Inflammmatory Proteases, Sheddases & Cardiomyocyte Death

描述（由申请人提供）：心肌肥厚进展过程中最早的事件之一被认为涉及炎症反应，炎症细胞及其蛋白酶协调心肌修复。尽管炎症细胞在心肌损伤后的早期阶段是有益的，但它们在心肌内释放自由基和蛋白水解酶，这可能导致心肌细胞死亡，并随后改变心脏的几何和力学特性。我们发现，在主动脉腔瘘（ACF）诱导后6-12小时内，肥大细胞和中性粒细胞浸润和蛋白酶激活（溶酶、组织蛋白酶G (CG)）与MMP激活和ECM降解相关，并持续15周。在注射CG 5天的大鼠中也观察到类似的心室扩张和心脏收缩力下降，这表明丝氨酸蛋白酶可能在容量过载引起的心脏重构的早期阶段发挥作用。我们进一步在体外发现，新生儿大鼠心室肌细胞（NRVM）急性暴露于CG可促进肝素结合表皮生长因子（HB-EGF）的脱落和刺激表皮生长因子受体（EGFR）的激活。EGFR刺激介导CG的大部分作用，导致局灶性黏附去磷酸化和半胱天蛋白酶激活，最终导致NRVM脱离基质和死亡（也称为anoikis）。这导致了一种假设，即炎症蛋白酶是心肌细胞死亡的关键介质，心肌细胞死亡发生在心力衰竭进展的早期，通过调节前hb - egf的膜脱落和随后的局灶粘连信号的破坏。目的1将确定sheddases和HB-EGF在CG效应中的作用。目的2将确定在cg诱导的心肌细胞病变过程中，EGFR与局灶黏附破坏和随后启动物半胱天酶激活之间的下游信号通路。目的3将确定膜脱落在容量过载引起的心脏重构中的作用。外结构域产物和脱落酶活性将通过微透析结合使用药理学干预和转基因方法的标准生化分析来测量。总的来说，这项研究将确定膜脱落和跨膜蛋白如何在炎症区域的心脏重塑过程中促进炎症过程，以及丝氨酸蛋白酶是否应被视为治疗干预的直接新靶点。

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.