Adipose Inflammation Initiates Recruitment of Leukocytes to Mouse Femoral Artery: Role of Adipo-Vascular Axis in Chronic Inflammation

Background: Although inflammation within adipose tissues is known to play a role in metabolic syndrome, the causative connection between inflamed adipose tissue and atherosclerosis is not fully understood. In the present study, we examined the direct effects of adipose tissue on macro-vascular inflammation using intravital microscopic analysis of the femoral artery after adipose tissue transplantation. Methods and Results: We obtained subcutaneous (SQ) and visceral (VIS) adipose tissues from C57BL/6 mice fed normal chow (NC) or a high fat diet (HF), then transplanted the tissues into the perivascular area of the femoral artery of recipient C57/BL6 mice. Quantitative intravital microscopic analysis revealed an increase in adherent leukocytes after adipose tissue transplantation, with VIS found to induce significantly more leukocyte accumulation as compared to SQ. Moreover, adipose tissues from HF fed mice showed significantly more adhesion to the femoral artery. Simultaneous flow cytometry demonstrated upregulation of CD11b on peripheral granulocyte and monocytes after adipose tissue transplantation. We also observed dominant expressions of the inflammatory cytokine IL-6, and chemokines MCP-1 and MIP-1b in the stromal vascular fraction (SVF) of these adipose tissues as well as sera of recipient mice after transplantation. Finally, massive accumulations of pro-inflammatory and dendritic cells were detected in mice with VIS transplantation as compared to SQ, as well as in HF mice as compared to those fed NC

Cell density impacts epigenetic regulation of cytokine-induced E-selectin gene expression in vascular endothelium.

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 × 10(3) cells/cm(2) and 29.2 × 10(3) cells/cm(2), 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

Effect of cell density on histone H3 acetylation at the E-selectin promoter.

<p>Schema of the E-selectin gene and the location of the primers used in the ChIP assay. Chromatin was prepared from sparse and confluent HUVECs monolayers treated with (+) or without (−) 1 ng/ml TNFα for 30 min, and the ChIP assay was performed using the anti-acetyl histone H3 (AcH3) antibody. Real-time PCR analysis of the ChIP samples was performed using primers designed to amplify the E-selectin promoter region (−195 to −67), which contains the cytokine response region (CRR) and the E-selectin extreme 3′ region (+13914 to +13977) (*<i>p</i><0.01). The values are expressed as mean ± SD of three independent experiments.</p

Effect of cell density on E-selectin protein expression levels in HUVECs.

<p>(<b>A</b>) HUVECs were cultured as described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0090502#s2" target="_blank">Materials and Methods</a>. Photomicrographs were obtained using phase-contrast microscopy (20× magnification). (<b>B</b>) E-selectin protein expression levels under the sparse (S) and confluent (C) conditions. HUVECs cultured under both the conditions were treated with (+) or without (−) 1 ng/ml TNFα for 4 h, and total cell extracts were prepared. Equal amounts of protein were separated using SDS-PAGE, and western blot analysis was performed using the anti-E-selectin antibody (Santa Cruz Biotechnology). (<b>C</b>) Flow cytometric analysis of E-selectin cell surface expression. HUVECs were stimulated with or without 1 ng/ml TNFα for 4 h, and flow cytometry was performed using the anti-E-selectin antibody (clone 7A9) (*<i>p</i><0.05). The values are expressed as mean ± SD of three independent experiments.</p

7-Ketocholesterol enhances leukocyte adhesion to endothelial cells via p38MAPK pathway.

7-Ketocholesterol is a major dietary cholesterol oxidation product found in high concentrations in atherosclerotic plaques, which contribute to the development of atherosclerosis. This study aimed to investigate the effects of 7-ketocholesterol on endothelial inflammation, as well as the underlying mechanisms. Pretreatment of human umbilical vein endothelial cells (HUVEC) with 7-ketocholesterol significantly enhanced the total interactions between human monocytic cells (THP-1 cell line) and TNFα-activated HUVECs under physiological flow conditions, compared to pretreatment with cholesterol (TNFα+50 μM cholesterol: 13.1 ± 0.54 cells/CPF, TNFα+50 μM 7-ketocholesterol: 18.9 ± 0.35 cells/CPF, p < 0.01). 7-Ketocholesterol enhanced the expression of E-selectin, ICAM-1, and VCAM-1 proteins. It also activated p38 mitogen-activated protein kinase (MAPK), and treatment with a p38 MAPK inhibitor inhibited both E-selectin expression via ATF-2 activation and 7-ketocholesterol-induced THP-1 adhesion to HUVECs. These findings suggest that 7-ketocholesterol enhances leukocyte-endothelial interactions by upregulating the expression of adhesion molecules, presumably via the p38 MAPK-dependent pathway

Effect of cell density on TNFα-induced MAPK and NF-kB activation in HUVECs.

<p>Western blot of phosphorylated (p) and total JNK, p38 MAPK (<b>A</b>), and NF-κB p65 (<b>B</b>). HUVECs were stimulated with (+) or without (−) 1 ng/ml TNFα for 15 min. (<b>C</b>) p65 was detected using western blot analysis of the nuclear and cytoplasmic fractions prepared from HUVECs treated with or without 1ng/ml TNFα for 30 min. Representative western blots are shown.</p

Chromatin accessibility differed between sparse and confluent HUVEC monolayers.

<p>CHART-PCR of the E-selectin promoter region. The location of primers used in CHART-PCR assays is shown. Nuclei were isolated from sparse and confluent HUVECs monolayers treated with (+) or without (−) 1 ng/ml TNFα for 60 min. The isolated nuclei were incubated with 10 U of micrococcal nuclease for 10 min at room temperature. Genomic DNA was extracted and quantified using real-time PCR relative to DNA prepared from undigested nuclei (*<i>p</i><0.05, **<i>p</i><0.01). The values are expressed as mean ± SD of three independent experiments. CRR indicates cytokine response region.</p

Effect of cell density on E-selectin mRNA expression and mRNA stability in HUVECs.

<p>(<b>A</b>) Relative E-selectin mRNA levels measured using quantitative RT-PCR. RNA was isolated 2 h after stimulation with 1 ng/ml TNFα and 5 ng cDNA was used in each reaction (*<i>p</i><0.05 vs sparse). (<b>B</b>) E-selectin mRNA stability in TNFα-activated HUVECs. HUVECs were stimulated with 1 ng/ml TNFα for 2 h and treated with actinomycine D (5 µg/ml) for the indicated time periods. RNA was subjected to real-time RT-PCR. For each condition, E-selectin expression levels were normalized against those of GAPDH. The values were expressed proportional to that at baseline (time zero). The values are expressed as mean ± SD of three independent experiments.</p