Molecular Pathogenesis of Chronic Kidney Disease-Dependent Vascular Calcification

描述（由申请方提供）：本研究计划的长期目标是确定血管钙化的分子机制，以确定治疗慢性肾脏病（CKD）依赖性血管钙化的新靶点。血管钙化与CKD患者的心血管发病率和死亡率密切相关。事实上，CKD受试者中超过一半的死亡可归因于心血管疾病。我们假设CKD依赖性血管钙化发病机制的中心事件是磷酸化转录激活因子4（ATF 4）表达增加。ATF 4是cAMP反应元件结合蛋白（CREB）家族的一员，该家族的基本含拉链的转录因子调节骨生成，还介导内质网（ER）中的未折叠蛋白反应（UPR）。我们的假设是基于以下来自我们实验室的一系列初步结果的证据：1）在血管平滑肌细胞（VSMCs）中，总的和磷酸化的ATF 4蛋白水平由许多血管钙化的正性调节因子诱导，例如无机磷酸盐、炎性细胞因子（TNFa）和饱和脂肪酸，通过激活UPR的PERK-eIF 2a轴; 2）腺病毒介导的ATF 4过表达可诱导VSMCs矿化; 3）ATF 4敲低可减轻血管钙化; 4）毛喉素激活PKA可诱导ATF 4的丝氨酸磷酸化（p-ATF 4），促进血管钙化; 5）PKA和ERK抑制剂可抑制ATF 4的磷酸化，从而减少血管钙化; 6）在动脉粥样硬化钙化的小鼠模型如ApoE敲除小鼠5/6肾切除（5/6 nx）和中膜钙化的DBA 2/J小鼠5/6 nx和klotho敲除小鼠中，总ATF 4和p-ATF 4蛋白增加; 7）在这些模型中，ATF 4靶点（CHOP和GADD 34）增加，8）ATF 4调节VSMC中主要磷酸盐转运蛋白Pit-1的表达。为了确定ATF 4在血管钙化发病机制中的关键作用，我们提出了三个具体目标。具体目标1：确定是否全球ATF 4的缺陷和过度表达调节CKD依赖性中膜和动脉粥样硬化钙化。具体目标2：确定VSMC特异性激活和ATF 4抑制是否影响CKD依赖性中膜和动脉粥样硬化钙化。具体目标3：阐明ATF 4调控VSMCs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.