Matrix Metalloproteinase-2 Proteolysis of Calponin-1 Contributes to Vascular Hypocontractility in Endotoxemic Rats

Abstract

Objective-Matrix metalloproteinase (MMP)-2 is activated in aorta during endotoxemia and plays a role in the hypocontractility to vasoconstrictors. Calponin-1 is a regulator of vascular smooth muscle tone with similarities to troponin, a cardiac myocyte protein that is cleaved by MMP-2 in myocardial oxidative stress injuries. We hypothesized that calponin-1 may be proteolyzed by MMP-2 in endotoxemia-induced vascular hypocontractility. Methods and Results-Rats were given a nonlethal dose of bacterial lipopolysaccharide (LPS) or vehicle. Some rats were given the MMP inhibitors ONO-4817 or doxycycline. Six hours later, plasma nitrateϩnitrite increased Ͼ15-fold in LPS-treated rats, an effect unchanged by doxycycline. Both ONO-4817 and doxycycline prevented LPS-induced aortic hypocontractility to phenylephrine. LPS activated MMP-2 in the aorta by S-glutathiolation. Calponin-1 levels decreased by 25% in endotoxemic aortae, which was prevented by doxycycline. Calponin-1 and MMP-2 coimmunoprecipitated and both exhibited uniform cytosolic staining in medial vascular smooth muscle cells. In vitro incubation of calponin-1 with MMP-2 led to calponin-1 degradation and appearance of its cleavage product. Conclusion-Calponin-1 is a target of MMP-2, which contributes to endotoxemia-induced vascular hypocontractility. Key Words: metalloproteinases Ⅲ calponin-1 Ⅲ endotoxemia Ⅲ lipopolysaccharide Ⅲ vascular hypocontractility S epsis remains one of the most common causes of death worldwide. 1 Its cardiovascular manifestations include myocardial dysfunction and severe arterial hypotension caused in part by vascular hyporeactivity to vasoconstrictors. 2 Several mechanisms 3 including lipopolysaccharide (LPS)-and interleukin-1␤-mediated activation of inducible nitric oxide (NO) synthase, excess biosynthesis of NO, 4,5 and peroxynitrite (ONOO Ϫ ) 6,7 in the vascular wall are important mediators of the vascular dysfunction in sepsis. ONOO Ϫ directly activates matrix metalloproteinases (MMPs), 8,9 a group of zinc-dependent endopeptidases best known for their ability to degrade extracellular matrix proteins, causing vascular dysfunction and remodeling in many cardiovascular diseases. 10 -16 MMPs are synthesized as inactive zymogens in several cells, including vascular smooth muscle that express 72 kDa MMP-2 abundantly. 10 It is activated by proteolytic removal of the autoinhibitory propeptide domain 10 or, alternatively, by S-glutathiolation of a critical cysteine in this propeptide, on reaction with ONOO Ϫ and glutathione, resulting in 72 kDa S-glutathiolated MMP-2. 9 However, whether MMP-2 activation occurs by S-glutathiolation in the vasculature is unknown.Studies have implicated MMPs and the beneficial effects of MMP inhibition in experimental models of sepsis. 17-23 MMP-2 plays an important role in LPS-induced vascular hypocontractility in rats. In vivo treatment with the MMP inhibitor doxycycline attenuated LPS-induced increase in vascular MMP-2 activity and protected against the loss of vascular contractile tone. 18 Doxycyclin...

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