SMAD1/5 binding regions and expression data of human umbilical vein endothelial cells (HUVECs) and pulmonary arterial smooth muscle cells (PASMCs) treated with BMPs
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ABSTRACT: This SuperSeries is composed of the SubSeries listed below.
Project description:Smad1/5 are transcription factors that engage in BMP-induced transcription. We determined and analyzed Smad1/5 binding sites by ChIP-sequencing. We used expression microarrays to compare the Smad1/5 binding sites identified by ChIP-seq to BMP-induced gene expressions.
Project description:Smad1/5 binding regions were identified by ChIP-seq. HUVECs were treated with BMP for 1.5 h and anti-SMAD1/5 ChIP-seq analyses were performed using Illumina genome analyzer.
Project description:Angiogenesis is primarily attributed to the excessive proliferation and migration of endothelial cells. Targeting the vascular endothelial growth factor (VEGF) is therefore significant in anti-angiogenic therapy. Although these treatments have not reached clinical expectations, the upregulation of alternative angiogenic pathways (endoglin/Smad1) may play a critical role in drug (VEGF-neutralizing agents) resistance. Enhanced endoglin expression following a VEGF-neutralizing therapy (semaxanib®) was noted in patients. Treatment with an endoglin-targeting antibody augmented VEGF expression in human umbilical vein endothelial cells (HUVECs). Therefore, approaches that inhibit both the androgen and VEGF pathways enhance the HUVECs cytotoxicity and reverse semaxanib resistance. The purpose of this study was to find natural-occurring compounds that inhibited the endoglin-targeting pathway. Curcuminoids targeting endoglin were recognized from two thousand compounds in the Traditional Chinese Medicine Database@Taiwan (TCM Database@Taiwan) using Discovery Studio 4.5. Our results, obtained using cytotoxicity, migration/invasion, and flow cytometry assays, showed that curcumin (Cur) and demethoxycurcumin (DMC) reduced angiogenesis. In addition, Cur and DMC downregulated endoglin/pSmad1 phosphorylation. The study first showed that Cur and DMC demonstrated antiangiogenic activity via the inhibition of endoglin/Smad1 signaling. Synergistic effects of curcuminoids (i.e., curcumin and DMC) and semaxanib on HUVECs were found. This might be attributed to endoglin/pSmad1 downregulation in HUVECs. Combination treatment with curcuminoids and a semaxanib is therefore expected to reverse semaxanib resistance.
Project description:Human umbilical cords were obtained from the Lucille Packard Children Hospital. In this study, we devised a rapid isolation scheme to preserve the in vivo phenotype of each endothelial subtype. ECs were first isolated from umbilical cords by collagenase perfusion through the vein or artery as described. Cells were further purified using a Percoll density gradient (Amersham Biosciences, Piscataway, NJ) to remove residual erythrocytes and platelets. 1-5 x 106 ECs were then cultured overnight on gelatin-coated T12.5 flasks in Clonetics EGM-2 media (Cambrex Bio Science, Walkersville, MD) in which the ECs generally reached confluence the next morning. The confluent EC were trypsinized and subjected to two rounds of immuno-magnetic beads purification, adapted from a published purification protocol. There was a negative selection step using CD14, CD45 and CD64 to remove residual contaminating leukocytes, followed by positive selection using a mouse anti-endothelial cell monoclonal antibody (anti-CD146/clone P1H12 purchased from Chemicon, Temecula, CA). Total processing time was limited to 20 to 24 hours. The homogeneous, viable, primary ECs were used immediately to construct the library. The construction of SAGE libraries was performed with the I-SAGE kit (Invitrogen, Carlsbad, CA) per manufacturer’s instructions Keywords: other
Project description:Human umbilical cords were obtained from the Lucille Packard Children Hospital. In this study, we devised a rapid isolation scheme to preserve the in vivo phenotype of each endothelial subtype. ECs were first isolated from umbilical cords by collagenase perfusion through the vein or artery as described. Cells were further purified using a Percoll density gradient (Amersham Biosciences, Piscataway, NJ) to remove residual erythrocytes and platelets. 1-5 x 106 ECs were then cultured overnight on gelatin-coated T12.5 flasks in Clonetics EGM-2 media (Cambrex Bio Science, Walkersville, MD) in which the ECs generally reached confluence the next morning. The confluent EC were trypsinized and subjected to two rounds of immuno-magnetic beads purification, adapted from a published purification protocol. There was a negative selection step using CD14, CD45 and CD64 to remove residual contaminating leukocytes, followed by positive selection using a mouse anti-endothelial cell monoclonal antibody (anti-CD146/clone P1H12 purchased from Chemicon, Temecula, CA). Total processing time was limited to 20 to 24 hours. The homogeneous, viable, primary ECs were used immediately to construct the library. The construction of SAGE libraries was performed with the I-SAGE kit (Invitrogen, Carlsbad, CA) per manufacturer’s instructions Keywords: other
Project description:Smad1/5 are transcription factors that engage in BMP-induced transcription. We determined and analyzed Smad1/5 binding sites by ChIP-sequencing. We used expression microarrays to compare the Smad1/5 binding sites identified by ChIP-seq to BMP-induced gene expressions. PASMCs were treated with 50 ng/ml BMP-4 for 0, 2, or 24 hrs. RNA was extracted and hybridized on Affymetrix microarrays.
Project description:To explore whether stent procedure may influence transcriptional response of endothelium, we applied different physical (flow changes) and/or mechanical (stent application) stimuli to human endothelial cells in a laminar flow bioreactor (LFB) system. Gene expression analysis was then evaluated in each experimental condition. Human umbilical vein endothelial cells (HUVECs) were submitted to low and physiological (1 and 10 dyne/cm(2)) shear stress in absence (AS) or presence (PS) of stent positioning in a LFB system for 24 h. Different expressed genes, coming from Affymetrix results, were identified based on one-way ANOVA analysis with p values <0.01 and a fold changed >3 in modulus. Low shear stress was compared with physiological one in AS and PS conditions. Two major groups include 32 probes commonly expressed in both 1AS versus 10AS and 1PS versus 10PS comparison, and 115 probes consisting of 83 in addition to the previous 32, expressed only in 1PS versus 10PS comparison. Genes related to cytoskeleton, extracellular matrix, and cholesterol transport/metabolism are differently regulated in 1PS versus 10PS condition. Inflammatory and apoptotic mediators seems to be, instead, closely modulated by changes in flow (1 versus 10), independently of stent application. Low shear stress together with stent procedure are the experimental conditions that mainly modulate the highest number of genes in our human endothelial model. Those genes belong to pathways specifically involved in the endothelial dysfunction.
Project description:This SuperSeries is composed of the following subset Series: GSE15482: The effect of PPARa siRNA on endothelial cells treated with fenofibrate GSE15483: The effect of fenofibrate on endothelial cells Refer to individual Series