Cyclooxygenase (COX)-2 is expressed in the heart in animal models of ischemic injury. Recent studies have suggested that COX-2 products are involved in inflammatory cell infiltration and fibroblast proliferation in the heart. Using a mouse model, we questioned whether 1). myocardial infarction (MI) in vivo induces COX-2 expression chronically, and 2). COX-2 inhibition reduces collagen content and improves cardiac function in mice with MI. MI was produced by ligation of the left anterior descending coronary artery in mice. Two days later, mice were treated with 3 mg/kg NS-398, a selective COX-2 inhibitor, or vehicle in drinking water for 2 wk. After the treatment period, mice were subjected to two-dimensional M-mode echocardiography to determine cardiac function. Hearts were then analyzed for determination of infarct size, interstitial collagen content, brain natriuretic peptide (BNP) mRNA, myocyte cross-sectional area, and immunohistochemical staining for transforming growth factor (TGF)-beta and COX-2. COX-2 protein, detected by immunohistochemistry, was increased in MI versus sham hearts. MI resulted in increased left ventricular systolic and diastolic dimension and decreased ejection fraction, fractional shortening, and cardiac output. NS-398 treatment partly reversed these detrimental changes. Myocyte cross-sectional area, a measure of hypertrophy, was decreased by 30% in the NS-398 versus vehicle group, but there was no effect on BNP mRNA. The interstitial collagen fraction increased from 5.4 +/- 0.4% in sham hearts to 10.4 +/- 0.9% in MI hearts and was decreased to 7.9 +/- 0.6% in NS-398-treated hearts. A second COX-2 inhibitor, rofecoxib (MK-0966), also decreased myocyte cross-sectional area and interstitial collagen fraction. TGF-beta, a key regulator of collagen synthesis, was increased in MI hearts. NS-398 treatment reduced TGF-beta immunostaining by 40%. NS-398 treatment had no effect on infarct size. These results suggest that COX-2 products contribute to cardiac remodeling and functional deficits after MI. Thus selected inhibition of COX-2 may be a therapeutic target for reducing myocyte damage after MI.

Upon induction of cyclooxygenase-2 (COX-2), neonatal ventricular myocytes (VMs) mainly synthesize prostaglandin E2 (PGE2). The biological effects of PGE2 are mediated through four different G protein-coupled receptor (GPCR) subtypes (EP(1-4)). We have previously shown that PGE2 stimulates cAMP production and induces hypertrophy of VMs. Because the EP4 receptor is coupled to adenylate cyclase and increases in cAMP, we hypothesized that PGE2 induces hypertrophic growth of cardiac myocytes through a signaling cascade that involves EP4-cAMP and activation of protein kinase A (PKA). To test this, we used primary cultures of VMs and measured [3H]leucine incorporation into total protein. An EP4 antagonist was able to partially block PGE2 induction of protein synthesis and prevent PGE2-dependent increases in cell surface area and activity of the atrial natriuretic factor promoter, which are two other indicators of hypertrophic growth. Surprisingly, a PKA inhibitor had no effect. In other cell types, G protein-coupled receptor activation has been shown to transactivate the epidermal growth factor receptor (EGFR) and result in p42/44 mitogen-activated protein kinase (MAPK) activation and cell growth. Immunoprecipitation of myocyte lysates demonstrated that the EGFR was rapidly phosphorylated by PGE2 in VMs, and the EP4 antagonist blocked this. In addition, the selective EGFR inhibitor AG-1478 completely blocked PGE2-induced protein synthesis. We also found that PGE2 rapidly phosphorylated p42/44 MAPK, which was inhibited by the EP4 antagonist and by AG-1478. Finally, the p42/44 MAPK inhibitor PD-98053 (25 micromol/l) blocked PGE2-induced protein synthesis. Altogether, we believe these are the first data to suggest that PGE2 induces protein synthesis in cardiac myocytes in part via activation of the EP4 receptor and subsequent activation of p42/44 MAPK. Activation of p42/44 MAPK is independent of the common cAMP-PKA pathway and involves EP4-dependent transactivation of EGFR.

Mesangial cells, but not podocytes, contain a cytoskeleton capable of contraction that is disorganized in long-term diabetes. Together with previous observations, the distribution of this cytoskeleton suggests that mesangial cell contraction may be involved in the redistribution of glomerular capillary blood flow, but not substantially in the modulation of glomerular distention. Disorganization of stress fibers may be a cause of hyperfiltration in diabetes.

Interleukin-1beta (IL-1beta), a proinflammatory cytokine, induces cyclooxygenase-2 (COX-2) in cultured neonatal ventricular myocytes (NVMs), resulting in the preferential production of prostaglandin E(2) (PGE(2)). To explain the preferential PGE(2) release by myocytes, we studied whether its specific synthase, PGE(2) synthase (PGES), is also induced by IL-1beta. Because COX-2 has been extensively associated with cell growth, we questioned whether PGE(2) plays a role in cardiac cell growth. IL-1beta--treated myocytes showed induction of PGES protein and mRNA by Western blot and reverse transcription--polymerase chain reaction, respectively. Immunofluorescence studies revealed perinuclear localization of COX-2 and PGES in IL-1beta--treated myocytes. Exogenous PGE(2) increased protein synthesis in NVMs, as indicated by a 1.6-fold increase in [(3)H]leucine incorporation, comparable to the known hypertrophic factor phenylephrine (1.6-fold). Because PGE(2) exerts different effects through 4 receptor subtypes (EP(1), EP(2), EP(3), and EP(4)), we investigated whether these receptors are functional in myocytes. Treatment of NVMs with the selective EP(1)/EP(3) agonist sulprostone significantly increased protein synthesis (1.7-fold), whereas the EP(1)/EP(2) antagonist AH6809 blocked this effect by 43%. In contrast, AH6809 had no effect on PGE(2)-induced protein synthesis. Regarding second messengers, sulprostone had no effect on cAMP, whereas PGE(2) increased it. We concluded that (1) PGE(2) production requires the induction of its specific synthase; (2) in myocytes, the inducible enzymes COX-2 and PGES are perinuclear; and (3) PGE(2) and sulprostone induce cardiac myocyte growth but seem to activate a different subset of EP receptors.

Abstract-Peroxisome proliferator-activated receptors (PPARs) are ligand-activated transcription factors belonging to the nuclear receptor superfamily. They regulate lipid metabolism, glucose homeostasis, cell proliferation, and differentiation and modulate inflammatory responses. We examined whether PPAR␥ is functional in cultured neonatal ventricular myocytes and studied its role in inflammation. Western blots revealed PPAR␥ in myocytes. When myocytes were transfected with a PPAR response element reporter plasmid (PPRE-TK-luciferase), the PPAR␥ activator 15-deoxy-⌬ 12,14 -prostaglandin J 2 (15dPGJ 2 ) increased promoter activity, whereas cotransfection of a dominant negative PPAR␥ inhibited it. To determine the role of 15dPGJ 2 in expression of proinflammatory genes, we tested its effect on interleukin-1␤ induction of cyclooxygenase-2 (COX-2). 15dPGJ 2 decreased interleukin-1␤ stimulation of COX-2 by 40% and PGE 2 production by 73%. We next questioned whether 15dPGJ 2 was modulating the expression of inducible prostaglandin E 2 synthase (PGES) and found that it completely blocked interleukin-1␤ induction of PGES. Use of a second PPAR␥ agonist, troglitazone, and the selective PPAR␥ antagonist GW9662 demonstrated that the effects seen were PPAR␥-dependent. In addition, we found that 15dPGJ 2 blocked interleukin-1␤ stimulation of inducible nitric oxide synthase (iNOS). We concluded that 15dPGJ 2 may play an anti-inflammatory role in a PPAR␥-dependent manner, decreasing COX-2, PGES, and PGE 2 production, as well as iNOS expression. Key Words: myocytes Ⅲ nitric oxide synthase Ⅲ cyclooxygenase Ⅲ prostaglandins P eroxisome proliferator-activated receptors (PPARs) are a family of 3 nuclear hormone receptors, PPAR␣, PPAR␤ (also named PPAR␦), and PPAR␥, which are members of the steroid receptor superfamily. PPARs bind to cognate DNA elements called peroxisome proliferator response elements (PPRE) as obligate heterodimers with the retinoid X receptor (RXR). After ligand activation, they work as transcription factors. 1,2 There are two PPAR␥ subtypes, PPAR␥ 1 and PPAR␥ 2 , which are derived from alternative splicing and promoter usage. 3,4 PPAR␥ 2 is highly expressed in adipose tissue, whereas PPAR␥ 1 has been found in the kidney, heart, liver, and activated monocytes. 5,6 PPAR␥ can be activated by docosahexanoic acid and certain prostaglandins. 7 Other PPAR␥ ligands include the natural prostaglandin metabolite 15-deoxy-⌬ 12,14 -prostaglandin J 2 (15dPGJ 2 ), 8,9 polyunsaturated fatty acids, 10 nonsteroidal anti-inflammatory drugs, 11 and members of the thiazolinedione family. 12 PPAR␥ has been associated with control of inflammation by inhibiting cytokine stimulation of COX-2 and inducible nitric oxide synthase (iNOS) expression in different cell types, 13-15 decreasing release of proinflammatory cytokines 16 and inhibiting vascular smooth cell migration. [17][18][19] PPAR␥ has also been shown to have antineoplastic and antigrowth properties. 18 However, the precise mechanisms defining these effects are unknown.We recentl...