Acid-sensing GPCRs in vascular inflammation and growth

项目摘要/摘要 心血管疾病(CVD)仍然是美国发病率和死亡率的主要原因 国家和世界范围内，心血管疾病发病的关键基础包括血管内皮细胞 血管内皮细胞(VEC)炎症、粘连和血管平滑肌(VSM)异常生长。在疾病中 组织局部微环境因细胞新陈代谢改变而变得酸性，并受到损害 血流量，但酸性pH对疾病过程的确切贡献，特别是对血管内皮细胞和 VSM功能障碍，潜在意义重大，但尚未得到很好的理解。耐人寻味的是，一个pH感应G家族 蛋白质偶联受体(GPCRs)已被发现，包括GPR4，主要存在于血管内皮细胞，以及 GPR68，主要定位于VSM，最近的发现表明，这些可能在诱导 VEC和VSM并发症是CVD发生的基础。这项研究计划的总体目标是确定 GPR4和GPR68在引发病理性血管内皮细胞炎症中的确切作用和机制 黏附和VSM生长。这项研究直接解决了心血管疾病的健康问题，是 潜在的临床重要性。该项目的假设是酸中毒激活了对pH敏感的血管内皮细胞。 GPR4和VSM GPR68，从而刺激环状AMP驱动的EPAC，抑制抗炎和 保护生长的AMPK，反过来促进血管内皮细胞炎症和黏附，以及有害的VSM生长 作为心血管疾病血管功能障碍的基础。使用野生型(WT)、GPR4基因敲除(KO)和GPR68 KO小鼠以及通过功能获得/功能丧失干预来验证体外和体内方法 机制，三个具体的目标将检验我们的假设：目标1将检查细胞信号的反应 对酸中毒包括cAMP含量、cAMP依赖蛋白激酶(PKA)和 环磷酸腺苷降解磷酸二酯酶(PDE)及其下游效应分子的表达和活性 EPAC和代谢AMP依赖的蛋白激酶(AMPK)。目标2将确定监管影响 GPR4和GPR68信号在血管内皮细胞炎症、黏附和VSMC迁移中的作用 增殖，以及Aim 3确定能够控制动脉生长的离散GPR4或GPR68过程 以及活体条件下的重塑。这一综合研究设计将决定pH传感 GPR4和GPR68及其细胞内效应因子EPAC和AMPK在血管内皮细胞炎症中的作用 而黏附、VSM迁移和增殖是CVD的基本因素。预期的调查结果有望 改变我们目前对血管细胞信号转导的理解，并将为基础和 临床研究，希望为治疗干预确定新的、更具选择性的靶点 在脑血管病患者中。

PROJECT SUMMARY/ABSTRACT Cardiovascular disease (CVD) remains the primary cause of morbidity and mortality in the United States and worldwide, and key underpinnings in CVD pathogenesis include vascular endothelial cell (VEC) inflammation and adhesion and abnormal growth of vascular smooth muscle (VSM). In diseased tissue the local microenvironment becomes acidic from altered cellular metabolism and compromised blood flow, yet the exact contributions of acidic pH to the disease process and in particular, to VEC and VSM dysfunction, is potentially significant yet not well understood. Intriguingly, a family of pH-sensing G protein-coupled receptors (GPCRs) has been identified including GPR4, primarily found in VECs, and GPR68, predominantly localized to VSM, and recent findings suggest these may be crucial in eliciting VEC and VSM complications foundational to CVD. The broad goal of this research plan is to determine precise roles and mechanisms of GPR4 and GPR68 in soliciting pathologic VEC inflammation and adhesion and VSM growth. This line of study directly addresses the health concerns of CVD and is of potential clinical importance. The hypothesis of this project is that acidosis activates pH-sensing VEC GPR4 and VSM GPR68, thereby stimulating cyclic AMP-driven Epac and inhibiting anti-inflammatory and growth-protective AMPK, in turn promoting VEC inflammation and adhesion and deleterious VSM growth as foundations of vascular dysfunction in CVD. Using wild type (WT), GPR4 knockout (KO) and GPR68 KO mice and in vitro and in vivo approaches with gain-of-function/loss-of-function interventions to validate mechanisms, three Specific Aims will test our hypothesis: Aim 1 will examine cellular signals in response to acidosis including cyclic AMP content, activities of cyclic AMP-dependent protein kinase (PKA) and cyclic AMP-degrading phosphodiesterase (PDE), and expression and activities of downstream effectors Epac and metabolic AMP-dependent protein kinase (AMPK). Aim 2 will determine the regulatory impacts of GPR4 and GPR68 signals on VEC inflammation and adhesion and VSM cell (VSMC) migration and proliferation, and Aim 3 identify discrete GPR4 or GPR68 processes capable of controlling arterial growth and remodeling under in vivo conditions. This integrated research design will determine pH-sensing GPR4 and GPR68 and their intracellular effectors Epac and AMPK as instrumental in VEC inflammation and adhesion and VSM migration and proliferation elemental to CVD. Anticipated findings promise to shift our current understanding of vascular cell signaling and will provide new avenues for basic and clinical investigation with the hopes of identifying novel, more selective targets for therapeutic intervention in CVD patients.