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Abstract
Intestinal microbiota is involved in the atherosclerotic process by development of an atheromatous core with foam cells in carotid arteries. It has reported that lipopolysaccharide (LPS) from Escherichia colilocalizes in human atherosclerotic plaque and causes inflammation via interaction with toll like receptor 4. However, there is no evidence that whether LPS-activated macrophages regulate endothelial cell (EC) function. We evaluated whether LPS-activated macrophage acts as one of the stimulants activating EC and its underlying signaling pathways. Using Western blotting and quantitative reverse transcription-polymerase chain reaction (qRT-PCR), we confirmed that intraperitoneal injection with LPS increases iNOS protein and inflammatory cytokine, TNF-α and IL-6 mRNA expressions. To determine whether LPS-mediated macrophage inflammatory condition affects EC activation and inflammation, human umbilical vein endothelial cells (HUVECs) were incubated with isolated peritoneal macrophages from LPS-injected mice. Interestingly, p90RSK Serine 380 phosphorylation and protein expression were significantly increased by macrophage treatment in EC. Messenger RNA levels of vascular cell adhesion molecule 1 and p90RSK was increased, but endothelial nitric oxide synthase was decreased. In addition, NF-κ B promoter activity, which plays an important role in the pathogenesis of inflammation, was strongly enhanced by the macrophage treatment in EC. We further evaluated the effects of LPS on EC function in the mouse aorta using en facestaining. In agreement with in vitroresult, p90RSK expression was strongly increased in the steady laminar flow region of the mouse aorta in mice injected with LPS. Together, our study demonstrates that p90RSK might be a one of the major therapeutic candidates for the prevention of vascular diseases mediated by LPS.
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Figure 1.
The expression of iNOS and inflammatory cytokines in LPS-activated peritoneal macrophages. LPS was i.p.injected with indicated doses in C57BL/6 mice. After 6 hrs, peritoneal macrophages were isolated and then assessed iNOS protein expression by western blotting using specific antibody (A) and TNF-α and IL-6 mRNAs by qRT-PCR using specific primers (B). Bar graph represents the quantification of relative fold changes of iNOS protein expression compared to the control in three independent experiments (0, A, lower panel). Data present mean ± SEM. ∗p< 0.05 and ∗∗p< 0.01, compared with control (0, PBS treated) by 1-way ANOVA followed by Bonferroni's post hoc test.
Figure 2.
The effect of LPS-activated macrophages on EC functions. The HUVECs were co-cultured with macrophages isolated from 5 mg/kg LPS-injected mice for indicated times. (A-B) Phospho-p90RSK and total-p90RSK were determined by western blotting using specific antibody, respectively. (C-D) Total RNAs were isolated and qPT-PCR for VCAM-1, p90RSK, and eNOS mRNA levels were performed. (E) HUVECs were transfected with NF-κ B-lucand pRT-tkusing Lipofectamine 2000. The cells were co-cultured with the LPS-activated macrophages for indicated times and then promoter assay was performed. Data present mean ± SEM. ∗p< 0.05 and ∗∗p< 0.01, compared with control (0, PBS treated) by 1-way ANOVA followed by Bonferroni's post hoc test.
Figure 3.
LPS stimulation increases p90RSK expression in aortic arch. After stimulation with 0 (PBS), 5, and 10 mg/kg LPS for 6 hrs, en facepreparations of the aortic arch of 7-week C57BL/6 mice were processed. The samples were double-stained with anti-VE-Cad (as an EC marker) and anti-p90RSK. Images were recorded using a confocal microscope equipped with a Planapo x60 1.42 NA oil objective lens. Scale bars: 10 μm. Bar graph indicates quantification of percentage of p90RSK fluorescence intensity. Data present mean ± SEM. ∗∗p< 0.01, compared with control (PBS treated) by 1-way ANOVA followed by Bonferroni's post hoc test.
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