Human umbilical vein endothelial cells: Control vs. Uninfected Groups
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ABSTRACT:
ORGANISM(S): Homo sapiens
PROVIDER: GSE206320 | GEO | 2022/06/17
REPOSITORIES: GEO
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Project description:Human umbilical vein endothelial cells: Control vs. Uninfected Groups
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Project description:Comparison of mRNA expression in human EPC vs. HUVEC vs. human monocytes. Cell-type specific gene expression under basal cell culture conditions (no stimulation). The hybridization was performed with three samples of EPC vs. three samples of HUVEC vs. 3 samples of CD14+ monocytes. • The origin of the biological sample: Human endothelial progenitor cells (EPC): EPC were ex vivo cultivated from human peripheral blood-derived mononuclear cells (PBMC). PBMC were isolated by density gradient centrifugation from healthy human volunteers as previously described (Dimmeler et al., 2001). Pooled human umbilical vein endothelial cells (HUVEC) were purchased from Cambrex (Verviers, Belgium). CD14+ monocytes were purified from PBMC by positive selection with anti-CD14-microbeads (Miltenyi Biotec, Bergisch-Gladbach, Germany). Purity assessed by FACS analysis was greater than 95%. • Manipulation of biological samples and protocols used: for example, growth conditions, treatments, separation techniques: EPC: 8000000 PBMC/ml were plated on human fibronectin (Sigma, Taufkirchen, Germany) and maintained in endothelial basal medium (Cambrex) with EGM SingleQuots and 20% fetal calf serum (FCS). After 3 days, nonadherent cells were removed and adherent cells were incubated in fresh medium for 24 h before starting experiments. HUVEC: HUVEC were cultured in endothelial basal medium (Cambrex) supplemented with hydrocortisone, bovine brain extract, gentamicin, amphotericin B, epidermal growth factor, and 10% FCS until the third passage according to the manufacturer’s recommendations. CD14+ monocytes: No culture. • Protocol for preparing the hybridization extract: for example, the RNA or DNA extraction and purification protocol. Total RNA was extracted from EPC, HUVEC, and CD14+ monocytes using the RNeasy cleanup system (Qiagen, Hilden, Germany) according to the manufacturer's protocol. Quantity and quality of total RNA was analyzed by the 2100 Bioanalyzer system (Agilent Technologies, Waldbronn, Germany) and agarose gel electrophoresis. • Labeling protocol(s): The detailed protocol for the sample preparation and microarray processing is available from Affymetrix (Santa Clara, CA). Briefly, 10 µg of purified total RNA was reverse transcribed by Superscript II reverse transcriptase (Life Technologies, Grand Island, NY) using T7-(dT)24 primer containing a T7 RNA polymerase promoter. After synthesis of the second complementary DNA (cDNA) strand, this product was used in an in vitro transcription reaction to generate biotinylated complementary RNA (cRNA). • The protocol and conditions used during hybridization, blocking and washing: Fifteen micrograms of fragmented, biotinylated cRNA were hybridized to a HG-U95Av2 microarray (Affymetrix Inc.) for 16 hours at 45° C with constant rotation at 60 rpm. This high-density oligonucleotide array targets 9,670 human genes as selected from the National Center for Biotechnology Information (NCBI) Gene Bank database with a total of 12,000 oligonucleotide sets. Each microarray was used to assay a single sample. After hybridization, the microarray was washed and stained on an Affymetrix fluidics station and scanned with an argon-ion confocal laser, with a 488 nm emission and detection at 570 nm. • GeneChip image analysis was performed using the Microarray Analysis Suite 5.0 (Affymetrix, Inc.). Expression data were analyzed utilizing the GeneSpring™ software version 4.2 (Silicon Genetics Inc., San Carlos, CA). Keywords: parallel sample
2004-12-09 | GSE2040 | GEO
Project description:A comparison was made between the THP-1(Human monocytic leukemia cells - TIB-202; ATCC) transcriptional responses of; (i) uninfected versus Coxiella burnetii NMII infected and (ii) uninfected versus Coxiella burnetii NMII infected THP-1 cells transiently treated with bacteriostatic levels (10μg/ml) of chloramphenicol (CAM). Briefly, infections were initiated and cultured in parallel with uninfected cells. At 48 hours post infection (hpi), media containing CAM (10μg/ml) was added to one set of cells (uninfected and infected THP-1 cells) and culturing was continued. The other set of cells were mock treated with normal media. Total RNA was isolated at 72 hpi from all conditions. Microarrays were performed for both condition sets and the results from each of the two microarrays were compared to define the host genes modulated by de novo C. burnetii NMII protein synthesis.
2010-10-01 | GSE23665 | GEO
Project description:BONCAT-MS analysis was performed against several HSV-1 infected and uninfected Hela cells.
PPI analysis of HSV-1 protein was performed against Flag-tagged HSV-1 protein expressed 293T cells.
2021-06-04 | PXD015712 | JPOST Repository
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.
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Project description:We profiled global gene expression in primary human umbilical vein endothelial cells to determine the gene expression changes associated with knocking down PKM2 and p53. We identified a p53 dependent transcriptional response that remodels metabolism in cells lacking p53, thus limiting cell growth.
2016-07-27 | GSE84877 | GEO
Project description:The conventional photosensitizers used in photodynamic therapy (PDT), such as haematoporphyrin (HP), have not yet reached satisfactory therapeutic effects on port-wine stains (PWSs), due largely to the long-term dark toxicity. Previously we have showed that hypericin exhibited potent photocytotoxic effects on Roman chicken cockscomb model of PWSs. However, the molecular mechanism of hypericin-mediated photocytotoxicity remains unclear. In this study, we employed human umbilical vein endothelial cells (HUVECs) to investigate the hypericin-photolytic mechanism. Our study showed that hypericin-PDT induced reactive oxygen species (ROS), resulting in cell killings and an activation of the inflammatory response. Importantly, we have also discovered that photoactivated hypericin induced apoptosis by activating the mitochondrial caspase pathway and inhibiting the activation of the vascular endothelial growth factor-A (VEGF-A)-mediated PI3K/Akt pathway. Notably, we found that hypericin exhibited a more potent photocytotoxic effect than HP, and largely addressed the inconvenience issue associated with the use of HP. Thereby, hypericin may be a better alternative to HP in treating PWSs.
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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.
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