Treatment of stroke with a selective inhibitor of phosphodiesterase type 5 (PDE5), e.g.sildenafil, significantly promotes functional recovery concomitant with significant increases of neurogenesis and angiogenesis when the treatment is initiated 1 or 7 days after stroke onset. In this application, we seek to elucidate fundamental mechanisms of neurogenesis, neuroblast migration and the coupling of neurogenesis and angiogenesis after stroke with and without treatment with sildenafil as the neurorestorative agent. Our hypotheses are that sildenafil mediated increases of cGMP levels in stroke brain: 1) promote proliferation of neural progenitor cells via shortening the length of the cell cycle which results from a decreased expression of cyclin-dependent kinase inhibitors; 2) enhance neuroblast migration towards the ischemic boundary regions via increasing expression of stromal-derived factor-la (SDF-1a), CXCR4 and matrix metalloproteinases (MMPs); 3) foster angiogenesis which generates a permissive niche to promote neurogenesis and to guide neuroblast migration towards the ischemic boundary regions; and 4) the PI3K/Akt signaling pathway mediates sildenafil-enhanced neurogenesis and neuroblast migration. The proposed experiments have been designed to test these hypotheses. We will investigate whether sildenafil amplifies cell cycle kinetics and the orientation of cell division that regulate the proliferation and differentiation of neural progenitor and stem cells in the subventricular zone. Using retroviral vector, siRNA, and time-lapse microscopy, we will delve into the molecular mechanisms, e.g. SDF-1/CXCR4 and MMPs, which promote neuroblast migration in the ischemic brain after treatment with sildenafil. To examine the effects of sildenafil-induced angiogenesis on neurogenesis and neuroblast migration, we will measure the interaction between angiogenesis and neurogenesis and proteins secreted by the endothelial cells. By blocking SDF-1a and MMPs, we will examine whether SDF-1a and MMPs secreted by endothelial cells mediate neuroblast migration. Using specific inhibitors, we will investigate whether blocking the PI3K/Akt signaling pathway attenuates the effects of sildenafil on neurogenesis, neuroblast migration and angiogenesis. These basic scientific studies will elucidate the mechanisms how the brain remodels itself after stroke and how cGMP amplifies this process in the adult brain, which may lead to novel methods to facilitate brain remodeling during stroke recovery.
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