Inhibition of matrix metalloproteinase activity in vivo protects against vascular hyporeactivity in endotoxemia

Abstract

Persistent arterial hypotension is a hallmark of sepsis and is believed to be caused, at least in part, by excess nitric oxide (NO). NO can combine with superoxide to produce peroxynitrite, which activates matrix metalloproteinases (MMPs). Whether MMP inhibition in vivo protects against vascular hyporeactivity induced by endotoxemia is unknown. Male Sprague-Dawley rats were administered either bacterial lipopolysaccharide (LPS, 4 mg/kg ip) or vehicle (pyrogen-free water). Later (30 min), animals received the MMP inhibitor doxycycline (4 mg/kg ip) or vehicle (pyrogen-free water). After LPS injection (6 h), animals were killed, and aortas were excised. Aortic rings were mounted in organ baths, and contractile responses to phenylephrine or KCl were measured. Aortas and plasma were examined for MMP activity by gelatin zymography. Aortic MMP and inducible nitric oxide synthase (iNOS) were examined by immunoblot and/or immunohistochemistry. Doxycycline prevented the LPS-induced development of ex vivo vascular hyporeactivity to phenylephrine and KCl. iNOS protein was significantly upregulated in aortic homogenates from endotoxemic rats; doxycycline did not alter its level. MMP-9 activity was undetectable in aortic homogenates from LPS-treated rats but significantly upregulated in the plasma; this was attenuated by doxycycline. Plasma MMP-2 activities were unchanged by LPS. Specific MMP-2 activity was increased in aortas from LPS-treated rats. This study demonstrates the in vivo protective effect of the MMP inhibitor doxycycline against the development of vascular hyporeactivity in endotoxemic rats.

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

“…Some rats were given the MMP inhibitors ONO-4817 (100 mg/kg by gavage, diluted in 0.5% carboxymethylcellulose) 2 hours prior to LPS or doxycycline (4 mg/kg IP, Sigma) 30 minutes after LPS. 18 Doxycycline preferentially inhibits MMP-2, -9, and -8 activities and is a much weaker inhibitor of MMP-1. 39,40 ONO-4817 is a more selective MMP inhibitor with a K i in the nanomolar range for MMP-2 and MMP-9 and almost no inhibitory activity up to 100 mol/L against several other proteases.…”

“…Rings were exposed to increasing concentrations of phenylephrine (10 Ϫ9 to 10 Ϫ5 mol/L) as previously described. 18 …”

“…18 To investigate whether LPS induces MMP-2 activation by S-glutathiolation, aortic homogenates were incubated without or with dithiothreitol (0.1, 0.3 or 1.0 mmol/L) and then coimmunoprecipitation analysis was performed with specific antibodies against glutathione and MMP-2 (online-only Data Supplement). Coimmunoprecipitation studies of MMP-2 with calponin-1 in aorta were also performed.…”

“…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 Doxycycline or another MMP inhibitor GM6001 also improved LPS-induced hypocontractility to phenylephrine in isolated aorta. 17 The mechanism by which MMPs contribute to this vascular hypocontractility remains unknown.…”

“…[17][18][19][20][21][22][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.…”