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Inhibition of the PDGF ß-receptor tyrosine phosphorylation and its downstream intracellular signal transduction pathway in rat and human vascular smooth muscle cells by different catechins¹

AGAPIOS SACHINIDIS², ROMANITA ADRIANA SKACH^*, CLAUDIA SEUL^*, YON KO^*, JÜRGEN HESCHELER, HEE-YUL AHN^,3 and JÜRGEN FINGERLE^,3

Center of Physiology and Pathophysiology, University of Cologne, 50931 Cologne, Germany;
^* Medizinische Universitäts-Poliklinik, 53111 Bonn, Germany;
Department of Pharmacology, College of Medicine, Chungbuk National University, South Korea; and
Hoffmann-La Roche, Preclinical Pharma Research, Vascular and Metabolic Disease, CH-4070 Basel, Switzerland

²Correspondence: University of Cologne, Center of Physiology and Pathophysiology, Robert-Koch-Str. 39, 50931 Cologne, Germany. E-mail:A.Sachinidis{at}uni-koeln.de

SPECIFIC AIMS

Abnormal vascular smooth muscle cell (VSMC) proliferation and the platelet-derived growth factor (PDGF) play an important role in the development and progression of proliferative cardiovascular diseases. In the present study, we examined the effects of natural plant-derived catechins (isolated from green tea leafs) with different chemical structure on cell growth and on the intracellular signal transduction pathway of PDGF-BB in rat and human aortic VSMC.

PRINCIPAL FINDINGS

1. Treatment of rat aortic VSMC for 24 h with 20 µM epigallocatechin-3 gallate (EGCG), catechin-3 gallate (CG), and epicatechin-3 gallate (ECG), which possess a galloyl group in the 3 position of the catechin structure, caused an 70% inhibition as well as complete inhibition of the (50 ng/ml) PDGF-BB-induced DNA synthesis, respectively.

Epigallocatechin (EGC) that possess a galloyl group in the 2 position also caused a 70% inhibition of the DNA synthesis. Treatment of VSMC with 50 µM CG, EGCG, ECG, and EGC resulted in complete inhibition of the PDGF-BB-induced increase in cell number of rat aortic VSMC. 2-(3,4-dihydroxyphenyl)-3,4-dihydro-2H-1-benzopyran-3,5,7-triol (catechin) or epicatechin (EC) (50 µM) failed to inhibit the PDGF-BB-induced VSMC growth.

2. After preincubations of rat aortic VSMC with various concentrations of the catechins, VSMC were stimulated with 50 ng/ml PDGF-BB.

Stimulation of the ECG-treated VSMC with PDGF-BB resulted in a dose-dependent inhibition of the PDGF-BB-induced tyrosine phosphorylation of PDGF ß-receptor (PDGF-Rß) (Fig. 1 A). A complete inhibition was observed by 20–50 µM ECG. Remarkably, treatment of the cells with 10–50 µM EGC had no effects on PDGF-Rß tyrosine phosphorylation (Fig. 1A ). The effect of PDGF-BB on PDGF-Rß tyrosine phosphorylation was almost completely abolished in 50 µM CG-treated cells whereas in 10 and 20 µM CG-treated cells it was slightly reduced (Fig. 1B ). Again, 50 µM ECG caused an almost complete inhibition of PDGF-Rß tyrosine phosphorylation (Fig. 1B ). Laser densitometric analysis of the band densities of the tyrosine phosphorylated PDGF-Rß obtained by three separate experiments is presented in Fig. 1C . Treatment of VSMC with 20 µM CG and ECG resulted in a 80% and 30% inhibition of the PDGF-BB-induced tyrosine phosphorylation of the PDGF-Rß in untreated cells (=100%), respectively. Treatment of the cells with 50 µM ECG or CG resulted in a complete inhibition of the PDGF-BB effect. As shown in Fig. 1A, B , tyrosine phosphorylation of PLC-1 and PI 3'-K was reduced in a dose-dependent manner in ECG- and CG-treated cells. As expected, 10–50 µM EGC failed to abolish the PDGF-BB-induced tyrosine phosphorylation of PLC-1 and PI 3'-K (Fig. 1A ). Treatment with 50 µM EGC did not influence phosphorylation of ERK1/2 whereas treatment with 50 µM ECG caused an almost complete inhibition (Fig. 1D ). Laser densitometric analysis of the band densities obtained from three independent experiments revealed that treatment of the cells with 50 µM CG and ECG resulted in an 83 ± 13 and 90 ± 16% inhibition of the PDGF-BB-induced phosphorylation of the PLC-1. PI 3'-K phosphorylation was completely inhibited in 50 µM CG- and ECG-treated cells. No significant effects were observed by 1–20 µM CG. Treatment of cells with 20 µM ECG caused a 50 ± 17% inhibition of the phosphorylated PI 3'-K and a 43 ± 24% reduction of the phosphorylated PLC-1. Catechin or EC (both 50 µM) failed to inhibit the PDGF-Rß-mediated intracellular signaling transduction pathway.

View larger version (49K): [in this window] [in a new window]   Figure 1. Effect of PDGF-BB on tyrosine phosphorylation of PDGF-Rß, PLC-1 and PI 3'-K in VSMC treated with ECG (A) and CG (B). Confluent cells in 75 cm2 were preincubated in serum-free medium consisting of DMEM in the presence and absence of different concentrations of the ECG and CG for 24 h. The medium was replaced with serum-free medium without catechins, and VSMC were stimulated with 50 ng/ml PDGF-BB for 5 min. Cell lysates were mixed with 80 µl Sepharose-coupled anti-phosphotyrosine antibody to immunoprecipitate PDGF-Rß, PLC-1, and PI 3'-K. Tyrosine phosphorylated kinases were eluted with lysis buffer containing 5 mM phenylphosphate. Tyrosine phosphorylated PDGF-Rß (180 kDa), PLC-1 (150 kDa), and PI 3'-K (85 kDa) were detected by the enhanced chemiluminescence Western blotting method. C) Laser densitometric analysis of the band densities obtained by three separate experiments showing the effect of 20 and 50 µM ECG and CG on PDGF-Rß tyrosine phosphorylation (mean±SD, n=3, *P<0.05 for ECG or CG effect vs. PDGF-BB effect). D) VSMC were treated with 50 µM EGC or 50 µM ECG for 24 h, then stimulated with PDGF-BB for 5 min. After lysis, phosphorylated ERK1/2 (44 and 42 kDa, respectively) were detected by enhanced chemiluminescence Western blotting.

3. PDGF-BB (50 ng/ml) induced in rat aortic VSMC a maximal increase in cytosolic free Ca²⁺ concentration ([Ca²⁺]_i) from 65 ± 12 to 255 ± 25 nM (mean±SD, n=3) within 50 s.

The effect of PDGF-BB was abolished in 20 µM ECG-treated VSMC and significantly attenuated to 130 ± 12 nM in 20 µM EGCG-treated cells.

4. PDGF-BB caused a 48 ± 4% increase in the number of human aortic VSMC.

Catechin and EC did not influence the PDGF-BB-induced increase in cell number. In contrast, 50 µM EGCG, ECG, and EGC induced a complete inhibition of the PDGF-BB-induced increase in cell number.

5. Treatment of human aortic VSMC cells with 50 µM EGCG resulted in a marked inhibition of PDGF-Rß, PLC-1, and PI 3'-K tyrosine phosphorylation.

In contrast to EGCG or ECG (50 µM), catechin, EC, and EGC had no effect on PDGF-Rß tyrosine phosphorylation. Laser densitometric analysis of the band densities obtained from three separate experiments showed that treatment of VSMC with 50 µM EGCG and ECG resulted in a 75 ± 5% and 88 ± 5% inhibition of the PDGF-BB-induced tyrosine phosphorylation of the PDGF-Rß in untreated cells (=100%).

6. Stimulation of the human aortic VSMC with PDGF-BB resulted in an elevation of [Ca²⁺]_i from 53 ± 8 to 226 ± 17 nM (mean±SD, n=3), with a maximum after 50 s.

Treatment of the ECG- and EGCG-treated (50 µM) cells significantly inhibits the maximal effect of PDGF-BB to 106 ± 27 nM and 137 ± 23 nM, respectively. Catechin, EC, and EGC (each 50 µM) had no significant effect on the PDGF-BB-induced increase in [Ca²⁺]_i.

7. To show that the inhibitory effects of the active catechins on cultured VSMC can also be attributed to the in vivo situation left common arteries from male Wistar Kyoto rats were injured by a catheter.

Animals were treated with EGCG for 7 days. EGCG was injected once daily at 10 mg/kg s.c. Before death (-18 h and -1 h), rats were treated with bromodeoxyuridine (BrdU). After immunocytochemistry of carotid arteries, BrdU-positive cells were counted and expressed as percentage of total cells per cross section for intima and media. As shown in Fig. 2 , in placebo-treated rats the intimal and medial BrdU labeling was 73 ± 8% and 7.31 ± 2.7% days after injury (mean±SD, n=5). EGCG-treated rats showed labeling indices in intima and medial smooth muscle cells that were reduced significantly by 41.4 ± 18% and 1.84 ± 3.4%, respectively, as judged from 2-way analysis of variance (P<0.05, n=8). These results demonstrate that the S-phase nuclei in the intima and media was significantly reduced by 43% and 75%, respectively, after treatment of the animals with EGCG for 7 days.

View larger version (74K): [in this window] [in a new window]   Figure 2. Cross-sections of rat carotid arteries 7 days after balloon injury labeled with BrdU to visualize the presence of S-phase nuclei in the presence (A) and absence (B) of EGCG 10 mg·kg-1·day-1. A) Treatment with EGCG clearly reduced the number of nuclei in S-phase, particularly in the intima, whereas untreated cells (placebo) show the expected high number of labeled nuclei (B). C) Percent of the S-phase nuclei in the intima (mean±SD, n=5, untreated animals; n=8, EGCG-treated animals, *P<0.05 for treated vs. untreated animals). D) Percent of S-phase nuclei in the media (mean±SD, n=5, untreated animals; n=8, EGCG-treated animals, *P<0.05 for treated vs. untreated animals). 40x.

CONCLUSIONS AND SIGNIFICANCE

The present results suggest that catechins possessing the galloyl group in the 3 position of the chemical structure of the catechin can inhibit the intracellular signal transduction pathway of PDGF-Rß in cultured rat and human VSMC. These compounds inhibit VSMC growth in vitro as well as in vivo. Catechins with the galloyl group in the 2 position inhibit VSMC growth without affecting the PDGF-Rß signal transduction pathway. The galloyl group in the 3 position in the catechin structure is essential for inhibiting the PDGF-Rß-mediated intracellular signal transduction pathway in VSMC (see Fig. 3 ).

View larger version (43K): [in this window] [in a new window]   Figure 3. Schematic diagram.

Catechins that do not possess a galloyl group had no effect on the PDGF-Rß-mediated signal transduction pathway or VSMC growth. We conclude that the galloyl group of several plant-derived flavonoids is essential for the preventive action of these compounds on the development of proliferative cardiovascular diseases.

A recent hypothesis that catechins act as scavengers of reactive oxygen species has been favored as an explanation for their beneficial effects on the pathogenesis of cardiovascular diseases. Results show that in the aqueous phase, the order of effectiveness as radical scavengers is ECG > EGCG > EGC > EC congruent to catechin. The effectiveness of the catechins as radical scavengers correlates well with our findings regarding the inhibitory effect of the catechins on the PDGF-Rß phosphorylation (ECG>EGCG; EGC, EC, and catechin: no effect). The anti-proliferative effects of catechins on VSMCx cell growth (ECG>EGCG=ECG; EC, and catechin: no effects) correlate equally with the ability of catechins to scavenge reactive oxygen species. Since enhanced activity of PDGF-Rß is involved in the development of proliferative cardiovascular diseases, we may postulate that catechins are active by their efficacy to inhibit tyrosine phosphorylation of PDGF-Rß. Indeed, consumption of catechins acts protectively against cardiovascular disease by several mechanisms.

In summary, we postulate that the galloyl group containing plant-derived flavonoids may act as natural PDGF-Rß tyrosine kinase inhibitors. These findings could be helpful for the development new natural drugs to prevent and treat proliferative cardiovascular diseases. We presume that chemical modification of the galloyl group can lead to new drugs against cardiovascular diseases. Finally, these findings may explain the preventive effects of plant-derived flavonoids on cardiovascular diseases observed in several epidemiological studies. Although some findings suggest that the galloyl group in the 3 position may prevent binding of PDGF-BB to PDGF-Rß, the mechanisms of inhibition of the PDGF-Rß phosphorylation remain to be elucidated.

FOOTNOTES

¹ To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.01-0799fje; to cite this article, use FASEB J. (April 23, 2002) 10.1096/fj.01-0799fje.

³ Contributed equally to the present work.

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