BACKGROUND:Acute ischemic stroke triggers endothelial activation that disrupts vascular integrity and increases hemorrhagic transformation leading to worsened stroke outcomes. rt-PA (recombinant tissue-type plasminogen activator) is an effective treatment; however, its use is limited due to a restricted time window and hemorrhagic transformation risk, which in part may involve activation of MMPs (matrix metalloproteinases) mediated through LOX-1 (lectin-like oxLDL [oxidized low-density lipoprotein] receptor 1). This study’s overall aim was to evaluate the therapeutic potential of novel MMP-9 (matrix metalloproteinase 9) ± LOX-1 inhibitors in combination with rt-PA to improve stroke outcomes.METHODS:A rat thromboembolic stroke model was utilized to investigate the impact of rt-PA delivered 4 hours poststroke onset as well as selective MMP-9 (JNJ0966) ±LOX-1 (BI-0115) inhibitors given before rt-PA administration. Infarct size, perfusion, and hemorrhagic transformation were evaluated by 9.4-T magnetic resonance imaging, vascular and parenchymal MMP-9 activity via zymography, and neurological function was assessed using sensorimotor function testing. Human brain microvascular endothelial cells were exposed to hypoxia plus glucose deprivation/reperfusion (hypoxia plus glucose deprivation 3 hours/R 24 hours) and treated with ±tPA and ±MMP-9 ±LOX-1 inhibitors. Barrier function was assessed via transendothelial electrical resistance, MMP-9 activity was determined with zymography, and LOX-1 and barrier gene expression/levels were measured using qRT-PCR (quantitative reverse transcription PCR) and Western blot.RESULTS:Stroke and subsequent rt-PA treatment increased edema, hemorrhage, MMP-9 activity, LOX-1 expression, and worsened neurological outcomes. LOX-1 inhibition improved neurological function, reduced edema, and improved endothelial barrier integrity. Elevated MMP-9 activity correlated with increased edema, infarct volume, and decreased neurological function. MMP-9 inhibition reduced MMP-9 activity and LOX-1 expression. In human brain microvascular endothelial cells, LOX-1/MMP-9 inhibition differentially attenuated MMP-9 levels, inflammation, and activation following hypoxia plus glucose deprivation/R.CONCLUSIONS:Our findings indicate that LOX-1 inhibition and ± MMP-9 inhibition attenuate negative aspects of ischemic stroke with rt-PA therapy, thus resulting in improved neurological function. While no synergistic effect was observed with simultaneous LOX-1 and MMP-9 inhibition, a distinct interaction is evident.

中文翻译：

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LOX-1 和 MMP-9 抑制可减轻延迟 rt-PA 治疗的不利影响并改善急性缺血性中风后的预后

背景：急性缺血性中风触发内皮激活，破坏血管完整性并增加出血转化，导致中风结果恶化。 rt-PA（重组组织型纤溶酶原激活剂）是一种有效的治疗方法；然而，由于时间窗口有限和出血性转化风险，其使用受到限制，部分可能涉及通过 LOX-1（凝集素样 oxLDL [氧化低密度脂蛋白]受体 1）介导的 MMP（基质金属蛋白酶）的激活。本研究的总体目的是评估新型 MMP-9（基质金属蛋白酶 9）± LOX-1 抑制剂与 rt-PA 联合使用的治疗潜力，以改善卒中结果。方法：利用大鼠血栓栓塞性卒中模型来研究rt-PA 在中风发作后 4 小时给药，并在 rt-PA 给药前给药选择性 MMP-9 (JNJ0966) ±LOX-1 (BI-0115) 抑制剂。通过 9.4-T 磁共振成像评估梗死面积、灌注和出血转化，通过酶谱法评估血管和实质 MMP-9 活性，并使用感觉运动功能测试评估神经功能。将人脑微血管内皮细胞暴露于缺氧加葡萄糖剥夺/再灌注（缺氧加葡萄糖剥夺3小时/R 24小时），并用±tPA和±MMP-9±LOX-1抑制剂处理。通过跨内皮电阻评估屏障功能，通过酶谱测定 MMP-9 活性，并使用 qRT-PCR（定量逆转录 PCR）和蛋白质印迹测量 LOX-1 和屏障基因表达/水平。 结果：中风和随后的 rt -PA 治疗增加水肿、出血、MMP-9 活性、LOX-1 表达，并使神经系统结果恶化。 LOX-1 抑制可改善神经功能、减少水肿并改善内皮屏障完整性。 MMP-9 活性升高与水肿增加、梗塞体积和神经功能下降相关。 MMP-9 抑制降低了 MMP-9 活性和 LOX-1 表达。在人脑微血管内皮细胞中，LOX-1/MMP-9 抑制会不同程度地减弱缺氧加葡萄糖剥夺/R 后的 MMP-9 水平、炎症和激活。结论：我们的研究结果表明，LOX-1 抑制和 ± MMP-9 抑制通过 rt-PA 治疗减轻缺血性中风的负面影响，从而改善神经功能。虽然同时抑制 LOX-1 和 MMP-9 没有观察到协同效应，但明显存在明显的相互作用。