PML- and Smyd1-dependent regulatory mechanisms and effects on inflammatory responses of vascular endothelial cells

内皮细胞（ECs）呈单层排列在淋巴管和血管的内表面。正常内皮功能的丧失会导致潜在的危及生命的疾病。它们可能表现为具有特定解剖定位的慢性炎症性疾病，如动脉硬化，但也可能出现急性，如败血症或缺血-再灌注损伤，与败血症相比，败血症通常局限于单个器官。在这些完全不同的情况下，内皮的炎症改变对受影响血管内皮的功能完整性起关键作用。该项目的初步实验揭示了Smyd1 （SET And MYND Domain Containing 1）和PML（早幼粒细胞白血病蛋白）的蛋白-蛋白相互作用，以及ECs中Symd1的PML依赖性summoylation。基因转染诱导的ECs中Smyd1过表达显示PML和NFB表达水平升高。值得注意的是，Smyd1对NFB表达的影响是由Smyd1的组蛋白甲基转移酶活性引起的。Smyd1和PML的过表达导致主要组织相容性复合体II类反激活因子CIITA的表达增加。内皮细胞MHC II类表达增加与自身免疫性疾病和内皮功能障碍相关。脂多糖可增强ECs中PML和Smyd1的表达。缺氧也有类似的效果。相反，动脉粥样硬化血流谱降低了PML和Smyd1的表达。这些发现为PML和Smyd1相互作用影响血管内皮细胞的炎症反应提供了实验证据。在目前的提案中，我们希望更详细地探讨PML和Smyd1在血管内皮细胞炎症反应、血管和器官灌注中的作用。将采用特殊的刺激方案来模拟炎症反应的特征，以引发动脉硬化，败血症和缺血再灌注损伤。这些刺激方案的系统比较应该加深对所研究的内皮功能障碍的多样性和相似性的理解。潜在的细胞机制，包括通过smyd1依赖性组蛋白修饰对假定的靶基因的转录调节，PML核小体介导的蛋白质的sumo化，以及两种蛋白质的相互作用，将被鉴定和详细表征。对内皮细胞功能的影响将通过实验量化。最后，将通过分析获得的发现，smyd1抑制剂的开发，包括与心血管数据库的比较，为转化应用做准备。

Endothelial cells (ECs) line as a single layer the interior surface of lymphatic and blood vessels. Loss of proper endothelial functions lead to potentially life-threatening conditions. They may emerge as a chronic inflammatory disease with specific anatomical localization, as in the case of arteriosclerosis, but can also arise acute, as in the case of a sepsis or of an ischemic-reperfusion injury, which in contrast to sepsis is typically restricted to single organs. In these, per se very different situations, inflammatory alterations of the endothelium play a pivotal role for the functional integrity of the endothelium of affected blood vessels. Preliminary experiments of the proposed project revealed a protein-protein-interaction of Smyd1 (SET And MYND Domain Containing 1) and PML (Promyelocytic Leukemia Protein) and a PML-dependent SUMOylation of Symd1 in ECs. Overexpression of Smyd1 in ECs induced by gene transfection showed increased expression levels of PML and NFB. Remarkably, the effect of Smyd1 on NFB expression was caused by the histone methyltransferase activity of Smyd1. Overexpression of both, Smyd1 and PML, resulted in an increased expression of the major histocompatibility complex class II transactivator CIITA. An increased endothelial MHC class II expression is associated with autoimmune diseasesand endothelial dysfunction. The expression of PML and Smyd1 in ECs was augmented by treatment with liposaccharide. Hypoxia showed a similar effect. In contrast, atherogenic flow profiles decreased PML and Smyd1 expression. These findings provided experimental evidence that PML and Smyd1 interact with each other to influence inflammatory responses of vascular endothelial cells.In the current proposal, we want to explore in more detail the role of PML and Smyd1 in inflammatory reactions of vascular endothelial cells, blood vessels and organ perfusion. Special stimulation protocols will be applied to mimic inflammatory reactions characteristic for the initiation of arteriosclerosis, sepsis and ischemic-reperfusion injury. The systematic comparison of these stimulation protocols ought to deepen the understanding for varieties and similarities in the investigated endothelial dysfunctions. The underlying cellular mechanisms, which include transcriptional regulation of putative target genes via Smyd1-dependent histone modifications, PML nuclear bodies-mediated SUMOylation of proteins, as well as interaction of both proteins, will be identified and characterized in detail. Implications for endothelial cell function will be experimentally quantified. Finally, translational applications will be prepared by analysis of the obtained findings, the development of Smyd1-inhibitors including the comparison with cardiovascular databases.