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Gene expression in immortalized versus primary isolated cardiac endothelial cells.

ABSTRACT: Endothelial cells take pivotal roles in the heart and the vascular system and their differentiation, subspecification and function is determined by gene expression. A stable, in vitro cardiac endothelial cell line could provide high cell numbers as needed for many epigenetic analyses and facilitate the understanding of molecular mechanisms involved in endothelial cell biology. To test their suitability for transcriptomic or epigenetic studies, we compared the transcriptome of cultured immortalized mouse cardiac endothelial cells (MCEC) to primary cardiac endothelial cells (pEC). Whole transcriptome comparison of MCEC and pEC showed a correlation of 0.75-0.77. Interestingly, correlation of gene expression declined in endothelial cell-typical genes. In MCEC, we found a broad downregulation of genes that are highly expressed in pEC, including well-described markers of endothelial cell differentiation. Accordingly, systematic analysis revealed a downregulation of genes associated with typical endothelial cell functions in MCEC, while genes related to mitotic cell cycle were upregulated when compared to pEC. In conclusion, the findings from this study suggest that primary cardiac endothelial cells should preferably be used for genome-wide transcriptome or epigenome studies. The suitability of in vitro cell lines for experiments investigating single genes or signaling pathways should be carefully validated before use.

SUBMITTER: Deng L

PROVIDER: S-EPMC7010830 | biostudies-literature | 2020 Feb

REPOSITORIES: biostudies-literature

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Gene expression in immortalized versus primary isolated cardiac endothelial cells.

Deng Lisa L Pollmeier Luisa L Zhou Qian Q Bergemann Stella S Bode Christoph C Hein Lutz L Lother Achim A

Scientific reports 20200210 1

Endothelial cells take pivotal roles in the heart and the vascular system and their differentiation, subspecification and function is determined by gene expression. A stable, in vitro cardiac endothelial cell line could provide high cell numbers as needed for many epigenetic analyses and facilitate the understanding of molecular mechanisms involved in endothelial cell biology. To test their suitability for transcriptomic or epigenetic studies, we compared the transcriptome of cultured immortaliz ...[more]

PMID: 32042042

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Project description:Wound healing is a complex biological process, and imbalances of various substances in the wound environment may prolong healing and lead to excessive scarring. Keloid is abnormal proliferation of scar tissue beyond the original wound margins with excessive deposition of extracellular matrix (ECM) and chronic inflammation. Despite numerous previous research efforts, the pathogenesis of keloid remains unknown. Vascular endothelial cells (VECs) are a major type of inductive cell in inflammation and fibrosis. Despite several studies on vascular morphology in keloid formation, there has been no functional analysis of the role of VECs. In the present study, we isolated living VECs from keloid tissues and investigated gene expression patterns using microarray analysis. We obtained 5 keloid tissue samples and 6 normal skin samples from patients without keloid. Immediately after excision, tissue samples were gently minced and living cells were isolated. Magnetic-activated cell sorting of VECs was performed by negative selection of fibroblasts and CD45+ cells and by positive selection of CD31+cells. After RNA extraction, gene expression analysis was performed to compare VECs isolated from keloid tissue (KVECs) with VECs from normal skin (NVECs). After cell isolation, the percentage of CD31+ cells as measured by flow cytometry ranged from 81.8%-98.6%. Principal component analysis was used to identify distinct molecular phenotypes in KVECs versus NVECs and these were divided into two subgroups. In total, 15 genes were upregulated, and 3 genes were downregulated in KVECs compared with NVECs using the t-test (< 0.05). Quantitative RT-PCR and immunohistochemistry showed 16-fold and 11-fold overexpression of SERPINA3 and LAMC2, respectively. SERPINA3 encodes the serine protease inhibitor, α1-antichymotripsin. Laminin γ2-Chain (LAMC2) is a subunit of laminin-5 that induces retraction of vascular endothelial cells and enhances vascular permeability. This is the first report of VEC isolation and gene expression analysis in keloid tissue. Our data suggest that SERPINA3 and LAMC2 upregulation in KVECs may contribute to the development of fibrosis and prolonged inflammation in keloid. Further functional investigation of these genes will help clarify the mechanisms of abnormal scar tissue proliferation.

Dataset Information

Gene expression in immortalized versus primary isolated cardiac endothelial cells.

Publications

Gene expression in immortalized versus primary isolated cardiac endothelial cells.

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