TNF mRNA stability and restenosis

描述（由申请人提供）：炎症在血管损伤和修复中起重要作用。单核细胞吞噬细胞在这些过程中是重要的贡献者，部分通过粘附相互作用和分泌促炎细胞因子。抗炎细胞因子IL-10通过单核细胞/巨噬细胞的失活和炎性细胞因子表达的抑制来抑制这种应答。然而，IL-10抑制作用的机制尚未完全表征。我们最近发表的数据和后续初步研究表明，小鼠颈动脉剥脱后全身IL-10治疗减弱了炎性细胞浸润、动脉TNF表达和内膜增生，同时增强了再内皮化。在分子水平上，IL-10介导的抑制通过以富含A + U的元件依赖性方式增强mRNA的去稳定化而发生。IL-10抑制mRNA稳定蛋白HuR的表达及其与TNF-ARE序列结合的能力。这项建议将集中我们的努力在调和我们在体外机制的观察，在体内生理模型。具体而言，我们将尝试：a）利用其中TNF的ARE部分已被特异性缺失的转基因小鼠，阐明TNF mRNA不稳定作为体内IL-10敏感性的潜在机制（TNF δ ARE）从而导致TNF水平显著升高，B）理解IL-10通过其转录后抑制TNF mRNA稳定性的分子事件，特别是IL-10对与控制mRNA半衰期的序列结合的反式因子/蛋白质的调节，和c）这些事件对颈动脉剥脱小鼠模型中动脉损伤后新生内膜增厚衰减的影响。我们的中心假设是体内TNF-ARE的缺失减弱了IL-10对TNF表达的抑制作用，导致损伤诱导的动脉炎症和新生内膜增生增强。本研究的目的是：1）确定TNF-ARE结合蛋白作为IL-10下游靶点的作用，阐明IL-10介导的TNF mRNA不稳定性的信号转导机制; 2）确定IL-10缺失对IL-10缺陷小鼠损伤后内膜增生的影响;和3）在TNF-ARE小鼠体内建立完整TNF-ARE对IL-10抑制内膜增生和抑制TNF mRNA稳定性的需要。由于IL-10的功能和信号传导是控制炎症反应的重要组成部分，了解IL-10在拟议的生理相关小鼠模型中的炎症基因调控的分子机制，可能会提供必要的见解，以制定策略，调节血管修复再狭窄和其他加速动脉病变，包括移植血管病变和静脉移植物增生。公共卫生相关性：通过球囊血管成形术打开堵塞的血管缓解了心脏病的症状，然而，该手术在手术后一年内会因清除的动脉再狭窄（再狭窄）而受到影响。鉴于仅在美国就有超过120万例此类手术，这是一个重大的健康和成本问题。该项目旨在了解再狭窄的机制，从而潜在地确定治疗这一重大健康问题的方法和手段。

DESCRIPTION (provided by applicant): Inflammation plays an essential role in vascular injury and repair. Mononuclear phagocytes are important contributors in these processes, in part via adhesive interactions and secretion of pro-inflammatory cytokines. The anti-inflammatory cytokine IL-10 suppresses such responses via deactivation of monocytes/macrophages and repression of inflammatory cytokine expression. The mechanisms of IL-10 suppressive action are, however, incompletely characterized. Our recently published data and follow-up preliminary studies indicate that systemic IL-10 treatment following carotid artery denudation in mice blunts inflammatory cell infiltration, arterial TNF expression and intimal hyperplasia while augmenting re- endothelialization. At molecular level, IL-10-mediated suppression occurs via enhanced de-stabilization of mRNA in A+U rich elements dependent manner. IL-10 inhibits the expression of mRNA stabilizing protein HuR and its ability to bind to TNF-ARE sequences. This proposal will focus our efforts upon reconciling our in vitro mechanistic observations to the in vivo physiological models. Specifically we will attempt to: a) elucidating TNF mRNA destabilization as underlying mechanism of IL-10 sensitivity in vivo, utilizing transgenic mice in which ARE portion of TNF has specifically been deleted (TNFdeltaARE) thereby resulting in significantly elevated TNF levels, b) understanding the molecular events through which IL-10 post- transcriptionally suppresses TNF mRNA stability, specifically IL-10 modulation of trans factors/proteins binding to sequences controlling mRNA half-life, and c) the implication of these events on attenuation of neointimal thickening after arterial injury in mouse models of carotid artery denudation. Our central hypothesis is that loss of TNF-ARE in vivo attenuates the inhibitory effect of IL-10 on TNF expression resulting in enhanced injury-induced arterial inflammation and neointimal hyperplasia. This proposal will test hypotheses organized according to the following 3 specific aims: 1) Determine the role of TNF-ARE binding proteins as downstream target of IL-10 and elucidate signaling mechanisms involved in IL-10- mediated TNF mRNA instability; 2) Determine the effect of the loss of IL-10 on post-injury intimal hyperplasia in IL-10 deficient mice; and 3) Establish the requirement of intact TNF-ARE for IL-10 inhibition of intimal hyperplasia and inhibition of TNF mRNA stability, in vivo in TNFdeltaARE mice. Because IL-10 function and signaling are important components for control of inflammatory response, understanding the molecular mechanisms of inflammatory gene regulation by IL-10 in proposed physiologically relevant mouse models may provide insights necessary to develop strategies for modulating vascular repair in restenosis and other accelerated arteriopathies, including transplant vasculopathy and vein graft hyperplasia. PUBLIC HEALTH RELEVANCE: The opening of clogged blood vessels by balloon angioplasty relieves the symptoms of heart disease, however, the procedure is compromised by re-narrowing of the cleared arteries (restenosis) within a year of the procedure. Given that over 1.2 million such procedures are done in the US alone, it is significant health and cost issue. This projects aims to understand the mechanisms of restenosis and thereby potentially identify ways and means to treat this significant health problem.