Evidence for the involvement of matrix metalloproteinases in the cardiovascular effects produced by nicotine.

Nicotine plays a role in smoking-associated cardiovascular diseases, and may upregulate matrix metalloproteinase (MMP)-2 and MMP-9. We examined whether nicotine induces the release of MMP-2 and MMP-9 by rat smooth muscle cells (SMC), and whether doxycycline (non-selective MMP inhibitor) inhibits the vascular effects produced by nicotine. SMC were incubated with nicotine 0, 50, and 150 nM for 48 h. MMP-2 and MMP-9 levels in the cell supernatants were determined by gelatin zymography. The acute changes in mean arterial pressure caused by nicotine 2 micromol/kg (or saline) were assessed in rats pretreated with doxycycline (or saline). We also examined whether doxcycline (30 mg/Kg, i.p., daily) modifies the effects of nicotine (10mg/kg/day; 4 weeks) on the endothelium-dependent relaxations of rat aortic rings. Aortic MMP-2 levels were assessed by gelatin zymography. Aortic gelatinolytic activity was assessed using a gelatinolytic activity kit. MMP-2 and MMP-9 levels increased in the supernatant of SMC cells incubated with nicotine 150 nM (P<0.05) but not with 50 nM. Nicotine (2 micromol/kg) produced lower increases in the mean arterial pressure in rats pretreated with doxycycline than those found in rats pretreated with saline (26+/-4 vs. 37+/-4 mm Hg, respectively; P<0.05). Nicotine impaired of the endothelium-dependent responses to acetylcholine, and treatment with doxycycline increased the potency (pD2) by approximately 25% (P<0.05). While we found no significant differences in aortic MMP-2 levels, nicotine significantly increased gelatinolytic activity (P<0.05). These findings suggest that nicotine produces cardiovascular effects involving MMPs. It is possible that MMPs inhibition may counteract the effects produced by nicotine.

Lercanidipine decreases vascular matrix metalloproteinase-2 activity and protects against vascular dysfunction in diabetic rats.

Abnormal matrix metalloproteinases (MMPs) activity causes cardiovascular diseases. Because hyperglycemia increase MMPs activities through increased oxidative stress, we hypothesized that antioxidant effects produced by lercanidipine could attenuate the increases in MMP-2 expression/activity in diabetic rats. Control and diabetic (alloxan-induced diabetes) rats received lercanidipine 2.5 mg/kg/day (or tap water) starting three weeks after alloxan (or vehicle) injections. Blood pressure was monitored weekly. After six weeks of treatment, vascular reactivity and structural changes were assessed in aortic rings. MMP-2 levels were determined by gelatin zymography, and MMP-2/tissue inhibitor of metalloproteinases (TIMP)-2 mRNA levels were determined by quantitative real time RT-PCR. Plasma thiobarbituric acid reactive substances concentrations were determined by fluorimetry. Lercanidipine produced antihypertensive effects (201+/-5 vs. 163+/-7 mm Hg in diabetic rats untreated and treated with lercanidipine, respectively; P<0.01) and reversed the impairment in endothelium-dependent vasorelaxation in diabetic rats. Increased MMP-2 and Pro-MMP-2 levels were found in the aortas of diabetic rats (both P<0.001). Lercandipine attenuated the increases in oxidative stress and in MMP-2 (both P<0.05). While diabetes induced no major structural changes, it caused a 16-fold increase in the ratio of MMP-2/TIMP-2 mRNA expression, which was completely reversed by lercanidipine (both P<0.001). These results show that antioxidant and beneficial vascular effects produced by lercanidipine in diabetic rats are associated with reversion of the imbalance in vascular MMP-2/TIMP-2 expression.

Lercanidipine reduces matrix metalloproteinase-2 activity and reverses vascular dysfunction in renovascular hypertensive rats.

Increased expression/activity of matrix metalloproteinases (MMPs), especially MMP-2, plays a role in the vascular alterations induced by hypertension, and increased oxidative stress is a major factor activating MMPs. Here, we hypothesized that lercanidipine, a calcium channel blocker, could attenuate the increases in oxidative stress and MMP-2 expression/activity in the two-kidney, one-clip (2K-1C) hypertensive rats. Sham-operated or 2K-1C hypertension rats were treated with lercanidipine 2.5 mg/kg/day (or vehicle) starting three weeks after hypertension was induced. Systolic blood pressure was monitored weekly. After five weeks of treatment, aortic rings were isolated to assess endothelium-dependent and independent relaxations. Quantitative morphometry of structural changes in the aortic wall were studied in hematoxylin/eosin sections. Aortic MMP-2 levels were determined by gelatin zymography. Aortic MMP-2/tissue inhibitor of metalloproteinases (TIMP)-2 mRNA levels were determined by quantitative real-time RT-PCR. Plasma thiobarbituric acid reactive substances concentrations were determined using a fluorometric method. Lercanidipine attenuated 2K-1C hypertension (224+/-12 versus 183+/-11 mm Hg in 2K-1C rats and 2K-1C + Lercandipine rats, respectively; P<0.01) and prevented the reduction in endothelium-dependent vasorelaxation found in 2K-1C rats. Increased MMP-2 and Pro-MMP-2 levels were found in the aortas of 2K-1C rats (all P<0.05). Lercandipine attenuated 2K-1C-induced increases in MMP-2 by more than 60% and blunted 2K-1C-induced increases in oxidative stress (both P<0.001). While hypertension-induced significant aortic wall hypertrophy and approximately 9-fold increases in the ratio of MMP-2/TIMP-2 mRNA expression (both P<0.05), lercandipine did not affect these changes. These results suggest that lercanidipine produces antihypertensive effects and reverses the endothelial dysfunction associated with 2K-1C hypertension, probably through mechanisms involving antioxidant effects leading to lower MMP-2 activation.

Lercanidipine reduces matrix metalloproteinase-9 activity in patients with hypertension.

Increased levels of metalloproteinase (MMP)-9 have been shown in hypertensive patients. Lercanidipine is a calcium channel blocker with antioxidant actions. We examined whether lercanidipine produces antioxidant effects and reduces MMP-9 activity in hypertensive patients in a placebo-controlled, crossover, single-blinded design study including 18 healthy volunteers (control group), and 14 hypertensive patients without (N = 7) or with (N = 7) diabetes mellitus. Hypertensive patients were randomized to treatment with placebo (15 days) or lercanidipine 20 mg/d (15 days). Arterial blood pressure was evaluated with ambulatory blood pressure monitoring. Plasma thiobarbituric acid reactive species (TBA-RS) levels were measured to assess oxidative stress, and plasma MMP-2 and MMP-9 were assayed by gel zymography before and after treatment with placebo or lercanidipine. Plasma concentrations of tissue inhibitor of metalloproteinases (TIMP)-1 were measured by ELISA. Lercanidipine reduced mean arterial pressure by 7% in hypertensive patients without diabetes (P < 0.05), but not in hypertensive patients with diabetes. It significantly decreased plasma TBA-RS levels in hypertensive patients without and with diabetes (95% confidence interval [CI], -26 to -46%, P = 0.048, and -22 to -33%, P = 0.036, respectively). In addition, lercanidipine decreased activated MMP-9 in hypertensive patients without and with diabetes (95% CI, -19 to -47%, P = 0.047, and -80 to -96%, P = 0.010, respectively). No effects were seen on MMP-2. No significant differences or changes in plasma TIMP-1 concentrations were found. Therefore, we demonstrate for the first time that lercanidipine consistently decreased MMP-9 activity and reduced oxidative stress in hypertensive patients, thus suggesting a mechanism probably involved in the pleotropic actions of lercanidipine.