P368Activation and susceptible targets of matrix metalloproteinase (MMP)-2 in the vasculature of endotoxemic rats

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Abstract

Purpose

Increased MMP-2 activity in aorta from rats exposed to lipopolysaccharide (LPS) contributes to sepsis-induced vascular hypocontractility. Calponin-1 and smoothelin-B are contractile proteins capable of regulating vascular tone. They are structurally similar to troponin T, a protein involved in cardiac myocyte contraction, which is cleaved by MMP-2 in oxidative stress injuries. Calponin-1 and smoothelin-B may be intracellular targets of MMP-2 in vascular smooth muscle, thus contributing to sepsis-induced vascular hypocontractility and arterial hypotension.

Methods and Results

To determine if MMP-2 co-localizes with calponin-1 or smoothelin-B in the vasculature, rat aorta was analyzed by co-immunoprecipitation studies and by double-immunofluorescence labeling followed by confocal microscopy. Both calponin-1 and smoothelin-B co-immunoprecipitated with MMP-2. Calponin-1 exhibited uniform cytosolic staining with MMP-2 in medial vascular smooth muscle cells. Rats were given either an i.p. injection of a non-lethal dose of lipopolysaccharide (LPS) or vehicle control. Two h before or 30 min after LPS, some rats were given the MMP inhibitors ONO-4817 or doxycycline. At 6 h after LPS, plasma nitrate + nitrite levels rose > 15-fold in LPS-treated rats, an effect unchanged by MMP inhibitors. LPS enhanced MMP-2 S-glutathiolation in aorta which was reversed by dithiothreitol. By tail-cuff plethysmography, ONO-4817 inhibited the LPS-induced reduction in systolic blood pressure (p < 0.01). Aortic rings were mounted in organ baths and their contractile response to phenylephrine was measured. ONO-4817 prevented LPS-induced aorta hypocontractility to phenylephrine (p < 0.01). While calponin-1 levels decreased 25% in endotoxemic aortae at 6 h, smoothelin-B increased by 30%, and both were prevented by MMP inhibitors. In vitro incubation of purified calponin-1 with MMP-2 (100:1 molar ratio) for 2 h at 37°C led to calponin-1 degradation and the appearance of putative cleavage products (identified by mass spectrometry), which was prevented by the MMP inhibitors GM6001 or ONO-4817. Calponin-1 is degraded by MMP-2 in the vasculature after LPS and may contribute to endotoxemia-induced vascular hypocontractility. In contrast, increased aortic smoothelin-B levels after LPS may represent its enhanced turnover.

Conclusions

Either calponin-1 or smoothelin-B may be targets of MMP-2 inside vascular smooth muscle cells following LPS, thus contributing to sepsis-induced vascular hypocontractility.

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