Vascular adhesion protein-1 (VAP-1) as a therapeutic target in stroke

描述（由申请人提供）：血管粘附蛋白-1（VAP-1），也称为氨基脲敏感性胺氧化酶（SSAO），据报道在多种白细胞亚群（即，嗜中性粒细胞、单核细胞、淋巴细胞）。我们实验室最近报道，在短暂性前脑缺血（TFI）后的再灌注期间，药物阻断VAP-1/SSAO可防止中性粒细胞 在动物中，中性粒细胞渗出增加（开始于~ 6小时再灌注）与缺血后6小时的治疗窗相关。这些结果强烈表明TFI后中性粒细胞浸润和神经病理学相关。然而，已知该研究中使用的大鼠（糖尿病、雌激素治疗的卵巢切除雌性）表现出夸大的缺血后中性粒细胞浸润反应，因此，这些发现可能不一定表明VAP-1/SSAO抑制在广泛的啮齿动物缺血/再灌注模型中的有益作用。在本项目中，我们将检查VAP-1/SSAO的高选择性抑制剂LJP-1586的作用。在再灌注0- 24小时时开始对成年雄性大鼠进行处理，然后进行1小时MCAo（腔内缝合）和1-14天恢复。该提案由两个特定目的指导，旨在测试以下假设：（1）VAP-1/SSAO阻断将提供长期神经保护（即，减少梗死体积;改善的神经行为功能），其具有延长的治疗窗，这可能部分与所选粘附分子的表达/活化减少有关。(2)神经保护作用主要涉及对非中性粒细胞亚群的干扰。实验将涉及中性粒细胞（抗PMNL抗体）和单核细胞（脂质体包封的氯膦酸盐）的选择性耗竭，以及使用免疫组织化学和FACS分析分析MCAo后中性粒细胞、造血单核细胞和T淋巴细胞的脑内表达。初步证据支持这两种假设，表明中性粒细胞的作用有限，但有利于非中性粒细胞亚群的显著贡献（即，单核白细胞）在伴随暂时性MCAo的神经病理学中。已发表的证据表明，单核白细胞在中性粒细胞出现后数小时（甚至数天）才显示脑内粘附和迁移增加，这可能是我们在这些早期实验中观察到的长治疗窗（6- 12小时）的原因（基于使用2种不同但选择性的VAP- 1/SSAO阻断剂的结果）。对影响多个白细胞亚群但具有有限脱靶作用的经验证药理学方法的表征具有实质性的翻译意义，因为它可能被证明在广泛的临床卒中和缺血性表现中有效。 公共卫生相关性：缺血后脑炎症在卒中后发生的脑神经病理学中起主要作用。在炎症攻击中一个潜在的关键参与者是白细胞。这些血液来源的细胞可能在中风后进入大脑并释放神经毒性物质。迄今为止，临床试验中抗白细胞策略的失败可能部分与多种白细胞亚群的存在以及负责其进入大脑的途径中的冗余有关。本项目利用一种选择性药理学方法，靶向一种新的蛋白质，该蛋白质在所有白细胞亚群的运输中似乎很重要，因此可能证明具有广泛的临床疗效。

DESCRIPTION (provided by applicant): Vascular adhesion protein-1 (VAP-1), also called semicarbazide-sensitive amine oxidase (SSAO), is reported to play an important role in the adhesion and endothelial transmigration of multiple leukocyte subsets (i.e., neutrophils, monocytes, lymphocytes) in peripheral vascular beds. Our laboratory recently reported that pharmacologic blockade of VAP-1/SSAO, during the reperfusion period following transient forebrain ischemia (TFI), prevented neutrophil infiltration into the brain, and was associated with a 6h post-ischemic therapeutic window in animals where heightened neutrophil diapedesis (which begins at ~6h reperfusion) had been documented. These results strongly suggested that post-TFI neutrophil infiltration and neuropathology were linked. However, the rats used in that study (diabetic, estrogen-treated ovariectomized females) are known to exhibit an exaggerated post-ischemic neutrophil infiltration response and, therefore, such findings may not necessarily be taken to suggest a beneficial role for VAP-1/SSAO inhibition over a wide spectrum of rodent models of ischemia/reperfusion. In the present project, we will examine the effect of a highly- selective inhibitor of VAP-1/SSAO, LJP-1586. Treatments will be initiated at 0-24h reperfusion in adult male rats subjected to 1h MCAo (intraluminal suture) and 1-14 days recovery. The proposal is guided by two Specific Aims designed to test the following hypotheses: (1) VAP-1/SSAO blockade will provide long-term neuroprotection (i.e., reduced infarct volumes; improved neurobehavioral function) with extended therapeutic windows that may, in part, be linked to diminished expression/activation of selected adhesion molecules. (2) The neuroprotection will largely involve interference with non- neutrophil leukocyte subsets. Experiments will involve selective depletion of neutrophils (anti-PMNL antibody) and monocytes (liposome-encapsulated clodronate), coupled with analysis of post-MCAo intracerebral expression of neutrophils, hematogenous monocytes, and T-lymphocytes using immunohistochemistry and FACS analyses. Preliminary evidence supports both hypotheses, suggesting a restricted role for neutrophils, but favoring a significant contribution from non-neutrophil subsets (i.e., mononuclear leukocytes) in the neuropathology accompanying temporary MCAo. Published evidence that mononuclear leukocytes do not display increased intracerebral adhesion and transmigration until many hours (even days) following the appearance of neutrophils may account for the long therapeutic windows (6-12h) we have observed in these early experiments (based upon results using 2 different, but selective, VAP- 1/SSAO blockers). The characterization of a validated pharmacologic approach that affects multiple leukocyte subsets, but with limited off-target actions, has substantial translational implications, since it may prove to be efficacious over a wide range of clinical stroke and ischemic presentations. PUBLIC HEALTH RELEVANCE: Post-ischemic brain inflammation can play a major role in the cerebral neuropathology that occurs following stroke. One potentially key player in that inflammatory attack is the leukocyte. These blood-derived cells may enter the brain after a stroke and release neurotoxic agents. The failure of anti-leukocyte strategies in clinical trials to date may, in part, relate to the presence of multiple leukocyte subsets and redundancy in the pathways responsible for their passage into the brain. The present project utilizes a selective pharmacologic approach that targets a novel protein which appears to be important in the trafficking of all leukocyte subsets and, therefore, may prove to have wide-ranging clinical efficacy.