Matrix metalloproteinases, oxidative stress and the acute response to acute myocardial ischaemia and reperfusion

Matrix metalloproteinases, oxidative stress and the acute response to acute myocardial ischaemia and reperfusion Cherry L Wainwright Matrix metalloproteases (MMPs) are responsible for the breakdown of extracellular matrix materials, including collagen and elastin. There is substantial evidence that, although the activity of MMPs in normal tissue is low, there is an increase in activity under a range of disease states that contributes to the chronic pathology of the disease. In cardiovascular disease, MMPs have been implicated in the development of ventricular remodelling post-infarction, and also in the degradation of the fibrous cap of atherosclerotic plaques, thereby contributing to plaque rupture. Recent attention has turned to using the presence of circulating MMPs in patients with recent acute myocardial infarction or unstable angina as a prognostic indicator for eventual chronic outcome. In addition, an emerging role for MMPs in contributing to the early consequences of acute myocardial infarction, such as cardiac dysfunction, has been identified. Addresses School of Pharmacy, The Robert Gordon University, Schoolhill, Aberdeen AB10 1FR, UK e-mail: [email protected]

Current Opinion in Pharmacology 2004, 4:132–138 This review comes from a themed issue on Cardiovascular and renal Edited by Kamal Badr and Cherry Wainwright 1471-4892/$ – see front matter ß 2004 Elsevier Ltd. All rights reserved. DOI 10.1016/j.coph.2004.01.001

Abbreviations AMI acute myocardial infarction CAD coronary artery disease EPA eicosapentanoic acid IL interleukin I/R ischaemia-reperfusion LOX-1 low-density lipoprotein receptor MAPK mitogen-activated protein kinase MMP matrix metalloproteinase OONOS peroxynitrite Ox-LDL oxidized low-density lipoprotein PKC protein kinase C TIMP tissue inhibitor of metalloproteinases

Introduction Matrix metalloproteinases (MMPs) are a structurally related family of endoproteinases that share some common functional features, despite differences in their cellular source, substrate specificity and inducibility [1–3]. Although approximately 20 different MMPs have been identified, they can be subdivided into four groups Current Opinion in Pharmacology 2004, 4:132–138

according to which components of the extracellular matrix they degrade. Group 1 contains the collagenases (MMP-1, MMP-8 and MMP-13), group 2 the gelatinases (MMP-2 and MMP-9) and group 3 the stromelysins (MMP-3, MMP-10 and MMP-11), whereas group 4 contains the membrane-type MMPs, which degrade various extracellular matrix components. Group 4 MMPs also have the ability to activate other MMPs. The activity of MMPs is normally low in healthy tissue, but the increased expression and activity of several MMPs in a range of pathological processes, including inflammation, tumour metastasis and cardiac remodelling, has led to the suggestion that they play a role in the pathophysiology and progression of these diseases. MMPs are regulated under normal conditions at the transcriptional level through control of the activation of the latent enzymes, and through inhibition by endogenous tissue inhibitors of metalloproteinases (TIMPs). The presence of disease, however, can induce MMP production at the transcriptional level (e.g. through inflammatory mediators such as cytokines and growth factors), activation of the latent enzymes and a reduction in the levels of endogenous inhibitors, resulting in excessive breakdown of extracellular material [4–7]. Much of the knowledge we have gained about the role of MMPs in the pathogenesis of disease comes from a wide range of studies with MMP inhibitors and in gene-deleted mice. These have collectively demonstrated the ability of MMP inhibitors or MMP knockout to prevent tumour cell invasion and metastasis [8,9], prevent cartilage breakdown in rheumatoid arthritis [10], improve wound strength [11] and promote healing [12], reduce neointimal formation following balloon angioplasty [13,14] and prevent remodelling of the ventricles, which all indicate a role for MMPs in chronic processes. For example, in the evolving myocardial infarct, there is direct evidence for a detrimental role for increased MMP-9 activity during infarct healing and left ventricular remodelling; further studies show that overexpression of TIMP-1 can prevent left ventricular rupture post-infarct [15–17]. Thus, MMPs have been classically implicated in the matrix remodelling that occurs over a time scale of hours to days (or even weeks). More recently, however, we are beginning to see the emergence of additional roles for MMPs through rapid stimulation of cellular transduction processes. Thus, there has been rising interest in the potential detrimental role of MMPs in the acute stages of acute myocardial infarction, before their involvement in the chronic infarction process. Furthermore, because of their early activation following an acute myocardial infarction (AMI) and www.sciencedirect.com

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their appearance in the circulating plasma, there is also interest in their potential use as prognostic markers of chronic outcome. This review focuses on this emerging picture of the prognostic value of MMPs and their acute role in determining the outcome of AMI. The chronic role of MMPs in cardiac diseases is reviewed excellently by Creemers et al. [18].

MMPs as clinical markers Atherosclerotic plaque rupture

The observation that MMP expression is induced in atherosclerotic plaques prone to rupture (for review, see [19]) suggests that patients with atherosclerotic disease may show enhanced plasma levels of MMPs as an indicator of the severity of disease or as a prognostic marker of ongoing inflammatory processes contributing to ultimate plaque rupture. MMP-9 is increased in patients with three-vessel coronary artery disease (CAD) compared with controls or patients with one or two vessel disease, implying that elevated levels of MMP-9 reflect severe coronary stenosis [20]. Furthermore, MMP-9 (but not MMP-2) was elevated in post-myocardial infarction patients when compared with patients with no history of athero-thrombotic disease [21], suggesting that MMP-9 serum levels may be a novel marker of inflammation in patients with CAD and could provide an index of plaque activity and propensity to rupture. In support of this, elevated levels of both MMP-9 and TIMP-1 have been detected in the coronary circulation of acute coronary syndrome patients (AMI and unstable angina) compared with control patients and those with stable angina [22], suggesting that these act as markers of active plaque rupture. In contrast, Kai et al. [23] found that both MMP-9 and MMP-2 levels were elevated for up to seven days in patients with unstable angina and AMI, suggesting a role for both of these MMPs in the pathology of plaque rupture. Acute myocardial infarction

A study on the time course of plasma concentrations of MMP-1 as a potential prognostic factor in determining the extent of left ventricular remodelling following an AMI revealed that raised MMP-1 levels were associated with a lower left ventricular ejection fraction in post-AMI patients for up to two weeks after admission, leading to the conclusion that high concentrations of MMP-1 in the sub-acute phase after AMI predicts left ventricular remodelling [24]. A further study showed that MMP-2 levels in plasma and the generation of MMP-1 by peripheral blood mononuclear cells post-AMI correlated with left ventricular end-diastolic volume index [25]. Furthermore, a study of the time course of MMP-1, MMP-9 and TIMP-1 appearance in plasma in patients post-myocardial infarction demonstrated that the ratio of MMP-9 to TIMP-1 was raised on admission and returned to baseline by 48 hours [26], supporting the hypothesis that MMP-9 plays a pathophysiological role in the early phase of AMI. www.sciencedirect.com

A further study to determine the effect of Mg2þ at reperfusion demonstrated that those patients receiving Mg2þ had less reperfusion injury and lower MMP-1 and interleukin (IL)-6 levels, whereas MMP-1 and IL-6 levels were elevated in untreated patients, particularly those with coronary heart failure [27]. Although these findings suggest that Mg2þ may protect the myocardium from injury by preventing the production of MMP-1 and IL-6, the converse argument that the decreased levels of MMP-1 and IL-6 might be a consequence of decreased injury, rather than the cause of the protection, must not be ignored.

Evidence for a role for MMPs in the early consequences of acute myocardial infarction MMP-1

MMP-1, a collagenase found within the interstitium, has been implicated in chronic ventricular remodelling after AMI. Increased activity of MMP-1, co-localised with cellular necrosis, has been reported following several hours of reperfusion in pigs [28], suggesting a potential link between MMPs and cell death. A more recent study in rats demonstrated that coronary occlusion for one hour, followed by a period of reperfusion as short as one hour, resulted in upregulation of MMP-1, cardiac dysfunction and tissue necrosis [29]. Furthermore, in the same study, active MMP-1 induced cell death in isolated cardiomyocytes, which was prevented by a specific MMP inhibitor, providing the first evidence that MMP-1 can directly cause cardiomyocyte cell death whereas, interestingly, recombinant transforming growth factor-b decreased necrosis and dysfunction, and inhibited upregulation of MMP-1 in the in vivo model, suggesting that this protection against myocardial injury might, in part, be mediated by inhibition of MMP-1 upregulation. On the basis of the observation that docosahexanoic acid, a o-3 polyunsaturated fatty acid, can suppress MMP activity [30,31] the same group determined the effects of eicosapentanoic acid (EPA) on MMP-1 expression and activity in cultured myocytes subjected to hypoxia and reperfusion [32]. EPA attenuated myocyte injury (measured by lactate dehydrogenase release) and suppressed the hypoxia-induced increase in MMP-1 expression and activity. Furthermore, the hypoxia/reperfusion-induced increases in phosphorylation of p38 mitogen-activated protein kinase (MAPK) and lipid peroxidation were both attenuated by EPA. These findings suggest that EPA inhibits MMP-1 either by preventing p38MAPK phosphorylation or through an antioxidant mechanism, which would indirectly prevent p38MAPK activation. MMP-2

MMP-2, a gelatinase enzyme responsible for degrading collagen type IV, has been found in the endocardial and subendocardial layers of the heart [33]. It is released rapidly from human platelets after MMP-induced platelet aggregation [34,35], suggesting that it may be involved in Current Opinion in Pharmacology 2004, 4:132–138

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rapid stimulation of cellular signal transduction processes before the development of changes in collagen matrix. A study in isolated rat hearts demonstrated the presence of pro-MMP-2, MMP-2 and an unidentified 75-kDa gelatinase in the coronary effluent of aerobically perfused hearts [36]. When hearts were then subjected to ischaemia-reperfusion (I/R), the release of pro-MMP-2 into the coronary effluent was markedly increased within minutes of reperfusion, and the levels of pro-MMP-2 correlated negatively with cardiac functional recovery. Furthermore, infusion of semi-purified MMP-2 worsened functional recovery, whereas the non-selective MMP inhibitors doxycycline and o-phenanthroline prevented the contractile dysfunction associated with I/R, providing collective evidence for a direct link between acute cardiac dysfunction and MMP-2 levels. A role for decreased proMMP-2 activation as a mechanism underlying ischaemic preconditioning has also been explored in studies in isolated rat hearts, where the rise in MMP-2 levels following I/R was prevented by ischaemic preconditioning [37]. However, this study did not investigate whether this effect on MMP-2 actually contributes to the protective effect of preconditioning, thus leaving open an intriguing question. Because there is evidence that MMP-2 is closely associated with the sarcomeres in dilated cardiomyopathy [38], it has been proposed that MMP-2 induces cardiac dysfunction through digestion of the contractile proteins. A study by Wang et al. [39] demonstrated that MMP-2 cleaves troponin I (but not troponin T or troponin C) when challenged with intact troponin complex in cardiac myocytes in vitro. Additional studies in isolated rat hearts demonstrated MMP-2 localisation in the sarcomeres, in close proximity to thin myofilaments, and co-localisation with troponin I, providing the first evidence for an intracellular action of MMP-2 on cardiomyocytes. The mechanism by which pro-MMP-2 is activated so rapidly following reperfusion has not been fully elucidated, but it could involve peroxynitrite generated during the first few minutes of reperfusion, either through direct activation of pro-MMP [40] or through inhibition of TIMP-1 [41]. Schulze et al. [42] investigated the latter possibility by assessing the effect of I/R injury on the balance between MMP and TIMP. In vitro studies in rats hearts showed that TIMP-4 (but not TIMP-1, -2 or -3) was increased within the first minute of reperfusion following 20 min of ischaemia, and was concurrent with reduced contractile function. Interestingly, this relationship was time-dependent; when periods of ischaemia were insufficient to reduce contractile function, there was no elevation of TIMP-4, and vice versa. In addition, there was a close association of TIMP-4 with cardiomyocyte sarcomeres, which was reduced in hearts subjected to injurious periods of I/R. Furthermore, the loss of TIMP-4 from the cardiomyocyte was associated with an increased gelatinolytic activity. Although other studies have shown that Current Opinion in Pharmacology 2004, 4:132–138

TIMPs are reduced in long-term diseased hearts (e.g. post-AMI, heart failure and dilated cardiomyopathy) and possibly involved in remodelling [43–45], this is the first study to demonstrate a loss of TIMP within the first minutes of reperfusion that can be correlated with a reduced recovery of mechanical function. The Schulze study agrees with a patient study by Mayers et al. [46] in which a reduction in TIMP-4 was observed immediately after artery bypass in tissue biopsies from patients undergoing by-pass surgery. However, not all studies agree that an imbalance between MMPs and TIMPs plays an early role following I/R. In pigs subjected to 90 min moderate ischaemia and 90 min reperfusion, although the total content of latent and active forms of MMP-9 were raised, MMP-2 levels (either latent or active) were not altered and MMP-3 and MMP-7 were absent [47]. Furthermore, only TIMP-2 and TIMP-3 were present in the hearts, with no differences between ischaemic and non-ischaemic regions. Although hearts were histologically normal, there was no evidence for disruption of collagen ultrastructure; however, there was depressed myocardial function and incomplete recovery, which was unaffected by an MMP inhibitor (GM2487). This led the authors to conclude that in moderate ischaemia there is elevation of MMP-9, but this is not caused by downregulation of TIMP expression. Thus, although there is clear evidence that an imbalance exists between endogenous MMP inhibition and MMP activity following a prolonged and severe I/R insult, suggesting a protective role for TIMP-4 and a detrimental role for MMPs in mediating contractile dysfunction and injury, this is not responsible for the contractile dysfunction experienced following a mild episode of ischaemia without tissue necrosis. MMP-9

MMP-9, like MMP-2, is a gelatinase that is upregulated following permanent coronary occlusion in a range of species [28,43,47,48]. Recently, MMP-9 has been shown to be upregulated in failing human heart, suggesting a role in cardiomyopathy [49]. Moreover, MMP-9 knockout mice have reduced left ventricular enlargement following permanent coronary artery ligation, indicative of a role for MMP-9 in ventricular remodelling [16]. However, despite evidence that MMP-9 is upregulated in the ischaemic-reperfused heart, the functional contribution of this to early injury is not known. A recent study in hearts from MMP-9 knockout mice subjected to in vivo coronary occlusion and reperfusion for 24 h revealed that both pro-MMP-9 and active MMP-9 were elevated after 30 min ischaemia and further elevated by reperfusion in wild-type mice [50]. This elevation was attenuated in MMP-9þ/ mice but abolished in MMP-9/ mice, concomitant with enhanced TIMP-1 expression in the knockout mice. In wild-type mice, MMP-9 expression was greatest in infiltrating neutrophils, whereas the www.sciencedirect.com

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MMP-9/ mice had smaller infarcts, thus providing the first evidence to delineate a pivotal role for MMP-9 in acute myocardial injury. These findings agree with results from another study [51] showing neutrophils as the major source of MMP-9 in I/R. Overall, evidence points to a role for MMP-9 in the inflammatory phase of the infarction process that occurs within the first few hours of the insult, and suggests that MMP-9 inhibition could be an approach to inhibiting acute injury, partly through attenuation of neutrophil-mediated injury and partly through improving the balance between proteinase activity and the endogenous anti-proteinases such as TIMP-1.

Possible signaling pathways for MMP activation in the acute phase following myocardial infarction LDL receptor

The low-density lipoprotein receptor (LOX-1) is the newly described lectin-like receptor for oxidized lowdensity lipoprotein (ox-LDL), which has been shown to mediate ox-LDL-induced cell injury through activation of the oxidative-stress-sensitive p38MAPK [52]. LOX-1 is upregulated by a variety of mechanisms, including free radicals, inflammatory cytokines and shear stress, and can be stimulated by advanced glycation end products. In human coronary artery endothelial cells, oxLDL induces expression of CD40 and CD40L, an effect that is mediated by LOX-1 and protein kinase C (PKC)-a, resulting in induction of pro-inflammatory genes such as those signaling for tumour necrosis factor-a production and P-selectin expression [53–55]. Ox-LDL, acting through LOX-1, has also been shown to decrease myocardial contractility [56]. A series of recent studies has investigated whether LOX-1 is involved in determining the extent of I/R injury and, if so, whether this involves MMP-1 upregulation and inflammatory cell recruitment. In the first study [57], induction of I/R in in vivo rats demonstrated that mRNA and immunocytochemistry expression of LOX-1 was increased in the endocardium and subendocardium, and was co-expressed with MMP-1 and adhesion molecules (P-selectin, vascular cell adhesion molecule-1 and intercellular adhesion molecule-1). Expression of LOX-1, MMP-1 and the adhesion molecules were all attenuated by a neutralising antibody to LOX-1, suggesting that activation of LOX-1 was the first step in the pathway leading to MMP activation and adhesion molecule expression. The LOX-1 antibody also blocked the I/R-induced increase in leukocyte presence in and around the coronary arteries within the ischaemic zone, decreased infarct size and blocked the rise in oxidative-stress-induced p38MAPK activity in the ischaemic-reperfused tissue. The authors hypothesised that oxLDL acting on LOX-1 could induce the generation of reactive oxygen species (although they provided no direct evidence for this) that then activate p38MAPK. Although this hypothesis does align with a previous observation that ox-LDL is localised within the ventricular walls of www.sciencedirect.com

patients with CAD [58], the studies described here were performed in normocholesterolaemic rats where the levels of ox-LDL within the myocardial wall would be expected to be very low. Therefore, it is unlikely that the endogenous ligand activating LOX-1 in the rat studies is ox-LDL. In a subsequent study by the same group [59], exploration of the intracellular mechanism mediating the interaction between LOX-1 and MMPs in human coronary arterial endothelial cells revealed that ox-LDL increased the expression (mRNA and protein) of both MMP-1 and MMP-3, with only a slight increase in TIMP-1 and TIMP-2, and increased collagenase and PKC activity. These effects were blocked by a LOX-1 antibody and the PKC-b inhibitor hispidin (but not other PKC inhibitors), suggesting that the induction of MMP-1 and -3 by ox-LDL is mediated through PKC-b in endothelial cells. Reactive oxygen species

Peroxynitrite (ONOO) is the reactive product of nitric oxide and superoxide, and has been recognised to play a key role in a range of cardiovascular pathologies, including I/R injury and myocardial dysfunction [60]. Several studies have shown that ONOO can activate MMPs and, because the time course of generation of ONOO and MMP activation post-reperfusion are similar, a relationship between the contractile dysfunction induced by ONOO and MMP activation has emerged. Recent studies in isolated rat hearts [61] have shown that infusion of ONOO into the coronary circulation results in a rapid increase in the release of MMP-2 into the coronary effluent, concomitant with a decline in contractile function. The ability of both a selective MMP-2 inhibitor (PD-166793) and detoxification of ONOO with glutathione to abrogate the effects of ONOO on contractile function strongly suggests that the pathway of ONOOinduced myocardial injury is mediated through MMP-2, most likely by degradation of the contractile apparatus.

Role of MMPs in transplant-related injury Although most of this review has focused on both the prognostic value of MMP activation and an early role for MMPs in contributing to injury post-AMI, it is worth mentioning a potential role for these enzymes in the injury sustained by the heart during cardiac transplantation. A recent experimental study [62] into the regulation of MMPs in transplanted rat hearts investigated the effect of the MMP inhibitor batimastat on transplant-induced injury. The key finding of this study was an increased active MMP-2 expression in allografted, but not isografted, hearts, despite all groups having equal levels of pro-MMP-2 expression, which was blocked by batamistat. Furthermore, with doses of batimastat that completely inhibited MMP-9 production, there was less basement membrane damage (laminin and collagen IV), reduced myeloperoxidase activity and lower tumour necrosis factor-a levels. Taken together, these findings Current Opinion in Pharmacology 2004, 4:132–138

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suggest that transplantation results in activation of MMP-2 through an inflammatory mechanism, and that this contributes to transplant-induced damage. Thus, inhibition of MMP activation might offer a novel approach to preventing damage and rejection.

Conclusions Although it is generally accepted that activation of the MMPs contributes to the chronic processes of ventricular remodelling, plaque rupture and dilated cardiomyopathy, as well as a range of non-cardiovascular diseases, there is now emerging evidence that these enzymes might also play a much earlier role in the progression of cardiovascular disease. In the setting of acute myocardial ischaemia, three MMPs (MMP-1, MMP-2 and MMP-9) appear to be of greatest importance, with each enzyme being gener-

ated from different sources and most likely responsible for different aspects of the pathological process of tissue necrosis and healing (Figure 1). MMP-1, which is activated through p38MAPK (either directly or indirectly), can induce cardiomyocyte death that might contribute to the immediate lethal injury that is observed within the first few minutes of coronary reperfusion. MMP-2, which could be present intracellularly or possibly released from platelets activated by ischaemia, appears to play a very early role following myocardial reperfusion, where it appears to orchestrate the breakdown of the contractile apparatus, resulting both in cellular injury and in the functional consequence of impaired myocardial contractility. Finally, MMP-9 is most closely associated with neutrophils, which are known to infiltrate injured tissue within a few hours of reperfusion, where it is likely to

Figure 1

Ischaemia/reperfusion injury

Platelet activation

LOX-1 expression and activation

MMP-2 release

Oxidative stress ONOO–?

PMN attachment/ tissue infiltration

P38 MAPK activation

TIMP-1

Troponin I degradation

Adhesion molecule expression

MMP-9 release MMP-1 activation Mechanical dysfunction

Tissue necrosis ECM breakdown

Cardiomyocyte death

Ventricular remodeling

Mechanical dysfunction Current Opinion in Pharmacology

Schematic diagram illustrating the potential role for MMPs in mediating the acute injury response to myocardial ischaemia and reperfusion.

Table 1 Potential pharmacological approaches to improving the acute outcome of myocardial I/R by interfering with pathways involving MMP activation. MMP

Cellular effects

Potential inhibitors

Outcome

MMP-1

Induction of cardiomyocyte death (acute) Breakdown of extracellular matrix (chronic) Troponin degradation

Selective MMP-1 inhibitors PUFAs Antioxidants Inhibitors of p38MAPK Inhibitors of platelet activation MMP-2 inhibitors (e.g. doxacycline) Antibodies to adhesion molecules? Antioxidants?

Reduced tissue injury and improved mechanical function (acute) Reduced ventricular remodelling

MMP-2 MMP-9

Contributes to inflammatory phase of tissue necrosis following reperfusion?

Improved functional recovery following reperfusion Reduced infarct size

PUFA, polyunsaturated fatty acid.

Current Opinion in Pharmacology 2004, 4:132–138

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contribute to the extension of cellular death. Taken together, this picture offers several potential new targets for preventing the acute cellular and functional consequences of myocardial reperfusion (Table 1).

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