Manoj M. Lalu

Preeclampsia: current understanding of the molecular basis of vascular dysfunction.

Preeclampsia is a pregnancy-specific disorder characterised by hypertension and proteinuria occurring after the 20th week of gestation. Delivery of the placenta results in resolution of the condition, implicating the placenta as a central culprit in the pathogenesis of preeclampsia. In preeclampsia, an inadequate placental trophoblast invasion of the maternal uterine spiral arteries results in poor placental perfusion, leading to placental ischaemia. This could result in release of factors into the maternal circulation that cause widespread activation or dysfunction of the maternal endothelium. Factors in the maternal circulation might induce oxidative stress and/or elicit an inflammatory response in the maternal endothelium, resulting in the altered expression of several genes involved in the regulation of vascular tone. This review addresses the potential circulating factors and the molecular mechanisms involved in the alteration of vascular function that occurs in preeclampsia.

Increased Activities of Cardiac Matrix Metalloproteinases Matrix Metalloproteinase (MMP)-2 and MMP-9 Are Associated with Mortality during the Acute Phase of Experimental Trypanosoma cruzi Infection

The strong inflammatory reaction that occurs in the heart during the acute phase of Trypanosoma cruzi infection is modulated by cytokines and chemokines produced by leukocytes and cardiomyocytes. Matrix metalloproteinases (MMPs) have recently emerged as modulators of cardiovascular inflammation. In the present study we investigated the role of MMP-2 and MMP-9 in T. cruzi-induced myocarditis, by use of immunohistochemical analysis, gelatin zymography, enzyme-linked immunosorbent assay, and real-time polymerase chain reaction to analyze the cardiac tissues of T. cruzi-infected C57BL/6 mice. Increased transcripts levels, immunoreactivity, and enzymatic activity for MMP-2 and MMP-9 were observed by day 14 after infection. Mice treated with an MMP inhibitor showed significantly decreased heart inflammation, delayed peak in parasitemia, and improved survival rates, compared with the control group. Reduced levels of cardiac tumor necrosis factor-alpha, interferon-gamma, serum nitrite, and serum nitrate were also observed in the treated group. These results suggest an important role for MMPs in the induction of T. cruzi-induced acute myocarditis.

Preconditioning decreases ischemia/reperfusion-induced release and activation of matrix metalloproteinase-2.

Release and activation of matrix metalloproteinases (MMPs) significantly contribute to myocardial stunning injury immediately after ischemia and reperfusion, however, their role in preconditioning remains unknown. We therefore examined the effects of preconditioning and subsequent ischemia/reperfusion on MMP activity in isolated rat hearts. Hearts were subjected to a preconditioning protocol (three consecutive 5-min periods of global ischemia interspersed with 5 min of reperfusion) followed by 30 min ischemia and 5 min reperfusion. To measure MMP release, coronary effluent was collected: (a) during aerobic perfusion, (b) in reperfusion following each preconditioning ischemia, and (c) during the final reperfusion following test ischemia. MMP-2 activities could be detected by gelatin zymography in the ventricles and coronary effluent samples from the perfused hearts. The levels of MMP-2 activity in the effluent were markedly increased in effluent following test ischemia from control hearts without preconditioning. This was accompanied by a decrease in corresponding tissue MMP activities. Preconditioning significantly decreased the MMP-2 activity in the coronary effluent following test ischemia/reperfusion and preserved the MMP-2 protein content and activity in the myocardium. Our results demonstrate that classic preconditioning inhibits ischemia/reperfusion induced release and activation of MMP-2. These results suggest that preconditioning may exert part of its cardioprotective effects through the reduction of MMP-2 release.

Matrix metalloproteinase inhibitors attenuate endotoxemia induced cardiac dysfunction: A potential role for MMP-9

Enhanced cardiac generation of peroxynitrite contributes to septic cardiomyopathy. Since matrix metalloproteinases (MMPs) are activated in vitro by peroxynitrite, we hypothezised that MMPs may contribute to cardiac mechanical dysfunction in sepsis. Rats were injected (i.p.) with either lipopolysaccharide (LPS, 4mg/kg) or vehicle. MMP inhibitors, either Ro 31-9790 (20 mg/kg), doxycycline (4mg/kg), or vehicle were administered i.p. 30 min after LPS. At 6 h, when the symptoms of endotoxemia peak, hearts were excised and perfused as working hearts with Krebs-Henseleit buffer at 37°C. Cardiac work (cardiac output x peak systolic pressure product) was measured. Perfusate and ventricle samples were analyzed by gelatin zymography to quantify MMP activity.Cardiac function was significantly depressed in LPS-treated rats compared to control rats (control: 55 ± 4, LPS: 26 ± 6 mmHg*mL*min−1). LPS also caused a loss of 72 kDa MMP-2 activity in the ventricles and the perfusate. Although MMP-9 activity was not detected in the ventricles, LPS resulted in an increase in perfusate 92 kDa MMP-9 activity. The MMP inhibitors significantly improved cardiac function of LPS-treated rats (Ro31-9790: 38 ± 3, doxycycline: 51 ± 3 mmHg*mL*min−1), had no effect on the loss of MMP-2 activity, and significantly reduced the MMP-9 activity in the perfusate. These results demonstrate, for the first time, that LPS induced cardiac dysfunction is associated with a loss in ventricular MMP-2 activity and the release of MMP-9 from the heart. MMP inhibitors can significantly preserve cardiac mechanical function during septic shock.

Matrix metalloproteinases contribute to endotoxin and interleukin‐1β induced vascular dysfunction

The acute vascular inflammatory dysfunction associated with endotoxaemia may reflect an imbalance between matrix metalloproteinases (MMPs) and their natural inhibitors (TIMPs), induced by the endotoxin. This possibility was tested in rat aortic tissue.

Peroxynitrite in Myocardial Ischemia-Reperfusion Injury

Peroxynitrite is a highly reactive oxidant which is produced during reperfusion of the ischemic heart. The role that this molecule plays in reperfusion injury has been controversial. Many investigations have demonstrated toxic effects of peroxynitrite, whereas others have found it to be protective during reperfusion. This review surveys evidence supporting both sides and proposes that peroxynitrite is a dichotomous molecule with beneficial and detrimental effects on the reperfused heart. Its toxic effects are mediated by modification and activation of a variety of targets (including poly (ADP) ribose synthetase and matrix metalloproteinases) while its beneficial effects are primarily mediated through its reaction with thiols, resulting in the formation of NO donor compounds (S-nitrosothiols).

Endothelial dependence of matrix metalloproteinase-mediated vascular hyporeactivity caused by lipopolysaccharide

Septic shock remains the leading cause of death in intensive care units in North America. Recent evidence implicates matrix metalloproteinases (MMP) in the pathogenesis of sepsis. MMP activity is upregulated in blood vessels exposed to bacterial lipopolysaccharide (LPS) or pro-inflammatory cytokines and contributes to vascular hyporeactivity to vasoconstrictors. The exact mechanism of MMP-mediated vascular hyporeactivity is unknown. We investigated the contribution of the endothelium in the MMP response to LPS-mediated vascular hyporeactivity in vitro. Tone induced by phenylephrine in isolated rat aortic rings with either intact or denuded endothelium was measured in the presence of LPS for 6 h. These rings were incubated with the nitric oxide (NO) synthase inhibitor, N(G)-nitro-l-arginine methyl ester (l-NAME), to determine whether NO synthase was involved in the response, or the MMP inhibitors, doxycycline or GM6001. MMP activity was measured after 6 h. LPS caused a greater reduction of phenylephrine-induced tone in endothelium-intact rings versus endothelium-denuded rings, indicating both endothelium-independent and -dependent mechanisms for LPS-induced vascular hyporeactivity. l-NAME abolished the response to LPS in both endothelium-intact and endothelium-denuded rings. MMP inhibitors prevented the LPS-induced loss of tone in endothelium-intact but not endothelium-denuded rings. LPS caused significantly greater MMP-2 activity in endothelium-intact aortae which was attenuated by doxycycline. MMP-2 activity in endothelium-denuded aortae was unchanged by LPS. The vascular endothelium contributes to MMP-mediated vascular dysfunction induced by LPS. The protective effect of MMP inhibition is endothelium-dependent and is a novel mechanism by which MMPs contribute to vascular dysfunction.

Matrix metalloproteinases: control of vascular function and their potential role in preeclampsia.

Preeclampsia is a pregnancy specific disorder characterised by hypertension and proteinuria occurring after the twentieth week of gestation. Preeclampsia induced hypertension is the result of increased vascular reactivity and endothelial dysfunction, however, the mechanisms underlying this state remain elusive. One possible mediator may be the matrix metalloproteinases (MMPs), a family of proteinases typically recognized for long term tissue remodelling. This review examines the evidence suggesting a role for MMPs in acutely regulating vascular function. Studies have shown that MMPs can activate vasoconstrictors (e.g. endothelin), inactivate vasodilators (e.g. calcitonin gene related peptide) and transactivate cell surface receptors responsible for vasoconstriction (e.g. epidermal growth factor receptor). The potential role of these proteinases in preeclampsia will then be discussed.

Effect of peroxynitrite scavenging on endothelial cells stimulated by plasma from women with preeclampsia: a proteomic approach.

Hypothesis: Increased peroxynitrite formation in endothelial cells incubated with plasma from women with preeclampsia alters the expression of specific endothelial proteins. Methods: We used a proteomic approach to examine the effect of a peroxynitrite scavenger (FeTPPS, 5 μmol/L) on the expression of endothelial proteins in response to plasma from women with preeclampsia. After treatment, cells were subjected to two-dimensional gel electrophoresis and proteins were identified using mass spectrometry. Results: Peroxynitrite scavenging decreased the expression of structural proteins, cytokeratin, actin-γ-1, and moesin, and the calcium-binding protein, calumenin. Conclusions: This study has identified novel proteins that are regulated by peroxynitrite in response to plasma from women with preeclampsia.

Proteasome inhibition decreases inflammation in human endothelial cells exposed to lipopolysaccharide.

Background: The proteasome degrades ubiquitinated proteins and is the major pathway for intracellular protein degradation. The role of the proteasome in endothelial dysfunction observed in septic shock remains unknown. We stimulated primary cultures of human umbilical vein endothelial cells with lipopolysaccharide (LPS) and investigated effects on the proteasome. We hypothesized that proteasome inhibition would decrease endothelial cell activation, oxidative stress, and alter the proteome. Methods: Endothelial cells were exposed to LPS (100 ng/mL) for 6 hours with or without lactacystin (5 mM), a proteasome inhibitor. Proteasome content and ubiquitinated proteins were measured by enzyme-linked immunosorbent assay and immunoblot, respectively. Markers of cellular activation, vascular cell adhesion molecule-1 and intercellular adhesion molecule-1, were measured by immunoblot and immunoassay. Superoxide anion production was determined by dihydroethidium assay, and nitrotyrosine (a marker of peroxynitrite) was visualized by immunofluoresence. The endothelial cell proteome was analyzed by 2D gel electrophoresis. Results: LPS stimulation of endothelial cells significantly increased proteasome content, whereas the total levels of ubquitinated proteins decreased. This suggests that LPS activates the proteasome system in endothelial cells. LPS increased total content and cell surface expression of vascular cell adhesion molecule-1 and intercellular adhesion molecule-1, whereas proteasome inhibition ameliorated these increases. LPS increased both superoxide anion production and nitrotyrosine staining. Proteasome inhibition decreased both markers of cellular oxidative stress. Proteomic analysis identified two novel proteins upregulated by LPS and normalized with proteasome inhibition as follows: guanine nucleotide binding protein-1 and heterogeneous ribonucleoprotein K transcript variant. Conclusions: These results suggest that inhibition of the proteasome diminishes a number of markers of cellular stress induced by LPS. The proteasome may be a promising therapeutic target in clinical situations of severe pro-inflammatory stress.