p-Cresyl sulfate promotes the formation of atherosclerotic lesions and induces plaque instability by targeting vascular smooth muscle cells

Abstract

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

Introduction

Cardiovascular disease (CVD) is the leading cause of death among patients with chronic kidney disease (CKD), particularly patients undergoing long-term dialysis [ 1]. Coronary atherosclerosis is a major cardiovascular complication, which is of urgent concern and is predictive of a dramatically elevated mortality risk [ 2]. Researchers and clinicians have been coordinating to explore the nature of this disorder in patients with CKD. However, the pathogenesis of this complex process, which is often a consequence of the mutual promotion of renal and cardiovascular dysfunction, is not fully understood [ 3].

The discovery of protein-bound uremic toxins enhances the understanding of the complex relationship between CKD and coronary atherosclerosis [ 4, 5]. p-Cresyl sulfate (PCS) is a protein-bound solute that accumulates in CKD patients [ 6]. Routine dialysis is inefficient in removing this toxin because approximately 94% of PCS is bound to plasma proteins in circulation [ 7]. Organic anion transporters (OATs) play a key role in mediating the intracellular influx of PCS, which tends to facilitate various imbalances and dysfunctions in several types of cells [ 8]. The effects of PCS on the cardiovascular system have also been the focus of considerable attention because clinical investigations have repeatedly proven that the serum level of PCS is related to cardiovascular mortality in CKD [ 9, 10]. However, the underlying mechanisms remain to be elucidated.

The migration and proliferation of vascular smooth muscle cells (VSMCs) is an early event during the initiation and progression of atherosclerosis, which is the underlying etiology of most CKD-related CVDs [ 11]. In addition, the apoptosis of VSMCs is a potential contributor to atherosclerotic plaque erosion and rupture at advanced stages of the disease [ 12]. OATs are highly expressed in smooth muscle cells and may mediate excess influx of PCS into VSMCs during advanced stages of CKD [ 13, 14]. The intracellular accumulation of PCS might provoke malfunctions of VSMCs, thus accelerating atherogenesis and destabilizing mature plaques.

A series of in vivo and in vitro experiments were conducted to investigate the effects of excess PCS, a prototype of protein-bound uremic toxins, on the formation and vulnerability of atherosclerotic plaques.

Materials and methods

Chemicals and materials

PCS was synthesized using the method previously described by Feigenbaum and Neuberg [ 15]. The identity and purity (>99%) of PCS were confirmed by nuclear magnetic resonance spectroscopy. Antibodies for the detection of the proliferating cell nuclear antigen (PCNA), α-smooth muscle actin (α-SMA), matrix metalloproteinase-2 (MMP-2), MMP-9, tissue inhibitor of metalloproteinase-1 (TIMP-1), TIMP-2, monocyte/macrophage (MOMA-2), B cell lymphoma 2 (Bcl-2), Bcl-2-associated X protein (Bax), and β-actin were purchased from Abcam (Cambridge, UK). Radioimmunoprecipitation assay buffer (RIPA lysis buffer), phenylmethanesulfonyl fluoride (PMSF), and BCA protein assay kit were purchased from Beyotime (Shanghai, China).

Animals

Pathogen-free ApoE^−/− mice (C57BL/6 background, male, eight weeks old) were purchased from the Model Animal Research Center of Nanjing University (Nanjing, China). All mice were conventionally housed in standard cages and kept at 21±2 °C and 50%±15% relative humidity under a 12 h light/dark cycle. All animals were fed with a high-fat diet (0.25% cholesterol and 15% cocoa butter) and sterile water ad libitum throughout the entire experiment.

All experimental mice underwent 5/6 nephrectomy with a two-step surgical procedure or a sham operation after one week of accommodation. Anesthetized mice underwent removal of approximately two thirds of the left kidney and right nephrectomy after one week. Sham-operated mice underwent laparotomy in parallel by decapsulating the kidney before wound closure. Then, the mice were randomly divided into two groups, namely, vehicle-treated group (n = 20) with oral gavage of water and PCS-treated group (n = 20) with oral gavage of PCS in water. The concentration of PCS was adjusted for a daily intake of 100 mg/kg. Mice were starved for 12 h after 8 or 24 weeks of high-fat diet and then killed. Blood samples were taken from the inferior vena cava. The hearts and aortas were dissected for en face analysis and cryosections. Tissue samples for Western blot analysis were immediately placed in liquid nitrogen and then kept at −80 °C until use.

Histopathology and immunohistochemistry

The aortas were en face stained with oil red O. The percentage of lesion coverage was calculated by dividing the positively stained area by the total aortic surface. Sections (5 µm thick) of the aortic roots were used for hematoxylin and eosin (H&E), oil red O, and Sirius red staining, or immunohistochemical analysis with the following antibodies: anti-MOMA-2 (1:50), anti-α-SMA (1:50), anti-MMP-2 (1:50), and anti-MMP-9 (1:50) antibodies. Sections were incubated with 3,3′-diaminobenzidine after incubation with horseradish peroxidase (HRP)-conjugated secondary antibodies (1:100). The expression of PCNA was assayed by immunofluorescence staining. The sections were immunostained with anti-PCNA antibody (1:100) for 12 h at 4 °C and incubated with Alex 488-conjugated secondary antibody (1:1000).

Images were captured with an Olympus microscope and quantified using Image-Pro Plus 6.0 (Media Cybernetics, Bethesda, Maryland, USA). The relative content of lipids, VSMCs, collagen, macrophages, and MMPs were quantified as the ratio of positively stained area to the total plaque section in at least 10 high-power fields (400×). The vulnerability index was calculated by the following formula: (macrophage staining% + lipid staining%)/(VSMC staining% + collagen staining%).

Cell culture

Rat aortic VSMCs were isolated as described previously [ 16] and cultured in Dulbecco’s modified Eagle’s medium (DMEM) high glucose (Gibco, Invitrogen, Grand Island, NY, USA) containing 10% fetal bovine serum (FBS), 100 U/ml penicillin, 10 mg/ml streptomycin, and 2 mmol/L glutamine at 37 °C under 5% CO₂. Cells from passages 4 to 8 at 80% confluence in culture wells were used after 24 h of serum depletion.

Biochemical investigation

The serum lipid profile of total cholesterol (TC), triglycerides (TG), low-density lipoprotein cholesterol (LDL-C), and high-density lipoprotein cholesterol (HDL-C) were detected enzymatically by kits (Shanghai Rongsheng Biotech Co., Ltd., Shanghai, China).

The serum PCS concentration was analyzed using high-performance liquid chromatography tandem mass spectrometry as described previously [ 17].

Measurement of cell proliferation

Cell Counting Kit-8 (Dojindo, Kumamoto, Japan) was used to measure VSMC proliferation in accordance with the manufacturer’s instructions. The cells were plated onto 96-well plates and divided into two groups, namely, vehicle group treated with phosphate buffer solution (PBS) and PCS group treated with 500 µmol/L PCS. The concentration of PCS was selected based on the results of a previous work [ 18]. Cell Counting Kit-8 solution (10 µl) was added to the medium after 24 h of incubation. The resulting solution was incubated for 2 h in an incubator with 5% CO₂. The amount of orange formazan dye produced was calculated by measuring the absorbance at 450 nm in a microplate reader.

Measurement of cell migration

Cell migration was assessed using Transwell plates (Millipore, Billerica, MA, USA), with 6.5 mm diameter and 8 µm pore filters. VSMCs were divided into the following groups: vehicle-treated group with serum-free DMEM; PCS-treated group with 500 µmol/L PCS; and FBS-treated group with 10% FBS. Equal numbers of VSMCs (1.0 × 10⁶ cells) were added to Transwell plates for each group. Cells that did not migrate from the upper side of the filter after 24 h of incubation at 37 °C were scraped off with a cotton swab. The filters were fixed and stained with crystal violet. The number of cells that migrated to the lower side of the filter was determined under a light microscope at a magnification of 400×in five randomly selected fields.

Flow cytometry analysis

Cell apoptosis was measured with fluorescein isothiocyanate-labeled human recombinant annexin V (annexin V-FITC) and propidium iodide (PI) using a detection kit (BD Pharmingen, San Diego, CA, USA), according to the manufacturer’s instructions. The cells were harvested, washed once with cold PBS, and resuspended in binding buffer at a concentration of 1.0 × 10⁶ cells/ml. Then, 100 ml of the solution (1.0 × 10⁵ cells) was incubated with 5 ml annexin V-FITC and 5 ml of PI solution for 15 min in the dark at room temperature. Samples were analyzed using flow cytometry (BD FACSCalibur; BD Biosciences, San Jose, CA, USA) within 1 h after the addition of 400 µl of binding buffer was added to the samples. Apoptotic cells (stained positive for annexin V-FITC and stained negative or positive for PI) were counted and presented as a percentage of the total cell counts.

Western blot analysis

Cell and tissue lysates were prepared in RIPA lysis buffer containing 1% PMSF. The homogenates were centrifuged at 14 000×g for 30 min at 4 °C. Then, the supernatant was collected, and the protein concentrations were assayed using a BCA protein assay kit. The supernatant was mixed with loading buffer and heated in a boiling water bath for 10 min. Equal amounts of prepared proteins were subjected to SDS-PAGE and blotted onto polyvinylidene fluoride membranes. The membranes were blocked and probed overnight at 4 °C with antibodies against MMP-2 (1:1000), MMP-9 (1:1000), Bcl-2 (1:1000), Bax (1:1000), and β-actin (1:2000), followed by incubation with HRP-conjugated secondary antibodies (1:5000) for 1 h at room temperature. Immunoreactive bands were detected using an enhanced chemiluminescense (ECL) system (Millipore, Billerica, MA, USA) and quantified by Image-Pro Plus 6.0.

Gelatin zymography

The activities of MMP-2 and MMP-9 were analyzed using gelatin zymograms. Equal amounts of prepared proteins were separated by electrophoresis on 10% SDS-PAGE gels containing 1 mg/ml gelatin. The gels were renatured by washing in 2.5% Triton X-100 solution twice for 30 min after electrophoresis. The gels were incubated in 50 mmol/L Tris-HCl (pH 7.4), 5 mmol/L CaCl₂, and 1 µmol/L ZnCl₂ at 37 °C overnight. After incubation, the gels were stained with 0.05% Coomassie brilliant blue R-250 for 30 min at room temperature and then destained in distilled water.

Statistical analysis

Continuous data are expressed as mean±standard error of the mean (SEM). Statistical significance was determined with one-way ANOVA followed by Student–Newman–Keuls post hoc analysis. P<0.05 was considered statistically significant. Data were analyzed using GraphPad Prism 5 (GraphPad Software Inc., San Diego, CA, USA) and SPSS software (version 13.0; IBM Corp., Armond, NY, USA).

Results

PCS administration had no effect on body weight or serum lipid profiles

No significant differences in body weight and serum lipid profiles between the vehicle-treated and PCS-treated mice during the in vivo study (8 and 24 weeks) were observed. This result indicates that long-term administration of PCS did not markedly disturb these parameters (Table 1).

PCS promoted the growth of aortic atherosclerotic plaques

En face analysis was conducted on the aortic surface after eight weeks of high-fat diet. The oil red O-positive area was significantly larger in PCS-treated mice than in vehicle-treated mice (vehicle, 6.53±0.75; PCS, 19.23±0.98; P<0.05; Fig. 1A and 1C). Similarly, the relative cross-sectional area of the aortic lesion also showed a dramatic increase in the PCS-treated group (vehicle, 16.43±2.89; PCS, 48.48±3.21; P<0.05; Fig. 1B and 1D). These results indicate that the accumulation of PCS may promote atherosclerotic lesion formation in ApoE^−/− mice.

PCS enhanced plaque instability

The aortic plaque components on the sections of the aortic roots were assessed after 24 weeks of treatment. PCS-treated mice possessed aortic plaques with fewer VSMCs and less collagen than vehicle-treated mice. However, PCS-treated mice had more lipids and macrophages; thus, they had a higher plaque vulnerability index. This result indicates that long-term administration of PCS had altered plaque phenotype toward an unstable form (Fig. 2).

PCS activated the migration and proliferation of VSMCs

The migration of VSMCs was measured using Transwell plates. The number of migrated VSMCs was greater in the PCS-treated group than that in the vehicle-treated group (vehicle, 4.67±1.45 cells/high-power field; PCS, 38.67±3.53 cells/high-power field; P<0.05). The FBS group, as positive control, reached 54.33±3.18 cells/high-power field (Fig. 3A and 3C).

PCNA was measured after eight weeks of treatment using immunofluorescence staining in the aortic plaques. The relative expression of PCNA in the PCS-treated mice was significantly higher than that in the vehicle-treated mice (vehicle, 20.67%±3.18%; PCS, 45.33%±2.96%; P<0.05; Fig. 3B and 3D).

VSMC proliferation was also measured in vitro using a colorimetric assay. The absorbance at 450 nm was greater in the PCS-treated VSMCs than that in the vehicle-treated cells (vehicle, 1.47±0.13; PCS, 2.10±0.12; P<0.05; Fig. 3E).

PCS caused an imbalance between MMPs and TIMPs

PCS stimulation in the in vivo study markedly upregulated MMP-2 and MMP-9 but downregulated TIMP-1 and TIMP-2 in the aortic samples (Fig. 4A and 4D). Similarly, the protein expressions of MMP-2 and MMP-9 within the aortic plaques were markedly higher in the PCS-treated mice than that in the vehicle-treated mice after 24 weeks of treatment (MMP-2: vehicle, 4.04±0.89; PCS, 11.87±0.88; P<0.05; MMP-9: vehicle, 6.57±0.72; PCS, 9.97±1.30; P<0.05; Fig. 4B, 4E, and 4F). In addition, gelatin zymographic analysis showed that PCS significantly increased the activities of MMP-2 and MMP-9 (Fig. 4C, 4G, and 4H).

PCS facilitated the apoptosis of VSMCs

The effect of PCS on apoptosis in VSMCs was assessed using flow cytometry. Annexin V-FITC and PI were used to determine whether the cells were viable (negative for both annexin V-FITC and PI), damaged (positive for annexin V-FITC only), or dead (positive for both annexin V-FITC and PI or for PI only). Quantitative analysis showed that PCS significantly increased the percentage of apoptotic VSMCs after 24 h of treatment (vehicle, 8.69%±0.77%; PCS, 12.39%±0.62%; P<0.05; Fig. 5A and 5C).

The balance between death agonists (Bax and Bak) and antagonists (Bcl-2 and Bcl-Xl) in the Bcl-2 protein family plays a pivotal role in apoptosis. Blots from the present study revealed a significant upregulation of Bax and downregulation of Bcl-2 expression following exposure to PCS (Fig. 5B and 5D).

Discussion

Continuous accumulation of PCS accelerated the development of atherosclerotic lesions in the mouse model of atherosclerosis by promoting the migration and proliferation of VSMCs. Moreover, long-term oral gavage of PCS disrupted the stability of plaques by inducing the imbalance of MMPs/TIMPs and apoptosis in VSMCs with the progress of atherosclerosis. To our knowledge, this study is the first to provide experimental evidence that the atherogenic effects of PCS involve targeting VSMCs.

The pathogenesis of atherosclerosis is a chronic inflammatory response within the arterial wall [ 19, 20]. This process involves the migration of VSMCs from the media into the vascular intima where these cells proliferate as the local inflammation persists. The mobilized VSMCs transitions into the synthetic state on a massive scale, exacerbating local inflammation and thus creating a positive feedback cycle [ 21]. In the present study, PCS was observed to promote this course of development. The accelerated plaque growth might be partially attributed to the increasing infiltration of synthetic VSMCs into the arterial intima. In the synthetic state, VSMCs may synthesize large quantities of extracellular matrix (ECM) protein [ 22]. The arrival of VSMCs and their elaboration of ECM probably produce fibro-fatty lesions in places with simple accumulation of macrophage-derived foam cells [ 23].

PCS also markedly disturbed the balance between MMPs and TIMPs within the plaques. The group of calcium-dependent, zinc-containing endopeptidases, MMPs, whose activities are inhibited by specific endogenous TIMPs, can degrade all kinds of ECM proteins [ 24]. MMPs/TIMPs play a vital role in regulating the degradation of ECM proteins [ 25]. Thus, our results indicate that the composition of atherosclerotic plaques was altered by PCS administration.

Tissue remodeling within atherosclerotic lesions is a feature of the progression of vulnerable plaques, the erosion and rupture of which contribute to the incidence of acute coronary syndrome [ 26]. Histological analysis of plaque components in the aortic sections showed that PCS treatment resulted in a significant increase in macrophages and lipids as well as a substantial decrease in collagen and VSMCs in the aortic plaques. Thus, PCS-treated mice exhibited increased vulnerability indices, which have been recognized as a histological marker of vulnerable plaques [ 27, 28]. These observations were consistent with clinical findings that patients with advanced-stage CKD are more likely to suffer acute cardiovascular events than the general population [ 29]. In addition to evoking the migration and proliferation of VSMCs, PCS also facilitates the apoptosis of these cells, which is considered an important mode for the loss of VSMCs in plaques [ 30]. Thus, PCS was observed to disturb plaque components and contribute to plaque instability.

In conclusion, the uremic toxin PCS accelerated the progression of atherosclerosis and weakened the stability of formed plaques by targeting VSMCs. Thus, VSMCs migrated into atherosclerotic lesions and continuously proliferated locally. Subsequent alteration of plaque contents and increased apoptosis of VSMCs further harmed the stability of formed plaques. This investigation may provide a novel perspective on the mechanisms of CKD-related cardiovascular complications.

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