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，如三甲基胺-N-氧化物（TMAO） 作为心血管疾病（CVD）的一个新的危险因素。TMAO，一种肠道微生物来源的膳食代谢物 磷脂酰胆碱/肉毒碱在CVD患者的循环中升高， 动脉粥样硬化和心血管疾病的进展。尽管有这种惊人的联系， TMAO如何诱导动脉粥样硬化和CVD进展的机制仍不清楚。在这份赠款中， 因此，我们试图阐明一种早期的细胞内分子机制，即Nlrp 3炎性体， 激活，这可能会通过其炎症或非炎症途径打开内皮损伤 导致内皮功能障碍并最终导致动脉粥样硬化。有趣的是，我们的初步研究 证明TMAO诱导Nlrp 3炎性小体活化并有助于内皮细胞的增殖。 损伤和微血管损伤，也表明除了炎症， 炎性小体直接作用于内皮细胞。这可能代表了一种新的致病性 炎性小体激活机制超出炎症。基于这些观察，我们假设 肠道微生物代谢物如TMAO释放到循环中作为内源性危险 通过Nlrp 3炎性体激活来信号传导并诱导炎性和非炎性应答 导致内皮功能障碍和血管损伤，其结果表现为动脉粥样硬化形成， 动脉壁为了检验这一假设，我们将首先确定TMAO诱导的Nlrp 3炎性小体是否 活化导致紧密连接破坏、血管通透性改变、内皮功能障碍和 使用Nlrp 3-/-小鼠、内皮特异性Nlrp 3敲除小鼠（EC-Nlrp 3-/-）和它们的 野生型同窝仔。然后，我们将研究TMAO如何在内皮细胞中激活Nlrp 3炎性小体 重点介绍了NADPH氧化酶介导的氧化还原信号转导的作用及其机制 调解其行动。最后，我们将确定TMAO的非炎症和炎症作用 活化的Nlrp 3炎性小体对内皮功能障碍和动脉粥样硬化的影响， 在CAEC和颈动脉的原代培养物中， Nlrp 3-/-和Nlrp 3 +/+小鼠。这些研究将揭示CVD发病机制的新见解 由微生物代谢产物如TMAO诱导，并将为临床相关， 用于治疗动脉粥样硬化和其它心血管疾病的新的治疗策略。

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.