TNF mRNA stability and restenosis

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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.