Estradiol-Induced Sialyation Of NCAM Prevents Leucocyte:Endothelial Adhesion

描述(由申请人提供)：本提案涉及广泛的挑战领域(04)临床研究和特定挑战主题，04-HL-103：评估白细胞与血小板、红细胞和内皮细胞相互作用在心肺和血液疾病发病机制中的作用。钙粘蛋白神经细胞黏附分子(NCAM)在白细胞与内皮细胞黏附中起关键作用。在趋化因子如MCP1的作用下，白细胞被吸引到内皮细胞。来自白细胞和内皮细胞的NCAM分子之间的同位相互作用(拉链)在它们之间形成了一条纽带，并允许白细胞的细胞表面抗原与内皮细胞的表面抗原相互作用。紧随其后的是细胞与细胞之间的相互作用，如汇合作用。由此产生的内皮下炎症过程可导致动脉粥样硬化的形成。然而，这一序列可以通过唾液酸修饰的称为多唾液酸化NCAM(PSA-NCAM)的异构体插入而被阻断。它们在胞外区域具有多水合2，8唾液酸链，干扰拉链与NCAM的粘连。我们记录了在所有被研究的器官(10个器官系统)的动脉、静脉和淋巴管中的人血管内皮细胞的糖基化反应中存在PSA-NCAM。我们还发现，两种必需的聚唾液酸转移酶(ST8Sia II/STX和ST8Sia IV/PST)的表达是由雌激素受体介导的。因此，用PSA-NCAM酶替代NCAM会耗尽母体NCAM分子，从而使黏附(NCAM)：非黏附(PSA-NCAM)平衡得到高度利用。对于血管内皮细胞，其结果可能是最大限度地减少白细胞黏附和内皮下炎症，从而减少动脉粥样硬化和动脉粥样硬化的形成。在以往的研究中，我们已经证明雌二醇诱导大鼠脑组织中PST和STX，并在人脐静脉内皮细胞(HUVEC)中以NCAM为代价增加PSA-NCAM。雌激素受体阻滞剂FUVESTRANT则有相反的作用。在这个方案中，我们将使用心血管相关细胞[人白细胞(THP1)和人动脉内皮细胞(HCAEC)]来研究雌激素调节的白细胞黏附：我们将确定雌激素和福维斯特诱导的NCAM：PSA-NCAM比率，它们对唾液酸酶的影响，以及它们对白细胞-内皮黏附的影响。我们将把结果与RAS通路诱导剂丙戊酸的效果进行比较，丙戊酸是已知的诱导PSA-NCAM的药物。此外，还将研究抑制STX和PST的siRNA对NCAM：PSA-NCAM平衡和白细胞与内皮细胞黏附的影响。通过这种方式，我们将测试雌激素的心脏保护作用是否可能是由于NCAM唾液酸化增加，导致白细胞黏附减少和内皮下炎症。影响：心脏病是导致死亡的唯一最大原因，并导致惊人的医疗支出和个人痛苦。为了国民的健康，找到预防动脉粥样硬化的新方法是当务之急。由于其在动脉粥样硬化形成中的重要性，了解白细胞-内皮细胞相互作用的调节具有最重要的临床意义。这项建议将测试雌激素在防止白细胞粘连方面的作用，这是导致动脉粥样硬化形成的先兆步骤。这样的作用将与实验和临床研究中雌激素的心脏保护作用相一致。如果这项提议成功揭示了雌激素影响白细胞与内皮细胞黏附的机制，就有可能改变现有的治疗方法以避免动脉粥样硬化，并开发专门针对临床心脏保护中调节NCAM唾液酸化的新的预防性治疗方法。动脉硬化性心脏病是美国的头号杀手，它始于血管炎症，最终使个人面临心脏病发作的风险。这项提案将探索雌激素是否通过产生特定的、雌激素调节的酶来抑制炎症细胞进入血管来预防动脉粥样硬化。这一建议可能会促进抗动脉粥样硬化药物的应用，并导致开发新的药物来保护血管免受动脉粥样硬化及其并发症的影响。 公共卫生相关性：动脉硬化性心脏病是美国的头号杀手，它始于血管炎症，最终使个人面临心脏病发作的风险。这项提案将探索雌激素是否通过产生特定的、雌激素调节的酶来抑制炎症细胞进入血管来预防动脉粥样硬化。这一建议可能会促进抗动脉粥样硬化药物的应用，并导致开发新的药物来保护血管免受动脉粥样硬化及其并发症的影响。

DESCRIPTION (provided by applicant): This proposal addresses broad Challenge Area (04) Clinical Research and specific Challenge topic, 04- HL-103: Assess the role of leukocyte interaction with platelets, erythrocytes, and endothelium in the pathogenesis of heart, lung, and blood diseases. The cadherin Neural Cell Adhesion Molecule (NCAM) is key in leukocyte adhesion to endothelial cells. Upon signaling by chemokines such as MCP1 leukocytes are drawn toward endothelial cells. Appositional interactions ("zippering") between NCAM molecules from the leukocyte and endothelial cell forms a tether between them and allows the leukocyte's cell surface antigens to interact with those of the endothelial cell. This is followed by cell:cell interactions such as diapedesis. The resulting sub-endothelial inflammatory processes can result in atheroma formation and atherosclerosis. However, this sequence may be blocked by the insertion of the NCAM by sialic acid decorated isoforms termed polysialated NCAM (PSA-NCAM). These have poly hydrated ¿2,8 sialic acid chains in the extracellular domain that interfere with the zipper adhesion to NCAM. We have documented the presence of PSA-NCAM in the glycocalyx of human vascular endothelial cells in arteries, veins and lymphatics of all organs (10 organ systems) studied. We also have shown that expression of the two required polysialyltransferases (ST8Sia II/STX and ST8Sia IV/PST) is estrogen receptor-mediated. Thus, the enzymatic replacement of NCAM with PSA-NCAM depletes the parent NCAM molecules, so that the adhesion (NCAM):non- adhesion (PSA-NCAM) balance is highly leveraged. In the case of the vascular endothelium, the result may be minimization of leukocyte adhesion and sub endothelial inflammation, which would diminish the formation of atheromas and atherosclerosis. In previous studies we have shown that estradiol induces PST and STX in rat brain tissue and increases PSA- NCAM at the expense of NCAM in human umbilical vein endothelial cells (HUVEC). The estrogen receptor blocker fulvestrant had the opposite effect. In this proposal, using cardiovascular-relevant cells [human leukocytes (THP1) and human arterial endothelial cells (HCAEC)] we will examine estrogen-regulated leukocyte adhesion: We will determine the NCAM:PSA-NCAM ratio induced by estradiol and fulvestrant, their effects on sialylation enzymes and their effect on leukocyte-endothelial adhesion. We will compare the results with effects of the Ras pathway inducer valproic acid which is known to induce PSA-NCAM. Further, the effects of siRNA inhibition of STX and PST on the NCAM:PSA-NCAM balance and on leukocyte adhesion to endothelial cells will also be investigated. In this manner, we will test whether estrogen's cardioprotective effects could be due to increased NCAM sialylation, leading to decreased leukocyte adhesion and sub-endothelial inflammation. Impact: Heart disease is the single greatest cause of death and results in staggering healthcare expenditures and personal suffering. Finding new ways to protect against atherosclerosis is imperative for the health of the nation. Because of its importance to atherogenesis, understanding of the regulation of leukocyte-endothelial cell interactions is of the greatest clinical importance. This proposal will test the role of estrogen in preventing leukocyte adhesion that is a premonitory step toward atheroma formation. Such action would be consistent with the well- documented cardio-protective effects of estrogen in experimental and clinical studies. If this proposal is successful in exposing the mechanism by which estrogen affects leukocyte adhesion to endothelial cells it may be possible to alter already available treatments to avoid atherosclerosis and develop novel preventative treatments specifically aimed at regulating NCAM sialylation in clinical cardioprotection. Arteriosclerotic heart disease, the number one killer in the USA, starts from blood vessel inflammation that ultimately puts the individual at risk of a heart attack. This proposal will explore whether estrogen protects against atherosclerosis through production of specific, estrogen-regulated enzymes that inhibit inflammatory cells from entering into blood vessels. This proposal may improve the application of anti-atherosclerosis agents and lead to development of new agents to protect blood vessels from atherosclerosis and its complications. PUBLIC HEALTH RELEVANCE: Arteriosclerotic heart disease, the number one killer in the USA, starts from blood vessel inflammation that ultimately puts the individual at risk of a heart attack. This proposal will explore whether estrogen protects against atherosclerosis through production of specific, estrogen-regulated enzymes that inhibit inflammatory cells from entering into blood vessels. This proposal may improve the application of anti-atherosclerosis agents and lead to development of new agents to protect blood vessels from atherosclerosis and its complications.