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Introduction

Chronic kidney disease (CKD) carries a high risk of mortality, the immediate cause of which is usually cardiovascular complications (1,2). The main complications observed in patients with CKD include impaired angiogenesis, atherosclerosis, arterial stiffness, vascular calcifications and neointimal hyperplasia (3,4). Endothelial progenitor cell (EPC) dysfunction is a key contributor to this pathogenesis, as the potential of EPCs to promote vascular repair by differentiating into endothelial cells is impaired (5).

Among the factors potentially influencing EPC function in patients with CKD, the constituents of the uremic milieu, particularly protein-bound uremic toxins, are likely to be important, since they cannot be removed by dialysis therapy (6–8). Para-cresol (p-cresol) is a protein-bound uremic toxin that originates from bacterial amino acid L-tyrosine fermentation in the large intestine mucosa (9). P-cresol has been demonstrated to inhibit proliferation of various cell types in vitro, including EPCs (10–12), indicating that p-cresol may be among the factors mediating the high incidence of cardiovascular complications in patients with CKD. However, the mechanism remains unclear.

Reactive oxygen species (ROS) such as superoxide anion and hydrogen peroxide have been demonstrated to function as signaling molecules that regulate EPC proliferation (13). P-cresol has antioxidant activity, and a previous study has demonstrated that its antiproliferative effect in platelets was related to inhibition of ROS (14). Therefore, the hypothesis that p-cresol may inhibit EPC proliferation via its antioxidant activity was tested. The present study aimed to further elucidate the mechanism of p-cresol and its toxicity to the cardiovascular system.

Materials and methods

Culture and determination of human late EPCs

Culture and determination of human late EPCs were performed as reported previously (10). Briefly, 20 ml human peripheral blood was diluted 1:1 with phosphate-buffered saline (PBS) and suspended in an equal volume of Ficoll density-gradient media (Sigma-Aldrich; Merck Millipore, Darmstadt, Germany). Cells were centrifuged for 30 min at room temperature at a speed of 740 × g. Recovered cells were then washed twice with PBS and resuspended in Gibco Medium 199 (Thermo Fisher Scientific, Inc., Waltham, MA, USA) supplemented with 20% fetal bovine serum (FBS; Thermo Fisher Scientific, Inc.) and endothelial cell growth medium (EGM)-2 SingleQuot (Lonza, Basel, Switzerland). Late EPC colonies appeared following ~2–4 weeks of culture in a 5% CO2 incubator at 37°C. These cells were then harvested and cultured for later experiments. These cells were confirmed to be EPCs as cell determination demonstrated that these cells could incorporate acetylated low density lipoprotein and exhibited Ulex europaeus agglutinin I binding affinity. The cells were also negative for prominin 1 (CD133) expression, but expressed CD34 and vascular endothelial factor receptor 2.

Measurement of ROS production in EPCs

Intracellular oxidant formation was assessed in EPCs using 5-(and-6)-carboxy-2′, 7′-dichlorodihydrofluorescein diacetate (carboxy-H2DCFDA; Invitrogen; Thermo Fisher Scientific, Inc.), a non-fluorescent and cell-permeable analog of fluorescein that is converted into carboxy-2′,7′-dichlorodihydrofluorescein following intracellular deacetylation and is oxidized to highly fluorescent carboxy-dichlorofluorescein (carboxy-DCF). Following incubation with 10 µM carboxy-H2DCFDA in warm Hank's Buffered Salt Solution (HBSS, Invitrogen; Thermo Fisher Scientific, Inc.) for 15 min, cells were washed with PBS for 30 sec and immediately viewed by fluorescence microscopy or analyzed by fluorescence-activated cell sorting (FACS) analysis.

Cell proliferation assay

Effects of ROS inhibitors on growth factor-dependent EPC proliferation were determined using the water-soluble tetrazolium-1 (WST-1) assay. EPCs at a concentration of 10,000 cells per well were seeded on 96-well culture plates in Medium 199 (Thermo Fisher Scientific, Inc.) supplemented with 20% FBS (Thermo Fisher Scientific, Inc.) and EGM-2 SingleQuot (Lonza) in a 5% CO2 incubator at 37°C and cultured for 24 h. Cells were then washed once with warm HBSS (Invitrogen; Thermo Fisher Scientific, Inc.) and the media was subsequently replaced with Medium 199 (Thermo Fisher Scientific, Inc.) containing 0.5% FBS (Thermo Fisher Scientific, Inc.). Following culture for 6 h, EPCs were treated with 20 µM N-acetylcysteine (NAC) or 20 µM dibenziodolium (DPI) for 1 h, then stimulated for 24 h with different concentrations of growth factors [0–10 ng/ml, including epidermal growth factor (EGF), vascular endothelial growth factor (VEGF), fibroblast growth factor (FGF), and insulin-like growth factor (IGF); Lonza]. Cell proliferation assay reagent WST-1 (Roche Applied Science, Penzberg, Germany) was then added to each well (10 µl) and incubated for 4 h. Absorbance at 450 nm was measured using an enzyme-linked immunosorbent assay reader.

Reverse transcription-quantitative polymerase chain reaction (RT-qPCR)

Total cellular RNA was extracted using TRIzol reagent (Thermo Fisher Scientific, Inc.). RNA was then reverse transcribed to cDNA in a total volume of 20 µl using a Moloney-Murine Leukemia Virus Reverse Transcriptase kit (Promega Corporation, Madison, WI, USA). The mRNA levels of antioxidant proteins, including superoxide dismutase 1 (SOD-1), superoxide dismutase 2 (SOD-2), glutathione peroxidase 1 (Gpx-1), glutathione peroxidase 4 (Gpx-4), and catalase (CAT), were analyzed by RT-qPCR using specific primers, according to a previous report (15). The qPCR reaction was carried out using a commercial SYBR-Green reaction mix (Takara Bio, Inc., Otsu, Japan). Thermal cycling was performed in an ABI 5700 sequence detection system (Applied Biosystems; Thermo Fisher Scientific, Inc.). Reaction conditions were as follows: 95°C for 30 sec, followed by 40 cycles of 95°C for 5 sec and 60°C for 31 sec. mRNA expression levels were quantified using the 2∆∆Cq method as described previously (16). β-actin was used as internal control for each sample.

Preparation of cell lysates and western blotting

Following 72 h exposure to p-cresol in complete M199 medium, EPCs were rinsed twice with ice-cold PBS and proteins were extracted using the ProteoJET Mammalian Cell Lysis Reagent (Fermentas; Thermo Fisher Scientific, Inc.). The lysate solution was then centrifuged at 14,000 × g for 15 min at 4°C, and the supernatant (soluble fraction) was transferred into new tubes and stored at −80°C until needed. Equal amounts of protein sample (80 µg) were separated by 12 or 15% SDS-PAGE and transferred to polyvinylidene fluoride membranes. The membranes were incubated at room temperature for 1 h in TBS/0.1% Tween-20 (TBST) containing 5% milk to block nonspecific binding and subsequently incubated overnight at 4°C with the following primary antibodies: Anti-cytochrome b-245 α chain (CYBA; catalog no. sc-130550; 1:500), anti-cytochrome b-245 β chain (CYBB; catalog no. sc-130543; 1:500) and anti-nicotinamide adenine dinucleotide phosphate (NAPDH) oxidase 4 (NOX4; catalog no. sc-30141; 1:500), all purchased from Santa Cruz Biotechnology, Inc. (Dallas, TX, USA), in TBST containing 5% bovine serum albumin (Thermo Fisher Scientific, Inc.). The membranes were then washed three times with TBST and incubated for 1 h at room temperature in horseradish peroxidase-conjugated goat anti-mouse or goat anti-rabbit secondary antibodies (1:5,000 dilution in TBST containing 5% non-fat milk). The immunoreactive proteins were detected using an enhanced chemiluminescence western blotting detection system (Merck Millipore, Darmstadt, Germany). β-actin served as the loading control. Each experiment was repeated three times, with a representative blot shown for each experiment.

Statistical analysis

Results are expressed as the mean ± standard deviation from at least three independent experiments. Statistically significant differences among different treatment groups at a single point in time were determined by one-way analysis of variance, followed by two-tailed Student's t-tests. P<0.05 was considered to indicate a statistically significant difference.

Results

Effects of ROS on growth factor-induced EPC proliferation

To assess the role of ROS in growth factor-induced EPC proliferation, ROS levels were measured in EPCs treated with different concentrations of EGM-2 SingleQuot. The results demonstrated that ROS levels in EPCs were visibly increased following 6 h growth factor treatment, as measured by the carboxy-H2DCFDA fluorescence assay and FACS analysis (Fig. 1A). Decomposition of cellular ROS by treatment of EPCs with the antioxidants NAC or DPI significantly reduced cell viability compared with EPCs cultured without antioxidant in the same concentration of growth factors (P<0.05, Fig. 1B and C, respectively). These observations underscore the critical role of ROS in driving the proliferative properties of EPCs.

Figure 1. Analysis of ROS production and its effect on EPC cell proliferation. (A) DCF fluorescence in EPCs treated with different concentrations of growth factors for 6 h was measured by fluorescence-activated cell sorting analysis. A representative histogram of fluorescence intensity is presented here for 0 (control, magenta line), 2.5 (red line) and 5 (green line) ng/ml growth factors treatments. EPCs were pretreated with (B) NAC or (C) DPI for 1 h and then stimulated with different concentrations of growth factors for 24 h. Cell proliferation was then determined using a WST-1 assay. Data were expressed as mean ± standard deviation of three independent experiments (n=8 samples per group). *P<0.05, with comparisons indicated by lines. ROS, reactive oxygen species; EPC, endothelial progenitor cells; DCF, dichlorofluorescein; M1, unstressed cells; M2, cells with increased ROS production; GF, growth factors; NAC, N-acetylcysteine; DPI, dibenziodolium; WST-1, water-soluble tetrazolium-1.

Effects of p-cresol on ROS expression in human EPCs

Since growth factor stimulation induced ROS production in EPCs within 6 h of exposure, the effect of p-cresol on ROS production in the same timeframe was analyzed. Treatment of EPCs with p-cresol resulted in a dose-dependent decrease in ROS levels in EPCs, as demonstrated by a significant reduction in DCF-positive cells in the p-cresol-treated cells compared with the control cells (P<0.05, Fig. 2).

Figure 2. Effect of p-cresol on ROS production in EPCs. DCF fluorescence in EPCs treated with 0, 40 or 80 µg/ml p-cresol was visualized by fluorescence microscopy (upper panel) and measured by FACS analysis (lower panel). In the representative images displayed here, green denotes DCF signal, while blue denotes stained cell nuclei by DAPI. Data are presented as the mean + standard deviation (n=3). *P<0.05 vs. control. p-cresol, para-cresol; ROS, reactive oxygen species; EPC, endothelial progenitor cells; FACS, fluorescence-activated cell sorting; DCF, carboxy-dichlorofluorescein.

Effects of p-cresol on antioxidant gene expression

The antioxidant activity of p-cresol on EPCs was confirmed by RT-qPCR analysis of mRNA expression levels of known antioxidant markers. The mRNA expression of antioxidant genes SOD-1, SOD-2, Gpx-1, Gpx-4, and CAT was analyzed in EPCs treated with 40 or 80 µg/ml p-cresol (Fig. 3). Gpx-4 mRNA expression was significantly upregulated by 3-fold with 80 µg/ml p-cresol after 6 h (P<0.05 compared with control), while expression of the other genes was not significantly affected (Fig. 3).

Figure 3. Effect of p-cresol on antioxidant gene expression in EPCs. EPCs were treated with 0, 40 or 80 µg/ml p-cresol for 6 h and mRNA expression of known antioxidant markers was analyzed by reverse transcription-quantitative polymerase chain reaction. Data are presented as the mean + standard deviation (n=3). *P<0.05 vs. control (0 µg/ml). p-cresol, para-cresol; EPC, endothelial progenitor cells; SOD, superoxide dismutase; Gpx, glutathione peroxidase; CAT, catalase.

Effects of p-cresol on NOX in human EPCs

NOX is a major source of ROS in EPCs. The NOX complex consists of the membrane-associated catalytic CYBB and the regulatory CYBA subunits, plus cytosolic components, including neutrophil cytosolic factor 1 (p47phox), neutrophil cytosolic factor 2 (p67phox), neutrophil cytosolic factor 4 (p40phox), and the small GTPase Ras-related C3 substrate 1 (Rac1) (13). Since NOX is a major source of ROS, its subunits were investigated in EPCs treated with p-cresol. The results demonstrated that incubation of EPCs with p-cresol for 72 h resulted in a significant downregulation of CYBB and CYBA expression in a dose-dependent manner (P<0.05 compared with control; Fig. 4), but it did not inhibit NOX4 (Fig. 4).

Figure 4. Effect of p-cresol on protein expression levels of NADPH oxidase subunits. Following incubation of EPCs with 0 (control), 40 or 80 µg/ml p-cresol for 72 h, cell lysates were subjected to western blot analysis to detect protein expression of CYBA, CYBB and NOX4. β-actin served as a loading control. Representative blots are depicted in the upper panel, while a quantification of each protein signal relative to the β-actin control is depicted in the graph of the lower panel. *P<0.05 vs. control. p-cresol, para-cresol; NAPDH, nicotinamide adenine dinucleotide phosphate; EPC, endothelial progenitor cells; CYBA, cytochrome b-245 alpha chain; CYBB, cytochrome b-245 beta chain; NOX4, NAPDH oxidase 4.

Discussion

Protein-bound uremic toxins constitute a heterogeneous group of compounds that are retained in patients with CKD. The main common characteristic of this group of toxins is their difficult removal by dialysis. Vanholder et al termed them as ‘the forgotten toxins’ (8). According to the European Uremic Toxin (EUTox) group, 25 protein-bound toxins have been identified to date, including four phenols (17). In the present study, the antiproliferation effect of one such phenol, p-cresol, on EPCs was demonstrated to be related to its antioxidant activity.

Phenols are a class of chemical compounds consisting of a hydroxyl group (−OH) bonded directly to an aromatic hydrocarbon group. They are reactive species toward oxidation, thus considered to have antioxidant activity. Ujhelyi et al (18) have previously suggested that the decreased antioxidant capacity of plasma ultrafiltrate observed after a hemodialysis session is related to the removal of several protein-bound uremic toxins, including p-cresol. This indicates that phenolic uremic toxins, usually retained in patients with CKD, might have antioxidant activity. Although Chang et al (12) demonstrated that p-cresol increases ROS in endothelial and mononuclear cells, the present study demonstrated that p-cresol inhibits EPC proliferation via its antioxidant activity, suggesting that the three remaining phenols identified in the protein-bound uremic toxins in CKD patients might have similar activity.

There are two different types of EPCs derived from peripheral blood: The spindle-shaped early EPCs, which have limited proliferative potential and are unsuitable for long-term culture under in vitro conditions (19), and the cobblestone-shaped late EPCs, which are produced by prolonged culture of peripheral mononuclear cells in the presence of various growth factors and have potential for rapid growth (20,21). Previous studies have demonstrated that early and late EPCs make different contributions to angiogenesis (20); early EPCs contribute to angiogenesis primarily by secreting angiogenic cytokines that recruit resident mature endothelial cells and induce their proliferation and survival, and late EPCs enhance angiogenesis by providing a sufficient number of endothelial cells by virtue of their rapid growth. Thus, it is possible that the proliferative dysfunction of late EPCs induced by p-cresol could directly contribute to impaired angiogenesis following ischemic insult. An in vivo study would be required in order to further confirm this finding.

ROS serve an important role in normal cell growth, migration, differentiation, apoptosis, and senescence (22,23). Excess amounts of ROS are toxic and involved in stem/progenitor cell senescence and apoptosis (24). By contrast, ROS at low levels function as signaling molecules to regulate EPC proliferation and EPC-mediated angiogenesis (25). Signal transduction activated by ROS has been an emerging area of investigation. NOX is a major source of ROS in EPCs (26). The NOX complex consists of catalytic subunits (mainly CYBB or NOX4), the regulatory subunit, CYBA, and the cytosolic subunits p47phox, p67phox, and Rac1 (13). A previous study reported the role of CYBB-based NOX in the angiogenesis function of EPCs (27). The present study revealed that the antiproliferation effect of p-cresol on EPCs was associated with decreased ROS levels and decreased CYBB and CYBA expression. In conclusion, the present in vitro study indicated that a uremic toxin, p-cresol, inhibits EPC proliferation via its antioxidant activity. The results of the present study may facilitate understanding of uremic toxins toxicity on the cardiovascular system.

Acknowledgements

The present study was partly supported by grants from the Science and Technology Commission of Wenzhou Municipality, China (grant no. Y20140504).

References