PAR1 as a Therapeutic Target in Doxorubicin-induced Cardiotoxicity

即使采用目前旨在减少心脏损伤的治疗方案，Dox心脏毒性(DoxTox)仍然会发生。它的 预防和管理仍然是心脏病学家和肿瘤学家面临的一个主要问题，限制了最大 终生剂量，并危及患者的癌症结局。蛋白水解酶激活受体1(PAR1)是一种G-受体。 由心肌细胞(CM)和心脏成纤维细胞(CFs)表达的蛋白偶联受体(GPCR)。它 是人类血小板上的主要凝血酶受体，也是FDA批准的药物Vorapaxar的靶标。在……里面 与人类血小板相比，小鼠血小板不表达PAR1，这使它们成为一个很好的研究模型 PAR1的非血小板依赖性作用。PAR1被多种蛋白水解酶激活。与其他GPCR一样，PAR1 表现出有偏见的信号。例如，凝血酶或基质金属蛋白酶(MMPs)激活PAR1是 激活的蛋白C(APC)具有细胞毒性，而激活的蛋白C(APC)则具有细胞保护作用。重要的是，我们最近 凝血酶激动肽(AP)激活PAR1增强Dox诱导的细胞死亡 CMS和CFS的体外培养。此外，用伏拉帕沙治疗的PAR1缺陷小鼠和野生型小鼠也受到保护 死于急性DoxTox。我们假设服用Dox会增加凝血酶的生成和 其他激活CMS和CFs上PAR1的蛋白酶，如MMPs，会导致氧化应激，导致 DoxToxOur的提案有两个目标。1.确定不同的PAR1激活酶对DoxTox的影响。我们 假设凝血酶和MMPs激活的PAR1/G、α、Q/Ca~(2+)依赖的信号通路增强了Doxtox。在……里面 相反，我们假设APC/PAR1/β-arrestin2通路将减少DoxTox。具体目标2.阐明 参与DoxTox的细胞特异性PAR1和蛋白水解酶依赖途径。我们假设PAR1 CMS和CFs的表达都是通过共同的PAR1/G蛋白和不同的Cf特异性PAR1/β- 急、慢性DoxTox的抑制机制。我们假设凝血酶和基质金属蛋白对 而外源性APC会在体内减少DoxTox。在本提案中，我们将分析PAR1的角色 在CMS上表达，而在DoxTox上表达。此外，我们将使用药理学的方法来研究 凝血酶或MMPs对DoxTox的作用依赖于PAR1。最后，我们将测试PAR1抑制剂或 APC可降低小鼠急性和慢性DoxTox。我们认为，voapaxar将通过阻断所有 标准杆1。此外，APC治疗先于Dox治疗将通过增强细胞保护作用来减少DoxTox PAR1/β-arrestin2信号转导。最后，偏向的PAR1抑制剂Parmodin 2将减少主要的病理变化 PAR1/G蛋白同时增强DoxTox的保护性APC样信号。这项提案的总体目标是 为了(I)了解PAR1在DoxTox中的病理作用，以及(Ii)开发新的治疗方法来预防蒽环类药物- 致心脏损伤。

Even with current regimens designed to reduce cardiac injury, Dox cardiotoxicity (DoxTox) still occurs. Its prevention and management remain a major problem for both cardiologists and oncologists, limiting the maximal lifetime dosage and compromising the patient’s cancer outcomes. Protease-activated receptor 1 (PAR1) is a G- protein coupled receptor (GPCR) that is expressed by cardiac myocytes (CM) and cardiac fibroblasts (CFs). It is the main thrombin receptor on human platelets and is the target of the FDA-approved drug vorapaxar. In contrast to human platelets, mouse platelets do not express PAR1 making them an excellent model to study platelet-independent effects of PAR1. PAR1 is activated by a variety of proteases. As with other GPCRs, PAR1 exhibits biased signaling. For instance, PAR1 activation by thrombin or matrix metalloproteinases (MMPs) is cytotoxic whereas activation by activated protein C (APC) is cytoprotective. Importantly, we recently demonstrated that activation of PAR1 with a thrombin agonist peptide (AP) enhanced Dox-induced cell death of CMs and CFs in vitro. In addition, PAR1 deficient mice and wild-type mice treated with vorapaxar were protected from acute DoxTox. We hypothesize that administration of Dox leads to increased generation of thrombin and other proteases, such as MMPs, that activate PAR1 on CMs and CFs contributes to oxidative stress, leading to DoxToxOur proposal has 2 aims. 1. Determine the effect of different PAR1 activating proteases on DoxTox. We hypothesize that PAR1/Gαq/Ca2+-dependent signaling activated by thrombin and MMPs enhances DoxTox. In contrast, we hypothesize that the APC/PAR1/β-arrestin2 pathway will reduce DoxTox. Specific Aim 2. Elucidate the cell-specific PAR1- and protease-dependent pathways contributing to DoxTox. We hypothesize that PAR1 expressed by CMs and CFs both contribute via common PAR1/G-protein and distinct CF-specific PAR1/β- arrestin1 mechanisms to acute and chronic DoxTox. We hypothesize that thrombin and MMPs contribute to DoxTox whereas exogenous APC will reduce DoxTox in vivo. In this proposal, we will analyze the role of PAR1 expressed on CMs versus CFs on DoxTox. In addition, we will use a pharmacologic approach to investigate the PAR1-dependent contribution of thrombin or MMPs to DoxTox. Finally, we will test whether PAR1 inhibitors or APC reduce acute and chronic DoxTox in mice. We propose that vorapaxar will reduce DoxTox by blocking all PAR1. In addition, APC treatment prior to Dox treatment will reduce DoxTox by enhancing cytoprotective PAR1/β-arrestin2 signaling. Finally, the biased PAR1 inhibitor Parmodulin 2 will reduce primarily pathologic PAR1/G-protein while enhancing protective APC-like signaling in DoxTox. The overall goals of this proposal are to (i) understand the pathologic roles of PAR1 in DoxTox and (ii) develop new therapies to prevent anthracycline- induced cardiac injury.