Alcohol-induced Impairment of Endothelial Cell Recovery

项目总结： 保持血脑屏障(BBB)的完整性对健康的中枢神经系统至关重要。许多 病理情况，包括艾滋病毒感染、脑损伤和酒精使用障碍(AUD)，已知 损害了BBB的完整性。狂饮烈性酒是一种流行的饮酒方式 在青少年和青壮年中开展活动。我们的初步研究表明，酗酒者 酒精浓度高的饮料会造成严重的血脑屏障损害。因此，过量饮酒 对于那些已经容易受到血脑屏障功能障碍影响的人来说，消费带来了更大的风险。 脑镜血管内皮细胞(BMVECs)是血脑屏障的关键细胞成分。BMVEC包含 紧密连接(TJ)复合体，由occludin、claudins和zonula occludens(ZO)组成，限制 细胞旁物质通过血脑屏障，并确保结构和功能的完整性 BBB。BMVECs的死亡(细胞凋亡)是血脑屏障损伤的生物标志物，可由酒精滥用引起。 然而，酒精影响血脑屏障完整性恢复的机制尚不完全清楚，但 可能包括酒精延迟修复BMVEC衬里。而研究表明，骨髓内皮细胞的凋亡 可能会导致血脑屏障损坏，但对BMVEC衬里的修复和 恢复血脑屏障完整性。这主要是由于缺乏合适的实验动物模型。 其中BBB的完整性已经受到损害。 中间链球菌分泌的中间溶血素(ILY)可与该种细菌特异结合。 特异性人细胞膜蛋白CD59(HCD59)。我们之前证明了将ILY注射到一个 表达hCD59的转基因小鼠内皮细胞快速损伤的实验研究 呈剂量依赖方式。我们最近建立了Cre诱导的FloxedSTOP-hCD59转基因小鼠系 (IhCD59)，并将可诱导的hCD59与Tek-cre杂交，产生复合小鼠(ihCD59+/-/tek-cre+/-(+/-， 转基因的半合子)，其中Cre的表达驱动hCD59在 内皮细胞。因此，我们可以使用ILY给药来建立内皮细胞特异性消融模型。 内皮祖细胞可以分化为内皮细胞并修复受损的内皮细胞。 牢房衬里。根据文献和我们初步研究的数据，我们假设(A)酒精- 对已经受损的血脑屏障内皮细胞的损伤是乙醇浓度- 依赖；和(B)受损内皮细胞衬里的修复和恢复因暴饮暴食而延迟 饮酒。为了验证这一假设，我们建议利用我们的新型ILY介导的细胞消融 技术来生成已经受到威胁的BBB模型。然后，我们将检查乙醇浓度- 对该模型中受损的血脑屏障的依赖效应，包括血脑屏障的修复和恢复 内皮细胞衬里。这项建议的两个具体目标是： 目的1：探讨血脑屏障内皮细胞损伤的机制 在暴饮暴食不同浓度的乙醇后。 目的2：探讨血脑屏障内皮细胞修复的机制 在暴饮暴食不同浓度的乙醇后。 这项具有高度意义和临床相关性的研究结果将提供有关 酒精滥用进一步损害已经受损的血脑屏障的机制，以及 修复和恢复血脑屏障的完整性。

Project Summary: Maintaining blood-brain barrier (BBB) integrity is essential for a healthy central nervous system. Many pathological conditions, including HIV infection, brain trauma, and alcohol use disorders (AUD), are known to compromise the integrity of the BBB. Binge drinking of hard liquor [>40% alcohol by volume (ABV)] is a popular activity among adolescents and young adults. Our preliminary studies indicate that binge drinking of alcoholic beverages with a high ethanol (EtOH) concentration can cause severe BBB damage. Thus, excessive alcohol consumption poses an increased risk for those individuals who are already susceptible to BBB dysfunction. Brain mircrovascular endothelial cells (BMVECs) are key cellular components of the BBB. BMVECs contain tight junction (TJ) complexes, consisting of occludin, claudins, and zonula occludens (ZO), that restrict paracellular passage of substances across the BBB and ensure the structural and functional integrity of the BBB. The death (apoptosis) of BMVECs is a biomarker of BBB damage, and can be caused by alcohol abuse. However, the mechanisms by which alcohol affects the recovery of BBB integrity are not fully understood, but may include alcohol-delayed repair of the BMVEC lining. While studies have shown that apoptosis of BMVECs can cause BBB damage, there has been little, if any, investigation into the repair of the BMVEC lining and the restoration of BBB integrity. This is due, mainly, to the lack of an appropriate experimental animal model in which BBB integrity is already compromised. Intermedilysin (ILY), a toxin secreted by Streptococcus intermedius, binds exclusively to the species- specific human cell membrane protein, CD59 (hCD59). We previously demonstrated that injection of ILY into a transgenic mouse strain that expresses hCD59 on endothelial cells induces rapid endothelial cell damage in a dose-dependent manner. We recently established a Cre-inducible floxedSTOP-hCD59 transgenic mouse line (ihCD59) and crossed the inducible hCD59 with Tek-cre to generate compound mice (ihCD59+/-/Tek-cre+/- (+/-, hemizygous for the transgene) in which Cre expression drives the expression of hCD59 specifically in endothelial cells. Thus, we can generate an endothelial cell-specific ablation model using ILY administration. Endothelial progenitor cells (EPCs) can differentiate into endothelial cells and repair an injured endothelial cell lining. Based on the literature and the data from our preliminary studies, we hypothesize that (a) alcohol- induced damage to the endothelial lining of an already compromised BBB is EtOH concentration- dependent; and (b) repair and restoration of the damaged endothelial cell lining is delayed by binge alcohol consumption. To test this hypothesis, we propose to utilize our novel ILY-mediated cell ablation technique to generate an already compromised BBB model. Then, we will examine EtOH concentration- dependent effects on the damaged BBB in that model, including the repair and restoration of the BBB endothelial cell lining. The two Specific Aims of this proposal are: Aim 1: To determine the mechanisms underlying damage to the endothelial cell lining of the BBB following binge exposure to various concentrations of EtOH. Aim 2: To determine the mechanisms underlying the repair of the endothelial cell lining of the BBB following binge exposure to various concentrations of EtOH. The findings from this highly significant and clinically relevant study will provide valuable information on the mechanisms by which alcohol abuse further damages an already compromised BBB, and the timeframe of repair and restoration of BBB integrity.