Gut microbial metabolite- Trimethylamine-N-oxide and endothelial inflammasome signaling in cardiovascular injury

项目摘要 最近的研究已经确定了肠道微生物衍生的代谢物，如三甲胺-N-氧化物(TMAO) 作为一种新的心血管疾病危险因素。TMAO，一种肠道微生物衍生的膳食代谢物 CVD患者循环中的磷脂酰胆碱/肉碱升高，并与 啮齿动物和人类的动脉粥样硬化和心血管疾病进展。尽管存在这种惊人的联系，但分子 TMAO如何诱导动脉粥样硬化和CVD进展的机制仍不清楚。在这笔赠款中 我们试图阐明一个早期的细胞内分子机制，即Nlrp3炎症体 激活，可能通过炎症或非炎症途径启动内皮损伤 导致内皮功能障碍，最终导致动脉粥样硬化。有趣的是，我们的初步研究 已证实TMAO-诱导Nlrp3炎症体激活并促进内皮细胞 损伤和微血管损伤也表明，除了炎症，激活的 炎性小体对血管内皮细胞有直接作用。这可能代表了一种新的致病因素 炎症以外的炎性小体激活机制。基于这些观察结果，我们假设 肠道微生物代谢产物，如TMAO被释放到循环中，起到内源性危险的作用 通过Nlrp3炎症体激活信号并诱导炎性和非炎性反应 导致内皮功能障碍和血管损伤，从而表现为动脉粥样硬化。 动脉壁。为了验证这一假设，我们将首先确定TMAO诱导的Nlrp3炎症体 激活导致紧密连接破坏、血管通透性改变、内皮功能障碍和 利用Nlrp3-/-小鼠、内皮特异性Nlrp3基因敲除小鼠(EC-Nlrp3-/-)及其 野生型产仔。然后，我们将研究TMAO如何激活内皮细胞中的Nlrp3炎症体 重点介绍了NADPH氧化酶介导的氧化还原信号的作用和相应的机制 调解它的行为。最后，我们将确定TMAO的非炎性和炎症作用 活化的Nlrp3炎性小体在内皮功能障碍和动脉粥样硬化中的作用 血管内皮细胞和颈动脉原代培养中IL-1β、IL-18、上睑下垂和炎性物质等产物的研究 NLRP3-/-和Nlrp3+/+小鼠。拟议的研究将揭示心血管疾病发病机制的新见解。 由微生物代谢产物如TMAO诱导，将为临床相关疾病的发展铺平道路， 治疗动脉粥样硬化和其他心血管疾病的新治疗策略。

Project Summary Recent studies have identified intestinal microbe-derived metabolites such as Trimethylamine-N-oxide (TMAO) as a novel risk factor for cardiovascular diseases (CVDs). TMAO, a gut microbe-derived metabolite of dietary phosphatidylcholine/carnitine is elevated in the circulation of CVD patients and has been associated with atherosclerosis and CVD progression in rodents and humans. In spite of this striking association, the molecular mechanisms of how TMAO induces atherosclerosis and CVD progression are still unclear. In this grant proposal, we attempt to elucidate an early intracellular molecular mechanism, namely, the Nlrp3 inflammasome activation, which may switch on endothelial damage through its inflammatory or non-inflammatory pathway leading to endothelial dysfunction and ultimately atherosclerosis. Interestingly, our preliminary studies demonstrated that TMAO-induces the Nlrp3 inflammasome activation and contributes to the endothelial damage and microvascular injury and have also shown that beyond inflammation, the activated inflammasomes have direct actions on the endothelial cells. This may represent a novel pathogenic mechanism of inflammasome activation beyond inflammation. Based on these observations, we hypothesize that gut microbial metabolites such as TMAO which are released into the circulation act as endogenous danger signals and induce both inflammatory and non-inflammatory responses via Nlrp3 inflammasome activation leading to endothelial dysfunction and vascular injury which consequently manifests into atherogenesis in the arterial wall. To test this hypothesis, we will first determine whether TMAO-induced Nlrp3 inflammasome activation contributes to tight junction disruption, altered vascular permeability, endothelial dysfunction and atherosclerosis in vivo using Nlrp3-/- mice, endothelium-specific Nlrp3 knockout mice (EC-Nlrp3-/-) and their wild type littermates. We will then study how Nlrp3 inflammasomes are activated in endothelial cells by TMAO with a focus on the roles of NADPH oxidase mediated redox signaling and corresponding mechanisms mediating its actions. Finally we will determine the non-inflammatory and inflammatory effects of TMAO activated Nlrp3 inflammasomes on endothelial dysfunction and atherosclerosis by studying the various products such as IL-1β, IL-18, pyroptosis and DAMPs in primary cultures of CAECs and carotid arteries of Nlrp3-/- and Nlrp3+/+ mice. The proposed studies will reveal new mechanistic insights of CVD pathogenesis induced by microbial metabolites such as TMAO and will pave way to the development of clinically relevant, novel therapeutic strategies for treating atherosclerosis and other cardiovascular disorders.