Molecular Pathogenesis of Chronic Kidney Disease-Dependent Vascular Calcification

描述(申请人提供)：本研究方案的长期目标是确定血管钙化的分子机制，以寻找治疗慢性肾脏疾病依赖性血管钙化的新靶点(S)。血管钙化与慢性肾脏病患者心血管疾病的发病率和死亡率密切相关。事实上，在慢性肾脏病患者中，超过一半的死亡可以归因于心血管疾病。我们假设CKD依赖的血管钙化发病机制中的一个中心事件是磷酸化激活转录因子4(ATF4)的表达增加。ATF4是碱性拉链转录因子中cAMP反应元件结合蛋白(CREB)家族的成员，它调节成骨，也介导内质网(ER)中的未折叠蛋白反应(UPR)。我们的假说基于我们实验室的一系列初步结果：1)通过激活血管平滑肌细胞(VSMCs)中UPR的perk-elF2a轴，无机磷、炎性细胞因子(TNFa)和饱和脂肪酸等一系列积极的血管钙化调节因子诱导了ATF4蛋白的总水平和磷酸化水平；2)腺病毒介导的ATF4过表达诱导了VSMCs的矿化；3)ATF4基因被敲除，另一方面，血管钙化程度减弱；4)Forsklin激活PKA，诱导ATF4(p-ATF4)丝氨酸磷酸化，从而促进血管钙化；5)PKA和ERK抑制剂抑制ATF4的磷酸化，导致血管钙化减少；6)总ATF4和p-ATF4蛋白在动脉粥样硬化性钙化小鼠，如5/6肾切除(5/6 NX)的ApoE基因敲除小鼠，以及中层钙化小鼠，如DBA2/J小鼠，5/6 NX和Klotho基因敲除小鼠；7)ATF4靶标(CHOP和Gadd34)在这些模型中增加，8)ATF4调节VSMC中主要的磷酸盐转运体Pit-1的表达。为了确定ATF4在血管钙化发病机制中的关键作用，我们提出了三个特定的目标。具体目标1：确定全球ATF4缺乏和过度表达是否调节CKD依赖的中膜和动脉粥样硬化性钙化。具体目标2：确定VSMC特异性激活和抑制ATF4是否影响CKD依赖的中膜和动脉粥样硬化性钙化。具体目的3：阐明ATF4调控血管平滑肌细胞成骨分化和矿化的分子机制。

DESCRIPTION (provided by applicant): The long term objective of this research proposal is to determine the molecular mechanisms of vascular calcification in order to identify novel target(s) for the treatment of chronic kidney disease (CKD)-dependent vascular calcification. Vascular calcification is closely associated with cardiovascular morbidity and mortality in patients with CKD. In fact, more than half of all deaths in CKD subjects can be attributed to cardiovascular diseases. We hypothesized that a central event in the pathogenesis of CKD-dependent vascular calcification is increased expression of phosphorylated activating transcription factor 4 (ATF4). ATF4 is a member of the cAMP-responsive element-binding protein (CREB) family of basic zipper-containing transcription factors that regulates osteogenesis and also mediates unfolded protein response (UPR) in the endoplasmic reticulum (ER). Our hypothesis is based on the following evidence derived from a series of preliminary results from our lab: 1) total and phosphorylated ATF4 protein levels were induced by a number of positive regulatory factors for vascular calcification, such as inorganic phosphate, inflammatory cytokines (TNFa) and saturated fatty acids through the activation of the PERK-elF2a axis of the UPR in vascular smooth muscle cells (VSMCs); 2) adenovirus-mediated overexpression of ATF4 induced mineralization of VSMCs; 3) ATF4 knockdown, on the other hand, attenuated vascular calcification; 4) serine-phosphorylation of ATF4 (p-ATF4) was induced by PKA activation by forskolin, which is known to promote vascular calcification; 5) PKA and ERK inhibitors inhibited the phosphorylation of ATF4, resulting in the reduction of vascular calcification; 6) Total ATF4 and p-ATF4 proteins were increased in the aortas of murine models of atherosclerotic calcification such as ApoE knockout mice with 5/6 nephrectomy (5/6 nx), and medial calcification such as DBA2/J mice with 5/6 nx and klotho knockout mice; 7) ATF4 targets (CHOP and GADD34) increased in these models and 8) ATF4 regulates the expression of Pit-1, a major phosphate transporter in VSMCs. To determine the pivotal role of ATF4 in the pathogenesis of vascular calcification, we propose three specific aims. Specific Aim 1: Determine whether global ATF4 deficiency and overexpression modulate CKD-dependent medial and atherosclerotic calcification. Specific Aim 2: Determine whether VSMC-specific activation and inhibition of ATF4 influence CKD-dependent medial and atherosclerotic calcification. Specific Aim 3: Elucidate molecular mechanisms by which ATF4 regulates osteoblastic differentiation and mineralization of VSMCs.