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Abstract-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 ␤1 integrin (a receptor for OPN) protein expression in WT-AB group. Hypertrophic response as measured by increased heart weight-tobody 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␤ 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.

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.

Abstract-Sympathetic nerve activity increases in the heart during cardiac failure. Here, we hypothesized that ␤1 integrins play a protective role in chronic ␤-adrenergic receptor-stimulated cardiac myocyte apoptosis and heart failure. L-isoproterenol (iso; 400 g/kg per hour) was infused in a group of wild-type (WT) and ␤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 ␤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 Langendorffperfusion analyses were significantly higher in the WT-iso group (PϽ0.05 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 (PϽ0.001 versus WT-iso). Thus, ␤1 integrins play a crucial role in ␤-adrenergic receptor-stimulated myocardial remodeling with effects on cardiac myocyte hypertrophy, apoptosis, and left ventricular function.

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-1beta (IL-1beta), tumor necrosis factor (TNF-alpha), and interferon-gamma (IFN-gamma) had no effect. A combination of Ang II with IL-1beta or TNF-alpha, not IFN-gamma, 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-1beta-stimulated increases in OPN mRNA. Ang II activated ERK1/2 within 5 min of treatment, not JNKs. IL-1beta activated ERK1/2 and JNKs within 15 min of treatment. A combination of Ang II and IL-1beta activated ERK1/2 within 5 min of treatment. None of these stimuli activated p38 kinase. DPI almost completely inhibited Ang II + IL-1beta-stimulated activation of ERK1/2, while partially inhibiting JNKs. PD98059, ERK1/2 pathway inhibitor, and SP600125, JNKs inhibitor, partially inhibited Ang II + IL-1beta-stimulated increases in OPN mRNA. A combination of PD98059 and SP600125 almost completely inhibited Ang II + IL-1beta-stimulated increases in OPN mRNA. Thus, Ang II alone increases OPN expression, while IL-1beta and TNF-alpha 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.

We have shown that osteopontin (OPN), an extracellular matrix protein, plays an important role in post myocardial infarction (MI) remodeling by promoting collagen synthesis and accumulation. Interleukin-1␤ (IL-1␤), increased in the heart following MI, increases matrix metalloproteinase (MMP) activity in cardiac fibroblasts in vitro. Here, we show that OPN alone has no effect on MMP activity or expression. However, it reduces IL-1␤-stimulated increases in MMP activity and expression in adult rat cardiac fibroblasts. Pretreatment with bovine serum albumin had no effect on MMP activity or protein content, whereas GRGDS (glycinearginine-glycine-aspartic acid-serine)-pentapeptide (which interrupts binding of RGD-containing proteins to cell surface integrins) and monoclonal antibody m7E3 (a rat ␤ 3 integrins antagonist) inhibited the effects of OPN. Inhibition of PKC using chelerythrine inhibited the activities of both MMP-2 and MMP-9. Stimulation of cells using IL-1␤ increased phosphorylation and translocation of PKC to membrane fractions, which was inhibited by OPN. OPN inhibited IL-1␤-stimulated increases in translocation of PKC-from cytosolic to membrane fractions. Furthermore, the levels of phospho-PKC-were lower in the cytosolic fractions of OPN knock-out mice hearts as compared with wild type 6 days post-MI. Inhibition of PKC-using PKCpseudosubstrate inhibited IL-1␤-stimulated increases in MMP-2 and MMP-9 activities. These observations suggest that OPN, acting via ␤ 3 integrins, inhibits IL-1␤-stimulated increases in MMP-2 and MMP-9 activity, at least in part, via the involvement of PKC-. Thus, OPN may play a key role in collagen deposition during myocardial remodeling following MI by modulating cytokine-stimulated MMP activity.The dynamic synthesis and breakdown of extracellular matrix (ECM) 1 proteins may play an important role in myocardial remodeling (1, 2). Osteopontin (OPN) is a glycosylated and phosphorylated ECM protein (3, 4). Although first isolated from mineralized matrix, OPN has since been shown to be synthesized by cardiac fibroblasts, endothelial cells, and myocytes (3, 5-7). Using spontaneously hypertensive and aorticbanded rats, we have shown increased expression of OPN coincident with heart failure (8). OPN interacts with specific integrins, namely, and also with CD44 receptors and affects many cellular processes including cell attachment, spreading, and migration (3, 4, 9). Recently, we have documented an increased expression of OPN in remote and infarct regions of left ventricle following myocardial infarction (MI). Furthermore, using OPN knock-out mice and MI as a model of myocardial remodeling, we demonstrated that a lack of OPN results in greater left ventricle dilation and reduced collagen synthesis and accumulation 1 month post-MI, suggesting a role for OPN in ECM reorganization in the heart during myocardial remodeling (10). A critical role for OPN in the generation of interstitial fibrosis was observed in the skin incision model of wound healing and in the kidney following obstructive...

Mountain DJ, Singh M, Menon B, Singh K. Interleukin-1␤ increases expression and activity of matrix metalloproteinase-2 in cardiac microvascular endothelial cells: role of PKC␣/␤ 1 and MAPKs.

Objective: To study the role of b1 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 b1 integrin heterozygous knockout (hKO) mice one month after MI. MI increased b1 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: b1 integrins are crucial in post-MI remodelling with effects on LV function, hypertrophy and apoptosis.