Role of a novel IL18 binding protein in atherosclerosis

 描述(申请人提供)：钠氯共转运蛋白(NCC)是一种125 kDa的12跨膜钠离子依赖的阳离子-氯离子共转运蛋白，主要表达于肾脏远端曲管，以吸收肾脏中过滤的氯化钠负荷。在小鼠中，NCC缺乏会导致Gitelman综合征，这是一种遗传性低血钾性碱中毒，伴有明显的低钙尿和低镁血症，继发于盐吸收缺陷。白介素18(IL18)是一种炎性细胞因子，可促进炎性细胞因子/趋化因子(如干扰素-γ、白介素6、单核细胞趋化蛋白-1)的产生。在动脉粥样硬化病变中，巨噬细胞、平滑肌细胞(SMCs)和内皮细胞(ECs)中IL18及其同源受体IL18r水平升高。血清IL18水平在冠心病患者中升高，与CAD严重程度评分相关，与心血管危险因素相关，并预测未来的CAD事件。在实验性动脉粥样硬化中，IL18的过表达或腹腔注射通过干扰素-γ依赖的途径增加动脉粥样硬化病变的形成并增强易损斑块的表型，而IL18的失活或遗传缺陷则减缓病变的进展。我们的初步研究表明，IL18与NCC结合，并介导小鼠心脏内皮细胞下游蛋白酪氨酸的磷酸化。炎性细胞因子(如IL-18、肿瘤坏死因子-α、IL-1？)刺激巨噬细胞、T细胞、主动脉内皮细胞和血管内皮细胞可诱导NCC表达。在人类和小鼠的正常主动脉中，NCC和IL18r的表达可以忽略不计，但动脉粥样硬化病变中的SMC、ECs和巨噬细胞都表达高水平的NCC和IL18r。在易患动脉粥样硬化的载脂蛋白E缺陷(APOE-/-)小鼠中，IL18r或NCC的缺失并不能显著减缓动脉粥样硬化的发展。但IL18r和NCC的联合缺陷显著降低了主动脉弓巨噬细胞含量和病变面积，降低了胸腹主动脉脂质沉积，降低了血清细胞因子干扰素-γ和IL-6水平。在培养的巨噬细胞中，NCC和IL18r的联合缺失削弱了IL18介导的细胞信号转导，并显著减少了IL18细胞与表面的结合，从而减少了炎性细胞因子和趋化因子的产生。这些观察结果导致了我们的中心假设，即NCC和IL18R是炎性细胞和心血管细胞上可替代的IL18结合分子，它们在动脉粥样硬化形成中协同介导IL18活性，并可能在其他炎症性疾病中发挥作用。我们提出了三个目标：1)。研究NCC的抑制、激活或遗传缺陷是否影响动脉粥样硬化的形成；NCC对炎性细胞(如巨噬细胞)的活动是否对动脉粥样硬化的形成是必需的；以及是否与年龄有关；为了研究NCC是否与其他激酶受体一样发挥作用，NCC在盐吸收过程中的活性与IL18信号的相互作用，IL18对NCC和IL18r具有不同的亲和力，而NCC对IL18具有特异性；目的：检测NCC是否介导T细胞、血管内皮细胞和血管内皮细胞的IL18活性。

 DESCRIPTION (provided by applicant): Na-Cl co-transporter (NCC) is a 125-kDa 12-transmembrane Na+-dependent cation-chloride co-transporter that is primarily expressed in kidney distal convoluted tubules to resorb the filtered load of NaCl in the kidney. In mice, deficiency of NCC causes Gitelman syndrome, an inherited hypokalemic alkalosis with significant hypocalciuria and hypomagnesemia, which are secondary to the deficit in NaCl resorption. Interleukin 18 (IL18) is an inflammatory cytokine that promotes inflammatory cell cytokine/chemokine (e.g., IFN-γ, IL6, MCP-1) production. In atherosclerotic lesions, increased levels of IL18 and its cognate receptor IL18r are detected in macrophages, smooth muscle cells (SMCs), and endothelial cells (ECs). Serums IL18 levels are elevated in patients with CAD, correlate with CAD severity score, are associated with cardiovascular risk factors, and predict future CAD events. In experimental atherosclerosis, overexpression or intraperitoneal administration of IL18 increases atherosclerotic lesion formation and enhances vulnerable plaque phenotypes via an IFN-γ- dependent pathway, while inactivation or genetic deficiency of IL18 slows lesion progression. Our preliminary studies show that IL18 binds to NCC and mediates downstream protein tyrosine phosphorylation in mouse heart ECs. Stimulation of macrophages, T cells, and aortic SMCs and ECs with inflammatory cytokines (e.g., IL18, TNF-α, IL1ß) induces NCC expression. In both humans and mice, normal aortas express negligible NCC and IL18r, but SMCs, ECs, and macrophages in atherosclerotic lesions express high amounts of both NCC and IL18r. In atherosclerosis-prone apolipoprotein E-deficient (Apoe-/-) mice, the absence of either IL18r or NCC does not slow atherosclerosis development significantly. But combined deficiency of IL18r and NCC significantly reduces aortic arch macrophage content and lesion areas, and thoracic-abdominal aorta lipid deposition, along with reduced serum cytokines IFN-γ and IL6. From cultured macrophages, the combined absence of NCC and IL18r impairs IL18-mediated cell signaling, and significantly reduces IL18 cell-surface binding and consequent inflammatory cytokine and chemokine production. These observations lead to our central hypothesis that NCC and IL18r are alternative IL18-binding molecules on inflammatory cells and cardiovascular cells, and that they mediate IL18 activities cooperatively in atherogenesis and possibly in other inflammatory diseases. We propose three aims: 1). to examine whether inhibition, activation, or genetic deficiency of NCC affects atherogenesis; whether NCC activities on inflammatory cells (e.g. macrophages) are essential to atherogenesis; and age-dependent; 2). To examine whether NCC acts like other kinase receptor, NCC activities in salt uptake and IL18 signaling interplay with each other, IL18 has different affinity for NCC and IL18r, and NCC is specific for IL18; and 3). To examine whether NCC mediates IL18 activities in T cells and vascular SMCs and ECs.