Mahipal Singh

Osteopontin Modulates Myocardial Hypertrophy in Response to Chronic Pressure Overload in Mice

Osteopontin (OPN) expression increases in the heart during hypertrophy and heart failure. Here, we studied the role of OPN in pressure overload–induced hypertrophy and analyzed the signaling pathways involved in hypertrophy. Aortic banding (AB) was performed in a group of wild-type (WT) and OPN knockout (KO) mice to induce pressure overload. Left ventricular (LV) structural and functional remodeling was studied 1 month after AB. AB increased OPN and &bgr;1 integrin (a receptor for OPN) protein expression in WT-AB group. Hypertrophic response as measured by increased heart weight-to-body weight ratio and myocyte cross-sectional area was significantly increased in WT-AB and KO-AB groups when compared with their respective shams. However, the increase was significantly higher in WT-AB. Re-expression of atrial natriuretic factor was only detected in WT-AB group. LV end-diastolic pressure-volume curve obtained using Langendorff perfusion analysis exhibited a leftward shift in WT-AB group, not in KO-AB. LV-developed pressures measured over a range of LV volumes were significantly increased in WT-AB, not in KO-AB mice. Increased phosphorylation of c-Jun N-terminal kinases, p38 kinase, Akt, and glycogen synthase kinase-3&bgr; was significantly higher in WT-AB when compared with KO-AB group. Increased OPN expression may play an essential role in modulating compensatory cardiac hypertrophy in response to chronic pressure overload.

β1 Integrins Modulate β-Adrenergic Receptor–Stimulated Cardiac Myocyte Apoptosis and Myocardial Remodeling

Sympathetic nerve activity increases in the heart during cardiac failure. Here, we hypothesized that &bgr;1 integrins play a protective role in chronic &bgr;-adrenergic receptor–stimulated cardiac myocyte apoptosis and heart failure. l-isoproterenol (iso; 400 &mgr;g/kg per hour) was infused in a group of wild-type (WT) and &bgr;1 integrin heterozygous knockout (hKO) mice. Left ventricular structural and functional remodeling was studied at 7 and 28 days of iso-infusion. Western blot analysis demonstrated reduced &bgr;1 integrin levels in the myocardium of hKO-sham. Iso-infusion increased heart weight:body weight ratios in both groups. However, the increase was significantly higher in WT-iso. M-mode echocardiography indicated increased left ventricular end-diastolic diameter, percentage of fractional shortening, and ejection fraction in the WT-iso group. The percentage of fractional shortening and ejection fraction were significantly lower in hKO-iso versus hKO-sham and WT-iso. Peak left ventricular developed pressure and left ventricular end-diastolic pressure measured using Langendorff–perfusion analyses were significantly higher in the WT-iso group (P<0.05 versus WT-sham and hKO-Iso). The number of TUNEL-positive myocytes was significantly higher in hKO-iso hearts 7 and 28 days after iso-infusion. The increase in myocyte cross-sectional area and fibrosis was higher in the WT-iso group. Matrix metalloproteinase-9 protein levels were significantly higher in WT-iso, whereas matrix metalloproteinase-2 levels were increased in hKO-iso hearts. Iso-infusion increased phosphorylation of c-Jun N-terminal kinase and extracellular signal-regulated kinase 1/2 in both groups. The increase in c-Jun N-terminal kinase phosphorylation was significantly higher in hKO-iso (P<0.001 versus WT-iso). Thus, &bgr;1 integrins play a crucial role in &bgr;-adrenergic receptor–stimulated myocardial remodeling with effects on cardiac myocyte hypertrophy, apoptosis, and left ventricular function.

ERK1/2 and JNKs, but not p38 kinase, are involved in reactive oxygen species‐mediated induction of osteopontin gene expression by angiotensin II and interleukin‐1β in adult rat cardiac fibroblasts

Osteopontin (OPN), also called cytokine Eta‐1, expressed in the myocardium co‐incident with heart failure plays an important role in post myocardial infarction (MI) remodeling by promoting collagen synthesis and accumulation. Angiotensin II (Ang II) and inflammatory cytokines are increased in the heart following MI. We studied the involvement of mitogen‐activated protein kinases (ERK1/2, JNKs, p38 kinase) and reactive oxygen species (ROS) in Ang II‐ and cytokine‐induced OPN gene expression in adult rat cardiac fibroblasts. Ang II alone increased OPN mRNA (3.3 ± 0.3‐folds; P < 0.05; n = 7), while interleukin‐1β (IL‐1β), tumor necrosis factor (TNF‐α), and interferon‐γ (IFN‐γ) had no effect. A combination of Ang II with IL‐1β or TNF‐α, not IFN‐γ, increased OPN mRNA more than Ang II alone. Nitric oxide donor, S‐nitrosoacetylpenicillamine (SNAP), alone or in combination with Ang II had no effect. Diphenylene iodonium (DPI), inhibitor of NAD(P)H oxidase, and tiron, superoxide scavenger, inhibited Ang II‐ and Ang II+ IL‐1β‐stimulated increases in OPN mRNA. Ang II activated ERK1/2 within 5 min of treatment, not JNKs. IL‐1β activated ERK1/2 and JNKs within 15 min of treatment. A combination of Ang II and IL‐1β activated ERK1/2 within 5 min of treatment. None of these stimuli activated p38 kinase. DPI almost completely inhibited Ang II + IL‐1β‐stimulated activation of ERK1/2, while partially inhibiting JNKs. PD98059, ERK1/2 pathway inhibitor, and SP600125, JNKs inhibitor, partially inhibited Ang II + IL‐1β‐stimulated increases in OPN mRNA. A combination of PD98059 and SP600125 almost completely inhibited Ang II + IL‐1β‐stimulated increases in OPN mRNA. Thus, Ang II alone increases OPN expression, while IL‐1β and TNF‐α act synergistically with Ang II to increase OPN mRNA possibly via NO independent mechanisms. The synergistic increase in OPN mRNA involves ROS‐mediated activation of ERK1/2 and JNKs, not P38 kinase, pathways in cardiac fibroblasts. J. Cell. Physiol. 198: 399–407, 2004© 2003 Wiley‐Liss, Inc.

Osteopontin: role in extracellular matrix deposition and myocardial remodeling post-MI.

Remodeling after myocardial infarction (MI) associates with left ventricular (LV) dilation, decreased cardiac function and increased mortality. The dynamic synthesis and breakdown of extracellular matrix (ECM) proteins play a significant role in myocardial remodeling post-MI. Expression of osteopontin (OPN) increases in the heart post-MI. Evidence has been provided that lack of OPN induces LV dilation which associates with decreased collagen synthesis and deposition. Inhibition of matrix metalloproteinases, key players in ECM remodeling process post-MI, increased ECM deposition (fibrosis) and improved LV function in mice lacking OPN after MI. This review summarizes--1) signaling pathways leading to increased expression of OPN in the heart; 2) the alterations in the structure and function of the heart post-MI in mice lacking OPN; and 3) mechanisms involved in OPN-mediated ECM remodeling post-MI.

Deficiency of β1 integrins results in increased myocardial dysfunction after myocardial infarction

Objective: To study the role of β1 integrins in left ventricular (LV) remodelling after myocardial infarction (MI). Methods and results: LV structural and functional alterations were determined in wild-type (WT) and β1 integrin heterozygous knockout (hKO) mice one month after MI. MI increased β1 integrin expression in both groups; however, the increase was lower in hKO. Infarct size was similar in WT and hKO mice, whereas lung wet weight to dry weight ratio was increased in the hKO-MI mice (5.17 (SE 0.13) v 4.60 (0.15) in WT-MI, p < 0.01). LV end systolic and end diastolic diameters were significantly higher and percentage fractional shortening was significantly lower in hKO-MI. The ratio of peak velocity of early LV filling (E wave) to that of the late LV filling (A wave) and the isovolumic relaxation time (IVRT) were increased in both MI groups but the increase in IVRT was significantly higher in hKO-MI group than in WT-MI mice. Langendorff perfusion analysis indicated reduced peak LV developed pressure and increased LV end diastolic pressure in both MI groups. The reduction in peak LV developed pressure (36.7 (2.2) v 53.4 (1.9) mm Hg, p < 0.05) and increase in LV end diastolic pressure was higher in hKO-MI than in WT-MI. Increase in fibrosis was not different between the two MI groups. The increase in myocyte circumference was higher in the hKO-MI group (p < 0.001 v WT-MI). The number of apoptotic myocytes was significantly higher in hKO-MI than in WT-MI mice (p < 0.005) three days after MI. The number of necrotic myocytes was not different between the two MI groups. Conclusion: β1 integrins are crucial in post-MI remodelling with effects on LV function, hypertrophy and apoptosis.

β1 integrins expression in adult rat ventricular myocytes and its role in the regulation of β‐adrenergic receptor‐stimulated apoptosis

We have shown that the stimulation of β‐adrenergic receptors (β‐AR) increases apoptosis in adult rat ventricular myocytes (ARVMs). Integrins, a family of αβ‐heterodimeric cell surface receptors, are postulated to play a role in ventricular remodeling. Here, we show that norepinephrine (NE) increases β1 integrins expression in ARVMs via the stimulation of α1‐AR, not β‐AR. Inhibition of ERK1/2 using PD 98059, an inhibitor of ERK1/2 pathway, inhibited α1‐AR‐stimulated increases in β1 integrins expression. Activation of β1 integrins signaling pathway using laminin (LN) inhibited β‐AR‐stimulated apoptosis as measured by terminal deoxynucleotidyl transferase‐mediated nick end labeling (TUNEL)‐staining and flow cytometry. Likewise, ligation of β1 integrins with anti‐β1 integrin antibodies prevented β‐AR‐stimulated apoptosis. Treatment of cells using LN or anti‐β1 integrin antibodies activated ERK1/2 pathway. PD 98059 inhibited activation of ERK1/2 by LN, and prevented the anti‐apoptotic effects of LN. Thus (1) stimulation of α1‐AR regulates β1 integrins expression via the activation of ERK1/2, (2) β1 integrins signaling protects ARVMs from β‐AR‐stimulated apoptosis, (3) activation of ERK1/2 plays a critical role in the anti‐apoptotic effects of β1‐integrin signaling. These data suggest that β1 integrin signaling protects ARVMs against β‐AR‐stimulated apoptosis possibly via the involvement of ERK1/2. J. Cell. Biochem. 89: 381–388, 2003. Published 2003 Wiley‐Liss, Inc.

Osteopontin: a novel inflammatory mediator of cardiovascular disease.

Osteopontin, also called cytokine Eta-1, is a multifunctional protein containing Arg-Gly-Asp-Ser (RGDS) cell-binding sequence. It interacts with alpha(v)beta1, alpha(v)beta3 and alpha(v)beta5 integrins and CD44 receptors. OPN is suggested to play a role during inflammation via the recruitment and retention of macrophages and T-cells to inflamed sites. OPN regulates the production of inflammatory cytokines and nitric oxide in macrophages. In this review, we will discuss diverse roles of OPN related to cardiovascular diseases, including atherosclerosis, valvular stenosis, hypertrophy, myocardial infarction and heart failure.

Extracellular ubiquitin inhibits β-AR-stimulated apoptosis in cardiac myocytes: role of GSK-3β and mitochondrial pathways

AIMS Beta-adrenergic receptor (beta-AR) stimulation induces apoptosis in adult rat ventricular myocytes (ARVMs) via the activation of glycogen synthase kinase-3beta (GSK-3beta) and mitochondrial pathways. However, beta-AR stimulation induces apoptosis only in a fraction ( approximately 15-20%) of ARVMs. We hypothesized that ARVMs may secrete/release a survival factor(s) which protects 80-85% of cells from apoptosis. METHODS AND RESULTS Using two-dimensional gel electrophoresis followed by MALDI TOF and MS/MS, we identified ubiquitin (Ub) in the conditioned media of ARVMs treated with beta-AR agonist (isoproterenol). Western blot analysis confirmed increased Ub levels in the conditioned media 3 and 6 h after beta-AR stimulation. Inhibition of beta1-AR and beta2-AR subtypes inhibited beta-AR-stimulated increases in extracellular levels of Ub, whereas activation of adenylyl cyclase using forskolin mimicked the effects of beta-AR stimulation. Incubation of cells with exogenous biotinylated Ub followed by western blot analysis of the cell lysates showed uptake of extracellular Ub into cells, which was found to be higher after beta-AR stimulation (1.9 +/- 0.4-fold; P < 0.05 vs. control, n = 6). Pre-treatment with Ub inhibited beta-AR-stimulated increases in apoptosis. Inhibition of phosphoinositide 3-kinase using wortmannin and LY-294002 prevented anti-apoptotic effects of extracellular Ub. Ub pre-treatment inhibited beta-AR-stimulated activation of GSK-3beta and c-Jun N-terminal kinase (JNK) and increases in the levels of cytosolic cytochrome c. The use of methylated Ub suggested that the anti-apoptotic effects of extracellular Ub are mediated via monoubiquitination. CONCLUSION beta-AR stimulation increases levels of Ub in the conditioned media. Extracellular Ub plays a protective role in beta-AR-stimulated apoptosis, possibly via the inactivation of GSK-3beta/JNK and mitochondrial pathways.

Role of osteopontin in heart failure associated with aging

Cardiovascular disease is one of the leading causes of death in the elderly. Much of the morbidity and mortality in the elderly is attributable to acute ischemic events leading to myocardial infarction (MI) and death of cardiac myocytes. Evidence has been provided that aging associated with adverse remodeling post MI as demonstrated by less effective myocardial repair, greater infarct expansion, and septal hypertrophy. Expression of osteopontin (OPN) increases in the heart post MI. Transgenic mice studies suggest that increased expression of OPN plays a protective role in post-MI LV remodeling by modulating collagen deposition and fibrosis. OPN, a multifunctional protein, has the potential to influence the molecular and cellular changes associated with infarct healing. The post-MI infarct healing process involves temporarily overlapping phases that include the following—(1) inflammation with migration and adhesion of neutrophils and macrophages, phagocytosis and inflammatory gene expression; (2) tissue repair with fibroblast adhesion and proliferation, myofibroblast differentiation, extracellular matrix deposition and scar formation; and (3) structural and functional remodeling of infarcted and non-infarcted myocardium through cardiac myocyte apoptosis, hypertrophy and myocardial angiogenesis. This review is focused on the expression of OPN in the heart post MI and its role in various phases of infarct healing.

Impairment of myocardial angiogenic response in the absence of osteopontin.

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