Ang II-Induced Hypertension: Role of PGE2 and EP4 in End Organ Damage

高血压是心血管危险因素，可导致缺血性损伤、心肌梗死(Ml)和 心力衰竭。在这些疾病过程中，心脏经历肥大和纤维化，称为 心脏重塑。重塑是由机械因素、促炎细胞因子的释放、 神经激素制剂(p-肾上腺素能激素)和具有营养特性的血管活性激素，如 血管紧张素II(Ang II)。这些药物还调节与炎症反应有关的基因，包括 环氧合酶-2(COX-2)和PGE2合成酶mPGES-1，导致大量产生 心肌细胞表达前列腺素E_2。我们已经证明，环氧合酶-2在小鼠心脏中被诱导， 使用特定的COX-2抑制剂治疗2周可改善心功能，减轻肥厚和 纤维化症。使用心肌细胞的体外研究表明，PGE2与其受体EP4的结合导致 表皮生长因子受体(EGFR)的反式激活，p42/44 MAPK的激活和增加 蛋白质合成。肥大的b型利钠肽(BMP)的基因标志也受PGE2的调节。 我们假设PGE2(由mPGES-1产生)和EP4参与了终末性器官损伤(心脏 肥大和炎症)对血管紧张素Ⅱ诱导的高血压和缺血性损伤的反应 (由ML引起)通过激活EGFR和p42/44 MAPK。在Aim I中，我们将研究信号分子 参与EGFR反式激活和下游p42/44 MAPK的激活。我们还将过度表达EP4在 心肌细胞通过内吞作用研究其内化及其对p42/44 MAPK的作用 发信号。在AIM II中，我们将使用BMP启动子的瞬时转染和药物治疗 以及信号分子的分子抑制剂，如Src、小GTP酶和EGFR，以进一步阐明 心肌细胞中的EP4依赖事件。我们还将研究下游的其他激酶和转录因子。 来自p42/44的参与心肌细胞生长的MAPK，包括Egr-1和GATA-4。在目标三和目标四中，我们 将使用新的突变小鼠品系来研究心肌细胞特异性的过度表达和缺失 EP4和mPGES-1基因缺失对血管依赖性高血压和高血压大鼠肥厚的调节作用这个 COX-2产物PGE2及其受体EP4在慢性心脏病理生理中的作用 未被开发的。我们的研究将使用综合的方法来研究细胞和分子基础 炎性前列腺素E_2及其受体在体外和新生小鼠体内的毒副作用 活体模型。 缩写：ACM=成人心肌细胞；ADAM=去整合素和金属蛋白酶蛋白；Ang II= 血管紧张素II；p2AR=p2-肾上腺素能受体；BNP=B型利钠肽；COX=环氧合酶；EGFR =表皮生长因子受体；Egr-1=早期生长反应因子1；EOD=终末器官损害；EP= PGE2受体；EP4=PGE2受体4型；Grk=G蛋白偶联受体激酶；HB-EGF=肝素- 结合表皮生长因子；IL-1=白介素1p；KO或-/-=基因敲除；LAD=左前 冠状动脉降支；LV=左心室；ML=心肌梗死；mPGES-1=诱导膜- PGE2合酶定位；NVm=新生心肌细胞；P42/44 MAPK=P42/44丝裂原激活 蛋白激酶=ERK1/2；PGE2=前列腺素E2；PKA=蛋白激酶A；ROS=活性氧； SiRNA=小干扰RNA分子。 PHS 398/2590(09/04版，2006年4月4期)第236页延续格式页 首席研究员/项目主任(最后、第一、中间)：Lapointe，M.C.，Ph.D/Carretero，Oscar A.，M.D. 一个。

Hypertension is a cardiovascular risk factor that can lead to ischemic injury, myocardial infarction (Ml)and heart failure. During these disease processes, the heart undergoes hypertrophy and fibrosis, referred to as cardiac remodeling. Remodeling is stimulated by mechanical factors, release of pro-inflammatory cytokines, neurohormonal agents (p-adrenergic hormones) and vasoactive hormones with trophic properties, such as angiotensin II (Ang II). These agents also regulate the genes involved in the inflammatory response, including cyclooxygenase-2 (COX-2) and the PGE2 synthase mPGES-1, resulting in substantial production of the prostanoid PGE2 by cardiac myocytes. We have shown that COX-2 is induced in the mouse heart following Ml, and 2 wk treatment with a specific COX-2 inhibitor improves cardiac function and reduces hypertrophy and fibrosis. In vitro studies using cardiac myocytes indicate that binding of PGE2 to its receptor, EP4, results in transactivation of the epidermal growth factor receptor (EGFR), activation of p42/44 MAPK and increased protein synthesis. The gene marker of hypertrophy b-type natriuretic peptide (BMP) is also regulated by PGE2. We hypothesize that PGE2 (generated by mPGES-1) and EP4 contribute to end organ damage (cardiac hypertrophy and inflammation) in response to Ang ll-induced hypertension and ischemic injury (caused by Ml) via activation of EGFR and p42/44 MAPK. In Aim I we will study the signaling molecules involved in EGFR transactivation and downstream p42/44 MAPK activation. We will also overexpress EP4 in myocytes to study its internalization via endocytosis and the contribution of endocytosis to p42/44 MAPK signaling. In Aim II we will use transient transfection of the BMP promoter and treatment with pharmacological and molecular inhibitors of signaling molecules such as Src, small GTPases and EGFR to further elucidate EP4-dependent events in myocytes. We will also study other kinases and transcription factors downstream from p42/44 MAPK that are involved in myocyte growth, including Egr-1 and GATA-4. In Aims III and IV, we will use novel mutant mouse lines to examine how cardiac myocyte-specific overexpression and deletion of EP4 and deletion of mPGES-1 modulate hypertrophy in models of Ang ll-dependent hypertension and Ml. The contribution of the COX-2 product PGE2 and its receptor EP4 to chronic cardiac pathophysiology is virtually unexplored. Our studies will use an integrative approach to study the cellular and molecular basis for the deleterious effects of the inflammatory prostanoid PGE2 and its receptor both in vitro and in novel mouse models in vivo. Abbreviations: ACM = adult cardiac myocytes; ADAM = a disintegrin and metalloproteinase protein; Ang II = angiotensin II; p2AR = p2-adrenergic receptor; BNP = B-type natriuretic peptide; COX = cyclooxygenase; EGFR = epidermal growth factor receptor; Egr-1 = early growth response factor 1; EOD = end organ damage; EP = PGE2 receptor; EP4 = PGE2 receptor type 4; Grk = G-protein coupled receptor kinase; HB-EGF = heparin- binding epidermal growth factor; IL-1 = interleukin-1p; KO or -/- = gene knockout; LAD = left anterior descending coronary artery; LV = left ventricle; Ml = myocardial infarction; mPGES-1 = inducible membrane- localized PGE2 synthase; NVM = neonatal ventricular myocytes; p42/44 MAPK = p42/44 mitogen-activated protein kinase = Erk1/2; PGE2 = prostaglandin E2; PKA = protein kinase A; ROS = reactive oxygen species; siRNA = small interfering RNA molecule. PHS 398/2590 (Rev. 09/04, Reissued4/2006) Page 236 Continuation Format Page Principal Investigator/Program Director (Last, First, Middle): LaPointe, M.C., Ph.D/Carretero, Oscar A., M.D. A.