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Cited by 267Nitric Oxide and Endothelin EffectsAtherosclerosis and Cardiovascular DiseasesCell Adhesion Molecules Research

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Trimethylamine N-oxide in atherogenesis: impairing endothelial self-repair capacity and enhancing monocyte adhesion

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Ma, GuoHua, Pan, Bing, Chen, Yue, Guo, CaiXia, Zhao, MingMing, Zheng, LeMin, Chen, BuXing·2017·Bioscience Reports
DOI 10.1042/bsr20160244PMC PMC5333780
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Abstract

Several studies have reported a strong association between high plasma level of trimethylamine N-oxide (TMAO) and atherosclerosis development. However, the exact mechanism underlying this correlation is unknown. In the present study, we try to explore the impact of TMAO on endothelial dysfunction. After TMAO treatment, human umbilical vein endothelial cells (HUVECs) showed significant impairment in cellular proliferation and HUVECs-extracellular matrix (ECM) adhesion compared with control. Likewise, TMAO markedly suppressed HUVECs migration in transwell migration assay and wound healing assay. In addition, we found TMAO up-regulated vascular cell adhesion molecule-1 (VCAM-1) expression, promoted monocyte adherence, activated protein kinase C (PKC) and p-NF-κB. Interestingly, TMAO-stimulated VCAM-1 expression and monocyte adherence were diminished by PKC inhibitor. These results demonstrate that TMAO promotes early pathological process of atherosclerosis by accelerating endothelial dysfunction, including decreasing endothelial self-repair and increasing monocyte adhesion. Furthermore, TMAO-induced monocyte adhesion is partly attributable to activation of PKC/NF-κB/VCAM-1.

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Figure 1 The effect of TMAO on cell viability HUVECs were exposed to different TMAO levels (0, 10, 50 and 100 μmol/l) for 24 h, cell toxicity assay was tested by MTT assay (A) and LDH assay test (B). Results are presented as the mean ± S.D. of three independent experiments.
Figure 1 The effect of TMAO on cell viability HUVECs were exposed to different TMAO levels (0, 10, 50 and 100 μmol/l) for 24 h, cell toxicity assay was tested by MTT assay (A) and LDH assay test (B). Results are presented as the mean ± S.D. of three independent experiments.CC BY · PubMed Central
Figure 2 Self-repair capacity of TMAO-treated HUVECs (A) HUVECs were treated with different doses of TMAO (0, 10, 20, 40, 80, 100 and 200 μmol/l) for 48 h. Relative cell proliferation was examined by BrdU ELISA and expressed as fold of TMAO-treated cells compared with control. (B) HUVECs were exposed to different TMAO levels (0, 10, 50 and 100 μmol/l) for 24 h, TMAO-stimulated HUVECs attached to ECM was measured, n=3. (C, E) HUVECs were incubated with TMAO at the indicated concentrations in the upper chamber for 24 h. Transwell assay was used to determine the cell migration and pictures were shown in high power (200×). Cell migration was quantified by cell counting. (D, F) Monolayer HUVECs were wounded by manual scraping and incubated with TMAO at the indicated concentrations for 24 h and then photographed in high power (100×). Cells migrating into the gaps were quantified by cell counting. Results are presented as the mean ± S.D. of three independent experiments; (*P<0.05, **P<0.01, ***P<0.001).
Figure 2 Self-repair capacity of TMAO-treated HUVECs (A) HUVECs were treated with different doses of TMAO (0, 10, 20, 40, 80, 100 and 200 μmol/l) for 48 h. Relative cell proliferation was examined by BrdU ELISA and expressed as fold of TMAO-treated cells compared with control. (B) HUVECs were exposed to different TMAO levels (0, 10, 50 and 100 μmol/l) for 24 h, TMAO-stimulated HUVECs attached to ECM was measured, n=3. (C, E) HUVECs were incubated with TMAO at the indicated concentrations in the upper chamber for 24 h. Transwell assay was used to determine the cell migration and pictures were shown in high power (200×). Cell migration was quantified by cell counting. (D, F) Monolayer HUVECs were wounded by manual scraping and incubated with TMAO at the indicated concentrations for 24 h and then photographed in high power (100×). Cells migrating into the gaps were quantified by cell counting. Results are presented as the mean ± S.D. of three independent experiments; (*P<0.05, **P<0.01, ***P<0.001).CC BY · PubMed Central
Figure 3 THP-1 cells adherence to HUVECs induced by TMAO is attributed to VCAM-1 up-regulation (A, B) HUVECs were starved for 6 h and treated with TMAO (0, 10, 50 and 100 μmol/l) for 6 h. Images represent the mean of adherent monocytes per microscopic field. Scale bar =50 μm. The results were normalized to the number of control cells. (C) HUVECs were treated with TMAO at the indicated concentrations as above for 6 h. VCAM-1, ICAM-1 and E-selectin expression in HUVECs were examined by cell ELISA. Results are represented as fold of TMAO-treated cells in comparison with control. (D) HUVECs were incubated with no antibody, negative control antibody and anti-VCAM-1 antibody for 30 min at 37°C before the adhesion assay, the fold change is showed. The results are presented as the mean ± S.D. of three or more independent experiments; (*P<0.05, **P<0.01).
Figure 3 THP-1 cells adherence to HUVECs induced by TMAO is attributed to VCAM-1 up-regulation (A, B) HUVECs were starved for 6 h and treated with TMAO (0, 10, 50 and 100 μmol/l) for 6 h. Images represent the mean of adherent monocytes per microscopic field. Scale bar =50 μm. The results were normalized to the number of control cells. (C) HUVECs were treated with TMAO at the indicated concentrations as above for 6 h. VCAM-1, ICAM-1 and E-selectin expression in HUVECs were examined by cell ELISA. Results are represented as fold of TMAO-treated cells in comparison with control. (D) HUVECs were incubated with no antibody, negative control antibody and anti-VCAM-1 antibody for 30 min at 37°C before the adhesion assay, the fold change is showed. The results are presented as the mean ± S.D. of three or more independent experiments; (*P<0.05, **P<0.01).CC BY · PubMed Central
Figure 4 TMAO injection promotes THP-1 adhesion to the aortic wall and increases VCAM-1 expression (A) TMAO treatment increased THP-1 cell adhesion to the aorta vascular wall. Results were shown as fluorescence in confocal microscopic images. Scale bars =250 μm. Images represent the immunohistochemical staining of VCAM-1 (B), ICAM-1 (C) and E-selectin (D) in mice aorta vascular ECs. Arrows in (B) represent VCAM-1-positive areas. Scale bars =50 µm, (n=4). (E) Quantification of adhesion on aorta, the result was expressed as areas of monocytes attached to aorta. Data are expressed as mean ± S.D. (F) Quantification of adhesion molecules expression, the fold change is presented as mean ± S.D.; (*P<0.05, **P<0.01, ***P<0.001).
Figure 4 TMAO injection promotes THP-1 adhesion to the aortic wall and increases VCAM-1 expression (A) TMAO treatment increased THP-1 cell adhesion to the aorta vascular wall. Results were shown as fluorescence in confocal microscopic images. Scale bars =250 μm. Images represent the immunohistochemical staining of VCAM-1 (B), ICAM-1 (C) and E-selectin (D) in mice aorta vascular ECs. Arrows in (B) represent VCAM-1-positive areas. Scale bars =50 µm, (n=4). (E) Quantification of adhesion on aorta, the result was expressed as areas of monocytes attached to aorta. Data are expressed as mean ± S.D. (F) Quantification of adhesion molecules expression, the fold change is presented as mean ± S.D.; (*P<0.05, **P<0.01, ***P<0.001).CC BY · PubMed Central
Figure 5 TMAO activated NF-κB in HUVECs HUVECs were treated with TMAO (0, 10, 50 and 100 μmol/l) for 6 h in serum-free media. (A) Effect of TMAO on the protein level of p-NF-κB was determined by Western blot. p-NF-κB relative expression ratios were determined by grey-scale image analysis. (B) Results are presented as the mean ± S.D. of six independent experiments, (*P<0.05, **P<0.01). (C) p-NF-κB was also detected by fluorescent staining (red fluorescence) in HUVECs on coverslips. Blue fluorescence represented nucleus. Fluorescent images were detected by confocal laser scanning microscopy. Scale bars =25 µm.
Figure 5 TMAO activated NF-κB in HUVECs HUVECs were treated with TMAO (0, 10, 50 and 100 μmol/l) for 6 h in serum-free media. (A) Effect of TMAO on the protein level of p-NF-κB was determined by Western blot. p-NF-κB relative expression ratios were determined by grey-scale image analysis. (B) Results are presented as the mean ± S.D. of six independent experiments, (*P<0.05, **P<0.01). (C) p-NF-κB was also detected by fluorescent staining (red fluorescence) in HUVECs on coverslips. Blue fluorescence represented nucleus. Fluorescent images were detected by confocal laser scanning microscopy. Scale bars =25 µm.CC BY · PubMed Central
Figure 6 TMAO activated NF-κB in HUVECs TMAO-induced endothelial dysfunction is associated with PKC activiation (A) HUVECs were treated with TMAO (0, 10, 50 and 100 μmol/l) for 6 h in serum-free media. Cell lysates from TMAO-treated cells were analysed by the PepTag non-radioactive PKC assay. HUVECs were treated with 100 μmol/l TMAO in the presence or absence of staurosporine (2.5 nmol/l) for 6 h and then p-NF-κB expression (B), VCAM-1 expression (D) and monocyte adhesion to HUVECs (E) were measured. The results are presented as the mean ± S.D. of three or more independent experiments (*P<0.05, **P<0.01).
Figure 6 TMAO activated NF-κB in HUVECs TMAO-induced endothelial dysfunction is associated with PKC activiation (A) HUVECs were treated with TMAO (0, 10, 50 and 100 μmol/l) for 6 h in serum-free media. Cell lysates from TMAO-treated cells were analysed by the PepTag non-radioactive PKC assay. HUVECs were treated with 100 μmol/l TMAO in the presence or absence of staurosporine (2.5 nmol/l) for 6 h and then p-NF-κB expression (B), VCAM-1 expression (D) and monocyte adhesion to HUVECs (E) were measured. The results are presented as the mean ± S.D. of three or more independent experiments (*P<0.05, **P<0.01).CC BY · PubMed Central
Figure 7 The likely molecular mechanism of TMAO-induced monocyte adhesion This pathway suggests that TMAO affects vascular endothelial ECs and subsequently stimulates activation of PKC. Then, NF-κB is activated by phosphorylation and translocates to the nucleus. NF-κB activation induces VCAM-1 expression and monocyte adhesion to the vascular wall.
Figure 7 The likely molecular mechanism of TMAO-induced monocyte adhesion This pathway suggests that TMAO affects vascular endothelial ECs and subsequently stimulates activation of PKC. Then, NF-κB is activated by phosphorylation and translocates to the nucleus. NF-κB activation induces VCAM-1 expression and monocyte adhesion to the vascular wall.CC BY · PubMed Central

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Metadata

Authors
Ma, G., Pan, B., Chen, Y., Guo, C., Zhao, M., Zheng, L., & Chen, B.
Affiliations
  • Department of Cardiology, Beijing Tian Tan Hospital, Capital Medical University, Beijing 100050, China
  • Peking University (CN)
  • Capital Medical University (CN)
Corresponding author
  • Zheng, LeMin
  • Chen, BuXing
ORCID
Venue
Bioscience Reports
Publisher
Portland Press Ltd.
Published
2017-03-02
Volume
37
Issue
2
ISSN
  • 0144-8463 (ISSN-L)
  • 1573-4935
DOI
10.1042/bsr20160244
PMC
PMC5333780
OpenAlex
W2583372114
Cited by
267
References
46
Citations by year
2026: 14 · 2025: 35 · 2024: 29 · 2023: 45 · 2022: 36 · 2021: 31 · 2020: 35 · 2019: 18 · 2018: 16 · 2017: 8
FWCI
15.59
Type
journal article
Language
en
Open access
Licence
Locations
  • Bioscience Reports (publishedVersion, cc-by)
  • Europe PMC (PubMed Central) (publishedVersion, cc-by)
  • PubMed Central (publishedVersion, cc-by)
  • PubMed (publishedVersion)
  • Greater South Information System
Funders
  • Academy of Medical Sciences
  • National Natural Science Foundation of China (81172500, 81170101, 81570321, 81370235)
  • Chinese Academy of Sciences
  • Chinese Academy of Medical Sciences
Topics
  • Health Sciences › Medicine › Physiology (medical) › Nitric Oxide and Endothelin Effects
  • Life Sciences › Immunology and Microbiology › Immunology › Atherosclerosis and Cardiovascular Diseases
  • Health Sciences › Medicine › Immunology and Allergy › Cell Adhesion Molecules Research
Keywords
trimethylamine N-oxide, atherosclerosis, endothelial dysfunction, monocyte adhesion, VCAM-1, protein kinase C, NF-κB, endothelial cell migration, endothelial cell proliferation, extracellular matrix adhesion, human umbilical vein endothelial cells
MeSH terms
Cell Adhesion, Cells, Cultured, Humans, Methylamines, Monocytes, Protein Kinase C, Signal Transduction, NF-kappa B, Vascular Cell Adhesion Molecule-1, Atherosclerosis, Human Umbilical Vein Endothelial Cells

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