Ischemic stroke acutely targets the cerebral microvasculature. Within hours following proximal MCA occlusion, the microvessel endothelial permeability barrier opens causing ?vasogenic? edema in the corpus striatum. This process is not fully understood and has no treatment currently. Cerebral microvessel structure and signaling is pertinent to these acute changes. Interfering with the established adhesion of endothelial cell ?1-integrins to their underlying matrix acutely increases permeability, decreases inter-endothelial cell tight junction (TJ) expression, and alters F-actin conformation, without proteolysis or loss of endothelial cell viability. However, inhibition of myosin light chain (MLC) phosphorylation, within 6 hours in vitro, prevents the permeability increase when established ?1-integrin?matrix adhesion is blocked. Hence, modulating endothelial ?1-integrin signaling acutely could preserve the barrier and decrease striatal edema. The hypotheses to be tested in this proposal state that i) interactions of endothelial cells with matrix proteins of the basal lamina through ?1- integrin adhesion receptors are a major structural and signaling determinant of blood-brain barrier behavior, ii) focal ischemia disrupts stable ?1-integrin?matrix interactions, iii) disruption of these interactions increases microvessel permeability, and iv) this acutely increased permeability and edema can be prevented by inhibiting endothelial pMLC signaling caused by ?1-integrin loss.
The Specific Aims will demonstrate that: 1) rapid modulation of the MLC pathway of ?1-integrin signaling can prevent the acute increase in permeability caused by interference with ?1-integrin?matrix adhesion, 2) experimental ischemia disrupts endothelial cell ?1- integrin?matrix adhesion, induces endothelial signaling that increases permeability, and this can be prevented, and 3) focal ischemia, through tissue injury, decreases ?1-integrin expression or matrix adhesion, and increases endothelial permeability, which can be prevented. A central role for endothelial cell ?1-integrin? matrix adhesion as the determinate of acute ?vasogenic edema? in focal ischemia is conceptually novel and testable. With high-quality primary cerebral endothelial cell cultures from wild type and conditional endothelial ?1-integrin knockout (?1iKO) constructs to define MLC signaling events (Specific Aim 1), in vitro models to examine the effects of ischemia on endothelial cell MLC signaling (Specific Aim 2), and real-time assessment of acute edema formation in mouse stereotaxic injection and MCA occlusion models using dynamic imaging with 14T MRI to guide acute molecular assessments of permeability and its prevention with signaling inhibitors (Specific Aims 1 and 3), this Project will demonstrate that ?1-integrin?matrix adhesion is pivotal in edema prevention. These very feasible novel studies are a new direction in stroke research that will substantially further our understanding of acute blood-brain barrier behavior under focal ischemia. Understanding these signaling pathways and mechanisms is likely to identify novel specific targets to preserve or selectively change endothelial barrier function in other neurovascular disorders, with the aim ultimately to improve outcome.
Ischemic stroke is a devastating disorder that can quickly disrupt important bonds between the (endothelial) cells that line the small blood vessels of the brain; these bonds protect the brain from swelling, blood leakage, and increased injury. We have recently found that molecules (?1-integrins) that glue the endothelial cells to the scaffolding (matrix) they sit on within the vessel wall are essential for maintaining the bonds between endothelial cells; ischemic stroke quickly disrupts ?1-integrins, thereby causing leakage to occur in the first hours of stroke. This research project will define the exact mechanisms by which endothelial cell ?1-integrin? matrix interactions are broken, how the loss of endothelial ?1-integrins contributes to brain injury during ischemic stroke, and how these events can be prevented, thereby offering new acute treatment strategies for stroke.
