Stroke remains the leading cause of disability and a major cause of death among Americans. This grant seeks to illuminate fundamental processes driving development of the inflammatory and coagulant phenotype of cerebral microvessels in stroke. Preliminary data in murine stroke shows that upregulated expression of leukocyte adhesion receptors (ICAM-1, P- and E-selectin), GPIIb/IIIa-receptor mediated aggregation of platelets, and diminished endogenous fibrinolysis are all contributory. New data suggests that ischemic vascular stress initiates a pathway leading from protein kinase C (PKC) betaII activation to rapid induction of the early growth response-1 gene (Egr-1), a transcription factor which triggers expression of target genes which modulate the pathological ischemic vascular phenotype. These targets include inflammatory (cytokine/chemokine), leukocyte adhesion receptor, prothrombotic, antifibrinolytic, and permeability-inducing genes. As mice null for PKCbeta or Egr-1 are significantly protected from pulmonary ischemic injury, and because Egr-1 mRNA and protein are increased in the ischemic brain, we hypothesize that Egr-1 serves as a """"""""master switch,"""""""" whose induction during cerebral ischemia may trigger pathological downstream events leading to micro vascular failure and increased tissue injury in stroke.
We Aim (1) To elucidate the pathological role of Egr-1 in micro vascular failure in stroke, using Egr 1+/+ and Egr-1-/- mice to examine stroke outcomes as well as induction of Egr-1 target genes; (2) To determine whether an event upstream of Egr-1, activation of PKCbetaII, has a role in micro vascular failure in stroke. These studies will use PKCbeta null mice and unique transgenic mice with macrophage-targeted expression of wild type, dominant negative, or constitutively active PKCbetaII driven by the type A macrophage scavenger receptor; (3) To identify alternative mechanisms which may link coagulant and inflammatory axes in the setting of stroke. Microarrays will be used for gene expression profiling in ischemic brains from Egr-1+/+ and Egr-1-/- mice, a detailed promoter analysis will be undertaken for a prototypical hypoxia-inducible, Egr-1 responsive gene (plasminogen activator inhibitor-1), and a post-transcriptional link between inflammation and thrombosis in stroke will be examined in P-selectin null mice. Overall these studies will identify unifying molecular events which link micro vascular coagulation and inflammation in stroke.
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