Ischemic stroke is a significant public health concern in the United States. Current therapeutic approaches for stroke involve thrombolytic therapy with recombinant tissue plasminogen activator (r-tPA) or endovascular treatments; however, many patients still experience disability. The goal of improving post-stroke outcomes requires novel neuroprotective drugs for stroke treatment. While many such compounds have been identified in preclinical stroke studies, none of these have been successfully translated to the clinic. In contrast, 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitors (i.e., statins) are routinely given to stroke patients due to an inherent ability of these drugs to improve post-stroke functional outcomes. In this grant, we will test the mechanistic hypothesis that neuroprotection from statins results from transport across the blood-brain barrier (BBB) that is mediated by the critical uptake transporter organic anion transporting polypeptide 1a4 (Oatp1a4). This hypothesis will be tested by two aims.
Aim 1 : To investigate CNS delivery of statins mediated by Oatp1a4 in ischemic stroke. To evaluate the role of Oatp1a4 in CNS drug delivery during stroke, we will investigate Oatp1a4-mediated statin transport at the BBB using the transient middle cerebral artery occlusion (tMCAO) model. In these studies, age-matched male and female rats will be subjected to tMCAO for 90 min (Aim 1A). We will then demonstrate that Oatp1a4- mediated statin delivery improves both biomarkers of neuroprotection and BBB integrity (Aim 1B). We will also perform neurocognitive, sensorimotor, and motor performance studies (i.e., functional neurological tests) in animals administered statins and subjected to tMCAO with reperfusion times of up to 21 days (Aim 1C). In all studies, statins will be administered intravenously either at the time of reperfusion or after 2 h of reperfusion to show that early administration of neuroprotective drugs can improve post-stroke outcomes.
Aim 2 : Transforming Growth Factor-b (TGF-b) signaling can be targeted to control Oatp1a4- mediated CNS statin delivery in ischemic stroke. In these experiments, we will perform dose-response studies and multiple-dosing experiments in age-matched male and female Sprague-Dawley rats using the TGF- b/ALK1 agonist bone morphogenetic protein (BMP)-9 and the TGF-b/ALK5 antagonist SB431542. We will study the activation of specific Smad proteins that control TGF-b signaling in brain microvascular endothelial cells (Aim 2A). We will also determine the time course of Oatp1a4 expression and activity changes following BMP-9 or SB431542 treatment and their effects on CNS delivery of statins (Aim 2B). Additionally, we will measure indices of neuroprotection, markers of BBB protection, and neurological outcomes in rats subjected to tMCAO, administered BMP-9 or SB431542, and injected intravenously with a statin (Aim 2C). Overall, these studies are clinically relevant because they will demonstrate the effective BBB transport mechanisms are required to confer effectiveness of neuroprotective drugs in stroke.
Despite development of reperfusion therapies, many patients still experience considerable neurological disability following ischemic stroke, which indicates a critical need for neuroprotective treatments that can improve post-stroke functional outcomes in the setting of recanalization. In this grant renewal application, we will demonstrate that neuroprotective effectiveness of 3-hydroxy-3-methyllutaryl coenzyme A (HMG-CoA) reductase inhibitors (i.e., statins) is dependent upon functional expression of organic anion transporting polypeptide 1a4 (Oatp1a4), an endogenous uptake transporter expressed at the blood-brain barrier (BBB). Our work will directly impact public health in the United States by informing development of novel treatment paradigms for stroke by showing that transporter-mediated drug delivery is a critical mechanism that enables neuroprotective drugs to achieve efficacious concentrations in the ischemic brain.
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