Patients with chronic renal failure are at greater risk of developing atherosclerosis than healthy individuals, and recent data suggest that the putative uremic toxin indoxyl sulfate (IS) promotes the pathogenesis of atherosclerosis. The present study examined the effects of IS on vascular smooth muscle cells (VSMCs) with respect to reactive oxygen species (ROS), platelet-derived growth factor (PDGF) receptors, and mitogen-activated protein kinases (MAPKs). IS induced the migration and proliferation of VSMCs and synergistically enhanced their PDGF-induced migration as well as proliferation. The effects of PDGF were promoted after a 24-h incubation with IS despite the absence of IS during PDGF stimulation. Intracellular ROS levels were increased in the presence of IS, and PDGF-dependent ROS production was augmented by a prior 24-h incubation with IS even in the absence of IS during PDGF stimulation. These data suggest that IS increases the sensitivity of VSMCs to PDGF. IS also phosphorylated PDGF-β-receptors and upregulated PDGF-β receptor but not α-receptor protein expression in the absence of exogenous PDGF. The NADPH oxidase inhibitor diphenylene iodonium blocked IS-dependent increase in receptor expression. Administration of IS to nephrectomized rats also elevated receptor protein expression in arterial VSMCs. Inhibitors of NADPH oxidase, PDGF-β receptors, extracellular-regulated protein kinase (ERK), and p38 MAPK all inhibited IS-induced VSMCs migration and proliferation. Taken together, these findings indicate that IS induces the migration as well as proliferation of VSMCs through PDGF-β receptors and that ROS generation is critically involved in this process, which promotes the development of atherosclerosis.
- uremic toxin
- chronic renal failure
- reactive oxygen species
- platelet-derive growth factor-β
patients with chronic renal failure are at greater risk of developing atherosclerosis than healthy individuals, and cardiovascular diseases are the leading cause of mortality in such patients (1, 15, 27). Horl et al. (11) have recently suggested that high concentrations of uremic solutes in the patients' sera are likely candidates for the cause of atherosclerosis. The tryptophan metabolite indoxyl sulfate (IS) is a putative uremic toxin (3, 12, 17, 21) that accumulates in the serum of patients due to poor urinary clearance (3, 12, 21). Therefore, IS may promote atherosclerosis.
Until now, few reports have described the direct effect of IS on vascular cells (7, 8, 28). Dou et al. demonstrated that IS and p-cresol suppress the wound repair and proliferation of endothelial cells (ECs) derived from the human umbilical vein (7), and that IS enhances the production of reactive oxygen species (ROS), increases NAD(P)H oxidase activity, and decreases glutathione levels in these cells (8). Yamamoto et al. (11) showed that IS causes vascular smooth muscle cells (VSMCs) to proliferate via the activation of extracellular-regulated protein kinase (ERK), one of the mitogen-activated protein kinases (MAPKs), and that IS promotes the expression of platelet-derived growth factor (PDGF)-β receptors and PDGF-C chains. Because the pathogenesis of atherosclerosis involves both dysfunction of vascular ECs and proliferation of VSMCs (19, 20, 23–25), these data suggest that IS induces the initiation and/or progression of atherosclerosis. However, the molecular mechanisms of the IS actions in these cells are obscure.
In addition to VSMC proliferation, migration is also critically involved in the pathogenesis of atherosclerosis and restenosis (20, 23–25). Several vasoactive humoral factors such as angiotensin II and PDGF that play crucial roles in the progression of atherosclerosis generate ROS in VSMCs (4, 10, 25). Recent data indicating that IS produces ROS in renal mesangial cells (9) and in ECs (8) suggest that ROS also mediate IS actions in VSMCs. Accumulating evidence demonstrates that p38 MAPK as well as ERK is involved in the proliferation and migration of these cells (2, 13, 24, 25). Notably, p38 MAPK is activated by ROS (16). Moreover, Yamamoto et al. (28) have suggested the close relationship between IS and PDGF signaling pathways. Therefore, the present study examined the effect of IS on VSMC migration as well as proliferation with respect to ROS, PDGF signaling pathways, and the MAPKs ERK and p38.
MATERIALS AND METHODS
Antibodies were obtained from the following suppliers: anti-PDGF-β receptor antibody for Western blot analysis, Santa Cruz Biotechnology (Santa Cruz, CA); anti-PDGF-β receptor antibody for immunohistochemistry, Lab Version (Fremont, CA); phospho-ERK 1/2 and phospho-p38, Cell Signaling Technology (Beverly, MA), and anti-α-tubulin, Calbiochem (La Jolla, CA). IS was from Alfa Aesar (Lancashire, UK) and PDGF-BB was purchased from Austral Biologicals (San Ramon, CA). PD-98059, SB-203580, and N-acetylcysteine (NAC) were from Calbiochem (La Jolla, CA). Dulbecco's modified Eagle's medium (DMEM), fetal bovine serum (FBS), and penicillin and streptomycin were purchased from Sigma Chemical (St. Louis, MO).
VSMCs prepared from the thoracic aorta of adult Sprague-Dawley rats as described previously (24) were maintained in DMEM supplemented with 10% FBS, 100 U/ml penicillin, and 100 μg/ml streptomycin. The responses of VSMC to IS and PDGF were analyzed between passages 8 and 10 after a 48-h incubation in DMEM containing 0.1% FBS.
Cell migration assay.
Cell migration was determined using a modified Boyden chamber assay (Coaster). Polyvinylpyrrolidone-free polycarbonate filters with a pore size of 8 μm were coated with fibronectin. Serum-starved VSMCs (2 × 105 cells/well) preincubated with or without various reagents for 30 min were added to the upper chamber. The upper chambers with cells were inserted into the bottom chamber filled with medium and incubated in the presence and absence of PDGF or IS for 5 h. Cells that migrated to the bottom surface of the membrane were fixed in methanol, stained with hematoxylin and eosin, and counted in 10 representative fields.
Cell proliferation assay.
The proliferation was measured by using a 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt (MTS) reduction assay kit (Cell Titer 96 Aqueous Nonradioactive Cell Proliferation Assay, Promega). Serum-starved VSMCs (3 × 103 cells/well) in a 96-well plate were stimulated with IS or PDGF for 48 h. Thereafter, cells were subjected to the MTS assay according to the manufacture's instructions.
Measurement of intracellular ROS.
The generation of intracellular ROS was detected using the oxidant-sensitive probe 2′,7′-dichlorofluorescein diacetate (DCFH-DA) that is distributed throughout the cytosol and emits green fluorescence when oxidized with ROS. VSMCs (2 × 105 cells/dish) in 3.5-cm dishes were starved in DMEM containing 0.1% FBS for 48 h, washed, incubated with DCFH-DA (5 μmol/l) for 15 min, and left unstimulated or stimulated with IS or PDGF. Thereafter, fluorescence was detected by confocal laser scanning microscopy. Examination and mission wavelengths were 488 and 500–630 nm, respectively, for DCFH-DA. Images were collected and analyzed using confocal scanning system (TCP SP2, Leica).
Detection of activated ERK and p38 MAPK.
Cell lysates (15 μg of protein) were fractionated by SDS-PAGE on 10% polyacrylamide gels, and proteins were transferred to PVDF membranes. Activated ERK1/2 and p38 MAPK were detected using specific antibodies raised against their phosphorylated forms. The protein bands were visualized using an enhanced chemiluminescence kit (Santa Cruz, CA).
Experimental design of uremic rats and immunostaining of the aorta.
Sprague-Dawley male rats were anesthetized with an intraperitoneal injection of pentobarbital. Two-thirds of the renal branches of both kidneys were ligated through a lateral incision. One week later, the right kidney was removed, which was considered to be a 5/6 nephrectomy. The nephrectomized rats were pair matched based on serum creatinine concentrations and assigned to one group that received IS (150–200 mg/kg body wt) in drinking water for 2 wk or another that did not (control). Sections of the aorta were immunostained with an anti-PDGF-β receptor antibody. All experiments were approved by Animal Care Committee of Biomedical Research Laboratories of Kureha and proceeded according to the Guiding Principles for the Care and Use of Laboratory Animals of the Japanese Pharmacological Society.
All results were confirmed in multiple independent experiments. Data were analyzed by using the Student's t-test and are expressed as means ± SE. A value of P < 0.05 was considered statistically significant.
IS induces migration as well as proliferation of VSMCs.
VSMCs were incubated with increasing concentrations of IS as shown in Fig. 1. IS alone slightly but significantly induced cell proliferation (Fig. 1A), which agrees well with the data of Yamamoto et al. (28). We also found that IS significantly increased the migration of these cells (Fig. 1B). Since concentrations of IS in the serum of patients with end-stage renal disease range from 100 to 300 μmol/l (3, 12, 21), those used in the present study (25, 100, and 250 μmol/l) reflected pathophysiological conditions.
IS enhances PDGF-induced proliferation and migration of VSMCs.
We examined the effect of IS on VSMCs in the presence or absence of 20 ng/ml of PDGF, a crucial humoral factor for the pathogenesis of atherosclerosis. Figure 1A shows that PDGF caused VSMCs to proliferate, a process that IS enhanced at all indicated concentrations. Similarly, the same concentrations of IS augmented the migration induced by PDGF at 20 ng/ml as shown in Fig. 1B. Together with the effects of IS alone, these results demonstrate that IS initiates and promotes not only the proliferation, but also the migration of VSMCs.
IS increases the sensitivity of VSMCs to PDGF.
We speculated that in addition to the direct effect of IS on the proliferation and migration of VSMCs, IS might alter various properties of these cells such as sensitivity to several factors that initiate and/or promote atherosclerosis. To test this possibility, VSMCs were incubated with or without IS (250 μmol/l) for 24 h. IS was removed, and then the cells were stimulated with PDGF (20 ng/ml) without IS for 48 h. Prior incubation with IS significantly enhanced the PDGF-induced proliferation (Fig. 2A) and migration (Fig. 2B) of these cells in a manner similar to concurrent stimulation with IS and PDGF, indicating that IS increases the sensitivity of VSMCs to PDGF.
IS generates ROS and enhances PDGF-induced ROS production.
Since the past reports have demonstrated IS-stimulated ROS production in ECs (8) and in mesangial cells (9), we investigated whether stimulation with IS (250 μmol/l) generates ROS in VSMCs. Figure 3A shows that IS increased the content of intracellular ROS after 10 min and 24 h of IS stimulation, suggesting that the IS-dependent increase in intracellular ROS is durable. We next incubated the cells with IS (250 μmol/l) for 24 h, removed the reagent, then added PDGF (20 ng/ml), a powerful stimulator of ROS generation in VSMCs. Figure 3B reveals that IS significantly augmented PDGF-induced ROS production, demonstrating increased sensitivity of the cells to PDGF in terms of ROS production. These data closely correlate with the results of the cell proliferation and migration assays shown in Fig. 2. The fact that ROS are involved in PDGF-dependent proliferation and migration of these cells (13, 16, 25) suggests that the increased sensitivity to PDGF stimulated by IS that results in ROS production mediates the enhancement of cell proliferation and migration induced by PDGF.
Both NAC and diphenyleneiodonium inhibit IS-increased PDGF-β receptor protein levels.
To understand the molecular mechanisms through which the sensitivity of VSMCs to PDGF is increased, we measured the protein level of PDGF-β receptors that mainly mediate the PDGF-stimulated proliferation and migration of these cells. Figure 4, A and B, shows a significantly elevated protein level of PDGF-β receptors in cultured VSMCs at 24 h after adding IS (250 μmol/l). This increase was obviously reduced by the antioxidant NAC (5 mmol/l) and the NADPH oxidase inhibitor diphenyleneiodonium (DPI, 10 μmol/l), respectively. In contrast, IS did not increase the protein level of PDGF α-receptors (Fig. 4C), indicating that the effect of IS on PDGF-β receptors is specific. Thus the IS-dependent increase in sensitivity of these cells to PDGF is probably due at least, in part, to the IS-induced upregulation of PDGF-β receptors mediated by the NADPH oxidase-dependent generation of ROS.
To determine whether IS increases the PDGF-β receptor protein level in vivo, IS (150–200 mg/kg body wt) was given to nephrectomized rats for 2 wk to enhance the IS effect. Sections of the aorta were immunostained with an anti-PDGF-β receptor antibody. Figure 4D shows considerably more immunoreactivity of PDGF-β receptor protein in the media of IS-treated rats when compared with that of control rats. These findings confirmed that IS also increases the level of PDGF-β receptor protein in vivo.
Cell proliferation and migration stimulated by IS are inhibited by NAC and DPI.
To confirm that the generation of ROS correlates with IS-induced proliferation and migration of VSMCs, we investigated the effects of NAC and DPI on these cellular responses to IS (250 μmol/l). Figure 5, A and B, shows that NAC (1 mmol/l) and DPI (10 μmol/l) inhibited cell proliferation stimulated by IS, respectively. Similarly, NAC (3 mmol/l) and DPI (10 μmol/l) also blocked the cell migration induced by IS as shown in Fig. 5, C and D. These data indicate that IS-stimulated ROS production is mediated by NADPH oxidase and that ROS is mainly responsible for these effects of IS on VSMCs.
Inhibitors of the ERK pathway and p38 MAPK suppress IS-stimulated cell proliferation and migration.
Accumulating evidence indicates that p38 MAPK and ERK are activated by ROS and that they mediate VSMC proliferation and migration (2, 13, 24, 25). Figure 6A shows that IS (250 μmol/l) activated p38 MAPK as well as ERK. The p38 MAPK inhibitor SB-203580 (10 μmol/l) reduced both cell proliferation and migration as shown in Fig. 6, B and C. Figure 6C indicates that the MEK inhibitor PD-98059 (10 μmol/l) also suppressed IS-induced cell migration. Yamamoto et al. (28) demonstrated that IS-induced ERK activation is involved in IS-dependent proliferation of VSMCs. Consistent with the findings of Yamamoto et al., PD-98059 (10 μmol/l) suppressed IS-dependent cell proliferation as in Fig. 6B. These data suggest that IS induces the migration as well as proliferation of VSMCs via the activation of ERK and p38 MAPK.
Our data showed that IS increases the sensitivity of VSMCs to PDGF because exposure to IS augmented subsequent PDGF-induced cell proliferation and migration (Fig. 2). To confirm these findings, we investigated whether IS enhances the PDGF-dependent phosphorylation of ERK and p38 MAPK by incubating VSMCs with or without IS (250 μmol/l) for 24 h. After IS was removed, the cells were stimulated with PDGF (20 ng/ml) without IS for 10 min. Figure 6, D and E, shows that prior exposure to IS significantly increased the phosphorylation level of both ERK and p38 MAPK induced by PDGF.
IS increases phosphorylation levels of PDGF β-receptors and a receptor inhibitor suppresses IS-stimulated cell proliferation and migration.
To understand the role of PDGF-β receptors in the IS-induced intracellular signaling pathways, we examined the phosphorylation level of the receptor in IS-stimulated cells and the effect of AG1296, a PDGF receptor-specific inhibitor, on IS-dependent proliferation and migration of VSMCs. The cells are incubated with or without IS (250 μmol/l) for 15 min, and cell lysates were immunoprecipitated with anti-PDGF-β receptor antibody followed by Western blot analysis with anti-pTyr antibody. Figure 7A shows that the receptor phosphorylation level was significantly increased upon IS stimulation even in the absence of PDGF. Furthermore, adding AG1296 (10 μmol/l) inhibited IS-induced cell proliferation and migration as in Fig. 7, B and C. Because PDGF-β receptors are protein tyrosine kinases and their autophosphorylation is essential for stimulating their downstream signaling pathways, these data suggest that IS-dependent phosphorylation of PDGF-β receptors and the activity of the receptors are critically involved in these effects of IS.
The present study found that 1) IS alone induces the migration as well as proliferation of VSMCs via the phosphorylation of PDGF-β receptors and the activity of the receptors; 2) NADPH oxidase-dependent production of ROS with subsequent activation of ERK and p38 MAPK is also critically involved in these IS effects; 3) IS enhances PDGF-dependent migration and proliferation of VSMCs; and 4) IS increases the sensitivity of these cells to PDGF via the generation of ROS followed by upregulation of PDGF-β-receptor protein. Together with the report indicating that IS inhibits the wound repair and proliferation of ECs, the present findings support the notion that the uremic toxin IS promotes the development of atherosclerosis. Our findings also suggest that IS acts in concert with PDGF-β receptors to stimulate the pathogenesis of atherosclerosis.
We consider that the IS actions in VSMCs can be separated into short- and long-term signaling pathways. In the short-term pathway, IS phosphorylates PDGF-β receptors probably by inactivating protein tyrosine phosphatases (PTPs). Tyrosine phosphorylation levels of proteins are regulated by the coordinate activities of protein tyrosine kinases and PTPs, and the catalytic site of PTPs contains a Cys residue that is essential for their activity (6, 26). We consider that IS-induced ROS generation via NADPH oxidase inactivates some PTPs by oxidizing the Cys residue in the catalytic domain with subsequent increase in the phosphorylation level of PDGF-β receptors. In fact, Liu et al. (14) recently indicated that serotonin stimulates the growth of pulmonary VSMCs by activating PDGF-β receptors that may occur through oxidation of catalytic Cys of PTPs. Such activation of the receptor probably plays a key role in the proliferation and migration of the cells. In our study, the increased phosphorylation of the receptors may lead to additional ROS production followed by the activation of ERK and p38 MAPK that are also critically involved in the IS actions. In contrast, the long-term pathway might contribute to the increased sensitivity of VSMCs to PDGF. IS-induced ROS generation stimulates PDGF-β receptor gene expression with a subsequent increase in its protein level. Chen et al. (5) described that FBS-dependent gene expression of PDGF-β receptors is positively controlled by nuclear factor-κB (NF-κB) in hepatic stellate cells. This NF-κB effect was inhibited by the antioxidant l-epigallocatechin-3-gallate, which is the main component of Japanese green tea. Motojima et al. (18) reported that IS induces ROS and activates NF-κB in human renal proximal tubular (HK-2) cells. These findings suggest that IS-dependent ROS production enhances NF-κB activity with subsequent promotion of PDGF-β receptor expression in VSMCs. The upregulated receptors must be responsible for the increased sensitivity to PDGF. Yamamoto et al. (28) reported that PDGF-C chain expression is promoted by IS in VSMCs, and Rao et al. (22) showed that uric acid induces proliferation of VSMCs by promoting PDGF-A chain expression. Together with these results, the present study suggests that IS initiates a vicious cycle of accelerated atherosclerotic pathogenesis through the short-term and long-term pathways in VSMCs.
We found that IS enhanced PDGF-induced VSMC migration and proliferation in a synergistic, rather than an additive manner, because of the following reasons. IS (25 μmol/l) and PDGF (20 ng/ml) alone increased cell proliferation by 9.3 ± 1.1% and by 27.0 ± 2.2%, respectively, when compared with unstimulated controls (Fig. 1A). If these IS and PDGF effects were additive, simultaneous exposure to both would yield an increase of 36.3 ± 1.2%. However, simultaneous addition of IS and PDGF actually increased cell proliferation by 51.3 ± 4.1%, which is significantly higher than the additive value of 36.3 ± 1.2% (P = 0.044). Significant differences were also obtained with IS at 100 μmol/l (P = 0.038) and 250 μmol/l (P = 0.049). The actual 221.7 ± 2.4% increase in cell migration upon simultaneous IS (25 μmol/l) and PDGF (20 ng/ml) addition was also significantly higher than the value of 163.0 ± 9.6% obtained by adding 17.3 ± 1.9% for IS alone and 145.7 ± 11.1% for PDGF alone (P = 0.031) (Fig. 1B). Simultaneous stimulation with 100 μmol/l of IS and 20 ng/ml of PDGF elicited a similarly synergistic trend, although the difference did not reach significance. Therefore, IS synergistically enhances PDGF-induced proliferation and migration of VSMCs. Although the detailed molecular mechanism of this synergistic effect remains unclear, our findings indicate that IS stimulates the pathogenesis of atherosclerosis in concert with PDGF signaling pathways.
The present study showed that ROS production and PDGF-β receptor phosphorylation are critical events in the initiation of IS actions in VSMCs and that ROS comprise a key factor. Therefore, understanding the process of IS-dependent ROS generation is essential for elucidating the molecular mechanisms of IS actions not only in VSMCs but also in other cells such as ECs and mesangial cells. The removal of IS from the blood should be very important to prevent the pathogenesis of atherosclerosis in patients with chronic renal failure. Because it binds albumin, hemodialysis cannot effectively clear IS from the bloodstream. In addition to therapies that remove IS such as hemodialysis and oral charcoal absorbents, a diet rich in antioxidants should be recommended to attenuate the effects of IS.
This work was supported by a grant from the Kureha Corporation, Japan.
We thank Norma Foster for help in preparing the manuscript.
↵* H. Shimizu and Y. Hirose contributed equally to this work.
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