Hypoxia-Induced Changes in the Fibroblast Secretome, Exosome, and Whole-Cell Proteome Using Cultured, Cardiac-Derived Cells Isolated from Neonatal Mice,Journal of Proteome Research

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Hypoxia-Induced Changes in the Fibroblast Secretome, Exosome, and Whole-Cell Proteome Using Cultured, Cardiac-Derived Cells Isolated from Neonatal Mice
Journal of Proteome Research ( IF 3.8 ) Pub Date : 2017-07-06 00:00:00 , DOI: 10.1021/acs.jproteome.7b00144
Jake Cosme ₁ , Hongbo Guo ₂ , Sina Hadipour-Lakmehsari ₁ , Andrew Emili ₂ , Anthony O. Gramolini ₁

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Cardiac fibroblasts (CFs) represent a major subpopulation of cells in the developing and adult heart. Cardiomyocyte (CM) and CF intercellular communication occurs through paracrine interactions and modulate myocyte development and stress response. Detailed proteomic analysis of the CF secretome in normal and stressed conditions may offer insights into the role of CF in heart development and disease. Primary neonatal mouse CFs were isolated and cultured for 24 h in 21% (normoxic) or 2% (hypoxic) O₂. Conditioned medium was separated to obtain exosomes (EXO) and EXO-depleted secretome fractions. Multidimensional protein identification technology was performed on secreted fractions. Whole cell lysate data were also generated to provide subcellular context to the secretome. Proteomic analysis identified 6163 unique proteins in total. Statistical (QSpec) analysis identified 494 proteins differentially expressed between fractions and oxygen conditions. Gene Ontology enrichment analysis revealed hypoxic conditions selectively increase expression of proteins with extracellular matrix and signaling annotations. Finally, we subjected CM pretreated with CF secreted factors to hypoxia/reoxygenation. Viability assays suggested altered viability due to CF-derived factors. CF secretome proteomics revealed differential expression based on mode of secretion and oxygen levels in vitro.

中文翻译：

缺氧诱导的成纤维细胞分泌蛋白，外来体和全细胞蛋白质组的变化，使用的是从新生小鼠中分离出来的培养的，来源于心脏的细胞

心脏成纤维细胞（CF）代表发育中和成年心脏中的主要细胞亚群。心肌细胞（CM）和CF细胞间通讯通过旁分泌相互作用发生，并调节心肌细胞的发育和应激反应。在正常和压力条件下对CF分泌蛋白组进行详细的蛋白质组学分析，可能有助于了解CF在心脏发育和疾病中的作用。分离出新生儿原代小鼠CF，并在21％（常氧）或2％（低氧）O _2中培养24小时。分离条件培养基以获得外泌体（EXO）和耗尽EXO的分泌组份。对分泌级分进行了多维蛋白质鉴定技术。还产生了全细胞裂解物数据，以向分泌组提供亚细胞环境。蛋白质组学分析共鉴定出6163种独特蛋白质。统计（QSpec）分析确定了494种蛋白质在馏分和氧气条件之间差异表达。基因本体论富集分析显示，低氧条件选择性增加具有细胞外基质和信号注释的蛋白质的表达。最后，我们对用CF分泌因子预处理的CM进行了缺氧/复氧。生存力测定表明，由于CF衍生的因素，生存力发生了变化。

更新日期：2017-07-12

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