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Proteomic analysis of human umbilical vein endothelial cells exposed to PM2.5*

ABSTRACT: Exposure to ?ne ambient particulate matter (PM2.5) is known to be associated with cardiovascular disease. To uncover the molecular mechanisms involved in cardiovascular toxicity of PM2.5, we investigated alterations in the protein profile of human umbilical vein endothelial cells (HUVECs) treated with PM2.5 using two-dimensional electrophoresis in conjunction with mass spectrometry (MS). A total of 31 protein spots were selected as differentially expressed proteins and identi?ed by matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF) MS. The results demonstrated that DNA damage and cell apoptosis are important factors contributing to PM2.5-mediated toxicity in HUVECs. It is further proposed that PM2.5 can inhibit superoxide dismutase (SOD) activity and increase reactive oxygen species (ROS) and malonaldehyde (MDA) production in a concentration-dependent manner. Induction of apoptosis and DNA damage through oxidative stress pathways may be one of the key toxicological events occurring in HUVECs under PM2.5 stress. These results indicated that the toxic mechanisms of PM2.5 on cardiovascular disease are related to endothelial dysfunction.

SUBMITTER: Zhu J

PROVIDER: S-EPMC6011028 | biostudies-literature | 2018 Jun

REPOSITORIES: biostudies-literature

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Project description:Human umbilical vein endothelial cells (HUVECs) are a widely-used model system to study pathological and physiological processes associated with the cardiovascular system. An understanding of genes and proteins that are expressed in any cell type is a fundamental need which facilitates studies of molecular changes in disease states and response to various stimuli. In this study, we employed next generation sequencing and mass spectrometry to profile the transcriptome and proteome of primary HUVECs. Analysis of 145 million paired-end reads from next generation sequencing confirmed expression of 12,186 protein-coding genes (FPKM>0.1), 439 novel long non-coding RNAs and revealed 6,089 novel isoforms that were not annotated in GENCODE. A comparison of the transcripts against human gene expression data for 53 tissues catalogued by the Genotype-Tissue Expression (GTEx) project revealed a number of HUVEC-specific genes. Proteomics analysis identified 6,477 proteins including confirmation of N-termini for 1,091 proteins and isoforms for 149 proteins for which transcriptomic evidence was observed. Alternate translational start sites for seven proteins and alternate splicing in five proteins were also identified. A database search to specifically identify other post-translational modifications provided evidence for a number of modification sites on 117 proteins which included ubiquitylation, lysine acetylation and mono, di- and tri-methylation events. Based on the data from this study and a survey of other databases, we provide evidence for 11 “missing proteins,” which are proteins for which there was insufficient or no protein level evidence. Peptides supporting missing protein and novel events were validated by comparison of MS/MS fragmentation patterns with synthetic peptides. By creating a custom database of proteins containing sample-specific single amino acid variants (SAAV), we also identified 245 variant peptides derived from 207 expressed proteins. Overall, we believe that the integrated approach employed in this study is widely applicable to study any primary cell type for deeper molecular characterization.

Project description:Human umbilical vein endothelial cells (HUVECs) are a widely-used model system to study many pathological and physiological processes associated with the cardiovascular system. An understanding of genes and proteins that are expressed in any cell type is a fundamental need which facilitates study of molecular changes in disease states and response to various stimuli. In this study, we employed next generation sequencing and mass spectrometry to profile the transcriptome and proteome of primary HUVECs. Analysis of 145 million paired-end reads from next generation sequencing confirmed expression of 12,186 protein-coding genes (FPKM>0.1), 439 novel long non-coding RNAs and revealed 6,089 novel isoforms that were not annotated in GENCODE. A comparison of the transcripts against human gene expression data for 53 tissues catalogued by the Genotype-Tissue Expression (GTEx) project revealed a number of HUVEC-specific genes. Proteomics analysis identified 6,477 proteins including confirmation of N-termini for 1,091 proteins and isoforms for 149 proteins for which transcriptomic evidence was also observed. Alternate translational start sites for seven proteins and alternate splicing in seven proteins were also identified. A database search to specifically identify other post-translational modifications provided evidence for a number of modification sites on 117 proteins which include ubiquitylation, lysine acetylation, mono, di- and tri-methylation. Based on the data from this study and a survey of other databases, we provide evidence for 11 “missing proteins,” which are proteins for which protein level evidence is lacking or insufficient. Proteogenomic analysis identified novel N-termini and exon-exon junctions at both RNA and protein levels by searching against a translated database created from alternate transcript assembly. Peptides supporting the novel events of expressed proteins are further validated by synthesis and spectral comparison. Alternative allelic expression of genes arising from transcription of two alleles of a gene in chromosome pairs is observed. By creating a custom database of proteins incorporated with sample specific single amino acid variations (SAAV) we identified alternative alleles for 207 expressed proteins with 245 peptides with SAAV. Overall, we believe that the integrated approach employed in this study is widely applicable to study any primary cell type for deeper molecular characterization.

Project description:Background: In diabetes chronic hyperinsulinemia is responsible for the instability of the atherosclerotic plaque and stimulates cellular proliferation through the activation of the MAP kinases, which in turn regulate cellular proliferation. However, it is not known whether insulin itself could increase the transcription of specific genes for cellular proliferation in the endothelium. Hence, the characterization of transcriptional modifications in endothelium is an important step for a better understanding of the mechanism of insulin action and the relationship between endothelial cell dysfunction and insulin resistance. Methodology and principal findings: The transcriptional response of endothelial cells in the 440 minutes following insulin stimulation was monitored using microarrays and compared to a control condition. About 1700 genes were selected as differentially expressed based on their treated minus control profile, thus allowing the detection of even small but systematic changes in gene expression. Genes were clustered in 7 groups according to their time expression profile and classified into 15 functional categories that can support the biological effects of insulin, based on Gene Ontology enrichment analysis. In terms of endothelial function, the most prominent processes affected were NADH dehydrogenase activity, N-terminal myristoylation domain binding, nitric-oxide synthase regulator activity and growth factor binding. Pathway-based enrichment analysis revealed “Electron Transport Chain” significantly enriched. Results were validated on genes belonging to “Electron Transport Chain” pathway, using quantitative RT-PCR. Conclusions: As far as we know, this is the first systematic study in the literature monitoring transcriptional response to insulin in endothelial cells, in a time series microarray experiment. Since chronic hyperinsulinemia is responsible for the instability of the atherosclerotic plaque and stimulates cellular proliferation, some of the genes identified in the present work are potential novel candidates in diabetes complications related to endothelial dysfunction.

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Proteomic analysis of human umbilical vein endothelial cells exposed to PM2.5*

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