- Email: [email protected]
Cardiac stress adaptation and hyperlipidemia: Role of matrix metalloproteinase-2
ABSTRACTS / Journal of Molecular and Cellular Cardiology 40 (2006) 920 – 1015
to expose HL-5 cells to various durations of calcium overload. Calpain activation was investigated by means of immunoblot analyses of desmin degradation and fluorescence increases reflecting the hydrolysis of the synthetic peptide Suc-LLVY-AMC, a calpain substrate. Cell death was assessed as lactic dehydrogenase (LDH) release. Calpain activation became detectable after 20 min of calcium overload and was followed by the increase in LDH release, which approached to plateau after 40 min. The addition of EGTA after 30 min was no longer able to block the progression in cell death and calpain activation. More importantly, calpain inhibition by 10 AM PD150606 or 100 AM calpeptin reduced significantly LDH release although at a lesser extent than calpain-mediated proteolysis. In conclusion, the present findings suggest that (i) calpain activation precedes the onset of cell death; (ii) intracellular calcium overload hampers cell viability in a process that eventually becomes independent of Ca2+; (iii) calpain activation is causally related to cell death, although the severity of the present protocol limits the protective efficacy of calpain inhibition. doi:10.1016/j.yjmcc.2006.03.060
046. Protease-activated receptors and endothelial prostanoid production Caroline P.D. Wheeler-Jones. Royal Veterinary College, Royal College Street, London NW1 0TU, UK The principal mechanism through which serine proteases regulate cell behaviour is by activation of a unique family of G-protein coupled receptors, referred to as protease-activated receptors (PARs 1 – 4). The functional significance of PARs in endothelial cells is largely undefined and the intracellular consequences of their activation are poorly understood. We have shown that the serine protease thrombin, as well as PAR-1 and -2-selective peptides induces cyclooxygenase-2 (COX-2) protein and mRNA expression in human endothelial cells without modifying COX-1 expression. COX-2 induction was accompanied by sustained production of PGI2, and this was inhibited by the COX-2-selective inhibitor NS398. PAR-1 and PAR-2 stimulation rapidly activated both ERK1/2 and p38mapk and pharmacological or molecular blockade of MEK or p38mapk strongly inhibited thrombin- and PAR-2-induced COX-2 expression and PGI2 formation. Thrombin, and peptide agonists of PAR-1 and PAR-2, increased luciferase activity in HUVEC infected with an NF-nB-dependent luciferase reporter adenovirus and this, as well as PAR-induced PGI2 synthesis, was inhibited by coinfection with adenovirus encoding wild-type or mutated (Y42F) InBa. Thrombin- and PAR-2-induced COX-2 expression and 6-keto-PGF1a generation were markedly attenuated by the NF-nB inhibitor PG490 and partially inhibited by the proteasome pathway inhibitor MG-132. PAR-1 or PAR-2 stimulation caused nuclear translocation and phosphorylation of p65-NF-nB, and thrombin-, but not
937
PAR-2-induced p65-NF-nB phosphorylation was reduced by inhibition of MEK or p38mapk. Activation of PAR-4 increased phosphorylation of ERK1/2 and p38mapk without modifying NF-nB activation or COX-2 induction. These findings provide new insights into the molecular mechanisms used by proteases and PARs to regulate generation of the cytoprotective molecule PGI2 through changes in endothelial COX-2 expression. doi:10.1016/j.yjmcc.2006.03.061
047. Cardiac stress adaptation and hyperlipidemia: Role of matrix metalloproteinase-2 Pe´ter Ferdinandy. Pharmahungary 2000 Ltd., Szeged; and Cardiovascular Research Group, Department of Biochemistry, University of Szeged, Szeged, Hungary. www.pharmahungary. com. Ischemic heart disease in humans is a complex disorder caused by or associated with other systemic diseases such as e.g. hyperlipidemia. Therefore, it is of great interest that the cardioprotective effects of ischemic stress adaptation including preconditioning and postconditioning have been shown to be deteriorated in several models of experimental hyperlipidemia and in hyperlipidemic patients as well. The mechanisms by which hyperlipidemia interferes with cellular mechanisms of cardioprotection are unknown. We have previously reported that in normolipidemic rats preconditioning decreased ischemia-induced, peroxynitrite-mediated oxidative activation and release of myocardial matrix metalloproteinase-2 (MMP-2) into the coronary perfusate. We have recently shown that cardioprotection by preconditioning in normolipidemia but not in cholesterol diet-induced hyperlipidemia was associated with an approximately 20% inhibition ischemia/reperfusion-induced activation and release of myocardial MMP-2. However, myocardial protein levels of tissue inhibitors of MMPs (TIMP-2 and TIMP-4) were not changed. A reduction of infarct size in nonpreconditioned hearts from both control and cholesterol-fed group was produced by the MMPs inhibitor ilomastat at a concentration producing MMP-2 inhibition comparable to that of preconditioning in the control group. These results show that hyperlipidemia blocks preconditioning-induced cardioprotection and abolishes preconditioning-induced inhibition of myocardial MMP-2 activation and release; however, preconditioning-induced inhibition of MMP-2 activation and release is not mediated by TIMPs. Furthermore, pharmacological inhibition of MMPs produces cardioprotection in both normal and hyperlipidemic rats. MMP inhibition is an encouraging new therapeutic option for acute cardioprotection in both hyperlipidemic and normolipidemic conditions. doi:10.1016/j.yjmcc.2006.03.062
to expose HL-5 cells to various durations of calcium overload. Calpain activation was investigated by means of immunoblot analyses of desmin degradation and fluorescence increases reflecting the hydrolysis of the synthetic peptide Suc-LLVY-AMC, a calpain substrate. Cell death was assessed as lactic dehydrogenase (LDH) release. Calpain activation became detectable after 20 min of calcium overload and was followed by the increase in LDH release, which approached to plateau after 40 min. The addition of EGTA after 30 min was no longer able to block the progression in cell death and calpain activation. More importantly, calpain inhibition by 10 AM PD150606 or 100 AM calpeptin reduced significantly LDH release although at a lesser extent than calpain-mediated proteolysis. In conclusion, the present findings suggest that (i) calpain activation precedes the onset of cell death; (ii) intracellular calcium overload hampers cell viability in a process that eventually becomes independent of Ca2+; (iii) calpain activation is causally related to cell death, although the severity of the present protocol limits the protective efficacy of calpain inhibition. doi:10.1016/j.yjmcc.2006.03.060
046. Protease-activated receptors and endothelial prostanoid production Caroline P.D. Wheeler-Jones. Royal Veterinary College, Royal College Street, London NW1 0TU, UK The principal mechanism through which serine proteases regulate cell behaviour is by activation of a unique family of G-protein coupled receptors, referred to as protease-activated receptors (PARs 1 – 4). The functional significance of PARs in endothelial cells is largely undefined and the intracellular consequences of their activation are poorly understood. We have shown that the serine protease thrombin, as well as PAR-1 and -2-selective peptides induces cyclooxygenase-2 (COX-2) protein and mRNA expression in human endothelial cells without modifying COX-1 expression. COX-2 induction was accompanied by sustained production of PGI2, and this was inhibited by the COX-2-selective inhibitor NS398. PAR-1 and PAR-2 stimulation rapidly activated both ERK1/2 and p38mapk and pharmacological or molecular blockade of MEK or p38mapk strongly inhibited thrombin- and PAR-2-induced COX-2 expression and PGI2 formation. Thrombin, and peptide agonists of PAR-1 and PAR-2, increased luciferase activity in HUVEC infected with an NF-nB-dependent luciferase reporter adenovirus and this, as well as PAR-induced PGI2 synthesis, was inhibited by coinfection with adenovirus encoding wild-type or mutated (Y42F) InBa. Thrombin- and PAR-2-induced COX-2 expression and 6-keto-PGF1a generation were markedly attenuated by the NF-nB inhibitor PG490 and partially inhibited by the proteasome pathway inhibitor MG-132. PAR-1 or PAR-2 stimulation caused nuclear translocation and phosphorylation of p65-NF-nB, and thrombin-, but not
937
PAR-2-induced p65-NF-nB phosphorylation was reduced by inhibition of MEK or p38mapk. Activation of PAR-4 increased phosphorylation of ERK1/2 and p38mapk without modifying NF-nB activation or COX-2 induction. These findings provide new insights into the molecular mechanisms used by proteases and PARs to regulate generation of the cytoprotective molecule PGI2 through changes in endothelial COX-2 expression. doi:10.1016/j.yjmcc.2006.03.061
047. Cardiac stress adaptation and hyperlipidemia: Role of matrix metalloproteinase-2 Pe´ter Ferdinandy. Pharmahungary 2000 Ltd., Szeged; and Cardiovascular Research Group, Department of Biochemistry, University of Szeged, Szeged, Hungary. www.pharmahungary. com. Ischemic heart disease in humans is a complex disorder caused by or associated with other systemic diseases such as e.g. hyperlipidemia. Therefore, it is of great interest that the cardioprotective effects of ischemic stress adaptation including preconditioning and postconditioning have been shown to be deteriorated in several models of experimental hyperlipidemia and in hyperlipidemic patients as well. The mechanisms by which hyperlipidemia interferes with cellular mechanisms of cardioprotection are unknown. We have previously reported that in normolipidemic rats preconditioning decreased ischemia-induced, peroxynitrite-mediated oxidative activation and release of myocardial matrix metalloproteinase-2 (MMP-2) into the coronary perfusate. We have recently shown that cardioprotection by preconditioning in normolipidemia but not in cholesterol diet-induced hyperlipidemia was associated with an approximately 20% inhibition ischemia/reperfusion-induced activation and release of myocardial MMP-2. However, myocardial protein levels of tissue inhibitors of MMPs (TIMP-2 and TIMP-4) were not changed. A reduction of infarct size in nonpreconditioned hearts from both control and cholesterol-fed group was produced by the MMPs inhibitor ilomastat at a concentration producing MMP-2 inhibition comparable to that of preconditioning in the control group. These results show that hyperlipidemia blocks preconditioning-induced cardioprotection and abolishes preconditioning-induced inhibition of myocardial MMP-2 activation and release; however, preconditioning-induced inhibition of MMP-2 activation and release is not mediated by TIMPs. Furthermore, pharmacological inhibition of MMPs produces cardioprotection in both normal and hyperlipidemic rats. MMP inhibition is an encouraging new therapeutic option for acute cardioprotection in both hyperlipidemic and normolipidemic conditions. doi:10.1016/j.yjmcc.2006.03.062