Inflammmatory Proteases, Sheddases & Cardiomyocyte Death

炎症蛋白酶、脱落酶

• 批准号: 7095184
• 负责人: AbdelKarim Sabri
• 金额: $ 29.39万
• 依托单位: TEMPLE UNIV OF THE COMMONWEALTH
• 依托单位国家: 美国
• 财政年份: 2004
• 资助国家: 美国
• 起止时间: 2004-09-15 至 2008-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7095184
• 关键词: SDS polyacrylamide gel electrophoresis; apoptosis; biological signal transduction; cardiac myocytes; endopeptidases; enzyme activity; focal adhesion kinase; genetically modified animals; immunocytochemistry; immunoprecipitation; inflammation; laboratory mouse; laboratory rat; membrane proteins; microdialysis; myocardium; vascular endothelial growth factors; western blottings

DESCRIPTION (provided by applicant): One of the earliest events during the progression of cardiac hypertrophy is thought to involve an inflammatory response where inflammatory cells and their proteases orchestrate myocardial repair. Although beneficial at early stages after myocardial injury, inflammatory cells release free radicals and proteolytic enzymes within the myocardium that may contribute to myocyte death and subsequent alterations in both the geometry and mechanical properties of the heart. We have found mast cell and neutrophil infiltration and protease activation (chymase, cathepsin G (CG)) associated with MMP activation and ECM degradation within 6-12 hrs after induction of aortocaval fistula (ACF), which persisted for 15 wks. Similar ventricular dilatation and decreases in cardiac contractility were also observed in rats injected with CG for 5 days suggesting that serine proteases may play a role in the early stages of volume overload-induced cardiac remodeling. We have further found in vitro that acute exposure of neonatal rat ventricular myocytes (NRVM) to CG promotes shedding of heparin-binding epidermal growth factor (HB-EGF) and stimulation of epidermal growth factor receptor (EGFR) activation. EGFR stimulation mediates most of the effect of CG that lead to focal adhesion dephosphorylation and caspases activation that culminate in NRVM detachment from matrix and death (also termed anoikis). This led to the hypothesis that inflammatory proteases are critical mediators of cardiac myocyte death that occur early during the progression to heart failure by regulating membrane shedding of pro-HB-EGF and subsequent disruption of focal adhesion signaling. Aim 1 will establish the role of sheddases and HB-EGF in CG effect. Aim 2 will identify downstream signaling pathways that link EGFR to focal adhesion disruption and subsequent activation of initiator caspases during CG-induced myocyte anoikis. Aim 3 will determine the role of membrane shedding in volume overload-induced cardiac remodeling. Ectodomain products and sheddase activity will be measured by microdialysis combined with standard biochemical assays using pharmacological interventions and transgenic approaches. Collectively, this investigation will determine how membrane shedding and transmembrane proteins contribute to the inflammatory process during cardiac remodeling in areas of inflammation and whether serine proteases should be considered direct novel targets for therapeutic interventions.

描述（由申请人提供）：心脏肥大进展过程中最早的事件之一被认为涉及炎症反应，其中炎症细胞及其蛋白酶会策划心肌修复。尽管在心肌损伤后的早期阶段有益，但炎症细胞在心肌内释放自由基和蛋白水解酶，这可能导致心脏的几何形状和机械性能的随后改变。我们发现肥大细胞和中性粒细胞浸润和蛋白酶激活（Chymase，组织蛋白酶G（CG））与MMP激活和ECM降解相关，在诱导主动脉瘘（ACF）后6-12小时内，持续了15 wks。在注射CG的大鼠中，还观察到5天的大鼠也观察到类似的心室扩张和心脏收缩性的降低，这表明丝氨酸蛋白酶可能在体积过载过载诱导的心脏重塑的早期阶段起作用。我们在体外进一步发现，新生大鼠心室心肌细胞（NRVM）急性暴露以促进肝素结合表皮生长因子（HB-EGF）的脱落，并刺激表皮生长因子受体受体（EGFR）激活。 EGFR刺激介导了CG的大部分效应，从而导致局灶性粘附性去磷酸化和胱天蛋白酶激活，这些激活最终导致NRVM脱离矩阵和死亡（也称为AnoikiS）。这导致了以下假设：炎性蛋白酶是心肌病毒死亡的关键介质，通过调节pro-HB-EGF的膜脱落并随后破坏局灶性粘附信号传导，这是心脏肌细胞死亡的关键介体。 AIM 1将确定Sheddase和HB-EGF在CG效应中的作用。 AIM 2将识别将EGFR与CG诱导的肌细胞厌氧症期间引发液胱天蛋白酶的激活联系起来的下游信号传导途径。 AIM 3将确定膜脱落在体积过载诱导的心脏重塑中的作用。外生域的产物和SHEDDASE活性将通过微透析与使用药理学干预措施和转基因方法结合使用的标准生化测定法测量。总的来说，这项研究将决定膜脱落和跨膜蛋白如何在炎症区域进行心脏重塑期间的炎症过程以及丝氨酸蛋白酶是否应视为治疗干预措施的直接新靶标。