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Keywords:

  • atherosclerosis;
  • connective tissue growth factor;
  • extracellular matrix;
  • homocysteine;
  • vascular smooth muscle cell

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. Disclosure of Conflict of Interests
  9. References

Summary. Background: Increased homocysteine levels in blood might be an important risk factor for the development of cardiovascular diseases. Connective tissue growth factor (CTGF) was found to be involved in atherosclerotic plaque progression. So far, the possible connection between homocysteine and CTGF has not been studied.

Objective: This study was designed to test whether homocysteine could induce CTGF expression in vascular smooth muscle cells (VSMC).

Methods and results: Hyperhomocysteinemia was induced in Sprague–Dawley rats after 4 weeks of a high-methionine diet. CTGF mRNA and protein expression was detected in the aortas isolated from hyperhomocysteinemic rats, but not in the controls. The underlying mechanism of homocysteine-induced CTGF expression was investigated in cultured human umbilical vein smooth muscle cells (HUVSMC). CTGF mRNA expression was induced after treatment with dl-homocysteine (50 μmol L−1) for 1 h, which remained at the elevated level for up to 8 h. CTGF protein level increased after homocysteine treatment for 8 h, and the elevated status was maintained for up to 48 h. Several intracellular signals elicited by homocysteine are involved in CTGF synthesis, including protein kinase C (PKC) activation and reactive oxygen species (ROS). Transfection HUVSMCs with a CTGF small interference RNA (siRNA) plasmid, which specifically inhibited the expression of CTGF, decreased extracellular matrix (ECM) accumulation caused by homocysteine.

Conclusion: Our results demonstrate that homocysteine could increase the expression of CTGF in VSMC both in vivo and in vitro. The novel findings suggest that homocysteine might contribute to accelerated progression of atherosclerotic lesions by inducing CTGF expression.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. Disclosure of Conflict of Interests
  9. References

Homocysteine is a naturally occurring, sulfur-containing amino acid formed from the metabolism of methionine, an essential amino acid derived from the diet. The interconversion of methionine and homocysteine depends on the availability of the methyl donor 5-methyltetrahydrofolate, cofactors vitamin B12 and folate, and the enzyme activity of methionine synthase. Hyperhomocysteinemia may be caused by genetic and environmental factors. Despite previous meta-analyses suggesting that homocysteine is an independent risk factor for atherosclerosis and other cardiovascular diseases (CVD) [1], recently there has been a debate regarding the role of homocysteine in the development of CVD [2]. However, evidence indicates that an elevated plasma homocysteine level is still considered as an important risk factor for CVD, at least in high-risk patients [3,4]. It has been recently reported that total plasma homocysteine is a strong predictor of recurrence of unstable angina [5]. Although hyperhomocysteinemia may be regarded as a minor risk factor for CVD in low-risk patients, it can play a role in triggering new events in patients with known CVD, also by interacting with the ‘classical’ CVD risk factors [6].

Homocysteine may directly be involved in remodeling and destabilization of atherosclerotic plaques [7,8]. Several biological mechanisms have been proposed to explain cardiovascular pathological changes associated with hyperhomocysteinemia. These include: dysfunction of vascular endothelial cells, proliferation of vascular smooth muscle cell (VSMC), generation of reactive oxygen species (ROS) and activation of proinflammatory factors [7–10]. Yet the mechanisms by which homocysteine promotes atherosclerotic plaque formation are not clear. Although the precise molecular mechanisms by which homocysteine promotes the development of atherosclerosis need further investigation, the homocysteine-stimulated VSMC proliferation is considered to be one of the leading mechanisms contributing to atherogenesis [9,11]. It has been proposed that homocysteine may induce the proliferation and collagen deposition in VSMC [12,13]. However, the mechanism by which homocysteine induces deposition of extracellular matrix (ECM) components, such as collagen type I in VSMC, is largely unknown.

Recent studies have shown that connective tissue growth factor (CTGF) is a novel growth factor involved in the development and progression of atherosclerosis [14]. CTGF is a potent profibrotic factor implicated in fibroblast proliferation, angiogenesis and ECM synthesis [15] and its expression is regulated by several factors, such as transforming growth factor-β(TGF-β), high glucose, endothelin-1 and angiotensin II [16–18]. However, whether homocysteine plays a role in regulation of CTGF gene expression, or more generally in atherogenesis, remains unknown. The aim of the current study is to test the hypothesis that homocysteine can up-regulate the expression of CTGF in VSMC. We utilized both experimental hyperhomocysteinemia rat model and cultured human umbilical vein smooth muscle cells (HUVSMC) to investigate the correlation of high homocysteine level and CTGF expression. By using siRNA knockdown, we further demonstrated that CTGF expression was necessary for homocysteine-induced ECM component accumulation in HUVSMCs.

Methods

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. Disclosure of Conflict of Interests
  9. References

Dietary induction of hyperhomocysteinemia in an animal model

This study was approved by the medical ethics committee of the West China Hospital of Sichuan University and conformed to National Institutes of Health (NIH) guidelines regarding animal experimentation.

A dietary model of hyperhomocysteinemia was induced in Sprague–Dawley (SD) rats according to previous studies [10,19]. In brief, male SD rats (from the Animal Center of Sichuan University) aged 8 weeks were divided into four groups (= 10 for each group) and maintained for 4 weeks on the following diets before experiments: (i) regular diet, control chow containing 0.41%methionine (wt/wt), 0.0006% folate (wt/wt), 0.21% choline (wt/wt) and 0.27% cysteine (wt/wt); (ii) high-methionine diet, regular diet plus 1.7% methionine (wt/wt); (iii) high-methionine plus folate-rich diet, consisting of regular diet plus methionine (1.7% wt/wt) and folate (0.006% wt/wt); and (iv) high-cysteine diet, regular diet plus 1.2% cysteine (wt/wt). Food and water were provided ad libitum.

Measurement of homocysteine, methionine, cysteine and folate

Blood samples were drawn from the tail vein, promptly centrifuged, and stored at −20 °C. Plasma homocysteine levels were measured as the total homocysteine by the homocysteine EIA kit (Bio-Rad Laboratories, Hercules, CA, USA) in a micro-plate reader (Model 680, Bio-Rad Laboratories). Plasma concentrations of methionine and cysteine were measured by high-performance liquid chromatography using ninhydrin. Serum folate was measured with a commercially available radioimmunoassay kit from the Radioimmunoassay Center of Beijing (China).

In situ detection of CTGF expression in the rat aorta by immunohistochemistry

To detect the expression of CTGF in vivo, the thoracic aorta were isolated and immersion-fixed in 10% neutral-buffered formalin overnight and then embedded in paraffin. Sequential 5-μm paraffin-embedded cross-sections were prepared. Immunohistochemical analysis was performed as previously described [19] to detect CTGF expression using a rabbit polyclonal antibody against CTGF (Santa Cruz Biotechnology, Santa Cruz, CA, USA). Negative controls without the primary antibody or using an unrelated antibody were included to check for non-specific staining.

Cell culture

HUVSMCs were purchased from Technoclone (Vienna, Austria) and were grown in smooth muscle cell medium (Technoclone). For all experiments, confluent HUVSMC cells at passage 4 to 8 were washed and incubated with serum-free media for 48 h and then treated with homocysteine and/or other compounds. dl-homocysteine (Sigma, St Louis, MO, USA) was freshly prepared for all experiments.

Quantitative real-time reverse transcription PCR

The expression of CTGF, collagen type I and fibronectin (FN) gene was identified by quantitative RT-PCR. Total RNA extraction and real-time RT-PCR were performed as in our previous report [20].

Western blot analysis

Western-blot analysis of CTGF, collagen type I or FN protein expression was performed according to the method described before [20]. In brief, total protein extracts from the thoracic aorta of the rats (50 μg) or HUVSMC cell lysates (40 μg) were separated by denaturing 10% SDS–PAGE and then transferred to polyvinylidene difluoride membrane (Millipore, Bedford, MA, USA) using a MiniProtein III system (Bio-Rad). Transferred proteins were probed with the primary antibody (anti-CTGF, anti-collagen type I or anti-FN antibody; Santa Cruz Biotechnology, Inc.) and visualized using a horseradish peroxidase-conjugated secondary anti-rabbit (1:3000; Amersham Biosciences, Amersham, UK) antibody and ECL solution. Equal protein loading was verified by reprobing the membrane with an anti-β-actin antibody (Santa Cruz Biotechnology, Inc.). For quantification purposes, densitometric measurements were performed using quantity one image analysis software for Windows (Bio-Rad). All specific blot values were corrected for β-actin expression.

The CTGF-siRNA plasmid construction and transient transfection

CTGF-siRNA plasmid vectors were constructed as previously reported [20]. The pSilencer 3.1-H1 neo siRNA expression plasmid was purchased from Ambion (Austin, TX, USA). The CTGF-siRNA plasmid expressing short hairpin small interfering RNA under the control of the polymerase-III H1-RNA promoter was produced after inserting pairs of annealed DNA oligonucleotides between the BamHI and HindIII restriction sites. The targeted 21-nucleotide (nt) sequences derived from human CTGF mRNA (Genebank No. NM_001901; bp 762-781) were selected. A scrambled control siRNA with the same nucleotide composition as CTGF siRNA but lacking significant sequence homology to the human genome was also constructed. Transient transfection was performed by usage of the cationic lipid Lipofectamine 2000® (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s specifications. Transfection efficiency averaged between 45% and 55%, as measured using fluorescence microscopy in cells co-transfected with plasmid containing the green fluorescent protein gene (pEGFP-N1) from Clontech (San Jose, CA, USA).

Statistical analysis

The experimental data were expressed as means ± SEM. Group means were compared by one-way anova using the statistical software program spss 10.0 for Windows (Chicago, IL, USA), and a P value < 0.05 was considered significant in all cases.

Results

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. Disclosure of Conflict of Interests
  9. References

CTGF mRNA and protein detection in the rat aortas

It has been reported that feeding rats with a high-methionine diet for 4 weeks induced hyperhomocysteinemia [10,19]. In our hands, the high-methionine diet resulted in a significant increase in plasma homocysteine levels (22.28 ± 2.16 vs. 4.53 ± 0.5 μmol L−1 in control rats, Table 1). Cysteine is another thiol-containing amino acid and has been shown to have no effect on the expression of MCP-1, VCAM-1, ICAM-1 or NF-κB [10,19]. As a control, a high-cysteine diet group was included to test the specificity of homocysteine effect on CTGF induction in animals. There was no significant increase of plasma homocysteine levels observed in rats fed with a high-cysteine diet (Table 1). As a folate-rich diet has been shown to prevent hyperhomocysteinemia [10,19], another group of rats fed with the high-methionine plus folate-rich diet was also included to test whether lowering blood homocysteine levels by folate supplement could prevent hyperhomocysteinemia-induced CTGF expression. Plasma homocysteine levels were significantly lower in rats fed with the high-methionine plus folate-rich diet than in rats fed with the high methionine diet (Table 1).

Table 1.   Plasma homocysteine, methionine, cysteine levels and serum folate level in different diet groups (mean ± SEM)
GroupsTotal homocysteine (μmol L–1)Methionine (μmol L–1)Cysteine (μmol L–1) Folate (μg L–1)
  1. *< 0.05 vs. control; < 0.05 vs. high-methionine diet group.

  2. < 0.05 vs. control; §< 0.05 vs. control.

  3. < 0.05 vs. control; **< 0.05 vs. control.

Control (regular diet)4.53 ± 0.562.5 ± 3.53.2 ± 0.686.7 ± 5.7
High-methionine diet22.28 ± 2.16*241.6 ± 35.624.5 ± 4.182.5 ± 7.8
High methionine +folate diet7.8 ± 1.4221.7 ± 24.922.9 ± 3.0216.8 ± 18.5**
High cysteine diet4.86 ± 0.6 38.4 ± 4.8§25.4 ± 3.885.2 ± 6.4

Serum folate levels were significantly elevated in rats receiving the high-methionine plus high-folate diet. However, there were no significant differences in folate levels among the control, high-methionine-fed, and high-cysteine-fed groups. As expected, plasma methionine was markedly higher in the high-methionine groups, but there was a significant decrease in the high-cysteine group. There were no significant differences in plasma cysteine levels among rats fed with high-methionine, high-cysteine or high-methionine plus high-folate diets, all of which were significantly higher than the controls (Table 1). There were no significant differences in body weights among rats fed with different diets.

Immunohistochemistry staining was carried out to detect CTGF expression in the rat aortas. The aorta samples from control rats did not display significant staining of CTGF. In the aortas isolated from hyperhomocysteinemic rats (fed with a high-methionine diet), CTGF expression was induced, showing positive staining in VSMCs and in the endothelial cells. In contrast, little CTGF was detected in the aortas prepared from rats fed with a high-methionine plus folate-rich diet or a high-cysteine diet (Fig. 1A), indicating that folate in the diet prevents CTGF expression induced by a high-methionine diet.

image

Figure 1.  Homocysteine increases production of connective tissue growth factor (CTGF) in rat aortas. Thoracic aortas were isolated from rats fed with a regular diet (Control), high-methionine diet (Met), high-methionine plus folate-rich diet (Met+folate), or high-cysteine diet (Cysteine). (A) Cross-sections of thoracic aorta were prepared, and analyzed for CTGF expression by immunohistochemistry. Figures show a representative animal of 10 studied in each group. Magnification ×400. (B) Quantitative RT-PCR (Q-PCR) results: total RNA was extracted from aortas and CTGF mRNA levels were measured by Q-PCR. Graph is representative of relative CTGF mRNA levels in different groups. *< 0.05 vs. control. #< 0.05 vs. high-methionine diet group (Methionine). (C) Representative Western blot (top) and values of CTGF protein (means ± SEM of three experiments, bottom). Results of total CTGF protein production were obtained from densitometric analysis and expressed as ratio CTGF/β-actin. *< 0.05 vs. control. #< 0.05 vs. high-methionine diet group (Methionine).

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Furthermore, CTGF expression was also detected in the rat aortas by quantitative RT-PCR and Western blot. Aortic samples from control rats showed low CTGF mRNA expression, but in samples from hyperhomocysteinemic rats it was much higher (Fig. 1B). As shown in Fig. 1C, CTGF protein almost could not be detected in control rats; however, high CTGF expression was induced by a high-methionine diet. Consistent with immunohistochemistry results, CTGF expression in the aortas from rats fed with a high-methionine diet was significantly decreased by folate. The samples from rats fed with a high-cysteine diet showed little CTGF expression compared with the controls.

Effect of homocysteine on CTGF mRNA and protein expression in HUVSMC

To determine whether homocysteine modulates CTGF gene expression in cultured HUVSMCs, cells were treated with 50 μmol L−1dl-homocysteine, and total RNA was isolated at various times from 1 to 24 h. The concentration of dl-homocysteine used in this study has been reported to promote MCP-1 and IL-8 expression and secretion [21]. After 1 hour’s treatment, real-time quantitative RT-PCR revealed that homocysteine stimulation rapidly induced the expression of CTGF above basal levels, and the induction peaked at 4 h after treatment. The elevated CTGF mRNA level lasted for up to 8 h, then declined to near baseline by 24 h (Fig. 2A). We also examined the CTGF protein level change in homocysteine-treated HUVSMCs. Under serum-starvation condition, growth-arrested HUVSMCs expressed a low level of CTGF protein, which was detected by a specific antibody as a 38KD protein on Western blot. Total cellular CTGF protein levels began to increase 8 h after HUVSMCs treated with homocysteine, and the elevated CTGF level lasted for up to 48 h after treatment (Fig. 2B).

image

Figure 2.  Homocysteine increases connective tissue growth factor (CTGF) mRNA expression (A) and protein production (B) in cultured human umbilical vein smooth muscle cells (HUVSMCs). Growth-arrested HUVSMCs were stimulated with 50 μmol L−1dl-homocysteine or l-cysteine for different times. (A) Quantitative RT-PCR (Q-PCR) results: total cellular RNA was isolated from normal control, homocysteine or cysteine-treated HUVSMCs. After reverse transcription, the CTGF mRNA levels were determined by quantitative PCR analysis. Graph is representative of relative CTGF mRNA levels in the various conditions. Experiments were performed five times with similar results (= 5 in each group). *< 0.05 vs. control. (B) Representative Western blot (top) and values of total CTGF production (means ± SEM of four experiments, bottom). Results of total CTGF protein production were obtained from densitometric analysis and expressed as ratio CTGF/β-actin. *< 0.05 vs. control.

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To exclude the possibility that homocysteine-induced CTGF expression was caused by an unspecific thiol effect, we tested the effect of 50 μmol L−1l-cysteine on the CTGF mRNA and protein expression. Compared with cells in the control medium, there was no significant stimulatory effect on CTGF expression in HUVSMCs treated with l-cysteine (Fig. 2), confirming the specificity of the homocysteine response in stimulating the CTGF expression in HUVSMCs. These data suggest that homocysteine induces both CTGF mRNA and protein production in HUVSMCs.

Role of CTGF in homocysteine-induced ECM accumulation

Previous studies showed that homocysteine increased collagen synthesis in cultured smooth muscle cells [13]. We examined whether the homocysteine-induced up-regulation of CTGF expression in HUVSMCs correlated with enhanced ECM component deposition. Using real-time PCR and Western blot analysis, we confirmed the observations [13,22,23] that the accumulation of collagen type I and FN, two ECM components, significantly increased after homocysteine treatment (Fig. 3). To evaluate the contribution of increased thiol concentration to collagen type I and FN synthesis, we also examined the effect of 50 μmol L−1l-cysteine on ECM accumulation in HUVSMCs. The deposition of collagen type I and FN in cells treated with l-cysteine was not significantly different from that in the normal control medium. This result ruled out the possibility that the homocysteine-induced ECM synthesis was caused by an unspecific thiol effect (data not shown).

image

Figure 3.  Connective tissue growth factor (CTGF)-siRNA transfection reduces basal and homocysteine-induced CTGF, collagen type I and FN mRNA (A) and protein expression (B, C and D) in human umbilical vein smooth muscle cells (HUVSMC). (A) Q-PCR results: growth-arrested HUVSMCs were transfected with CTGF-siRNA plasmid for 24 h and then exposed to 50 μmol L−1 homocysteine for 4 h. CTGF, collagen type I and FN mRNA expression were assayed by Q-PCR. Experiments were performed five times with similar results (= 5 in each group). *< 0.05 vs. mock or scrambled-siRNA transfection without homocysteine treatment. **< 0.05 vs. mock or scrambled-siRNA transfection without homocysteine treatment. #< 0.05 vs. mock or scrambled-siRNA transfection with homocysteine treatment. (B) Representative Western blot (top) and values of total CTGF production (means ± SEM of three experiments, bottom). Results of total CTGF protein production were obtained from densitometric analysis and expressed as ratio CTGF/β-actin. *< 0.05 vs. mock or scrambled-siRNA transfection without homocysteine treatment. **< 0.05 vs. mock or scrambled-siRNA transfection without homocysteine treatment. #< 0.05 vs. mock or scrambled-siRNA transfection with homocysteine treatment. (C) Representative Western blot (top) and values of total collagen type I production (means ± SEM of three experiments, bottom). Results of total collagen type I protein production were obtained from densitometric analysis and expressed as ratio collagen type I/β-actin. *< 0.05 vs. mock or scrambled-siRNA transfection without homocysteine treatment. **< 0.05 vs. mock or scrambled-siRNA transfection without homocysteine treatment. #< 0.05 vs. mock or scrambled-siRNA transfection with homocysteine treatment. (D) Representative Western blot (top) and values of cellular fibronectin (FN) production (means ± SEM of three experiments, bottom). Results of FN protein production were obtained from densitometric analysis and expressed as ratio FN/β-actin. *< 0.05 vs. mock or scrambled-siRNA transfection without homocysteine treatment. **< 0.05 vs. mock or scrambled-siRNA transfection without homocysteine treatment. #< 0.05 vs. mock or scrambled-siRNA transfection with homocysteine treatment.

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In order to answer whether the CTGF is necessary for the homocysteine-stimulated ECM accumulation, we used a gene silencing approach to specifically block CTGF gene expression. A small inhibitory RNA (siRNA) expression plasmid targeting the CTGF gene (CTGF-siRNA) was constructed. In HUVSMCs, this CTGF-siRNA significantly inhibited both basal and homocysteine-induced CTGF gene expression, as evaluated by quantitative RT-PCR. Similarly, CTGF-siRNA also knocked-down CTGF protein as confirmed by Western- blot (Fig. 3B). The knockdown of CTGF also significantly reduced mRNA and protein levels of collagen type I and FN. In contrast, a scrambled siRNA showed no effect on CTGF, collagen I or FN expression (Fig. 3). Thus, our data suggest that in HUVSMCs, CTGF plays a role in homocysteine-induced ECM accumulation.

Molecular mechanisms of homocysteine-induced CTGF gene and protein production

Homocysteine activated several intracellular mediators, such as Ca2+ secondary messenger [24], protein kinase C (PKC) [25–27], phospholipids [25], nuclear factor-κB [26] and intracellular ROS [23]. In HUVSMCs, we were able to reproduce many observations made by others. We made the following observations.

1 PKC activator phorbol 12-myristate 13-acetate (PMA) induced a significant increase in the CTGF expression, and two PKC inhibitors, H-7 and bisindolymaleimide, reduced the homocysteine-induced CTGF production in HUVSMCs (Fig. 4A).

image

Figure 4.  Molecular mechanisms involved in homocysteine-induced connective tissue growth factor (CTGF) up-regulation. (A) Cells were pretreated with two inhibitors of protein kinase C (PKC) (H-7 and bisindolylmaleimide, 10−6 mol L−1), or anti-oxidants (Tiron, 5 mmol L−1) and treated with homocysteine (Hcy: 50 μmol L−1) for 24 h, and then studied by Western blot. PMA (10−7 mol L−1) was used as PKC activator. Figure shows a representative experiment of three performed (top) and values of total CTGF production (means ± SEM, bottom). *< 0.05 vs. control; #< 0.05 vs. Hcy. (B) Homocysteine-induced CTGF up-regulation is independent of TGF-β. Cells were co-treated with homocysteine (Hcy: 50 μmol L−1) and a TGF-β neutralizing antibody (Ab-TGF-β1; 10 μg mL−1) for 24 h. Figure shows a representative Western blot of three performed and values of total CTGF production (means ± SEM, bottom). *< 0.05 vs. control; #> 0.05 vs. Hcy. Hcy, homocysteine treatment.

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2 Exogenous H2O2 has been reported to increase CTGF mRNA expression and protein production in VSMCs [17]. In our study, we observed that the homocysteine-induced CTGF protein production was markedly abrogated when the O2– scavenger Tiron was incubated with homocysteine (Fig. 4A).

3 TGF-β1 up-regulated CTGF mRNA expression and protein synthesis. TGF-β1 caused a much higher increase in the CTGF gene and protein expression than that induced by homocysteine. To exclude the possibility that homocysteine-induced CTGF was through the TGF-β pathway, we simultaneously treated HUVSMC with homocysteine and a TGF-β1 neutralizing antibody, which was shown to specifically block the TGF-β1-induced signaling pathway. The homocysteine-induced CTGF mRNA expression and protein synthesis was almost unaffected (Fig. 4B), suggesting that CTGF up-regulated by homocysteine was independent of the TGF-β pathway.

Discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. Disclosure of Conflict of Interests
  9. References

In the present study, we have observed that diet-induced elevation of plasma homocysteine levels can stimulate the expression of CTGF in rat aortic VSMCs and endothelium. This observation was also confirmed in cultured HUVSMCs, in which homocysteine up-regulated CTGF expression. The data show that exposure of HUVSMCs to 50 μmol L−1dl-homocysteine, but not the same concentration of l-cysteine, leads to an increase of CTGF mRNA and protein expression. Using CTGF gene silencing and pharmacological approaches, the underlying mechanisms and downstream events were further investigated.

Consistent with previous reports [10,19], the experiments in vivo demonstrated that, after 4 weeks of dietary treatment, plasma homocysteine levels were significantly elevated in rats fed with the high-methionine diet. Although no visible atherosclerotic lesion was found in the aortas of hyperhomocysteinemic rats, a significant increase in the expression of CTGF in the aortic VSMCs and also in the endothelium was observed. Results obtained from the present study demonstrate that diet-induced chronic hyperhomocysteinemia stimulates the expression of CTGF. Furthermore, the observation in vivo was confirmed in the cultured HUVSMCs, but whether homocysteine could induce CTGF expression in cultured endothelial cells such as human umbilical vein endothelial cells was not investigated. It was reported that expression of CTGF in cultured human endothelial cells could be induced by lysophosphatidic acid and sphingosine-1-phosphate, and the enhanced synthesis of CTGF might contribute to the development of endothelial dysfunction [28]. Therefore, future studies in endothelial cells are necessary to determine the expression of CTGF induced by homocysteine.

In the present study, possible pathways leading to homocysteine-enhanced CTGF expression in HUVSMCs were investigated using a RNA interference approach. Knockdown of the CTGF gene by the CTGF-siRNA markedly reduced the synthesis of ECM proteins such as collagen type I and FN stimulated by homocysteine. The results indicate that CTGF is a mediator in the ECM accumulation induced by homocysteine in HUVSMCs.

The molecular mechanisms linked to homocysteine-induced ECM production in VSMCs involve several pathways. Others have previously demonstrated that homocysteine causes collagen synthesis and accumulation in VSMC [24,25]. Among the intracellular signals involved in homocysteine actions, the PKC activation plays an important role [25,26]. Homocysteine stimulates PKC activity in SMCs and PKC modulates collagen production in SMC-like cells [23]. The PKC pathway was previously shown to mediate CTGF up-regulation induced by angiotensin II in VSMCs [17]. The present observations demonstrate that homocysteine up-regulates CTGF via activation of PKC in HUVSMCs by using two specific PKC inhibitors, H-7 and bisindolylmaleimide.

Another pathway implicated in the homocysteine action is the generation of intracellular ROS [23]. It has been shown that hydrogen peroxide (H2O2) is a novel inducer of CTGF gene expression and JAK2/JAK3 seems to be involved in the enhanced CTGF mRNA expression stimulated by H2O2 [29]. Although the existence of ROS generated from homocysteine oxidation is proposed as a cause of the accumulation of collagen, this is not supported by data obtained in rabbit SMC [23]. However, our results show that the anti-oxidant Tiron, a cell permeable scavenger of ROS, partially blocks CTGF production elicited by homocysteine, suggesting that ROS act as intermediates of homocysteine-induced CTGF expression. The possible reasons for the difference between our data and theirs [23] might be explained by the different cell types, the different concentration of homocysteine (they used much higher concentration (300 μmol L−1) of homocysteine) and the different treatment time of homocysteine in the cultures (they treated the cells for 2 weeks). Altogether, our data suggest that homocysteine activates some intracellular signals, such as PKC pathway and ROS production, which contribute to CTGF up-regulation and ECM deposition in HUVSMCs.

TGF-β has been identified as a potent inducer of CTGF expression and it is also a very important regulator of ECM synthesis in different cell types [15,16]. Induction of CTGF expression by TGF-β was also confirmed in HUVSMCs. However, the blockade of TGF-β did not abrogate or reduce the expression of CTGF when compared with cultures incubated with homocysteine alone. These results suggest that TGF-β does not mediate the increase in CTGF expression stimulated by homocysteine. Intracellular ROS directly induces CTGF expression, through JAK activation, independently of TGF-β in human lens epithelial cells [29]. It has been shown that H2O2 up-regulates CTGF expression also in a TGF-β-independent manner in VSMCs [17]. Furthermore, TGF-β expression can not be stimulated by homocysteine treatment in human endothelial cells or smooth muscle cells in vitro [30]. The present experimental data suggest that homocysteine up-regulates CTGF by a mechanism independent of the TGF-β pathway.

In conclusion, our results demonstrate for the first time that homocysteine could induce the expression of CTGF in VSMC both in vivo and in vitro. Several pathways, such as PKC activation and ROS production, but not TGF-β, seem to be involved in the homocysteine-induced CTGF expression in VSMC. We propose that homocysteine might contribute to atherosclerosis by stimulating expression of CTGF.

Acknowledgements

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. Disclosure of Conflict of Interests
  9. References

This work was supported by grants from the Natural Science Foundation of China, No. 30470437 (X. Liu), No. 30500222 (F. Luo), No. 30600293 (J. Li), and from the Research Foundation of West China Hospital of Sichuan University, No.136050152 (X. Liu). We would like to acknowledge the assistance and critical advice provided by J. Lin (University of California, San Francisco) and R. Lin (Exelixis, Inc.) in the preparation of this manuscript.

Disclosure of Conflict of Interests

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. Disclosure of Conflict of Interests
  9. References

The authors state that they have no conflict of interest.

References

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. Disclosure of Conflict of Interests
  9. References
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