A novel microfluidic platform to study exosome biology in PAH.

一种用于研究多环芳烃外泌体生物学的新型微流体平台。

• 批准号: 10378161
• 负责人: VINICIO A DE JESUS PEREZ
• 金额: $ 19.68万
• 依托单位: STANFORD UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-03-25 至 2023-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10378161
• 关键词: Acoustics; Address; Animal Model; Antibodies; Bioinformatics; Biological; Biological Markers; Biology; Blood Vessels; CD81 gene; Cardiovascular system; Cell Culture Techniques; Cells; Characteristics; Chronic; Culture Media; Data Set; Disease; Endothelial Cells; Endothelium; Etiology; Experimental Models; Exposure to; Genetic; Goals; Inflammation; Inflammatory; Injury; Lab-On-A-Chips; Life; Lung; Machine Learning; Mediating; Methods; Microfluidics; Molecular; Molecular Genetics; Nucleic Acids; Outcome; Pathologic; Pathologic Processes; Pattern; Phase; Phenotype; Physiological; Physiological Processes; Population; Process; Production; Proteomics; Reporting; Reproducibility; Resolution; Role; Seeds; Signal Transduction; Sorting - Cell Movement; Stress; Surface; Techniques; Technology; Testing; Therapeutic Agents; Time; Vascular remodeling; Vision; angiogenesis; base; cytokine; design; endothelial dysfunction; exosome; extracellular vesicles; hemodynamics; lung microvascular endothelial cells; lung pressure; machine learning algorithm; magnetic beads; monolayer; multiple omics; new technology; novel; novel therapeutics; protein metabolite; pulmonary arterial hypertension; response; right ventricular failure; shear stress; stressor; technological innovation; tool; transcriptome sequencing

The endothelium is the cellular monolayer that covers the inner lining of the entire circulatory system. Endothelial dysfunction is a feature of pulmonary arterial hypertension (PAH), a life-threatening disease associated with abnormally high pulmonary pressures and chronic right heart failure. Due to the limitations of available static cell culture and animal models, our understanding of the mechanisms that orchestrate the initiation and perseverance of endothelial dysfunction in PAH remains incomplete. Given that endothelial dysfunction is a common finding in PAH, an understanding of the mechanism behind maladaptive endothelial responses could help accelerate the discovery of novel therapies for PAH. Presently, it is believed that endothelial derived exosomes contribute to PAH by carrying signals that trigger maladaptive endothelial responses in the setting of injury. Exosomes are cell-derived small (~30-150 nm) extracellular vesicles that carry proteins, metabolites and nucleic acids involved in a variety of physiological and pathological processes. While it is known that exosomes carry molecular and genetic factors associated with angiogenesis, inflammation and vasoreactivity, a comprehensive assessment of exosome cargo of healthy and dysfunctional PMVECs has been hindered by current low-yield exosome isolation techniques. These techniques cannot perform real-time dynamic exosome isolation from pulmonary microvascular endothelial cells (PMVECs) exposed to PAH-associated stressors. To address this unmet need, we have designed the MFES (Multifunctional Exosome Sorter) that can dissect the whole exosome population into subpopulations based on size and surface markers. MFES is the first lab-on-a-chip platform that integrates: 1) a vessel-on-a-chip module for real-time characterization of PMVEC functional responses across a wide range of physiological and pathological parameters, 2) a module for high-yield exosome size-based isolation, 3) a surface marker based exosome sorting using magnetic beads, and 4) multi-omics phenotyping of exosomes of PMVECs. Here, we are proposing a technology that can enable broadly to investigate the two main defining characteristics of exosomal subtypes, i.e., size and surface markers, both separately independently, and in combination sequentially. We will characterize changes in exosome cargo in healthy and PAH PMVECs exposed to shear stress-related conditions in the MFES. We will isolate subpopulations of exosomes based on size and surface markers and characterize them for their cargo (Aim 1). Then, we will determine whether exosomes derived from stressed PMVECs can induce pathological changes in healthy PMVECs cultured in a microfluidic culture chip (Aim 2). This technological innovation enables to study endothelial exosome biology in a setting that represents the flow dynamics associated with PAH. Further, the use of cutting-edge -omics technologies, bioinformatic analysis integrated with machine learning algorithms to analyze the purified exosomes is expected to yield a comprehensive dataset of exosome cargo profiles and open exciting opportunities for investigating the biological role of exosomes in PAH pathobiology and the testing of novel therapeutic agents.

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