TNF mRNA stability and restenosis

描述（申请人提供）：炎症在血管损伤和修复中起着至关重要的作用。单核吞噬细胞在这些过程中起重要作用，部分是通过粘附相互作用和促炎细胞因子的分泌。抗炎细胞因子IL-10通过单核/巨噬细胞的失活和炎症细胞因子表达的抑制来抑制这种反应。然而，IL-10抑制作用的机制尚未完全确定。我们最近发表的数据和后续的初步研究表明，小鼠颈动脉剥脱后全身IL-10治疗可以减弱炎症细胞浸润、动脉TNF表达和内膜增生，同时增强再内皮化。在分子水平上，il -10介导的抑制以A+U富元素依赖的方式通过增强mRNA的去稳定化发生。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。这一建议将把我们的努力集中在协调我们的体外机制观察到体内的生理模型。具体来说，我们将尝试：a)阐明TNF mRNA不稳定是体内IL-10敏感性的潜在机制，利用TNF ARE部分被特异性删除的转基因小鼠（TNFdeltaARE），从而导致TNF水平显著升高；b)了解IL-10转录后抑制TNF mRNA稳定性的分子事件，特别是IL-10调节与控制mRNA半衰期的序列结合的反式因子/蛋白质。c)这些事件对小鼠颈动脉剥脱模型动脉损伤后新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，导致损伤性动脉炎症和新生内膜增生增强。本研究将根据以下3个具体目标来检验假设：1)确定TNF- are结合蛋白作为IL-10的下游靶点的作用，阐明IL-10介导的TNF mRNA不稳定性的信号机制；2)测定IL-10缺失对IL-10缺陷小鼠损伤后内膜增生的影响；3)在TNFdeltaARE小鼠体内建立完整的TNF- are对IL-10抑制内膜增生和抑制TNF mRNA稳定性的要求。由于IL-10的功能和信号是控制炎症反应的重要组成部分，了解IL-10在提出的生理相关小鼠模型中炎症基因调控的分子机制可能为制定调节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增生）的血管修复策略提供必要的见解。公共卫生相关性：通过球囊血管成形术打开阻塞的血管可以缓解心脏病的症状，然而，手术后一年内清除的动脉再次狭窄（再狭窄）会危及该手术。考虑到仅在美国就有超过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.