Inflammmatory Proteases, Sheddases & Cardiomyocyte Death

描述(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，其中炎症细胞及其蛋白水解酶协调心肌修复。虽然炎症细胞在心肌损伤后的早期阶段是有益的，但它在心肌内释放自由基和蛋白水解酶，可能导致心肌细胞死亡和随后心脏几何和机械特性的改变。我们发现，在主动脉腔内瘘(ACF)诱导后的6-12小时内，肥大细胞和中性粒细胞的浸润和蛋白水解酶(Chymase，Cathepsin G，CG)的激活与基质的激活和ECM的降解有关，这种情况持续了15周。注射CG 5天的大鼠也观察到类似的心室扩张和心肌收缩能力下降，提示丝氨酸蛋白酶可能在容量超负荷诱导的心脏重构的早期阶段发挥作用。我们在体外进一步发现，新生大鼠心室肌细胞(NRVM)急性暴露于CG可促进肝素结合的表皮生长因子(HB-EGF)的脱落和刺激表皮生长因子受体(EGFR)的激活。EGFR刺激介导了CG的大部分作用，导致局部黏附、去磷酸化和半胱氨酸天冬氨酸酶的激活，最终导致NRVM脱离基质和死亡(也称为失巢)。这导致了一种假说，即炎症蛋白是心肌细胞死亡的关键介质，通过调节前HB-EGF的膜脱落和随后的局部黏附信号的破坏，心肌细胞死亡发生在心力衰竭的早期。目的1确定脱落酶和HB-EGF在CG效应中的作用。AIM 2将确定下游信号通路，将EGFR与CG诱导的心肌细胞失巢期间局部黏附破坏和随后激活的启动子caspase联系起来。目的3确定膜脱落在容量超负荷所致心脏重构中的作用。胞外区产物和脱落酶活性将通过微透析结合使用药理干预和转基因方法的标准生化分析来测量。总之，这项研究将确定在炎症区域心脏重塑过程中，膜脱落和跨膜蛋白如何促进炎症过程，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.