SUR1-TRPM4 channel activation and phasic secretion of MMP-9 induced by tPA in brain endothelial cells

Abstract

BackgroundHemorrhagic transformation is a major complication of ischemic stroke, is linked to matrix metalloproteinase-9 (MMP-9), and is exacerbated by tissue plasminogen activator (tPA). Cerebral ischemia/reperfusion is characterized by SUR1-TRPM4 (sulfonylurea receptor 1—transient receptor potential melastatin 4) channel upregulation in microvascular endothelium. In humans and rodents with cerebral ischemia/reperfusion (I/R), the SUR1 antagonist, glibenclamide, reduces hemorrhagic transformation and plasma MMP-9, but the mechanism is unknown. We hypothesized that tPA induces protease activated receptor 1 (PAR1)-mediated, Ca2+-dependent phasic secretion of MMP-9 from activated brain endothelium, and that SUR1-TRPM4 is required for this process.MethodsCerebral I/R, of 2 and 4 hours duration, respectively, was obtained using conventional middle cerebral artery occlusion. Immunolabeling was used to quantify p65 nuclear translocation. Murine and human brain endothelial cells (BEC) were studied in vitro, without and with NF-κB activation, using immunoblot, zymography and ELISA, patch clamp electrophysiology, and calcium imaging. Genetic and pharmacological manipulations were used to identify signaling pathways.ResultsCerebral I/R caused prominent nuclear translocation of p65 in microvascular endothelium. NF-κB-activation of BEC caused de novo expression of SUR1-TRPM4 channels. In NF-κB-activated BEC: (i) tPA caused opening of SUR1-TRPM4 channels in a plasmin-, PAR1-, TRPC3- and Ca2+-dependent manner; (ii) tPA caused PAR1-dependent secretion of MMP-9; (iii) tonic secretion of MMP-9 by activated BEC was not influenced by SUR1 inhibition; (iv) phasic secretion of MMP-9 induced by tPA or the PAR1-agonist, TFLLR, required functional SUR1-TRPM4 channels, with inhibition of SUR1 decreasing tPA-induced MMP-9 secretion.ConclusionstPA induces PAR1-mediated, SUR1-TRPM4-dependent, phasic secretion of MMP-9 from activated brain endothelium.

Role of Sulfonylurea Receptor 1 and Glibenclamide in Traumatic Brain Injury: A Review of the Evidence

“…Some upstream molecular mechanisms related to SUR1-TRPM4 upregulation have been characterized in hypoxia/ischemia, SAH, and mechanical stress ( Figure 2A) [67,[97][98][99]. Beyond mechanisms of edema and oncotic cell death, SUR1-TRPM4 signaling and inhibition by GLI have also been identified as relevant in apoptotic cell death pathways involving Bcl-associated X protein (BAX) and caspase-3 ( Figure 2A) [60,67,83], neuroinflammation (Figure 2A,B) [66,67,72,73,100,101], and BBB integrity (Figure 2A,C) via matrix metalloproteinase (MMP)-9 secretion, zona-occludens-1 (ZO-1) expression and redistribution [66,67,83,102] in different models of CNS injury, including SAH, TBI, ischemia-reperfusion (I/R), inflammation, and encephalomyelitis. However, the details are complex and remain incompletely understood, particularly in TBI.…”

“…Ongoing BBB breakdown perpetuates hemorrhage progression and contusion expansion after TBI. Although SUR1-TRPM4 contributes to BBB permeability via oncotic death of endothelial cells [14,15,76], it has also been implicated in this process in connection with other mechanisms involving ZO-1 [67,83], MMP-9 and/or tissue plasminogen activator (tPA) [91,95,102] (Figure 2A). MMP-9 is a zinc-dependent endopeptidase (collagenase) zymogen, widely implicated in extracellular matrix degradation and BBB disruption in many CNS diseases.…”

“…However, its overlap with SUR1-TRPM4 requires further investigation, particularly in TBI [14,106]. While neutrophils are a major source of MMP-9 [107], it is also secreted from neurons, microglia, pericytes, and endothelial cells; indeed, it is prominently expressed in the microvascular endothelium during the first 24 h after focal ischemia [102,108]. In a model of I/R, tPA induced SUR1-TRPM4 channel opening and MMP-9 secretion from activated brain endothelial cells ( Figure 2C) [102].…”

“…While neutrophils are a major source of MMP-9 [107], it is also secreted from neurons, microglia, pericytes, and endothelial cells; indeed, it is prominently expressed in the microvascular endothelium during the first 24 h after focal ischemia [102,108]. In a model of I/R, tPA induced SUR1-TRPM4 channel opening and MMP-9 secretion from activated brain endothelial cells ( Figure 2C) [102]. Conversion of plasminogen to plasmin (a serine protease) by tPA then induced canonical activation of protease activated receptor 1 (PAR1) by cleavage at Arg41.…”

“…SUR1-TRPM4 inhibition by GLI (and shRNA against Abcc8) unexpectedly reduced phasic MMP-9 secretion from activated endothelium. Although the precise mechanism of the benefit remains unestablished, it is thought to be related to increased phosphorylated-CamKII and subsequent desensitization and internalization of PAR1 [102]. Nonetheless, it remains to be determined whether/to what extent this mechanism and GLI modulates BBB permeability after TBI.…”