Matrix metalloproteinase (MMP)-2 belongs to a family of zinc-dependent proteases which are best known for their ability to proteolyse extracellular matrix proteins throughout the body, including the cardiovascular system. Increased MMP-2 activity has been demonstrated in myocardial ischaemia and reperfusion injury and the progression to congestive heart failure, with most evidence to date for its role in cardiac remodelling. Recent evidence, however, shows that MMP-2 also co-localizes with and proteolyses specific protein targets within the cardiomyocyte to cause acute, reversible contractile dysfunction, challenging the conventional wisdom on the 'extracellular matrix only' actions of this enzyme. In this review, we discuss the recent upsurge in MMP-2 research with regards to its activation by non-proteolytic pathways in the setting of enhanced oxidative stress in the heart. We will focus on the consequences of intracellular actions of MMP-2 within the cardiomyocyte and its regulation at several levels including its expression, post-translational modifications, and regulation by endogenous tissue inhibitors of metalloproteinases, caveolin, and small molecule MMP inhibitors. MMP-2 is emerging as an important signalling protease implicated in the proteolytic regulation of various intracellular proteins in myocardial oxidative stress injury.

Matrix metalloproteinases (MMPs) have been shown to play significant roles in a number of physiological as well as pathological processes. Best known to proteolyse components of the extracellular matrix, MMPs have recently been discovered to also target a growing list of proteins apart from these, both inside and outside the cell. MMPs have also been traditionally thought of as enzymes involved in chronic processes such as angiogenesis, remodelling and atherosclerosis on a days-week time-scale. However they are now understood to also act acutely in response to oxidative stress on a minutes time-scale on non-extracellular matrix substrates. This review focuses on the acute actions and both extracellular and intracellular targets of two prominent MMP family members, MMP-2 and -9, in cardiovascular diseases including ischaemia/reperfusion injury, inflammatory heart disease, septic shock and pre-eclampsia. Also discussed are various ways of regulating MMP activity, including post-translational mechanisms, the endogenous tissue inhibitors of metalloproteinases and pharmacological inhibitors. A comprehensive understanding of MMP biology is necessary for the development of novel pharmacological therapies to combat the impact of cardiovascular disease.

Matrix metalloproteinase-2 (MMP-2) is best understood for its biological actions outside the cell. However, MMP-2 also localizes to intracellular compartments and the cytosol where it has several substrates, including troponin I (TnI). Despite a growing list of cytosolic substrates, we currently do not know the mechanism(s) that give rise to the equilibrium between intracellular and secreted MMP-2 moieties. Therefore, we explored how cells achieve the unique distribution of this protease. Our data show that endogenous MMP-2 targets inefficiently to the endoplasmic reticulum (ER) and shows significant amounts in the cytosol. Transfection of canonical MMP-2 essentially reproduces this targeting pattern, suggesting it is the quality of the MMP-2 signal sequence that predominantly determines MMP-2 targeting. However, we also found that human cardiomyocytes express an MMP-2 splice variant which entirely lacks the signal sequence. Like the fraction of ER-excluded, full-length MMP-2, this variant MMP-2 is restricted to the cytosol and specifically enhances TnI cleavage upon hypoxia-reoxygenation injury in cardiomyocytes. Together, our findings describe for the first time a set of mechanisms that cells utilize to equilibrate MMP-2 both in the extracellular milieu and intracellular, cytosolic locations. Our results also suggest approaches to specifically investigate the overlooked intracellular biology of MMP-2.

No abstract

Total joint arthroplasty (TJA) patients often receive allogeneic blood transfusion. In this study we sought to create and validate a clinical prediction rule for transfusion in TJA using data that are easily available when scheduling the procedure. Logistic regression modeling was applied to retrospective data from all TJA procedures performed in Edmonton, Alberta in 2000 (n = 1875). The area under the receiver operating curve for the resulting model in the training and validation data sets was 0.80 and 0.76 respectively. By assigning a simple score based on six independent predictors (age, gender, weight, hemoglobin, ASA operative risk classification and whether revision surgery was planned), it was possible to classify a given subject's risk of receiving allogeneic transfusion. We conclude that accurate prediction of transfusion risk in TJA is possible using a rule based on simple preoperative clinical and laboratory data. Such prediction could allow transfusion prevention strategies to be applied selectively to those at greatest risk.

Heavy-body fast-set VPES experienced minimal contraction in vitro after prolonged storage, though surface detail scores were not as consistent as those of the VPS tested. The least contraction occurred when the material was examined immediately after ingot production.

Abstract:We previously reported a 50% reduction in cortical infarct volume following transient focal cerebral ischemia in rats preconditioned 3 days earlier with cortical spreading depression (CSD). The mechanism of the protective effect of prior CSD remains unknown. Recent studies demonstrate reversal of excitatory amino acid transporters (EAATs) to be a principal cause for elevated extracellular glutamate levels during cerebral ischemia. The present study measured the effect of CSD preconditioning on (a) intraischemic glutamate levels and (b) regulation of glutamate transporters within the ischemic cortex of the rat. Three days following either CSD or sham preconditioning, rats were subjected to 200 min of focal cerebral ischemia, and extracellular glutamate concentration was measured by in vivo microdialysis. Cortical glutamate exposure decreased 70% from 1,772.4 Ϯ 1,469.2 M-min in sham-treated (n ϭ 8) to 569.0 Ϯ 707.8 M-min in CSD-treated (n ϭ 13) rats ( p Ͻ 0.05). The effect of CSD preconditioning on glutamate transporter levels in plasma membranes (PMs) prepared from rat cerebral cortex was assessed by western blot analysis. Down-regulation of the glial glutamate transporter isoforms EAAT2 and EAAT1 from the PM fraction was observed at 1, 3, and 7 days but not at 0 or 21 days after CSD. Semiquantitative lane analysis showed a maximal decrease of 90% for EAAT2 and 50% for EAAT1 at 3 days post-CSD. The neuronal isoform EAAT3 was unaffected by CSD. This period of down-regulation coincides with the time frame reported for induced ischemic tolerance. These data are consistent with reversal of glutamate transporter function contributing to glutamate release during ischemia and suggest that down-regulation of these transporters may contribute to ischemic tolerance induced by CSD.

Anaplastic lymphoma kinase (ALK) is a receptor tyrosine kinase aberrantly expressed in neuroblastoma, a devastating pediatric cancer of the sympathetic nervous system. Germline and somatically acquired ALK aberrations induce increased autophosphorylation, constitutive ALK activation and increased downstream signaling. Thus, ALK is a tractable therapeutic target in neuroblastoma, likely to be susceptible to both small-molecule tyrosine kinase inhibitors and therapeutic antibodies–as has been shown for other receptor tyrosine kinases in malignancies such as breast and lung cancer. Small-molecule inhibitors of ALK are currently being studied in the clinic, but common ALK mutations in neuroblastoma appear to show de novo insensitivity, arguing that complementary therapeutic approaches must be developed. We therefore hypothesized that antibody targeting of ALK may be a relevant strategy for the majority of neuroblastoma patients likely to have ALK-positive tumors. We show here that an antagonistic ALK antibody inhibits cell growth and induces in vitro antibody-dependent cellular cytotoxicity of human neuroblastoma-derived cell lines. Cytotoxicity was induced in cell lines harboring either wild type or mutated forms of ALK. Treatment of neuroblastoma cells with the dual Met/ALK inhibitor crizotinib sensitized cells to antibody-induced growth inhibition by promoting cell surface accumulation of ALK and thus increasing the accessibility of antigen for antibody binding. These data support the concept of ALK-targeted immunotherapy as a highly promising therapeutic strategy for neuroblastomas with mutated or wild-type ALK.