Mac-1 coordinates PDGF-CC activation by microglia and promotes BBB opening

描述(由申请人提供)：大约700万美国人患有中风，导致严重的发病率和死亡率。大多数中风是由 血管阻塞(缺血性中风)。组织型纤溶酶原激活剂(TPA)是FDA批准的唯一一种治疗缺血性卒中的溶栓药物。然而，由于脑出血风险增加，其使用受到严重限制。众所周知，TPA通过促进血脑屏障(BBB)的开放来增加脑血管通透性，其机制依赖于脑中PDGF-CC活性形式的产生，进而激活血管周围星形胶质细胞上的PDGF受体(PDGFR)(Su，et al，Nat Med，2008)。越来越多的证据表明，tPA介导了潜伏的PDGF-CC的激活。然而，tPA在没有辅因子的情况下显示出较差的蛋白分解活性，并且不支持溶液中潜伏的PDGF-CC的有效激活。在缺血性卒中背景下，体内激活PDGF-CC的机制尚不清楚。我们发现，小胶质细胞通过依赖tPA、整合素Mac-1和低密度脂蛋白受体相关蛋白-1(LRP1)的机制促进潜伏的PDGF-CC的有效激活。事实上，我们发现tPA在体内诱导的BBB开放不仅依赖于LRP1，还依赖于Mac-1，而由PDGF-CC的裂解/激活形式诱导的BBB开放不需要Mac-1或LRP1。根据这些初步结果以及我们发表的Mac-1与tPA和LRP1形成复合体的观察(Cao等人，EMBO J，2006)，我们假设小胶质细胞对潜伏的PDGF-CC的激活依赖于tPA/Mac-1/LRP1多蛋白复合体的组装。为了验证这一假设，我们将进行结构功能分析，以确定Mac-1与tPA和LRP1相互作用的分子基础。在这些结构信息的指导下，我们将设计不与tPA或LRP1相互作用的特定Mac-1突变体，以及具有完全纤溶活性但缺乏Mac-1结合的tPA突变体。我们还将设计特定的拮抗剂 干扰tPA/Mac-1/LRP1复合体的形成。这些Mac-1和tPA突变体中断小胶质细胞介导的PDGF-CC激活的能力将使用基于细胞的PDGF-CC激活系统来确定。此外，将在实验性缺血性中风的小鼠模型中评估不同拮抗剂阻断PDGF-CC激活从而减少血脑屏障开放的可能性。该项目的完成将产生新的试剂，可以保护血脑屏障的完整性，而不会影响tPA的纤溶活性。这些试剂还有望在不干扰PDGFRs正常功能的情况下特异性地阻断活性PDGF-CC的产生。因此，这些抑制剂的副作用应该比PAN-PDGFR抑制剂，如格列卫更少，格列卫目前正在I-卒中临床试验中作为人类卒中患者tPA的辅助治疗进行评估。因此，这个项目所产生的信息可以帮助我们设计更好的策略来提高tPA的治疗效果，同时保持血脑屏障的完整性，从而对缺血性中风的临床治疗产生重大影响。

DESCRIPTION (provided by applicant): Approximately 7,000,000 Americans suffer from stroke, resulting in significant morbidity and mortality. The majority of strokes are caused by the blockade of blood vessels (ischemic stroke). Currently, tissue plasminogen activator (tPA) is the only thrombolytic drug approved by FDA for the treatment of ischemic stroke. However, its use is severely limited due to the increased risk of cerebral bleeding. tPA is known to increase cerebrovascular permeability by promoting the opening of the blood brain barrier (BBB) in a mechanism that depends on the generation of active forms of PDGF-CC in the brain, which in turn activate the PDGF receptor-¿ (PDGFR¿) on perivascular astrocytes (Su, et al, Nat Med, 2008). Emerging evidence suggests that tPA mediates the activation of latent PDGF-CC. However, tPA exhibits poor proteolytic activity without a cofactor and does not support efficient activation of latent PDGF-CC in solution. The mechanism that activates PDGF- CC in vivo under the setting of ischemic stroke is unknown. We discovered that microglia facilitate efficient activation of latent PDGF-CC in a mechanism that depends on tPA, integrin Mac-1 and the LDL receptor- related protein-1 (LRP1). Indeed, we found that the tPA-induced BBB opening in vivo depends not only on LRP1 but also on Mac-1, whereas the BBB opening induced by the cleaved/activated forms of PDGF-CC does not require either Mac-1 or LRP1. Based on these preliminary results and our published observation that Mac- 1 forms a complex with tPA and LRP1 (Cao, et al, EMBO J, 2006), we hypothesize that activation of latent PDGF-CC by microglia is dependent on the assembly of the tPA/Mac-1/LRP1 multi-protein complex. To test this hypothesis, we will perform structure-function analysis to define the molecular basis underlying Mac-1 interaction with tPA and LRP1. Guided by this structural information, we will design specific Mac-1 mutants that will not interact with tPA or LRP1, and tPA variants that possess full fibrinolytic activity but lack Mac-1 binding. We will also design specific antagonists to disrupt the formation of the tPA/Mac-1/LRP1 complex. The ability of these Mac-1 and tPA mutants to interrupt microglia-mediated PDGF-CC activation will be determined using a cell-based PDGF-CC activation system. In addition, the potential of the different antagonists to block PDGF- CC activation and thereby reduce BBB opening will be evaluated in a mouse model of experimental ischemic stroke. Completion of this project will generate new reagents that can protect the integrity of the BBB without compromising fibrinolytic activity of tPA. These reagents are also expected to specifically block the generation of active PDGF-CC without interfering with the normal functions of PDGFRs. As a result, these inhibitors should have fewer side effects than pan-PDGFR inhibitors, such as Gleevec, which is currently under evaluation in the I-STROKE clinical trial as an adjuvant therapy of tPA in human stroke patients. Thus, the information generated from this project could help us design better strategies to enhance the therapeutic efficacy of tPA while preserving the integrity of the BBB and therefore significantly impact clinical management of ischemic stroke.