A major problem with tissue plasminogen activator (tPA) therapy for ischemic stroke is the complication of hemorrhage and edema. In our previous grant, we established a quantitative model of tPA-induced hemorrhage in rodent embolic (clot-based) focal ischemia, and showed an overall role for oxidative damage. In this renewal application, we focus on novel signaling mechanisms that are involved. Our overall hypothesis is that after stroke, tPA binds the Low-density-lipoprotein Related Protein (LRP). LRP is a receptor present on neurons; astrocytes and endothelial cells which can trigger MAP kinase signaling cascades, resulting in upregulation of matrix metalloproteinase-9 (MMP-9) in the neurovascular unit (vascular/ astrocytic/neuronal compartments). MMP-9 degrades critical neurovascular matrix substrates that weaken blood vessel integrity and lead to leakage and rupture. To test this overall hypothesis, we will pursue 3 Specific Aims.
In Aim 1, we will examine LRP signaling pathways that upregulate MMP-9 activity after tPA therapy. To do this, we will use in vitro and in vivo models to document the following events: increased levels of LRP after hypoxia/ischemia, tPA-LRP triggering of MAP kinase, activation of AP1 promoter transcription of MMP-9. In vitro experiments will use neurons, astrocytes, and cerebral endothelial cells. In vivo experiments will use the tPA-induced hemorrhage model in rats.
In Aim 2, we wiI1 document a specific role for MMP-9. After focal ischemia, we will compare the severity of tPA-induced edema and hemorrhage in wildtype mice versus MMP-2 knockouts, MMP-7 knockouts, MMP-9 knockouts, and transgenic overexpressors of TIMP1 (an inhibitor of MMPs).
In Aim 3, we develop novel imaging methods to correlate the effects of tPA, MMP-9 activation, and vascular leakage. We will construct an MMP-9 specific probe that enables near-infrared fluorescence imaging of MMP-9 in vivo after tPA. Cranial window imaging experiments in rat and mouse embolic focal ischemia will correlate in real-time MMP-9 upregulation in the neurovascular unit with the onset of vascular leakage. At the NINDS Stroke Program Review Group, proteolytic pathology in the neurovascular unit was identified as a high priority research area. This proposal should fit the goals of the Stroke PRG because it examines novel mechanisms that link tPA, cell signaling, MMPs, and hemorrhagic transformation in the neurovascular unit. ? ? ?
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