Cellular Interactions in Vascular Calcification of Chronic Kidney Disease

慢性肾病血管钙化中的细胞相互作用

基本信息

批准号：
10525401
负责人：
Wei Chen
金额：
$ 12.53万
依托单位：
ALBERT EINSTEIN COLLEGE OF MEDICINE
依托单位国家：
美国
项目类别：
财政年份：
2022
资助国家：
美国
起止时间：
2022-09-01 至 2024-06-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10525401
关键词：
Address Age Amino Acids Arteries Attenuated Biological Blood Vessels Cardiovascular system Cell Communication Cell Culture Techniques Cells Chromatography Chronic Kidney Failure Coculture Techniques Complex Data Deposition Development End stage renal failure Endothelial Cells Environment Functional disorder General Population Hemodialysis High Prevalence Human Impairment Inorganic Phosphate Transporter Knowledge Literature Mediator of activation protein Metabolic Modeling Molecular Nitric Oxide Nitric Oxide Pathway Osteoblasts Osteogenesis Paracrine Communication Participant Pathogenesis Pathologic Pathway interactions Patients Phenotype Process Proteomics RNA Renal function Research Proposals Role Serum Signal Pathway Signal Transduction Small Interfering RNA Smooth Muscle Myocytes Stable Isotope Labeling System Testing Therapeutic Agents Toxin Translational Research Vascular Endothelial Cell Vascular Smooth Muscle Vascular calcification base bone health calcification calcium phosphate cell type design effective therapy healthy volunteer improved in vitro Model in vivo innovation inorganic phosphate insight mineralization mortality multidisciplinary new therapeutic target novel novel therapeutics osteogenic paracrine prevent promoter sex tool translational potential translational scientist treatment strategy uptake

项目摘要

ABSTRACT Patients with chronic kidney disease (CKD) have high cardiovascular mortality, and this could be partly due to the development of vascular calcification, which is characterized by a pathological deposition of calcium and phosphate in the arterial walls. Vascular calcification is common in patients with CKD due to the accumulation of uremic toxins and metabolic disturbances such as hyperphosphatemia. Unfortunately, there is no effective treatment to prevent or slow the progression of vascular calcification. Our overarching aim is to identify new therapeutic targets to treat vascular calcification by studying the interaction between two major vascular cell types—endothelial cells (ECs) and vascular smooth muscle cells (VSMCs). In the pathogenesis of vascular calcification, while osteogenic differentiation of VSMCs is the key process, EC-VSMC interaction also appears to be important, yet EC-VSMC interaction is not well-studied. To address this knowledge gap, we have assembled a multidisciplinary team of experts and established an insert co-culture system of primary human aortic ECs and human aortic VSMCs. Compared to mono-culture, this co-culture system allows us to model distinct aspects of the multi-cellular environment in vivo. We will test the central hypothesis that uremic serum from patients with severe CKD alters EC secretome, which in turn induces osteogenic differentiation of VSMCs and calcification. Studying uremic serum takes into account the complex metabolic alteration in CKD and has great translational potential. Our two aims are (1) to define the paracrine effects of ECs on phosphate induced calcification of VSMC culture; (2) to determine the effects of uremic serum via EC-VSMC signaling on osteogenic differentiation of VSMCs and calcification. In Aim 1, using medium containing high phosphate, VSMCs will be cultured with or without ECs, or with ECs transfected with small interfering RNA targeting phosphate transporters to block the intracellular uptake of phosphate. Then, we will elucidate the role of the nitric oxide pathway in EC-VSMC paracrine signaling and identify novel signaling pathways by examining cellular secretome using an unbiased approach of SILAC (stable isotope labeling of amino acids in cell culture)-based quantitative proteomics. In Aim 2, VSMCs will be cultured with or without ECs, and in the presence of uremic or normal serum. Uremic serum will be obtained from patients with end stage kidney disease receiving hemodialysis and compared to normal serum from age- and sex-matched healthy volunteers. Then, we will quantify cellular secretome using SILAC-based proteomics to identify specific paracrine factors, and fractionate serum using chromatography to isolate the components of uremic serum that promote calcification. Studying EC-VSMC signaling will help identify new therapies that can attenuate vascular calcification, making this proposal significant. With the use of human uremic serum, primary human cells in an insert co-culture system, and SILAC-based proteomics, this project is highly innovative and has great translational potential.

抽象的慢性肾脏病 (CKD) 患者心血管死亡率较高，部分原因可能是血管钙化的发展，其特征是钙和钙的病理性沉积由于磷酸盐的积累，血管壁钙化在 CKD 患者中很常见。不幸的是，目前还没有有效的治疗尿毒症毒素和高磷血症等代谢紊乱的方法。我们的首要目标是寻找新的治疗方法来预防或减缓血管钙化的进展。通过研究两个主要血管细胞之间的相互作用来治疗血管钙化的治疗目标血管发病机制中的两种类型——内皮细胞（EC）和血管平滑肌细胞（VSMC）。钙化过程中，VSMC成骨分化是关键过程，EC-VSMC相互作用也出现很重要，但 EC-VSMC 相互作用尚未得到充分研究。组建多学科专家团队，建立原代人类插入共培养体系主动脉 EC 和人类主动脉 VSMC 与单一培养相比，这种共培养系统使我们能够建模。我们将测试尿毒症血清的中心假设。来自严重 CKD 患者的 EC 分泌组发生改变，进而诱导 VSMC 成骨分化研究尿毒症血清考虑到 CKD 中复杂的代谢变化。我们的两个目标是（1）确定 EC 对磷酸盐诱导的旁分泌作用。 VSMC培养物的钙化；（2）确定尿毒症血清通过EC-VSMC信号传导对血管平滑肌细胞（VSMC）的影响。 VSMC 的成骨分化和钙化在目标 1 中，使用含有高磷酸盐的培养基， VSMC 将在有或没有 EC 的情况下进行培养，或者在 EC 转染有小干扰 RNA 靶向的情况下进行培养然后，我们将阐明磷酸盐转运蛋白阻断细胞内磷酸盐摄取的作用。 EC-VSMC 旁分泌信号传导中的一氧化氮途径，并通过检查确定新的信号传导途径使用 SILAC（细胞中氨基酸的稳定同位素标记）的无偏方法进行细胞分泌组分析在目标 2 中，将在有或没有 EC 的情况下培养 VSMC，并且在尿毒症或正常血清的存在将从终末期肾病患者获得。接受血液透析的疾病并与年龄和性别匹配的健康人的正常血清进行比较然后，我们将使用基于 SILAC 的蛋白质组学来量化细胞分泌组，以识别特定的细胞分泌组。旁分泌因子，并使用色谱法分离血清以分离尿毒症血清的成分研究 EC-VSMC 信号传导将有助于确定可减弱血管的新疗法。钙化，使得这一建议具有重要意义，因为使用了人尿毒症血清，原代人类细胞。插入共培养系统，以及基于SILAC的蛋白质组学，该项目具有高度的创新性和巨大的应用价值。翻译潜力。