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Harding P, Yang XP, Yang J, Shesely E, He Q, LaPointe MC. Gene expression profiling of dilated cardiomyopathy in older male EP4 knockout mice. Am J Physiol Heart Circ Physiol 298: H623-H632, 2010. First published December 11, 2009; doi: 10.1152/ajpheart.00746.2009.--Using a line of mice with cardiac-specific knockout (KO) of the EP4 receptor gene, experiments were designed to determine whether a cardiac phenotype developed with age. Cardiac function was assessed by echocardiography in 23- to 33-wk-old male and female KO and littermate controls (WT) mice. After echocardiography, hearts were removed to assess weight, and then some were further processed for histology [myocyte cross-sectional area (MCSA), interstitial collagen fraction (ICF), and macrophage infiltration] and some for extraction of total RNA and protein. Older male KO mice had reduced ejection fraction (EF) coupled with left ventricular dilatation. MCSA and infiltrating macrophages were not different between groups, but ICF increased by 39% in KO mice. In contrast to male KO mice, 30- to 32-wk-old female KO mice had only a slight reduction in EF. To understand gene expression differences between male WT and KO mice, we performed whole genome gene expression profiling (Illumina BeadChips) on hearts of 30-to 32-wk-old mice. Data indicated that 156 genes were overexpressed in the KO hearts more than twofold, including genes involved in remodeling, inflammation, and oxidative stress. Overexpressed chemokines/cytokines were further examined in hearts of 10- to 12-wk-old male KO mice, and we found that growth differentiation factor-15 (GDF-15) expression was higher in KO than in WT hearts. In conclusion, EP4 knockdown in cardiac myocytes in aged male KO mice is in part associated with increased fibrosis, reduced EF, and dilated cardiomyopathy. Early over-expression of GDF-15 in hearts of male KO mice may contribute to or be a marker of the disease phenotype. The absence of serious cardiac dysfunction in aged female mice suggests a sexual dimorphism in the phenotype. heart; remodeling; growth differentiation factor-15 doi: 10.1152/ajpheart.00746.2009

Am J Physiol Heart Circ Physiol 298: H136-H143, 2010. First published October 30, 2009; doi: 10.1152/ajpheart.00251.2009.--We have previously reported that 1) inhibition of cyclooxygenase-2 and [PGE.sub.2] production reduces hypertrophy after myocardial infarction in mice and 2) [PGE.sub.2] acting through its EP4 receptor causes hypertrophy of neonatal ventricular myocytes (NVMs) via ERK1/2. It is known that EP4 couples to adenylate cyclase, cAMP, and PKA. The present study was designed to determine interactions between the cAMP-PKA pathway and ERK1/2 and to further characterize events downstream of ERK1/2. We hypothesized that PKA and the small GTPase Rap are upstream of ERK1/2 and that 90-kDa ribosomal S6 kinase (p90RSK) is activated downstream. Treatment of NVMs with [PGE.sub.2] activated Rap, and this activation was inhibited in part by an EP4 antagonist and PKA inhibition. Transfection of a dominant negative mutant of Rap reduced [PGE.sub.2] activation of ERK1/2. [PGE.sub.2] activation of p90RSK was also dependent on EP4, PKA, and Rap. We also tested the involvement of Rap, ERK1/2, and p90RSK in [PGE.sub.2] regulation of gene expression. [PGE.sub.2] stimulation of brain natriuretic peptide promoter activity was blocked by either ERK1/2 inhibition or a dominant negative mutation of p90RSK. [PGE.sub.2] stimulation of c-Fos was dependent on EP4, PKA, ERK1/2, and p90RSK, whereas only the latter two kinases were involved in [PGE.sub.2] regulation of early growth response- 1. Finally, we tested the involvement of EP4-dependent signaling in the NVM growth response and found that the overexpression of EP4 increased NVM cell size. We conclude that EP4-dependent signaling in NVMs in part involves PKA, Rap, ERK1/2, and p90RSK and results in the increased expression of brain natriuretic peptide and c-Fos. prostaglandins; myocytes; prostaglandin E receptor 4

High blood pressure (HBP) is an important risk factor for cardiac, renal, and vascular dysfunction. Excess inflammation is the major pathogenic mechanism for HBP-induced target organ damage (TOD). N-acetyl-Ser-Asp-Lys-Pro (AcSDKP), a tetrapeptide specifically degraded by angiotensin converting enzyme (ACE), reduces inflammation, fibrosis, and TOD induced by HBP. Our hypothesis is that Ac-SDKP exerts its anti-inflammatory effects by inhibiting: 1) differentiation of bone marrow stem cells (BMSC) to macrophages, 2) activation and migration of macrophages, and 3) release of the proinflammatory cytokine TNF-[alpha] by activated macrophages. BMSC were freshly isolated and cultured in macrophage growth medium. Differentiation of murine BMSC to macrophages was analyzed by flow cytometry using F4/80 as a marker of macrophage maturation. Macrophage migration was measured in a modified Boyden chamber. TNF-[alpha] release by activated macrophages in culture was measured by ELISA. Myocardial macrophage activation in mice with ANG II-induced hypertension was studied by Western blotting of Mac-2 (galectin-3) protein. Interstitial collagen deposition was measured by picrosirius red staining. We found that Ac-SDKP (10 nM) reduced differentiation of cultured BMSC to mature macrophages by 24.5% [F4/80 positivity: 14.09 [+ or -] 1.06 mean fluorescent intensity for vehicle and 10.63 [+ or -] 0.35 for Ac-SDKP: P < 0.05]. Ac-SDKP also decreased galectin-3 and macrophage colony-stimulating factor-dependent macrophage migration. In addition, AcSDKP decreased secretion of TNF-[alpha] by macrophages stimulated with bacterial LPS. In mice with ANG II-induced hypertension, Ac-SDKP reduced expression of galectin-3, a protein produced by infiltrating macrophages in the myocardium, and interstitial collagen deposition. In conclusion, this study demonstrates that part of the anti-inflammatory effect of Ac-SDKP is due to its direct effect on BMSC and macrophage, inhibiting their differentiation, activation, and cytokine release. These effects explain some of the anti-inflammatory and antifibrotic properties of Ac-SDKP in hypertension. macrophages; inflammation: activation; angiotensin II

Several essential components of NADPH oxidase, including [p22.sup.phox], [gp91.sup.phox] (nox2) and its homologs nox1 and nox4, [p47.sup.phox], [p67.sup.phox], and rac1, are present in the vasculature. We previously reported that [p67.sup.phox] is essential for adventitial fibroblast NADPH oxidase [O.sup.-.sub.2] production. Thus we postulated that inhibition of adventitial [p67.sup.phox] activity would attenuate angioplasty-induced hyperplasia. To test this hypothesis, we treated the adventitia of carotid arteries with a control adenovirus (Ad-control), a virus expressing dominant-negative [p67.sup.phox] (Ad-p67dn), or a virus expressing a competitive peptide (gp91ds) targeting the [p47.sup.phox]-[gp91.sup.phox] interaction (Ad-gp91ds). Common carotid arteries (CCAs) from male Sprague-Dawley rats were transfected with Ad-control, Ad-p67dn, or Ad-gp91ds in pluronic gel. After 2 days, a 2-F (Fogarty) catheter was used to injure CCAs in vivo. After 14 days, CCAs were perfusion-fixed and analyzed. In 13 experiments, digital morphometry suggested a reduction of neointimal hyperplasia with Ad-p67dn compared with Ad-control; however, the reduction did not reach statistical significance (P = 0.058). In contrast, a significant reduction was achieved with Adgp91ds (P = 0.006). No changes in medial area or remodeling were observed with either treatment. Moreover, adventitial fibroblast proliferation in vitro was inhibited by Ad-gp91ds but not by Ad-p67dn, despite confirmation that Ad-p67dn inhibits NADPH oxidase in fibroblasts. These data appear to suggest that a multicomponent vascular NADPH oxidase plays a role in neointimal hyperplasia. However, inhibition of [p47.sup.phox] may be more effective than inhibition of [p67.sup.phox] at attenuating neointimal growth. NADPH oxidase; [p47.sup.phox]; adventitia; restenosis

Brain natriuretic peptide (BNP) produced by cardiac myocytes has antifibrotic and antigrowth properties and is a marker of cardiac hypertrophy. We previously showed that prostaglandin [E.sub.2] ([PGE.sub.2]) is the main prostaglandin produced in myocytes treated with proinflammatory stimuli and stimulates protein synthesis by binding to its [EP.sub.4] receptor. We hypothesized that [PGE.sub.2], acting through [EP.sub.4], also regulates BNP gene expression. We transfected neonatal ventricular myocytes with a plasmid encoding the human BNP (hBNP) promoter driving expression of a luciferase reporter gene. [PGE.sub.2] increased hBNP promoter activity 3.5-fold. An [EP.su.4] antagonist reduced the stimulatory effect of [PGE.sub.2] but not an [EP.sub.1] antagonist. Because [EP.sub.4] signaling can involve adenylate cyclase, cAMP, and protein kinase A (PKA), we tested the effect of H-89, a PKA inhibitor, on [PGE.sub.2] stimulation of the hBNP promoter. H-89 at 5 [micro]M decreased [PGE.sub.2], stimulation of BNP promoter activity by 100%. Because p42/44 MAPK mediates the effect of [PGE.sub.2], on protein synthesis, we also examined the role of MAPKs in the regulation of BNP promoter activity. [PGE.sub.2] stimulation of the hBNP promoter was inhibited by a MEK 1/2 inhibitor and a dominant-negative mutant of Rat, indicating that p42/44 MAPK was involved. In contrast, neither a p38 MAPK inhibitor nor a JNK inhibitor reduced the stimulatory effect of [PGE.sub.2]. Involvement of small GTPases was also studied. Dominant-negative Rap inhibited [PGE.sub.2] stimulation of the hBNP promoter, but dominant-negative Ras did not. We concluded that [PGE.sub.2] stimulates the BNP promoter mainly via [EP.sub.4] PKA, Rap, and p42/44 MAPK. EP receptor; cardiac myocytes; hypertrophy; signaling pathways

Angiotensin II (ANG II) plays a key role in regulating blood pressure and inflammation. Prostaglandin E2 (PGE2) signals through four different G protein-coupled receptors, eliciting a variety of effects. We reported that activation of the EP3 receptor reduces cardiac contractility. More recently, we have shown that overexpression of the EP4 receptor is protective in a mouse myocardial infarction model. We hypothesize in this study that the relative abundance of EP3 and EP4 receptors is a major determinant of end-organ damage in the diseased heart. Thus EP3 is detrimental to cardiac function and promotes inflammation, whereas antagonism of the EP3 receptor is protective in an ANG II hypertension (HTN) model. To test our hypothesis, male 10- to 12-wk-old C57BL/6 mice were anesthetized with isoflurane and osmotic minipumps containing ANG II were implanted subcutaneously for 2 wk. We found that antagonism of the EP3 receptor using L798,106 significantly attenuated the increase in blood pressure with ANG II infusion. Moreover, antagonism of the EP3 receptor prevented a decline in cardiac function after ANG II treatment. We also found that 10- to 12-wk-old EP3-transgenic mice, which overexpress EP3 in the cardiomyocytes, have worsened cardiac function. In conclusion, activation or overexpression of EP3 exacerbates end-organ damage in ANG II HTN. In contrast, antagonism of the EP3 receptor is beneficial and reduces cardiac dysfunction, inflammation, and HTN. [ABSTRACT FROM AUTHOR]