Gene expression profile at single cell level of aortic endothelial cells (ECs) from endothelial-specific Epas1 knockout mice (Epas1fl/fl, Cdh5Cre/+) and controls (Epas1fl/fl, Cdh5+/+)
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ABSTRACT:
ORGANISM(S): Mus musculus
PROVIDER: GSE274418 | GEO | 2024/08/27
REPOSITORIES: GEO
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Project description:Purpose: By integrating DNA methylation and gene expression of COPD lung tissues, we identified EPAS1 as a key regulator whose downstream genes significantly overlapped with multiple genes sets associated with COPD disease severity. EPAS1 is distinct in comparison with other key regulators in terms of methylation profiles and downstream target genes. Genes predicted to be regulated by EPAS1 were enriched for biological processes including signaling, cell communications, and system developement. As EPAS1 downstream genes were significantly enriched for hypoxia responsive genes in endothelial cells, we tested EPAS1 function in human and mouse endothelial cells HUVEC and C166. EPAS1 knockdown by siRNA in endothelial cells impacted genes that significantly overlapped with EPAS1 downstream genes in lung tissue including hypoxia responsive genes. Methods: The cell lines of HUVEC (Lonza, MD, USA) and C166 (American Type Culture Collection, VA, USA) were cultured in the appropriate media at 37M-BM-0C with 5% CO2. The cells were transfected with EPAS1 siRNA and non-targeting negative control siRNA (Life Technologies, CA, USA) using Lipofectamine RNAiMAX as recommended transfection protocols by the manufacturer. After the treatments with 5nM SilencerM-BM-. Select siRNA (s4700 for EPAS1, s65525 for Epas1; Life Technologies, USA) for 48 hours, the total RNA was purified with RNeasy Mini Kit (QIAGEN, Germany). The efficiencies of knocked down the EPAS1 expression were assessed by qPCR with 1.4% for HUVEC, 3.2% for C166. Approximately 250 ng of total RNA per sample were used for library construction by the TruSeq RNA Sample Prep Kit (Illumina) and sequenced using the Illumina HiSeq 2500 instrument with 100nt single read setting according to the manufacturer's instructions. Sequence reads were aligned to human genome assembly hg19 and mouse genome assembly mm10, respectively, using Tophat [96]. Total 23,228 human and 22,609 mouse genes were quantified using Cufflinks [96]. siRNA signatures were derived by comparing expression profiles of EPAS1 or Epas1 siRNAs with non-targeting siRNAs at paired t-test p-value cutoff 0.05 with resulting signature sizes of 2,796 and 3,730, and corresponding q-values [97] 0.11 and 0.07 for HUVEC and C166, respectively. Results: When comparing endothelial cells treated with EPAS1 siRNAs and scrambled siRNAs, we identified an EPAS1 siRNA signature consisting of 2796 and 3730 genes in human and mouse endothelial cell lines, respectively, whose expression levels significantly changed (t-test p-value<0.05), including EPAS1 itself (p-value = 0.002 and 0.02) and the EPAS1 downstream target gene VEGFA (p-value = 0.03 and 0.01). The EPAS1 siRNA signatures derived from human and mouse cell lines were highly consistent, with 695 genes in common to both signatures (p-value = 7.2x10-65). Both signatures not only significantly overlapped with EPAS1 downstream genes (p-value = 7.3x10-7 and 1.5x10-12), but also with hypoxia response genes in endothelial cells (FisherM-bM-^@M-^Ys Exact Test p-value = 5.8x10-8 and 1.2x10-12 in the human and mouse signatures, respectively). Moreover, the EPAS1 siRNA signatures consistently overlapped genes associated with the COPD severity phenotypes. These results together validate that EPAS1 causally regulates the downstream target genes we predicted, and that these genes in turn affect COPD development and progression. For each of human and mouse cell lines, 3 siRNA control cells, and 3 siRNA EPAS1 knockdown cells were used and analyzed. To identify EPAS1 signatures, paired t-test was performed between control siRNA and EPAS1 siRNA samples.
2015-01-16 | E-GEOD-62974 | biostudies-arrayexpress
Project description:Gene expression profile at single cell level of aortic endothelial cells (ECs) from endothelial-specific Epas1 knockout mice (Epas1fl/fl, Cdh5Cre/+) and controls (Epas1fl/fl, Cdh5+/+)
| PRJNA1146385 | ENA
Project description:Purpose: By integrating DNA methylation and gene expression of COPD lung tissues, we identified EPAS1 as a key regulator whose downstream genes significantly overlapped with multiple genes sets associated with COPD disease severity. EPAS1 is distinct in comparison with other key regulators in terms of methylation profiles and downstream target genes. Genes predicted to be regulated by EPAS1 were enriched for biological processes including signaling, cell communications, and system developement. As EPAS1 downstream genes were significantly enriched for hypoxia responsive genes in endothelial cells, we tested EPAS1 function in human and mouse endothelial cells HUVEC and C166. EPAS1 knockdown by siRNA in endothelial cells impacted genes that significantly overlapped with EPAS1 downstream genes in lung tissue including hypoxia responsive genes. Methods: The cell lines of HUVEC (Lonza, MD, USA) and C166 (American Type Culture Collection, VA, USA) were cultured in the appropriate media at 37°C with 5% CO2. The cells were transfected with EPAS1 siRNA and non-targeting negative control siRNA (Life Technologies, CA, USA) using Lipofectamine RNAiMAX as recommended transfection protocols by the manufacturer. After the treatments with 5nM Silencer® Select siRNA (s4700 for EPAS1, s65525 for Epas1; Life Technologies, USA) for 48 hours, the total RNA was purified with RNeasy Mini Kit (QIAGEN, Germany). The efficiencies of knocked down the EPAS1 expression were assessed by qPCR with 1.4% for HUVEC, 3.2% for C166. Approximately 250 ng of total RNA per sample were used for library construction by the TruSeq RNA Sample Prep Kit (Illumina) and sequenced using the Illumina HiSeq 2500 instrument with 100nt single read setting according to the manufacturer's instructions. Sequence reads were aligned to human genome assembly hg19 and mouse genome assembly mm10, respectively, using Tophat [96]. Total 23,228 human and 22,609 mouse genes were quantified using Cufflinks [96]. siRNA signatures were derived by comparing expression profiles of EPAS1 or Epas1 siRNAs with non-targeting siRNAs at paired t-test p-value cutoff 0.05 with resulting signature sizes of 2,796 and 3,730, and corresponding q-values [97] 0.11 and 0.07 for HUVEC and C166, respectively. Results: When comparing endothelial cells treated with EPAS1 siRNAs and scrambled siRNAs, we identified an EPAS1 siRNA signature consisting of 2796 and 3730 genes in human and mouse endothelial cell lines, respectively, whose expression levels significantly changed (t-test p-value<0.05), including EPAS1 itself (p-value = 0.002 and 0.02) and the EPAS1 downstream target gene VEGFA (p-value = 0.03 and 0.01). The EPAS1 siRNA signatures derived from human and mouse cell lines were highly consistent, with 695 genes in common to both signatures (p-value = 7.2x10-65). Both signatures not only significantly overlapped with EPAS1 downstream genes (p-value = 7.3x10-7 and 1.5x10-12), but also with hypoxia response genes in endothelial cells (Fisher’s Exact Test p-value = 5.8x10-8 and 1.2x10-12 in the human and mouse signatures, respectively). Moreover, the EPAS1 siRNA signatures consistently overlapped genes associated with the COPD severity phenotypes. These results together validate that EPAS1 causally regulates the downstream target genes we predicted, and that these genes in turn affect COPD development and progression.
2015-01-16 | GSE62974 | GEO
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2025-02-15 | GSE289628 | GEO
Project description:Arrhythmogenic cardiomyopathy (ACM) is frequently attributed to desmosomal mutations, such as those in the desmoplakin (DSP) gene. Patients with DSP- cardiomyopathy are predisposed to myocardial degeneration and arrhythmias. Despite advancements, the underlying molecular mechanisms remain incompletely understood, thus limiting therapeutic options. Here, we employed spatial transcriptomics on an explanted heart from a patient with a pathogenic DSP variant. Our transcriptional analysis revealed endothelial PAS domain-containing protein 1 (EPAS1) as a potential regulator of mitochondrial homeostasis in stressed cardiomyocytes. Elevated EPAS1 levels were associated with mitochondrial dysfunction and hypoxic stress in both human-relevant in vitro ACM models and additional explanted hearts with genetic cardiomyopathy. Collectively, cardiomyocytes bearing pathogenic DSP variants exhibit mitochondrial dysfunction, increased apoptosis, and impaired contractility, which are linked to the increased EPAS1 levels. These findings implicate EPAS1 as a key regulator of myocardial degeneration in DSP-cardiomyopathy, which expand to other forms of ACM.
2025-02-15 | GSE289630 | GEO
Project description:SAGE libraries were prepared from mRNA of cultured bovine glomerular and aortic endothelial cells. Cells both cell lines were positive for acetylated LDL uptake and vWF expression, and were proven to be free of non-endothelial cells. Cultures were studied at passage 9-10. The cells were confluent, cultured in parallel, under identical conditions. Cells were plated on gelatin coated cell culture plastic in RPMI 1640 medium containing 10% FBS and streptomycin/penicillin. The last medium change was 48 hours prior to mRNA harvest. Keywords = endothelial, glomerular, kidney, aorta, Keywords: other
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Project description:To understand the role of the hypoxia-inducible transcription factor EPAS1 in regulating spermatogonial stem cell (SSC) function, a conditional knockout mouse was generated (EPAS1-cKO) where Epas1 was ablated only in the germ cells (Ddx4-Cre, Epasfl/-). For single cell RNA sequencing analysis, control and EPAS1-cKO males were pre-treated with the chemotherapeutic reagent busulfan to instigate regenerative conditions, and a germ cell-enriched cell population was collected 14 days later. Transcriptome profiling revealed that EPAS1 deficiency alters gene expression and key cellular processes in undifferentiated spermatogonia, including metabolism and proteostasis. Overall, these transcriptomic changes resulted in an impaired capacity for SSCs to restore spermatogenesis after busulfan treatment.
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