Stat3 in Post-Ischemic Myocardial Inflammation

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Myocardial reperfusion results in the induction of pro-inflammatory genes through the activation of redoxsensitive nuclear transcription factors. We have found that signal transducer and activator of transcription-3 (Stat3) is activated after myocardial ischemia-reperfusion, in a manner dependent on the small GTPase Rac1, and binds to a GAS promoter element in the intercellular adhesion molecule-1 (ICAM-1) gene, a gene which codes for an important endothelial cell adhesion molecule which interacts with leukocytes. After phosphorylation of its serine 727 residue, StatS binds to the transcriptional activator Sp1. We hypothesize that this represents a novel mechanism for enhancing ICAM-1 transcription in endothelial cells (ECs) after ischemia-reperfusion. In support of this idea, inhibition of Stat3/Sp1 activity in cultured ECs significantly reduces the transcription of ICAM- 1 after hypoxia-reoxygenation. In this proposal, we will determine the role of StatS in the regulation of ICAM-1 and other pro-inflammatory genes in vascular endothelium after myocardial ischemia-reperfusion. In Aim 1 we will investigate the molecular mechanisms responsible for StatS interaction with Sp1, including the mechanisms involved in binding between the two molecules and the role of phosphorylation of the StatS S727 residue. We will determine whether inhibition of the Stat3-Sp1 interaction affects ICAM-1 gene regulation in ECs, and whether Stat3/Sp1 modifies post-ischemic microvascular inflammation (assessed by videomicroscopy) or myocardial ischemia-reperfusion injury, using in vivo murine models. In Aim 2 we will examine the mechanisms responsible for Rac1-dependent StatS activation after hypoxia-reoxygenation, including how the two molecules bind together, how this binding promotes the phosphorylation of the Y705 and S727 residues of StatS, and which kinases are involved. In Aim 3 we will determine whether activation of StatS in hypoxic-reoxygenated ECs is self-limited by a negative feedback loop involving StatS transcriptional regulation of T-cell lymphoma invasion and metastasis 2 (TIAM2), a guanine exchange factor, and RacGAPI, a GTPase-activating protein, both of which in turn downregulate the activation state of Rac1, and thereby of StatS itself.