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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.

摘要慢性肾脏病（CKD）患者的心血管死亡率较高，这可能部分是由于血管钙化的发展，其特征在于钙的病理性沉积，动脉壁中的磷酸盐血管钙化在CKD患者中很常见，尿毒症毒素和代谢紊乱如高磷血症。不幸的是，没有有效的治疗以防止或减缓血管钙化的进展。我们的首要目标是确定新的通过研究两种主要血管细胞之间的相互作用，类型-内皮细胞（EC）和血管平滑肌细胞（VSMCs）。在血管性心脏病的发病机制中钙化，而成骨分化是VSMC的关键过程，EC-VSMC之间也存在相互作用重要的是，但EC-VSMC相互作用没有得到很好的研究。为了弥补这一知识差距，我们组建了多学科专家团队，建立了原代人胚共培养体系，主动脉EC和人主动脉VSMC。与单一培养相比，这种共培养系统使我们能够模拟体内多细胞环境的不同方面。我们将检验尿毒症血清从严重CKD患者中提取的细胞因子改变EC分泌组，这反过来诱导VSMCs的成骨分化和钙化。研究尿毒症血清需要考虑CKD中复杂的代谢改变，巨大的翻译潜力。我们的两个目的是：（1）确定内皮细胞对磷酸盐诱导的细胞凋亡的旁分泌作用; （2）确定尿毒症血清通过EC-VSMC信号传导对VSMC钙化的影响， 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将与或不与EC一起培养，或与用小干扰RNA靶向转染的EC一起培养。磷酸盐转运蛋白，以阻止磷酸盐的细胞内摄取。然后，我们将阐明一氧化氮通路在EC-VSMC旁分泌信号传导中的作用，并通过检测使用SILAC（细胞中氨基酸的稳定同位素标记）的无偏方法的细胞分泌组培养）为基础的定量蛋白质组学。在目标2中，VSMC将与或不与EC一起培养，并且在目标2中，VSMC将与EC一起培养。存在尿毒症或正常血清。尿毒症血清将从终末期肾病患者中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志愿者然后，我们将使用基于SILAC的蛋白质组学定量细胞分泌组，以确定特定的旁分泌因子，以及使用色谱法分离尿毒症血清的组分，促进钙化。研究EC-VSMC信号传导将有助于确定新的治疗方法，钙化，使这个建议意义重大。通过使用人尿毒症血清，在体外培养的原代人细胞可以在体外培养。插入共培养系统和基于SILAC蛋白质组学，该项目具有高度创新性，平移势