Use of IL-1alpha to Promote Angiogenesis and Functional Recovery After Experiment

描述（由申请人提供）：治疗中风诱发的脑缺血（CI）失败的部分原因是缺乏对中风后神经炎症的疗效。然而，虽然急性炎症可能导致更差的患者结果，但慢性/延迟性炎症可以促进脑修复，并且可能是有吸引力的治疗靶点。特别是，我们已经证明了炎症细胞因子白细胞介素-1（IL-1）在CI中的重要作用。虽然IL-1 <$（CI后释放的IL-1的主要亚型）在CI中的有害作用已经确定，但IL-1 <$也至关重要，与IL-1 <$相反，可能发挥有益作用。我们最近发现IL-1可导致神经血管单位的成熟细胞产生层粘连蛋白球状结构域3（LG3），即细胞外基质成分串珠素的神经保护和促血管生成蛋白片段。重要的是，LG3在中风后持续（数周）在大脑中产生，但这是否是由IL-1引起的，甚至部分是由IL-1引起的，或者是任何病理生理或治疗益处，都是完全未知的。我们的初步数据现在表明，IL-1激活脑内皮，在体外是促血管生成的，在中风的大脑中保持长期升高，在那里它可能持续影响恢复，当缺乏时（IL-1敲除小鼠（KO））导致或有助于减少中风后血管生成。因此，我们假设IL-1是脑卒中诱导的脑梗死后通过生成LG3促进血管生成（脑修复机制）的关键增强剂。为了探讨这一假说，我们提出了以下具体目标：1）确定IL-1在正常条件下和CI后调节脑内皮血管生成中的作用，2）确定串珠素LG 3在正常条件下和CI后IL-1调节血管生成中的作用。我们将在野生型和IL-1基因敲除小鼠中使用一些体外血管生成试验和短暂大脑中动脉闭塞模型来确定IL-1基因在体外和体内的血管生成作用。还将进行功能/行为分析，以将血管生成的程度和梗死体积与功能恢复相关联。将使用表达10%正常总串珠素水平（pln-/-）的串珠素转基因小鼠来确定LG3对体外和体内（中风后）IL-1血管生成作用的重要性。最后，我们将采用各种遗传和生物化学方法来确定IL-1如何增加体外脑内皮细胞中LG3的产生，重点是关键的细胞蛋白酶，并确定这种破坏是否会影响IL-1血管调节活性。这项提案的成功完成将证明炎性细胞因子，特别是IL-1在中风后脑恢复中的新的有益作用。

DESCRIPTION (provided by applicant): Treatments for stroke-induced cerebral ischemia (CI) have failed in part due to lack of efficacy against post- stroke neuroinflammation. However, while acute inflammation may contribute to worse patient outcomes, w e h y p o t h e s i z e t h a t chronic/delayed inflammation could promote brain repair, and may b e an attractive therapeutic target. In particular, we have demonstrated a significant role for the inflammatory cytokine interleukin-1 (IL-1) in CI. Although the deleterious role of IL-1¿ (the main isoform of IL-1 released following CI) in CI is established, IL-1¿ is also critically important and, in contrast toIL-1¿, may play a beneficial role. We have recently shown that IL-1¿ causes c u l t u r e d cells of the neurovascular unit to generate laminin globular domain 3 (LG3), the neuroprotective and pro-angiogenic protein fragment of the extracellular matrix component perlecan. Importantly, LG3 is persistently (weeks) generated in the brain after stroke, but whether this is caused, even in part, by IL-1¿, or is of any pathophysiologic or therapeutic benefit, is completely unknown. Our preliminary data now suggest that IL-1¿ activates brain endothelium, is pro-angiogenic in vitro, remains chronically elevated in stroked brains where it could persistently impact recovery, and when absent (IL-1¿ knockout mice (KO)) results in or contributes to diminished post-stroke angiogenesis. Therefore, we hypothesize that IL-1¿ is a key enhancer of angiogenesis (a brain repair mechanism) after stroke-induced CI via generation of LG3. To explore this hypothesis, we propose the following specific aims: 1) Determine the role of IL-1¿ in modulating angiogenesis in brain endothelium under normal conditions and after CI, 2) Determine the role of perlecan LG3 in IL-1¿ regulation of angiogenesis under normal conditions and after CI. We will use a number of in vitro angiogenesis assays and a transient middle cerebral artery occlusion model in wild-type and IL-1¿ KO mice to determine the angiogenic effects of IL-1¿ in vitro and in vivo. Functional/behavioral analysis will also be performed to correlate the extent of angiogenesis and infarct volume with functional recovery. The importance of LG3 to IL-1¿ angiogenesis effects in vitro and in vivo (after stroke) will be determined using a perlecan transgenic mouse that expresses 10% of normal total perlecan levels (pln -/-). Finally, we will employ various genetic and biochemical methods to determine how IL-1¿ increases LG3 generation in brain endothelial cells in vitro with a focus on key cellular proteases, and determine whether disruption of this impacts IL-1¿ angiomodulatory activity. Successful completion of this proposal will demonstrate a novel beneficial effect of inflammatory cytokines, specifically IL-1¿, in brain recovery after stroke.