重组ADAMTS13促进脑缺血后tPA溶栓和血流再通的作用和机制

Thrombolytic therapy with tissue plasminogen activator (tPA) for acute ischemic stroke improves neurologic outcome, but the degree of improvement is still unsatisfactory. Thus, there remains a clear need to identify new therapeutic strategies to enhance the efficacy of tPA thrombolysis and improve cerebral blood flow restoration. Pericytes have recently come into focus as critical regulators of the blood-brain barrier integrity and microcirculatory blood flow. Recently, we have demonstrated that recombinant ADAMTS13 (rADAMTS13) reduced tPA-related cerebral hemorrhage after ischemic stroke. Our preliminary data show that rADAMTS13 significantly promoted tPA-induced thrombolysis and reduced ischemic brain injury in a mouse model of stroke. In this project, we will further assess the mechanism of rADAMTS13 and tPA combination therapy after ischemic stroke. We will employ mouse stroke model, cerebral pericytes and endothelial cells cultures, using VWF and ADAMTS13 knockout mice, VWF siRNA, ADAMTS13 siRNA, rADAMTS13, multi-photon confocal microscopy, high-field MRI, and molecular biological approaches to determine whether the inhibition of direct binding of tPA and NMDA receptor NR2B and consequent NR2B activation by treatment with rADAMTS13 is responsible for further changes in cell death signaling and cell survival pathway, and whether these effects contribute to the survival of pericytes and endothelial cells and function of microvessel after ischemic stroke. We will also determine the effects of rADAMTS13 and tPA combination therapy on tPA-induced cerebral blood flow restoration as well as on extending the therapeutic time window of tPA after ischemic stroke. These proposed studies may provide novel insights into improving thrombolytic therapy for the clinical treatment of acute ischemic stroke.

如何提高缺血性脑卒中后tPA溶栓和血流再通的效率，一直是困扰医学界的难题。我们前期研究证明，重组ADAMTS13可降低脑缺血后tPA溶栓引起的脑出血。我们近期预实验发现，重组ADAMTS13可促进tPA的溶栓效果并减轻脑缺血损伤，然而其作用途径和机制仍有待研究。血管周细胞-内皮系统对调控脑血管功能和脑血流发挥关键作用。基于前期研究结果，本项目拟采用脑缺血模型，结合体外脑血管周细胞和内皮细胞低氧再氧化模型，借助基因敲除、基因沉默、重组蛋白、多光子显微镜活体脑血管成像、核磁共振等多种手段，研究重组ADAMTS13促进tPA溶栓的有效途径，阐明重组ADAMTS13通过干预VWF途径，调控tPA-NR2B偶联介导的细胞生存和死亡信号传导，减轻tPA对周细胞-内皮系统的损害，保护脑微血管，从而增强tPA的溶栓效果并改善脑血流。这些研究将为延长tPA的治疗时间窗，扩大其临床应用提供新的思路和理论依据。

如何提高缺血性脑卒中后tPA的溶栓效率，一直是困扰医学界的难题。本项目的主要研究内容是揭示重组ADAMTS13是否通过VWF途径，保护血脑屏障并促进脑缺血后tPA溶栓的作用和机制。取得如下重要结果：1）重组ADAMTS13与tPA联合应用可促进血栓溶解。我们发现重组ADAMTS13单独或与tPA联合应用能够明显减少缺血脑半球中的纤维蛋白含量和血管内血栓形成，提示其本身具有溶栓作用，从而在延迟使用tPA不能溶解血栓时，发挥溶栓作用而促进血栓溶解。2）重组ADAMTS13通过VWF途径，减轻tPA引起的脑微血管和血脑屏障损伤。发现重组ADAMTS13能够逆转tPA引起的基底膜和紧密连接蛋白降解，减轻血脑屏障破坏。tPA导致血浆中VWF含量明显增加，而给予提纯的VWF可逆转重组ADAMTS13降低tPA破坏血脑屏障的作用。3）联合应用重组ADAMTS13与tPA保护缺血性脑损伤并改善小鼠的行为后果。重组ADAMTS13可减轻脑缺血后脑梗塞以及tPA引起的梗塞增加，增加血栓溶解和改善小鼠受损的神经功能，并延长tPA的治疗时间窗。4）重组ADAMTS13减轻脑缺血后tPA介导的细胞凋亡、坏死和自噬。重组ADAMTS13可减轻tPA引起的细胞凋亡和神经元变性坏死，抑制tPA引起的LC3-I向LC3-II转换，减少tPA导致的LC3阳性细胞增加。5）重组ADAMTS13通过调控tPA-NR2B偶联介导的NR2B和ERK1/2激活，减轻tPA引起的缺血性脑损伤。通过注射NMDA及其受体拮抗剂MK801，证明重组ADAMTS13通过NMDA受体减轻tPA引起的损伤。重组ADMTS13可降低tPA引起的ERK1/2磷酸化和NR2B表达升高，抑制NR2B与tPA之间的相互作用。tPA可恶化ADAMTS13基因敲除引起的梗塞增加。与重组ADAMTS13类似，NR2B拮抗剂ifenprodil也可减小tPA引起的梗塞增加。重组ADAMTS13可浓度依赖性降低OGD后tPA引起的内皮细胞损伤，减少内皮细胞分泌炎症因子，而VWF可阻断重组ADAMTS13的这些作用。重组ADAMTS13能够增加脑缺血后血管周细胞覆盖、减轻内皮细胞损伤、增加内皮细胞增值及血管长度和灌注。项目实施过程中培养多名研究生，获得国家重点研发计划课题一项，发表标注项目资助高质量论文。