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Gene expression profile in interleukin-4-stimulated human vascular endothelial cells.

ABSTRACT: Interleukin-4 (IL-4)-mediated pro-oxidative and pro-inflammatory vascular environments have been implicated in the pathogenesis of atherosclerosis. The cellular and molecular regulatory mechanisms underlying this process, however, are not fully understood. In the present study, we employed GeneChip microarray analysis to investigate global gene expression patterns in human vascular endothelial cells after treatment with IL-4. Our results showed that mRNA levels of a total of 106 genes were significantly up-regulated and 41 genes significantly down-regulated with more than a 2-fold change. The majority of these genes are critically involved in the regulation of inflammatory responses, apoptosis, signal transduction, transcription factors, and metabolism; functions of the remaining genes are unknown. The changes in gene expression of selected genes related to inflammatory reactions, such as vascular cell adhesion molecule-1 (VCAM-1), E-selectin, monocyte chemoattractant protein-1 (MCP-1), and interleukin-6 (IL-6), were verified by quantitative real-time reverse transcriptase-polymerase chain reaction (RT-PCR) and enzyme-linked immunosorbent assay (ELISA) analyses. IL-4 treatment also significantly increased the adherence of inflammatory cells to endothelial cell monolayers in a dose-dependent manner. These results may help determine the molecular mechanisms of action of IL-4 in human vascular endothelium. In addition, a better understanding of IL-4-induced vascular injury at the level of gene expression could lead to the identification of new therapeutic strategies for atherosclerosis.

SUBMITTER: Lee YW

PROVIDER: S-EPMC1431351 | biostudies-literature | 2004 Jan-Jun

REPOSITORIES: biostudies-literature

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Gene expression profile in interleukin-4-stimulated human vascular endothelial cells.

Lee Yong Woo YW Eum Sung Yong SY Chen Kuey Chu KC Hennig Bernhard B Toborek Michal M

Molecular medicine (Cambridge, Mass.) 20040101 1-6

Interleukin-4 (IL-4)-mediated pro-oxidative and pro-inflammatory vascular environments have been implicated in the pathogenesis of atherosclerosis. The cellular and molecular regulatory mechanisms underlying this process, however, are not fully understood. In the present study, we employed GeneChip microarray analysis to investigate global gene expression patterns in human vascular endothelial cells after treatment with IL-4. Our results showed that mRNA levels of a total of 106 genes were signi ...[more]

PMID: 15502879

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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.

Project description:Studies indicate that high-grade serous ovarian carcinoma (HGSOC), the most common epithelial ovarian carcinoma histotype, originates from the fallopian tube epithelium (FTE). Risk factors for this cancer include reproductive parameters associated with lifetime ovulatory events. Ovulation is an acute inflammatory process during which the FTE is exposed to follicular fluid containing both pro- and anti-inflammatory molecules, such as interleukin-1 (IL1), tumor necrosis factor (TNF), and cortisol. Repeated exposure to inflammatory cytokines may contribute to transforming events in the FTE, with glucocorticoids exerting a protective effect. The global response of FTE cells to inflammatory cytokines or glucocorticoids has not been investigated. To examine the response of FTE cells and the ability of glucocorticoids to oppose this response, an immortalized human FTE cell line, OE-E6/E7, was treated with IL1β, dexamethasone (DEX), IL1β and DEX, or vehicle and genome-wide gene expression profiling was performed. IL1β altered the expression of 47 genes of which 17 were reversed by DEX. DEX treatment alone altered the expression of 590 genes, whereas combined DEX and IL1β treatment altered the expression of 784 genes. Network and pathway enrichment analysis indicated that many genes altered by DEX are involved in cytokine, chemokine, and cell cycle signaling, including NFκΒ target genes and interacting proteins. Quantitative real time RT-PCR studies validated the gene array data for IL8, IL23A, PI3 and TACC2 in OE-E6/E7 cells. Consistent with the array data, Western blot analysis showed increased levels of PTGS2 protein induced by IL1β that was blocked by DEX. A parallel experiment using primary cultured human FTE cells indicated similar effects on PTGS2, IL8, IL23A, PI3 and TACC2 transcripts. These findings support the hypothesis that pro-inflammatory signaling is induced in FTE cells by inflammatory mediators and raises the possibility that dysregulation of glucocorticoid signaling could contribute to increased risk for HGSOC.

Project description:BackgroundInterleukin 6 (IL6) is a multifunctional cytokine produced by various cells, including vascular endothelial cells. IL6 has both pro- and non-/anti-inflammatory functions, and the response to IL6 is dependent on whether it acts via the membrane-bound IL6 receptor α (IL6Rα) (classic signaling) or the soluble form of the receptor (transsignaling). As human endothelial cells produce IL6 and at the same time express IL6Rα, we hypothesized that IL6 may have autocrine functions.MethodsKnockdown of IL6 in cultured human endothelial cells was performed using siRNA. Knockdown efficiency was evaluated using ELISA. RNA sequencing was employed to characterize the transcriptional consequence of IL6 knockdown, and Ingenuity Pathway Analysis was used to further explore the functional roles of IL6.ResultsKnockdown of IL6 in cultured endothelial cells resulted in a 84-92% reduction in the release of IL6. Knockdown of IL6 resulted in dramatic changes in transcriptional pattern; knockdown of IL6 in the absence of soluble IL6Rα (sIL6Rα) led to differential regulation of 1915 genes, and knockdown of IL6 in the presence of sIL6Rα led to differential regulation of 1967 genes (fold change 1.5, false discovery rate < 0.05). Pathway analysis revealed that the autocrine functions of IL6 in human endothelial cells are mainly related to basal cellular functions such as regulation of cell cycle, signaling, and cellular movement. Furthermore, we found that knockdown of IL6 activates functions related to adhesion, binding, and interaction of endothelial cells, which seem to be mediated mainly via STAT3.ConclusionIn this study, a large number of novel genes that are under autocrine regulation by IL6 in human endothelial cells were identified. Overall, our data indicate that IL6 acts in an autocrine manner to regulate basal cellular functions, such as cell cycle regulation, signaling, and cellular movement, and suggests that the autocrine functions of IL6 in human endothelial cells are mediated via IL6 classic signaling.

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Gene expression profile in interleukin-4-stimulated human vascular endothelial cells.

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Gene expression profile in interleukin-4-stimulated human vascular endothelial cells.

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