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Frontiers of Medicine

ISSN 2095-0217

ISSN 2095-0225(Online)

CN 11-5983/R

Postal Subscription Code 80-967

2018 Impact Factor: 1.847

Front. Med.    2016, Vol. 10 Issue (3) : 320-329     DOI: 10.1007/s11684-016-0463-x
p-Cresyl sulfate promotes the formation of atherosclerotic lesions and induces plaque instability by targeting vascular smooth muscle cells
Hui Han1,2,Yanjia Chen1,2,Zhengbin Zhu1,Xiuxiu Su1,Jingwei Ni1,Run Du1,Ruiyan Zhang1,2,*(),Wei Jin1,*()
1. Department of Cardiology, Ruijin Hospital Affiliated to Shanghai Jiao Tong University School of Medicine, Shanghai 200025, China
2. Institute of Cardiovascular Diseases, Shanghai Jiao Tong University School of Medicine, Shanghai 200025, China
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Coronary atherosclerosis is a major complication of chronic kidney disease. This condition contributes to the increased mortality in dialysis patients. p-Cresyl sulfate (PCS) is a prototype of protein-bound uremic toxins that cannot be efficiently removed through routine dialysis procedures. In the present study, ApoE−/− mice that underwent 5/6 nephrectomy were randomly divided into two groups, namely, vehicle-treated group (n = 20) and PCS-treated group (n = 20). Mice were sacrificed for en face and immunohistological analyses after 8 or 24 weeks of high-fat diet. Rat aortic vascular smooth muscle cells (VSMCs) were treated with phosphate buffer solution or 500 µmol/L PCS for in vitro evaluation. PCS-treated mice were observed to suffer increased atherosclerotic lesions after eight weeks of PCS administration. Moreover, 24 weeks of PCS administration also markedly increased the vulnerability index of aortic plaques. PCS was also observed to facilitate the migration and proliferation of VSMCs during the progression of the disease. Moreover, PCS disturbed the balance between matrix metalloproteinases and tissue inhibitor of metalloproteinases within the plaques. Thus, PCS played a vital role in promoting atherogenesis and disturbing the stability of formed plaques probably by targeting VSMCs.

Keywords p-cresyl sulfate      atherosclerosis      plaque stability      vascular smooth muscle cell     
Corresponding Authors: Ruiyan Zhang,Wei Jin   
Just Accepted Date: 19 July 2016   Online First Date: 12 August 2016    Issue Date: 30 August 2016
URL:     OR
Variables 8 weeks 24 weeks
Vehicle mice PCS mice Vehicle mice PCS mice
Body weight (g) 27.50±0.67 27.10±0.67 37.30±0.76 36.75±0.70
TG (mmol/L) 1.43±0.20 2.07±0.35 1.37±0.12 2.27±0.34
TC (mmol/L) 3.35±1.33 3.23±0.15 4.00±0.12 4.17±0.15
HDL-C (mmol/L) 1.70±0.11 1.75±0.97 1.54±0.16 1.62±0.19
LDL-C (mmol/L) 2.39±0.14 2.37±0.06 2.84±0.82 2.93±0.24
SCr (µmol/L) 66.00±8.11 72.33±8.58 75.67±6.44 86.17±7.98
PCS (µg/ml) 0.68 (0.63–0.74) 5.54 (4.29–6.94)# 0.72 (0.66–0.79) 5.83 (4.35–7.40)#
Tab.1  Characteristics of the animal models
Fig.1  Mice treated with p-cresyl sulfate (PCS) showed increased atherosclerotic plaque areas after eight weeks of PCS administration. (A) Representative aortic images of en face staining with oil red O in vehicle-treated (vehicle) and PCS-treated (PCS) mice. (B) Representative sectional images of aortic roots staining with H&E and oil red O in vehicle and PCS mice (bar= 200 µm). (C and D) Quantitative analysis of lesion areas for en face and cross-sectional staining. Data are presented as mean±SEM (n = 6–8). # P<0.05 compared with vehicle.
Fig.2  Mice treated with PCS show altered plaque contents and increased plaque vulnerability index after 24 weeks of treatment. (A) Representative sectional images of aortic roots stained with oil red O, MOMA-2, α-SMA, and Sirius red in vehicle-treated (vehicle) and PCS-treated (PCS) mice (bar= 100 µm). (B) Quantitative analysis of positively stained areas for oil red O, MOMA-2, α-SMA, and Sirius red in vehicle and PCS mice. (C) Quantification of vulnerability index in vehicle and PCS mice. Data are presented as mean±SEM (n = 6–8). # P<0.05 compared with vehicle. MOMA-2, monocyte/macrophage; α-SMA, α-smooth muscle actin.
Fig.3  PCS treatment promotes the migration and proliferation of VSMCs. (A) The number of cells that migrated through the pores was determined in high-power fields for each group. (B) PCNA expression in mice treated with vehicle and PCS (bar= 100 µm; blue, DAPI-labeled cell nuclei; green, PCNA; red, α-actin). (C) Quantitative analysis of migrated cells through the pores. (D) Quantitative analysis of PCNA-positive cells in the sections of aortic roots. (E) Colorimetric assay of the absorbance at 450 nm for each group. Data are presented as mean±SEM (n = 6). # P<0.05 compared with vehicle. PCNA, proliferating cell nuclear antigen; DAPI, 4′,6-diamidino-2-phenylindole.
Fig.4  PCS treatment disturbs the balance between MMPs and TIMPs. (A) Representative blots of aortic samples in vehicle-treated (vehicle) and PCS-treated (PCS) mice. (B) Representative sectional images of aortic roots staining with MMP-2 and MMP-9 in vehicle and PCS mice after 24 weeks of treatment (bar= 200 µm). (C) Representative gelatin zymography of aortic samples in vehicle and PCS mice. (D) Quantitative analysis of blots of aortic samples in vehicle and PCS mice. (E and F) Quantitative analysis of positively stained areas for MMP-2 and MMP-9 in vehicle and PCS mice. (G and H) Quantitative analysis of gelatin zymography of aortic samples in vehicle and PCS mice. Data are presented as mean±SEM (n = 6). # P<0.05 compared with vehicle. MMP, matrix metalloproteinase; TIMP, tissue inhibitor of metalloproteinase; VSMC, vascular smooth muscle cell.
Fig.5  PCS treatment facilitates the apoptosis of VSMCs in vitro. (A and C) Evaluation of apoptotic incidence by flow cytometry analysis via double staining with annexin V-FITC and PI. (B) Representative blots showing the expression of Bax and Bcl-2. (D) Quantitative analysis of blots in VSMCs. Data are presented as mean±SEM (n = 6). # P<0.05 compared with vehicle. Annexin V-FITC, fluorescein isothiocyanate-labeled human recombinant annexin V; PI, propidium iodide; Bcl-2, B cell lymphoma 2; Bax, Bcl-2-associated X protein.
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