Verapamil as an Adjunct Therapy to Reduce tPA Toxicity in Hyperglycemic Stroke: Implication of TXNIP/NLRP3 Inflammasome

Abstract

Thrombolytic therapy has remained quite challenging in hyperglycemic patients for its association with poor prognosis and increased hemorrhagic conversions. We recently showed that tissue plasminogen activator (tPA)-induced cerebrovascular damage is associated with thioredoxin-interacting protein (TXNIP) upregulation, which has an established role in the detrimental effects of hyperglycemia. In the present work, we investigated whether verapamil, an established TXNIP inhibitor, may provide protection against hyperglycemic stroke and tPA-induced blood-brain barrier (BBB) disruption. Acute hyperglycemia was induced by intraperitoneal administration of 20% glucose, 15 min prior to transient middle cerebral artery occlusion (tMCAO). Verapamil (0.15 mg/kg) or saline was intravenously infused with tPA at hyperglycemic reperfusion, 1 h post tMCAO. After 24 h of ischemia/reperfusion (I/R), mice were assessed for neurobehavioral deficits followed by sacrifice and evaluation of brain infarct volume, edema, and microbleeding. Alterations in TXNIP, inflammatory mediators, and BBB markers were further analyzed using immunoblotting or immunostaining techniques. As adjunctive therapy, verapamil significantly reduced tPA-induced BBB leakage, matrix metalloproteinase 9 (MMP-9) upregulation, and tight junction protein deregulation, which resulted in lesser hemorrhagic conversions. Importantly, verapamil strongly reversed tPA-induced TXNIP/NLRP3 (NOD-like receptor pyrin domain-containing-3) inflammasome activation and reduced infarct volume. This concurred with a remarkable decrease in high-mobility group box protein 1 (HMGB-1) and nuclear factor kappa B (NF-κB) stimulation, leading to less priming of NLRP3 inflammasome. This preclinical study supports verapamil as a safe adjuvant that may complement thrombolytic therapy by inhibiting TXNIP's detrimental role in hyperglycemic stroke.

Keywords: Acute hyperglycemia; Inflammasome; Stroke; Thioredoxin-interacting protein; Tissue plasminogen activator; Verapamil.

Figure 1.. Intravenous verapamil ameliorates ipsilateral edema and poor neurological outcomes in hyperglycemic stroke mice.

Adult C57Bl/6 mice underwent transient middle cerebral artery occlusion (tMCAO) 15 minutes after a single intraperitoneal injection of 20% glucose solution. Blood glucose check confirmed the hyperglycemic (HG) state both at occlusion and reperfusion during which some HG tMCAO animals received intravenous tPA (10 mg/kg) with or without verapamil (Ver) (0.15 mg/kg) (A). Intravenous tPA worsened neurological function in HG tMCAO animals (p=0.064). This was mitigated in verapamil treated animals (B). Infarct volume in HG tMCAO were not affected with tPA, but significantly decreased with verapamil (C). Verapamil also reduced tPA induced ipsilateral edema, an index of cerebrovascular damage (D). All values are presented as mean ± SEM, (n=6/group). * p<0.05, ** p<0.01.

Figure 2.. TXNIP inhibition by verapamil was sufficient to reverse tPA-induced GLUT-1 downregulation.

Following induction of acute hyperglycemia, adult C57Bl/6 mice were subjected to tMCAO and received intravenous (IV) tPA (10 mg/kg) with and without IV verapamil (0.15 mg/kg) at the time of reperfusion. Hyperglycemic tMCAO mice treated with tPA showed a marginal increase in brain TXNIP expression compared to untreated HG tMCAO mice. Verapamil induced a remarkable fall in TXNIP (A) but not TRX levels (B). Importantly, TXNIP alterations with tPA and/or verapamil concurred with reciprocal changes in GLUT-1 expression, confirming a strong effect of verapamil in repression of TXNIP and consequent GLUT-1 degradation (C). All values are presented as mean ± SEM, (n=6/group). * p<0.05, ** p<0.01. Ver, verapamil; tPA, tissue plasminogen activator; HG, hyperglycemic; tMCAO, transient middle cerebral artery occlusion; TXNIP, thioredoxin interacting protein; TRX, thioredoxin; GLUT-1, glucose transporter-1.

Figure 3.. tPA-induced TXNIP/NLRP3 inflammasome upregulation is blocked by verapamil co-administration.

Although tPA therapy had no effect on NLRP3 component expression, in hyperglycemic stroke (A), it did induce NLRP3 inflammasome priming, as indicated by ASC (B), and pro-caspase-1 upregulation (C), while also triggering inflammasome assembly, as indicated by its products, cleaved-caspase 1 (D) and IL-1β (E). Combination therapy with IV verapamil prevented NLRP3 inflammasome priming and activation. This could be explained by the remarkable TXNIP inhibition, illustrated in figure 2. Immunostaining of the tMCAO penumbral region further confirmed discernible TXNIP/IL-1β downregulation in HG animals given IV verapamil with tPA at reperfusion (F). All values are presented as mean ± SEM, (n=6/group). * p<0.05. Ver, verapamil; tPA, tissue plasminogen activator; HG, hyperglycemic; tMCAO, transient middle cerebral artery occlusion; NLRP3, NOD-like receptor pyrin domain-containing-3; ASC, apoptosis-associated speck-like protein; IL-1β, interleukin 1-β.

Figure 3.. tPA-induced TXNIP/NLRP3 inflammasome upregulation is blocked by verapamil co-administration.

Although tPA therapy had no effect on NLRP3 component expression, in hyperglycemic stroke (A), it did induce NLRP3 inflammasome priming, as indicated by ASC (B), and pro-caspase-1 upregulation (C), while also triggering inflammasome assembly, as indicated by its products, cleaved-caspase 1 (D) and IL-1β (E). Combination therapy with IV verapamil prevented NLRP3 inflammasome priming and activation. This could be explained by the remarkable TXNIP inhibition, illustrated in figure 2. Immunostaining of the tMCAO penumbral region further confirmed discernible TXNIP/IL-1β downregulation in HG animals given IV verapamil with tPA at reperfusion (F). All values are presented as mean ± SEM, (n=6/group). * p<0.05. Ver, verapamil; tPA, tissue plasminogen activator; HG, hyperglycemic; tMCAO, transient middle cerebral artery occlusion; NLRP3, NOD-like receptor pyrin domain-containing-3; ASC, apoptosis-associated speck-like protein; IL-1β, interleukin 1-β.

Figure 4.. Intravenous verapamil reduced tPA associated increases in HMGB-1 and NF-κB activation in hyperglycemic stroke.

Intravenous tPA (10 mg/kg) significantly upregulated HMGB-1 protein expression, in HG tMCAO animals (A) leading to consequent NF-κB activation, as determined by p-p65 NF-kB / p65 NF-kB ratio (B) and further confirmed by substantial TNF-α release (C). HMGB-1/NF-κB/TNF-α activation by tPA was strongly prevented with IV verapamil (0.15 mg/kg) combination therapy. All values are presented as mean ± SEM, (n=6/group). * p<0.05, ** p<0.01. Ver, verapamil; tPA, tissue plasminogen activator; HG, hyperglycemic; tMCAO, transient middle cerebral artery occlusion; HMGB-1, High Mobility Group Box Protein 1; NF-κB, nuclear factor kappa B; TNF-α, Tumor necrosis factor-α.

Figure 5.. BBB breakdown, following tPA therapy in hyperglycemic stroke, is attenuated by verapamil.

Verapamil combination with Intravenous tPA (10 mg/kg) in HG tMCAO animals profoundly reduced ipsilateral microbleeds (A) consistent with overall Hb content in brain parenchyma (B). Extravasation of endogenous IgG heavy and light chain also showed a discernible reduction in BBB permeability (C). In line with these alterations, verapamil combination therapy moderately repressed tPA-induced MMP-9 upregulation (D) and prevented accumulation of non-functional claudin 5 (E), while slightly preventing tPA/MMP-9 induced ZO-1 degradation (F). All values are presented as mean ± SEM, (n=6/group) * p<0.05, ** p<0.01. Ver, verapamil; tPA, tissue plasminogen activator; HG, hyperglycemic; tMCAO, transient middle cerebral artery occlusion; MMP, matrix metalloprotease; ZO-1, zonula occludens-1.

Figure 5.. BBB breakdown, following tPA therapy in hyperglycemic stroke, is attenuated by verapamil.

Verapamil combination with Intravenous tPA (10 mg/kg) in HG tMCAO animals profoundly reduced ipsilateral microbleeds (A) consistent with overall Hb content in brain parenchyma (B). Extravasation of endogenous IgG heavy and light chain also showed a discernible reduction in BBB permeability (C). In line with these alterations, verapamil combination therapy moderately repressed tPA-induced MMP-9 upregulation (D) and prevented accumulation of non-functional claudin 5 (E), while slightly preventing tPA/MMP-9 induced ZO-1 degradation (F). All values are presented as mean ± SEM, (n=6/group) * p<0.05, ** p<0.01. Ver, verapamil; tPA, tissue plasminogen activator; HG, hyperglycemic; tMCAO, transient middle cerebral artery occlusion; MMP, matrix metalloprotease; ZO-1, zonula occludens-1.

Figure 6.. Schematic diagram depicting verapamil therapeutic effects against tPA-induced toxicity in hyperglycemic stroke.

Verapamil’s protective role against hyperglycemic stroke, which underlines the TXNIP/NLRP3 inflammasome as a potential mediator of tPA-induced BBB breakdown. Abbreviations: GLUT-1, glucose transporter 1; HG: hyperglycemic; HMGB-1, High Mobility Group Box Protein 1; IL-1β, interleukin 1-β; I/R, ischemic reperfusion; MMP, matrix metalloprotease; NF-κB, nuclear factor kappa B; NLRP3, NOD-like receptor pyrin domain-containing-3; TJ proteins, tight junction proteins; MCAO, transient middle cerebral artery occlusion; TNF-α, Tumor necrosis factor-α; tPA, tissue plasminogen activator; TXNIP, thioredoxin interacting protein.