Venular leukocyte adhesion, impaired arteriolar vasoreactivity, and intestinal IR

描述（由申请方提供）：肠缺血/再灌注（I/R）发病机制中的最早期事件之一是微血管功能障碍，其特征为毛细血管后微静脉中的白细胞/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性NO介导的血管舒张反应（EDO）受损。尽管白细胞不会在缺血后肠内沿小动脉内皮沿着、粘附或迁移，但最近的研究表明，I/R诱导的毛细血管后微静脉中的LECA与小动脉中的EDO功能障碍有因果关系。然而，耦合I/R诱导的LECA在毛细血管后微静脉和缺血后EDO功能障碍在上游小动脉的机制是未知的。我们的总体假设是，I/R诱导的小动脉中的EDO功能障碍通过由毛细血管后小静脉中的LECA触发的机制发生，并且涉及由迁移的白细胞的蛋白水解活性引起的血管中信号的形成，其诱导肥大细胞依赖性的血管紧张素II的形成。随后在血管壁中NAD（P）H氧化酶的激活导致活性氧物质的形成，其使NO失活，从而损害小动脉中的EDO。我们将通过确定以下因素来检验这一假设：1）毛细血管后微静脉中的LECA是否在上游小动脉中EDO的发生中起着强制性作用; 2）白细胞衍生的蛋白酶（如基质金属蛋白酶-9（MMP-9））对小动脉中EDO的贡献; 3）小动脉中EDO功能障碍是否是由于肥大细胞衍生的食糜酶依赖性ALL形成所致。将使用活体显微镜方法检查小动脉EDO、小静脉LECA和肥大细胞对I/R的反应。本研究将采用多种基因敲除动物模型（P-选择素-/-、CD 11/CD 18-/-、ICAM-1-/-、MMP-9-/-、肥大细胞缺陷小鼠）进一步探讨缺血后肠组织中EDO小动脉受损的机制。总的来说，这些目标提出了一种新的机制来解释在组织中受到I/R的小动脉血管调节功能的深刻干扰。这项工作将确定LECA在毛细血管后微静脉，信号产生的白细胞迁移，和EDO功能障碍的小动脉在血小板之间的新的联系。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.