The only FDA-approved treatment for stroke is tPA, but it is used in <5% of stroke victims. Reasons for its limited use include severe, time-dependent, adverse effects, especially brain swelling, edema and hemorrhagic transformation (HT). tPA-triggered matrix metalloproteinase (MMP-9/2) activity is responsible for adverse effects, but our understanding is still incomplete, especially as regards upstream mechanisms responsible for tPA-induced activation and/or release of MMPs. The previous cycle of this grant established that the novel SUR1-regulated NCCa-ATP channel (hereinafter called the SUR1/TRPM4 channel) plays a critical role in edema, lesion expansion and cell death following brain ischemia. New preliminary data for this proposal, obtained with a severe ischemia/reperfusion models (6-h MCAo/48 h reperfusion) associated with high mortality, show that tPA exerts no protective effect when administered at 6 h, but that co-administration of glibenclamide with tPA at 6 h significantly reduces mortality and HT. Also, a completely novel finding is that tPA directly opens the SUR1/TRPM4 channel in brain microvascular endothelial cells, resulting in release of activated MMP-9/2 from cells undergoing oncotic lysis due to channel opening, and this effect of tPA is blocked by glibenclamide. This discovery suggests that the SUR1/TRPM4 channel may be a critical upstream regulator of MMP-9/2 release, and it may explain the strong protective effect of glibenclamide when administered with tPA. In this proposal, we will expand upon these novel preliminary data to consolidate our findings, establish the molecular pathway linking the channel with tPA-induced adverse effects, and develop novel treatment strategies to render tPA safer and more widely usable in patients with stroke. We will examine the hypotheses that: (i) preventing release of MMP-9/2 by inhibiting the SUR1/TRPM4 channel is superior to directly inhibiting MMP-9/2 after it is released;(ii) tPA-induced opening of the SUR1/TRPM4 channel in microvascular endothelium is due to signaling that involves protein kinase C phosphorylation of TRPM4, the pore- forming subunit of the channel;(iii) hypertension, an important risk factor for tPA-associated complications, is a risk factor because it results in NFkappaB-mediated upregulation of SUR1/TRPM4 channels.
In Specific Aims (SA) 1, using a rat model of severe ischemia/reperfusion injury, we will compare SUR1/TRPM4 channel inhibition using gene suppression and pharmacological strategies vs. direct MMP-inhibition on short-term and long-term sequelae. In SA2, we will characterize the molecular mechanism by which tPA opens SUR1/TRPM4 channels. In SA3, we will characterize the role of the transcription factor, NFkappaB, in predisposing to HT due to upregulation of the SUR1/TRPM4 channel. Demonstrating these concepts will advance our understanding of the fundamental cellular biology of stroke, and is anticipated to bring forth pharmaceutical treatments that will extend the treatment window and safety of tPA.

Public Health Relevance

tPA, the only FDA-approved treatment for stroke, is grossly underutilized in part because of severe adverse effects associated with delayed administration. In preliminary experiments for this proposal, we discovered a novel molecular mechanism involving direct activation of the SUR1/TRPM4 channel that accounts for tPA's adverse effects. The experiments in this proposal will expand upon these novel preliminary data to consolidate our findings, establish the molecular pathway linking the channel with tPA-induced adverse effects, and develop novel treatment strategies to render tPA safer and more widely usable in patients with stroke.

Agency
National Institute of Health (NIH)
Institute
National Heart, Lung, and Blood Institute (NHLBI)
Type
Research Project (R01)
Project #
5R01HL082517-09
Application #
8598491
Study Section
Brain Injury and Neurovascular Pathologies Study Section (BINP)
Program Officer
Kindzelski, Andrei L
Project Start
2006-01-19
Project End
2015-12-31
Budget Start
2014-01-01
Budget End
2014-12-31
Support Year
9
Fiscal Year
2014
Total Cost
$337,500
Indirect Cost
$112,500
Name
University of Maryland Baltimore
Department
Neurosurgery
Type
Schools of Medicine
DUNS #
188435911
City
Baltimore
State
MD
Country
United States
Zip Code
21201
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