Proteomic analysis of human umbilical vein endothelial cells exposed to PM2.5*
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ABSTRACT:
SUBMITTER: Zhu J
PROVIDER: S-EPMC6011028 | biostudies-literature | 2018 Jun
REPOSITORIES: biostudies-literature
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Project description:Understanding the molecular profile of every human cell type is essential for understanding its role in normal physiology and disease. Technological advancements in DNA sequencing, mass spectrometry, and computational methods allow us to carry out multiomics analyses although such approaches are not routine yet. Human umbilical vein endothelial cells (HUVECs) are a widely used model system to study pathological and physiological processes associated with the cardiovascular system. In this study, next-generation sequencing and high-resolution mass spectrometry to profile the transcriptome and proteome of primary HUVECs is employed. 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 6089 novel isoforms that were not annotated in GENCODE. Proteomics analysis identifies 6477 proteins including confirmation of N-termini for 1091 proteins, isoforms for 149 proteins, and 1034 phosphosites. A database search to specifically identify other post-translational modifications provide evidence for a number of modification sites on 117 proteins which include ubiquitylation, lysine acetylation, and mono-, di- and tri-methylation events. Evidence for 11 "missing proteins," which are proteins for which there was insufficient or no protein level evidence, is provided. Peptides supporting missing protein and novel events are validated by comparison of MS/MS fragmentation patterns with synthetic peptides. Finally, 245 variant peptides derived from 207 expressed proteins in addition to alternate translational start sites for seven proteins and evidence for novel proteoforms for five proteins resulting from alternative splicing are identified. Overall, it is believed that the integrated approach employed in this study is widely applicable to study any primary cell type for deeper molecular characterization.
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Project description:Vitamin C plays a protective role in oxidative damage by blocking the effects of free radicals. The present study investigated the mechanisms through which vitamin C partly mediates anti?apoptotic and antioxidant functions via the regulation of microRNAs (miRNAs or miRs). For this purpose, a global miRNA expression analysis on human umbilical vein endothelial cells (HUVECs) treated with vitamin C was conducted using microarrays containing human precursor and mature miRNA probes. The results revealed that there were 42 identical miRNAs among the differentially expressed miRNAs in the HUVEC group and H2O2 + vitamin C?treated HUVEC group compared to the H2O2?exposed HUVEC group, including 41 upregulated miRNAs and 1 down?regulated miRNA. Using bioinformatics analysis, differentially expressed miRNAs were investigated to identify novel target mRNAs and signaling pathways. Pathway enrichment analyses revealed that apoptosis, the mitogen?activated protein kinase (MAPK) signaling pathway, phosphoinositide 3?kinase (PI3K)/Akt signaling pathway and oxidative phosphorylation were significantly enriched. The results from western blot analysis demonstrated that the interleukin (IL)10, matrix metalloproteinase (MMP)2, cAMP?response element binding protein (CREB) and p?CREB protein expression levels in HUVECs transfected with hsa?miR?3928?5p and induced by H2O2 were significantly downregulated; the MAPK9, caspase?3 (CASP3) and p?CASP3 protein expression levels in HUVECs transfected with hsa?miR?323a?5p and induced by H2O2 were significantly downregulated. The present study therefore demonstrates that vitamin C partly exerts protective effects on HUVECs through the regulation of miRNA/mRNA axis expression.
| S-EPMC7595655 | biostudies-literature
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.
2019-10-28 | GSE115536 | GEO
Project description:BACKGROUND: In diabetes chronic hyperinsulinemia contributes to 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 contributes to 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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Project description:OBJECTIVES:Hypoxia leads to endothelial cell inflammation, apoptosis, and damage, which plays an important role in the complications associated with ischemic cardiovascular disease. As an oxidoreductase, p66Shc plays an important role in the regulation of reactive oxygen species (ROS) production and apoptosis. Ketamine is widely used in clinics. This study was designed to assess the potential protective effect of ketamine against hypoxia-induced injury in human umbilical vein endothelial cells (HUVECs). Moreover, we explored the potential mechanism by which ketamine protected against hypoxia-induced endothelial injury. METHODS:The protective effects of ketamine against hypoxia-induced injury was assessed using cell viability and adhesion assays, quantitative polymerase chain reaction, and western blotting. RESULTS:Our data showed that hypoxia reduced HUVEC viability, increased the adhesion between HUVECs and monocytes, and upregulated the expression of endothelial adhesion molecules at the protein and mRNA levels. Moreover, hypoxia increased ROS accumulation and upregulated p66Shc expression. Furthermore, hypoxia downregulated sirt1 expression in HUVECs. Alternatively, ketamine was shown to reverse the hypoxia-mediated reduction of cell viability and increase in the adhesion between HUVECs and monocytes, ameliorate hypoxia-induced ROS accumulation, and suppress p66Shc expression. Moreover, EX527, a sirt1 inhibitor, reversed the protective effects of ketamine against the hypoxia-mediated reduction of cell viability and increase in adhesion between HUVECs and monocytes. CONCLUSION:Ketamine reduces hypoxia-induced p66Shc expression and attenuates ROS accumulation via upregulating sirt1 in HUVECs, thus attenuating hypoxia-induced endothelial cell inflammation and apoptosis.
| S-EPMC7470431 | biostudies-literature
Project description:Angiogenesis is the process by which endothelial cells grow and disassemble into functional blood vessels. In this study, we examine the fundamental processes that control the assembly of endothelial cells into networks in vitro. Network assembly is known to be influenced by matrix mechanics and chemical signals. However, the roles of substrate stiffness and chemical signals in network formation is unclear. In this study, human umbilical vein endothelial cells (HUVECs) were seeded onto RGD or GFOGER functionalized polyacrylamide gels of varying stiffness. Cells were either treated with bFGF, VEGF, or left untreated and observed over time. We found that cells form stable networks on soft gels (Young's modulus 140 Pa) when untreated but that growth factors induce increased cell migration which leads to network instability. On stiffer substrates (Young's modulus 2500 Pa) cells do not assemble into networks either with or without growth factors in any combination. Our results indicate that cells assemble to networks below a critical compliance, that a critical cell density is needed for network formation, and that growth factors can inhibit network formation through an increase in motility.
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Project description:Primary human umbilical vein endothelial cells (HUVECs) are consistently the most reliable in vitro model system for studying the inner lining of blood and lymphatic vessels or the endothelium. Primary human cells originate from freshly isolated tissues without genetic manipulation and generally show a modal number of 46 chromosomes with no structural alterations, at least during early passages. We investigated the cytogenetic integrity of HUVECs with conventional (G-banding) and molecular cytogenetic methods (spectral karyotyping (SKY) and fluorescence in situ hybridization (FISH)). Our G-band data shows two X-chromosomes, confirming these HUVECs originate from a female donor. Notably, some cells consistently exhibit an unfamiliar banding pattern on one X chromosome toward the distal end of the long arm (Xq). Our FISH analysis confirms that approximately 50% of these HUVECs have a deletion of the Xq terminal region. SKY analysis indicates that the deleted region is apparently not integrated into any other chromosome. Finally, we demonstrated the presence of a similar Xq deletion in the daughter cell line, EA.hy926, which was generated by fusing HUVECs with A549 (a thioguanine-resistant clone of adenocarcinomic human alveolar basal epithelial cells). These findings will advance comprehension of HUVECs biology and will augment future endothelial studies.
| S-EPMC9222953 | biostudies-literature
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.
2019-04-24 | PXD009687 | Pride
Project description:To identify miRNAs that are involved in cell migration in human umbilical vein endothelial cells (HUVECs), we employed RNA sequencing under high glucose incubation and text mining within the databases miRWalk and TargetScanHuman using 83 genes that regulate HUVECs migration. From both databases, 307 predicted miRNAs were retrieved. Differentially expressed miRNAs were determined by exposing HUVECs to high glucose stimulation, which significantly inhibited the migratory ability of HUVECs as compared to cells cultured in normal glucose. A total of 35 miRNAs were found as differently expressed miRNAs in miRNA sequencing, and 4 miRNAs, namely miR-21-3p, miR-107, miR-143-3p, and miR-106b-5p, were identified as overlapping hits. These were subjected to hub gene analysis and pathway analysis using the Kyoto Encyclopedia of Genes and Genomes (KEGG), identifing 71 pathways which were influenced by all four miRNAs. The influence of all four miRNAs on HUVEC migration was phenomorphologically confirmed. miR21 and miR107 promoted migration in HUVECs while miR106b and miR143 inhibited migration. Pathway analysis also revealed eight shared pathways between the four miRNAs. Protein-protein interaction (PPI) network analysis was then performed to predict the functionality of interacting genes or proteins. This revealed six hub genes which could firstly be predicted to be related to HUVEC migration.
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Project description:Cryopreservation of human umbilical vein endothelial cells (HUVECs) facilitated their commercial availability for use in vascular biology, tissue engineering and drug delivery research; however, the key variables in HUVEC cryopreservation have not been comprehensively studied. HUVECs are typically cryopreserved by cooling at 1 °C/min in the presence of 10% dimethyl sulfoxide (DMSO). We applied interrupted slow cooling (graded freezing) and interrupted rapid cooling with a hold time (two-step freezing) to identify where in the cooling process cryoinjury to HUVECs occurs. We found that linear cooling at 1 °C/min resulted in higher membrane integrities than linear cooling at 0.2 °C/min or nonlinear two-step freezing. DMSO addition procedures and compositions were also investigated. By combining hydroxyethyl starch with DMSO, HUVEC viability after cryopreservation was improved compared to measured viabilities of commercially available cryopreserved HUVECs and viabilities for HUVEC cryopreservation studies reported in the literature. Furthermore, HUVECs cryopreserved using our improved procedure showed high tube forming capability in a post-thaw angiogenesis assay, a standard indicator of endothelial cell function. As well as presenting superior cryopreservation procedures for HUVECs, the methods developed here can serve as a model to optimize the cryopreservation of other cells.
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