Venular leukocyte adhesion, impaired arteriolar vasoreactivity, and intestinal IR

描述（由申请人提供）：肠缺血/再灌注（I/R）发病机制中最早的事件之一是微血管功能障碍，其特征是毛细血管后小静脉中的白细胞/内皮细胞粘附相互作用（LECA）和上游动脉和小动脉中内皮依赖、no介导的血管舒张反应（EDO）受损。尽管白细胞不会在缺血后的小肠中沿小动脉内皮滚动、粘附或迁移，但最近的研究表明，I/R诱导的毛细血管后小静脉LECA与小动脉EDO功能障碍有因果关系。然而，l/ r诱导的毛细血管后小静脉LECA与上游小动脉缺血后EDO功能障碍的耦合机制尚不清楚。我们的总体假设是，l/ r诱导的小动脉EDO功能障碍发生的机制是由毛细血管后小静脉的LECA触发的，并涉及迁出白细胞的蛋白水解活性引起的间质信号的形成，这些信号诱导肥大细胞依赖性血管紧张素II的形成。随后，血管壁上的NAD(P)H氧化酶被激活，导致活性氧的形成，使NO失活，从而损害小动脉内的EDO。我们将通过确定：1)毛细血管后小静脉的LECA是否在上游小动脉EDO的发展中发挥强制性作用来验证这一假设；2)白细胞来源的蛋白酶（如基质金属蛋白酶-9 (MMP-9)）在小动脉EDO中的作用；3)小动脉EDO功能障碍是否与肥大细胞源性乳糜依赖性All的形成有关。活体显微方法将用于检查小动脉EDO、静脉LECA和肥大细胞对I/R的反应。一些基因敲除动物模型（p -选择素-/-、CD11/CD18-/-、ICAM-1-/-、MMP-9-/-、肥大细胞缺陷小鼠）将被用于进一步探索从缺血后肠分离的EDO小动脉受损的机制。总的来说，这些目标解决了一个新的机制来解释受I/R影响的组织中动脉血管调节功能的深刻干扰。这项工作将确定毛细血管后小静脉中的LECA，迁移的白细胞在间质中产生的信号和小动脉中的EDO功能障碍之间的新联系。鉴于内皮在调节血管张力中的重要性，这些根本性的重要发现对我们理解I/R条件下血流失调具有巨大的意义。

DESCRIPTION (provided by applicant): One of the earliest events in the pathogenesis of intestinal ischemia/reperfusion (I/R) is microvascular dysfunction that is characterized by leukocyte/endothelial cell adhesive interactions (LECA) in postcapillary venules and impaired endothelium-dependent, NO-mediated vasodilatory responses (EDO) in upstream arteries and arterioles. Even though leukocytes do not roll along, adhere to, or emigrate across arteriolar endothelium in postischemic intestine, recent work indicates that I/R- induced LECA in postcapillary venules is causally linked to EDO dysfunction in arterioles. However, the mechanisms coupling l/R-induced LECA in postcapillary venules and postischemic EDO dysfunction in upstream arterioles are unknown. Our overall hypothesis is that l/R-induced EDO dysfunction in arterioles occurs by a mechanism that is triggered by LECA in postcapillary venules and involves the formation of signals in the interstitium elicited by the proteolytic activity of emigrated leukocytes that induce mast cell-dependent formation of angiotensin II. Subsequent activation of NAD(P)H oxidase in the vascular wall leads to the formation of reactive oxygen species which inactivates NO, thereby impairing EDO in arterioles. We will test this hypothesis by determining: 1) whether LECA in postcapillary venules play an obligatory role in the development of EDO in upstream arterioles; 2) the contribution of leukocyte-derived proteases (eg, matrix metalloproteinase-9 (MMP-9)) to EDO in arterioles; and 3) whether EDO dysfunction in arterioles is due to mast cell-derived chymase-dependent formation of All. Intravital microscopic approaches will be used to examine arteriolar EDO, venular LECA, and mast cell responses to I/R. A number of gene knockout animal models (P-selectin-/-, CD11/CD18-/-, ICAM-1-/-, MMP-9-/-, mast cell-deficient mice) will be used to further explore the mechanisms of impaired EDO arterioles isolated from postischemic intestine. Collectively, these aims address a novel mechanism to explain the profound disturbance in, arteriolar vasoregulatory function in tissues subjected to I/R. This work will identify new links between LECA in postcapillary venules, signals generated in the interstitium by emigrated leukocytes, and EDO dysfunction in arterioles. Given the importance of the endothelium in regulating vascular tone, these fundamentally important findings have enormous implications for our understanding of blood flow dysregulation in conditions characterized by I/R.