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Biomedical subjects

M S Marber

Publications and source records attributed to M S Marber.

At least 37 records · Page 2Linked to original sources
PubMed2000

Sustained activation of p42/p44 mitogen-activated protein kinase during recovery from simulated ischaemia mediates adaptive cytoprotection in cardiomyocytes.

Delayed cytoprotection (preconditioning) occurs 24 h after sublethal simulated ischaemia and reperfusion (SI/R) in neonatal rat ventricular cardiomyocytes. SI/R was used to investigate the role of activation of mitogen-activated protein kinases (MAPKs), stress-activated protein kinases (SAPKs) and phosphoinositide 3-kinase-dependent protein kinase B (PKB)/Akt in cytoprotection. SI resulted in transient dual (Thr/Tyr) phosphorylation of p42/p44-MAPK and p38-MAPK, weak phosphorylation of p46/p54-SAPK, but no phosphorylation of PKB. 'Reperfusion' caused further transient phosphorylation of p38-MAPK, but sustained phosphorylation of p42/p44-MAPK (lasting 4 h) and of Ser(473) of PKB (lasting 2 h). Furthermore, SI/R (24 h) induced delayed protection against lethal SI, as determined by an increase in cell viability ¿bioreduction of MTT [3-(4,5-dimethylthiazolyl-2)-2,5-diphenyltetrazolium bromide]¿ and a decrease in cell injury (release of creatine kinase). Both protection and phosphorylation of p42/p44-MAPK were blocked by the MEK-1/2 (MAPK/Erk kinase-1/2) inhibitor PD98059 (50 microM) when given during SI/R, but not when given during SI alone. The p38-MAPK inhibitor SB203580 (10 microM) blocked the p38-MAPK-dependent phosphorylation of activating transcription factor 2 in vitro, and the phosphoinositide 3-kinase inhibitor wortmannin (100 nM) blocked PKB phosphorylation on Ser(473). However, neither SB203580 nor wortmannin had any effect on delayed protection. Therefore sustained activation of p42/p44-MAPK during simulated 'reperfusion' following sublethal SI mediates preconditioning in cardiomyocytes independently of transient activation of p38-MAPK or sustained activation of PKB.

Adaptation, Physiological↗
PubMed2000

IGF-1 regulates cardiac fibroblast apoptosis induced by osmotic stress.

In this study we have determined the ability of IGF-1 to protect cardiac fibroblasts against osmotic-induced apoptosis and investigated the potential mechanism(s) underlying this protection. Treatment with IGF-1 (1-100 ng/ml) promoted a dose dependent increase in cell survival against osmotic cell death. Both Akt and ERK1/2 were rapidly phosphorylated by IGF-1 and blocked by wortmannin and PD98059, inhibitors of their upstream activators respectively. However, IGF-1-induced protection was mediated via a wortmannin-dependent but PD98059-independent pathway as determined by cell survival assay suggesting a role of PI3-K/Akt. Furthermore, IGF-1 appeared to reduce the activation of a number of early components in the apoptotic pathway in a wortmannin dependent manner including the osmotic stress-induced perturbation in mitochondrial membrane potential, cleavage and activation of caspase-3 and DNA fragmentation. Thus, the results suggest that IGF-1 regulates osmotic stress-induced apoptosis via the activation of the PI3-K/Akt pathway at a point upstream of the mitochondria and caspase-3.

Animals↗
PubMed2000

Activation of Akt during simulated ischemia/reperfusion in cardiac myocytes.

In the present study we have investigated whether Akt was activated during simulated ischemia (SI) and simulated ischemia/reperfusion (SI/R) in neonatal rat cardiomyocytes. Akt was phosphorylated on both S473 and T308 residues after 10 min of simulated SI/R and remained elevated for 60 min before returning to basal levels after 2 h. No phosphorylation was observed during SI alone. SI/R-stimulated Akt activation was inhibited by the phosphatidylinositol 3-kinase (PI3-K) inhibitor wortmannin, the tyrosine kinase inhibitor genistein and the Src tyrosine kinase inhibitor PP2, indicating a requirement for tyrosine kinase activity in Akt activation. Furthermore, SB203580, a p38 MAPK inhibitor, partially inhibited Akt activation. SI/R also induced the phosphorylation of PHAS-I, a downstream Akt target, in a wortmannin-dependent manner. These results demonstrate for the first time that SI/R stimulates Akt activation via PI3-K-and Src tyrosine kinase-dependent pathways, whereas p38 MAPK appears to be involved in maintaining Akt activation.

Androstadienes↗
PubMed2000

Ischemia-induced STAT-1 expression and activation play a critical role in cardiomyocyte apoptosis.

We show here that exposure of cardiac cells to simulated ischemia results in apoptosis and is accompanied by phosphorylation and increased expression and transcriptional activity of STAT-1. Similarly, interferon-gamma, which is known to induce STAT-1 activation, also induced apoptosis in cardiac cells. STAT-1-transfected cells were more susceptible to ischemia-induced cell death than cells transfected with a control plasmid lacking the STAT-1 coding sequence. Furthermore, an antisense STAT-1 vector reduced both ischemia- and overexpressed STAT-1-induced cell death in cardiac cells. Both STAT-1 overexpression and interferon-gamma treatment or exposure to ischemia activated the promoter of the pro-apoptotic caspase-1 gene in cardiomyocytes. Finally, ischemia/reperfusion also induced STAT-1 activation and caspase-1 processing in ventricular myocytes in the intact heart ex vivo. Immunofluorescent staining demonstrated an increase in STAT-1-positive staining in cardiomyocytes in response to ischemia/reperfusion that co-localized with terminal deoxynucleotidyl transferase dVTP nick end-labeling-positive apoptotic cells. These results suggest that STAT-1 plays a critical role in the regulation of ischemia/reperfusion-induced apoptosis in cardiac cells, acting at least in part via a caspase-1 activation-dependent pathway.

Animals↗
PubMed2000

Postinfarction left ventricular remodeling: a pathophysiological and therapeutic review.

Over the last 50 years, studies investigating the pathogenesis of left ventricular dysfunction have resulted in many potential therapeutic targets being identified and novel classes of drugs designed to treat this condition. Despite this, the long-term prognosis of patients with clinical heart failure remains poor with mortality rates equivalent to many terminal malignancies. This article reviews our present understanding of the pathophysiology of post-infarction left ventricular dysfunction and provides a rationale for current drug usage, drugs undergoing clinical trials and compounds still under pre-clinical development. In addition, the complexities involved in deciphering intra-cellular signalling pathways mediating ventricular hypertrophy which may form the basis of future treatments are also discussed.

Animals↗
PubMed2000

The role of differential activation of p38-mitogen-activated protein kinase in preconditioned ventricular myocytes.

Activation of protein kinase C (PKC) and more recently mitogen-activated protein kinases (MAPKs) have been associated with the cardioprotective effect of ischemic preconditioning. We examined the interplay between these kinases in a characterized model of ischemic preconditioning in cultured rat neonatal ventricular cardiocytes where ectopic expression of active PKC-delta results in protection. Two members of the MAPK family, p38 and p42/44, were activated transiently during preconditioning by brief simulated ischemia/reoxygenation. Overexpression of active PKC-delta, rather than augmenting, completely abolished this activation. We therefore determined whether a similar process occurred during lethal prolonged simulated ischemia. In contrast to ischemia, brief, lethal-simulated ischemia activated only p38 (2.8+/-0.45 vs. basal, P<0.01), which was attenuated by expression of active PKC-delta or by preconditioning (0.48+/-0.1 vs. ischemia, P<0.01). To determine whether reduced p38 activation was the cause or an effect of protection, we used SB203580, a p38 inhibitor. SB203580 reduced ischemic injury (CK release 38.0+/-3.1%, LDH release 77.3+/-4.0%, and MTT bioreduction 127.1+/-4.8% of control, n=20, P<0.05). To determine whether p38 activation was isoform selective, myocytes were infected with adenoviruses encoding wild-type p38alpha or p38beta. Transfected p38alpha and beta show differential activation (P<0.001) during sustained simulated ischemia, with p38alpha remaining activated (1.48+/-0.36 vs. basal) but p38beta deactivated (0.36+/-0.1 vs. basal, P<0.01). Prior preconditioning prevented the activation of p38alpha (0.65+/-0.11 vs. ischemia, P<0.05). Moreover, cells expressing a dominant negative p38alpha, which prevented ischemic p38 activation, were resistant to lethal simulated ischemia (CK release 82.9+/-3.9% and MTT bioreduction 130.2+/-6.5% of control, n=8, P<0.05). Thus, inhibition of p38alpha activation during ischemia reduces injury and may contribute to preconditioning-induced cardioprotection in this model.

Animals↗
PubMed2000

Nitric oxide-induced cardioprotection in cultured rat ventricular myocytes.

The aim of this study was to investigate the role of nitric oxide (NO) in a cellular model of early preconditioning (PC) in cultured neonatal rat ventricular myocytes. Cardiomyocytes "preconditioned" with 90 min of stimulated ischemia (SI) followed by 30 min reoxygenation in normal culture conditions were protected against subsequent 6 h of SI. PC was blocked by N(G)-monomethyl-L-arginine monoacetate but not by dexamethasone pretreatment. Inducible nitric oxide synthase (NOS) protein expression was not detected during PC ischemia. Pretreatment (90 min) with the NO donor S-nitroso-N-acetyl-L,L-penicillamine (SNAP) mimicked PC, resulting in significant protection. SNAP-triggered protection was completely abolished by 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ) but was unaffected by chelerythrine or the presence of glibenclamide and 5-hydroxydecanoate. With the use of RIA, SNAP treatment increased cGMP levels, which were blocked by ODQ. Hence, NO is implicated as a trigger in this model of early PC via activation of a constitutive NOS isoform. After exposure to SNAP, the mechanism of cardioprotection is cGMP dependent but independent of protein kinase C or ATP-sensitive K(+) channels. This differs from the proposed mechanism of NO-induced cardioprotection in late PC.

Alkaloids↗
PubMed2000

PKC-dependent delayed metabolic preconditioning is independent of transient MAPK activation.

In this study we used an in vitro model of delayed preconditioning to investigate activation of mitogen-activated protein kinases (MAPKs) and their potential role in protection. Neonatal rat cardiomyocytes were preconditioned using a buffer containing glycolytic inhibitors and low pH (minimal metabolic preconditioning; MMPC) consisting of modified Krebs buffer, 10 mM 2-deoxyglucose, and 20 mM lactate, pH 6.8, for 2 h followed by 24 h of simulated reperfusion before lethal simulated ischemia (LSI). MAPK activation during the MMPC protocol was determined using phospho-specific antisera and the effect on protection determined following LSI. Rapid, transient phosphorylation of extracellular signal-regulated kinases 1 and 2 (ERK1/2) and p38 MAPK was observed during each of the MMPC, reperfusion, and LSI phases; an effect blocked by MAPK inhibitors PD-98059 and SB-203580, respectively, but not by the protein kinase C (PKC) inhibitor Ro31-8220. However, although MMPC was blocked by Ro31-8220, treatment with the MAPK inhibitors during the preconditioning protocol did not block delayed protection conferred by MMPC. Thus the data suggest that, in this model of delayed preconditioning, protection appears to be PKC dependent but independent of ERK1/2 or p38 MAPK activation.

Animals↗
PubMed1999

CRH-like peptides protect cardiac myocytes from lethal ischaemic injury.

Simulated ischaemia causes both necrotic and apoptotic death of primary cultures of neonatal rat cardiac myocytes. Simulated ischaemia is associated with increased expression of urocortin mRNA and with the release of urocortin peptide into the medium. Exogenous urocortin is more potent than corticotropin releasing hormone (CRH) in protecting cardiac myocytes from necrotic and apoptotic death induced by ischaemia, and the cardioprotective effects of ischaemia-preconditioned media are abrogated by antagonists to the CRH family of peptides. Simulated ischaemia increases cardiac myocyte expression of CCAAT enhancer binding (C/EBP) transcription factors, and of the p65 subunit of NFkappaB, and reporter activity of a construct incorporating a fragment of the urocortin promoter containing a C/EBP consensus site is also enhanced by simulated ischaemia. The data suggest that ischaemia, acting partly through increased expression of C/EBP transactivators, increases expression of urocortin mRNA, which is rapidly translated to the mature form. The mature peptide is rapidly released, and exerts autocrine/paracrine protective effects through the cardiac CRH-R2 receptor which preferentially binds urocortin.

Animals↗
PubMed1999

Transmembrane signalling mechanisms regulating expression of cationic amino acid transporters and inducible nitric oxide synthase in rat vascular smooth muscle cells.

The signalling mechanisms involved in the induction of nitric oxide synthase and l-arginine transport were investigated in bacterial lipopolysaccharide (LPS)- and interferon-gamma (IFN-gamma)-stimulated rat cultured aortic smooth muscle cells (RASMCs). The expression profile of transcripts for cationic amino acid transporters (CATs) and their regulation by LPS and IFN-gamma were also examined. Control RASMCs expressed mRNA for CAT-1, CAT-2A and CAT-2B. Levels of all three transcripts were significantly elevated in activated cells. Stimulated CAT mRNA expression and l-arginine transport occurred independently of protein kinase C (PKC), protein tyrosine kinase (PTK) and p44/42 mitogen-activated kinases (MAPKs), but were inhibited by the p38 MAPK inhibitor SB203580, which at 3 microM caused maximum inhibition of both responses. Induction of NO synthesis was independent of p44/42 MAPK activation and only marginally dependent on PKC, but was attenuated markedly by the PTK inhibitors genistein and herbimycin A. SB203580 differentially regulated inducible NO synthase expression and NO production, potentiating both processes at low micromolar concentrations and inhibiting at concentrations of >/=1 microM. In conclusion, our results suggest that RASMCs constitutively express transcripts for CAT-1, CAT-2A and CAT-2B, and that expression of these transcripts is significantly enhanced by LPS and IFN-gamma. Moreover, stimulation of l-arginine transport and induction of NO synthesis by LPS and IFN-gamma appear to be under critical regulation by the p38 MAPK, since both processes were significantly modified by SB203580 at concentrations so far shown to have no effect on other signalling pathways. Thus, in RASMCs, the p38 MAPK cascade represents an important signalling mechanism, regulating both enhanced l-arginine transport and induced NO synthesis.

Amino Acid Transport Systems, Basic↗
PubMed1999

Heat shock proteins delivered with a virus vector can protect cardiac cells against apoptosis as well as against thermal or hypoxic stress.

Over expression of heat shock proteins (hsps) by transfection of plasmid constructs in vitro and in transgenic animals in vivo can protect primary cardiac cells from subsequent exposure to severe thermal or hypoxic stress. Here we show that such protection can also be achieved by over-expressing the hsps using herpes simplex virus (HSV) vectors capable of efficient gene delivery in vivo. Moreover, the convenience and high efficiency of this system has allowed us to show, for the first time, that over-expression of hsp27 or hsp70 can protect cardiac cells against three different apoptosis-inducing stimuli as well as against thermal or hypoxic stress whereas hsp56 has no protective effect. The potential therapeutic use of inducing the over-expression of specific hsps in cardiac cells in vivo using pharmacological or gene therapy procedures is discussed.

Animals↗