Cell Density Impacts Epigenetic Regulation of Cytokine-Induced E-Selectin Gene Expression in Vascular Endothelium

Abstract

Growing evidence suggests that the phenotype of endothelial cells during angiogenesis differs from that of quiescent endothelial cells, although little is known regarding the difference in the susceptibility to inflammation between both the conditions. Here, we assessed the inflammatory response in sparse and confluent endothelial cell monolayers. To obtain sparse and confluent monolayers, human umbilical vein endothelial cells were seeded at a density of 7.3×103 cells/cm2 and 29.2×103 cells/cm2, respectively, followed by culturing for 36 h and stimulation with tumor necrosis factor α. The levels of tumor necrosis factor α-induced E-selectin protein and mRNA expression were higher in the confluent monolayer than in the sparse monolayer. The phosphorylation of c-jun N-terminal kinase and p38 mitogen-activated protein kinase or nuclear factor-κB activation was not involved in this phenomenon. A chromatin immunoprecipitation assay of the E-selectin promoter using an anti-acetyl-histone H3 antibody showed that the E-selectin promoter was highly and specifically acetylated in the confluent monolayer after tumor necrosis factor α activation. Furthermore, chromatin accessibility real-time PCR showed that the chromatin accessibility at the E-selectin promoter was higher in the confluent monolayer than in the sparse monolayer. Our data suggest that the inflammatory response may change during blood vessel maturation via epigenetic mechanisms that affect the accessibility of chromatin.

Bovine pericardial extracellular matrix niche modulates human aortic endothelial cell phenotype and function

“…Interestingly, significant differences between expression levels were observed between serous and fibrous sides for TNF-α IL-6 and IL-8 at moderate densities, further indicating that cell density may play a role in modulating cellular response to ECM niche. This observation agrees with recent evidence of cell density impact on cytokines regulation and gene expression 49,50 . Further, TNF-α, IL-6 and IL-8 have been shown to upregulate laminin expression 48 , suggesting that their upregulation may be in response to basement membrane presence on the serous surface.…”

Crucial Role of the Aryl Hydrocarbon Receptor (AhR) in Indoxyl Sulfate-Induced Vascular Inflammation

“…To further elucidate the role of AhR in nuclear factor-B (NF-B) activation associated with E-selectin expression, we performed ChIP analysis of the E-selectin promoter region containing cytokine response region 34) . This region is located within the first 160 bp upstream of the transcriptional start site of the E-selectin gene and contains binding sites for NF-B and ATF-2/c-jun [41][42][43] .…”

“…Real-time quantitative reverse-transcriptase PCR was performed using a Thermal Cycler Dice Real Time System Single (Takara) 34) to quantitate the expression of Cyp1a1, Cyp1b1, PTGS2, CSF-2, and GAPDH mRNA using F, 5´-C| GAGCTC | GAGACAGAGTTTCGTCGAG-CCTTG-3´, NFELAM-AP1 F, 5´-C| GAGCTC | GAGA-CAGAGTTTCTGACTCATTG-3´, and NF-ELAM-CRE F, 5´-C| GAGCTC | GAGACAGAGTTTCTGAC-GTCATTG-3´, respectively (restriction sites are boxed and mutated nucleotides are underlined).…”

Humanβ-Defensin 3 Reduces TNF-α-Induced Inflammation and Monocyte Adhesion in Human Umbilical Vein Endothelial Cells

“…Further confirmation in animal models should be performed to uncover the underlying molecular mechanisms. In addition, cell density was identified to have an impact on the immune response of HUVECs [ 50 ].…”