Abstract
Intracranial hemorrhage remains the most feared complication in tissue plasminogen activator (tPA) thrombolysis for ischemic stroke. However, the underlying molecular mechanisms are still poorly elucidated. In this study, we reported an important role of caveolin-1 (Cav-1) s-nitrosylation in matrix metalloproteinase (MMP)-2 and 9 secretion from tPA-treated ischemic endothelial cells. Brain vascular endothelial cells (bEND3) were exposed to oxygen-glucose deprivation (OGD) for 2 h before adding recombinant human tPA for 6 h. This treatment induced a significant increase of MMP2 and 9 in the media of bEND3 cells and a simultaneous degradation of fibronectin and laminin β-1, the two main components of extracellular matrix (ECM). Inhibition of MMP2 and 9 with SB-3CT completely blocked the degradation of fibronectin and laminin β-1. ODG+tPA treatment led to Cav-1 shedding from bEND3 cells into the media. Notably, OGD triggered nitric oxide (NO) production and S-nitrosylationof Cav-1 (SNCav-1). Meanwhile tPA induced activation of ERK signal pathway and stimulates the secretion of SNCav-1. Pretreatment of bEND3 cells with C-PTIO (a NO scavenger) or U0126 (a specific ERK inhibitor) significantly reduced OGD-induced S-nitrosylation of Cav-1 in cells and blocked the secretion of Cav-1 and MMP2 and 9 into the media as well as the degradation of fibronectin and laminin β-1 in OGD and tPA-treated cells. These data indicate that OGD-triggered Cav-1 S-nitrosylation interacts with tPA-induced ERK activation to augment MMP2 and 9 secretion and subsequent ECM degradation, which may account for the exacerbation of ischemic blood brain barrier damage following tPA thrombolysis for ischemic stroke.
Highlights
Tissue plasminogen activator is a serine protease that catalyzes the conversion of plasminogen to plasmin to dissolve fibrin-based blood clots[1]
Our results demonstrated that the addition of Tissue plasminogen activator (tPA) to oxygen glucose deprivation (OGD)-treated endothelial cells promoted MMP2 and 9 secretion and Cav-1 shedding, which resulted in increased degradation of fibronectin and laminin β-1
The results showed that SB-3CT completely blocked fibronectin and laminin β-1 degradation in tPA and oxygen-glucose deprivation (OGD)-treated cells (Fig 1C)
Summary
Tissue plasminogen activator (tPA) is a serine protease that catalyzes the conversion of plasminogen to plasmin to dissolve fibrin-based blood clots[1]. Recombinant human tPA was approved by FDA to treat acute ischemic stroke in 1996 and remains to be the only stroke therapy almost two decades later. TPA thrombolysis is limited to less than 5% of stroke patients due to the narrow therapeutic time window (4.5 hrs after stroke onset) and the potentially devastating complication of intracerebral hemorrhage (ICH)[2,3,4]. TPA-associated ICH occurs as a consequence of severe blood brain barrier (BBB) disruption during thrombolytic reperfusion [5]. The molecular events of tPA application after ischemic stroke remain unknown
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