypertrophic remodeling in early hypertension.

Hypertension is characterized by maladaptive vascular remodeling and enhanced oxidative stress in the vascular wall. Peroxynitrite may directly activate latent matrix metalloproteinase (MMP)-2 in vascular smooth muscle cells (VSMC) by its S-glutathiolation. MMP-2 may then proteolyze calponin-1 in aortas from hypertensive animals, which stimulates VSMC proliferation and medial hypertrophy. Calponin-1 is an intracellular protein which helps to maintain VSMC in their differentiated (contractile) phenotype. The present study therefore investigated whether aortic MMP-2 activity is increased by oxidative stress in early hypertension and then contributes to hypertrophic arterial remodeling by reducing the levels of calponin-1. Male Wistar rats were submitted to the two kidney, one clip (2 K-1C) model of hypertension or sham surgery and were treated daily with tempol (18 mg/kg/day) or its vehicle (water) by gavage from the third to seventh day post-surgery. Systolic blood pressure (SBP) was daily assessed by tail-cuff plethysmography. After one week, aortas were removed to perform morphological analysis with hematoxylin and eosin staining and to analyze reactive oxygen­nitrogen species levels by dihydroethidium and immunohistochemistry for nitrotyrosine. MMP-2 activity was analyzed by in situ and gelatin zymography and its S-glutathiolation was analyzed by Western blot for MMP-2 of anti-glutathione immunoprecipitates. Calponin-1 levels were identified in aortas by immunofluorescence. SBP increased by approximately 50 mmHg at the first week in 2 K-1C rats which was unaffected by tempol. However, tempol ameliorated the hypertension-induced increase in arterial media-to-lumen ratio and hypertrophic remodeling. Tempol also decreased hypertension-induced aortic oxidative stress and the enhanced MMP-2 activity. S-glutathiolation may be a potential mechanism by which oxidative stress activates MMP-2 in aortas of 2 K-1C rats. Furthermore, calponin-1 was decreased in aortas from 2 K-1C rats and tempol prevented this. In conclusion, oxidative stress may contribute to the increase in aortic MMP-2 activity, possibly by S-glutathiolation, and this may result in calponin-1 loss and maladaptive vascular remodeling in early hypertension.

Ethanol withdrawal increases blood pressure and vascular oxidative stress: a role for angiotensin type 1 receptors.

We evaluated the possible mechanisms underlying the oxidative stress induced by ethanol withdrawal. With this purpose, we verified the role of AT1 receptors in such response. Male Wistar rats were treated with ethanol 3%-9% (vol./vol.) for 21 days. Ethanol withdrawal was induced by abrupt discontinuation of the treatment. Experiments were performed 48 hours after ethanol discontinuation. Increased plasma levels of angiotensin II were detected after ethanol withdrawal. Losartan (10 mg/kg; p.o. gavage), a selective AT1 receptor antagonist, impeded the increase in blood pressure induced by ethanol withdrawal. Increased lipoperoxidation and superoxide anion (O2-) levels were detected in aortas after ethanol withdrawal, and losartan prevented these responses. Decreased hydrogen peroxide and nitrate/nitrite concentration were detected in aortas after ethanol withdrawal, and losartan prevented these effects. Nitrotyrosine immunostaining in the rat aorta was increased after ethanol withdrawal, and AT1 blockade impeded this response. Increased expression of PKCδ and p47phox was detected after ethanol withdrawal, and treatment with losartan prevented these responses. Our study provides novel evidence that ethanol withdrawal increases vascular oxidative stress and blood pressure through AT1-dependent mechanisms. These findings highlight the importance of angiotensin II in ethanol withdrawal-induced increase in blood pressure and vascular oxidative damage.

Quercetin decreases the activity of matrix metalloproteinase-2 and ameliorates vascular remodeling in renovascular hypertension.

BACKGROUND AND AIMS: Increased activity of matrix metalloproteinase (MMP)-2 is observed in aortas of different models of hypertension, and its activation is directly mediated by oxidative stress. As quercetin is an important flavonoid with significant antioxidant effects, the hypothesis here is that quercetin will reduce increased MMP-2 activity by decreasing oxidative stress in aortas of hypertensive rats and then ameliorate hypertension-induced vascular remodeling. METHODS: Male two-kidney one-clip (2K1C) hypertensive Wistar rats and controls were treated with quercetin (10 mg/kg/day) or its vehicle for three weeks by gavage. Rats were then analyzed at five weeks of hypertension. Systolic blood pressure (SBP) was determined by tail-cuff plethysmography. Aortas were used to determine MMP activity by in situ zymography and reactive oxygen species (ROS) levels by dihydroethidium. Western blot was performed to detect focal adhesion kinase (FAK) and phosphorylated-FAK levels. RESULTS: SBP was increased in 2K1C rats and only a borderline reduction in SBP was observed after treating 2K1C rats with quercetin. Cross-sectional area and the number of vascular smooth muscle cells were significantly increased in aortas of hypertensive rats, and quercetin reduced them. Quercetin reduced ROS levels in aortas of 2K1C rats and the increased activity of gelatinases in situ. However, quercetin did not affect the levels of tissue inhibitor of MMP (TIMP)-2 and did not interfere with FAK and p-FAK levels in aortas of hypertensive rats. Furthermore, different concentrations of quercetin did not directly reduce the activity of human recombinant MMP-2 in vitro. CONCLUSIONS: Quercetin reduces hypertension-induced vascular remodeling, oxidative stress and MMP-2 activity in aortas.

Metabolic parameters and responsiveness of isolated iliac artery in LDLr-/- mice: role of aerobic exercise training.

Physical inactivity and dyslipidemia are considered risk factors for cardiovascular diseases. There are few studies evaluating the effects of physical exercise in small-caliber artery in a model that mimics familial hypercholesterolemia. The aim of this study was to examine the effect of exercise training, at moderate intensity, on metabolic parameters and iliac artery responsiveness in LDL-/- mice. Sedentary (SD) and trained (TR) mice performed AET (5 days/week, 60 minutes/day at 60-70% of maximum speed) during 8 weeks. Body weight gain (BWG), epididymal fat, blood glucose, total cholesterol and triglycerides were evaluated. Concentration-response curves to acetylcholine (ACh), sodium nitroprusside, phenylephrine and U46619 were obtained in isolated iliac artery. The production of nitric oxide (NO) and reactive oxygen species as well as the expression and activity of MMP-2 were assessed. AET was effective in preventing BWG and epididymal fat gain, whereas no changes were observed in glucose, total cholesterol and triglycerides levels. Improvement in responsiveness to ACh was found in TR (Emax = 85±3%) compared with SD group (Emax = 62±5%) without changes in the maximal vascular response or potency to SNP, PHE and U46619. The NO level was increased (10.8-fold) while ROS formation was decreased (3.7-fold) in iliac artery from TR, without changes in MMP-2 activity or its expression. AET was effective to improve endothelium-dependent relaxation that was accompanied by increased NO production and decreased ROS formation in iliac artery. The intensity of AET should be greater to modify metabolic disorders in this experimental model of dyslipidemia.

Matrix Metalloproteinase-2 Activity is Associated with Divergent Regulation of Calponin-1 in Conductance and Resistance Arteries in Hypertension-induced Early Vascular Dysfunction and Remodelling.

Matrix metalloproteinase (MMP)-2 participates in hypertension-induced maladaptive vascular remodelling by degrading extra- and intracellular proteins. The consequent extracellular matrix rearrangement and phenotype switch of vascular smooth muscle cells (VSMCs) lead to increased cellular migration and proliferation. As calponin-1 degradation by MMP-2 may lead to VSMC proliferation during hypertension, the hypothesis of this study is that increased MMP-2 activity contributes to early hypertension-induced maladaptive remodelling in conductance and resistance arteries via regulation of calponin-1. The main objective was to analyse whether MMP-2 exerts similar effects on the structure and function of the resistance and conductance arteries during early hypertension. Two-kidney, one-clip (2K-1C) hypertensive male rats and corresponding controls were treated with doxycycline (30 mg/kg/day) or water until reaching one week of hypertension. Systolic blood pressure was increased in 2K-1C rats, and doxycycline did not reduce it. Aortas and mesenteric arteries were analysed. MMP-2 activity and expression were increased in both arteries, and doxycycline reduced it. Significant hypertrophic remodelling and VSMC proliferation were observed in aortas but not in mesenteric arteries of 2K-1C rats. The contractility of mesenteric arteries to phenylephrine was increased in 2K-1C rats, and doxycycline prevented this alteration. The potency of phenylephrine to contract aortas of 2K-1C rats was increased, and doxycycline decreased it. Whereas calponin-1 expression was increased in 2K-1C mesenteric arteries, calponin-1 was reduced in aortas. Doxycycline treatment reverted changes in calponin-1 expression. MMP-2 contributes to hypertrophic remodelling in aortas by decreasing calponin-1 levels, which may result in VSMC proliferation. On the other hand, MMP-2-dependent increased calponin-1 in mesenteric arteries may contribute to vascular hypercontractility in 2K-1C rats. Divergent regulation of calponin-1 by MMP-2 may be an important mechanism that leads to maladaptive vascular effects in hypertension.