Korean J Physiol Pharmacol.  2018 Mar;22(2):193-201. 10.4196/kjpp.2018.22.2.193.

Fluvastatin inhibits advanced glycation end products-induced proliferation, migration, and extracellular matrix accumulation in vascular smooth muscle cells by targeting connective tissue growth factor

• Hwang AR
• Nam JO
• Kang YJ

• KMID: 2410109
• DOI: http://doi.org/10.4196/kjpp.2018.22.2.193

Abstract

Connective tissue growth factor (CTGF) is a novel fibrotic mediator, which is considered to mediate fibrosis through extracellular matrix (ECM) synthesis in diabetic cardiovascular complications. Statins have significant immunomodulatory effects and reduce vascular injury. We therefore examined whether fluvastatin has anti-fibrotic effects in vascular smooth muscle cells (VSMCs) and elucidated its putative transduction signals. We show that advanced glycation end products (AGEs) stimulated CTGF mRNA and protein expression in a time-dependent manner. AGE-induced CTGF expression was mediated via ERK1/2, JNK, and Egr-1 pathways, but not p38; consequently, cell proliferation and migration and ECM accumulation were regulated by CTGF signaling pathway. AGE-stimulated VSMC proliferation, migration, and ECM accumulation were blocked by fluvastatin. However, the inhibitory effect of fluvastatin was restored by administration of CTGF recombinant protein. AGE-induced VSMC proliferation was dependent on cell cycle arrest, thereby increasing G1/G0 phase. Fluvastatin repressed cell cycle regulatory genes cyclin D1 and Cdk4 and augmented cyclin-dependent kinase inhibitors p27 and p21 in AGE-induced VSMCs. Taken together, fluvastatin suppressed AGE-induced VSMC proliferation, migration, and ECM accumulation by targeting CTGF signaling mechanism. These findings might be evidence for CTGF as a potential therapeutic target in diabetic vasculature complication.

Keyword

Advanced glycation end products; Cell cycle arrest; Connective tissue growth factor; Extracellular matrix; Fluvastatin; Vascular smooth muscle cell

MeSH Terms

Cell Cycle
Cell Cycle Checkpoints
Cell Proliferation
Connective Tissue Growth Factor*
Connective Tissue*
Cyclin D1
Extracellular Matrix*
Fibrosis
Genes, Regulator
Glycosylation End Products, Advanced
Hydroxymethylglutaryl-CoA Reductase Inhibitors
Muscle, Smooth, Vascular*
Phosphotransferases
RNA, Messenger
Vascular System Injuries
Connective Tissue Growth Factor
Cyclin D1
Glycosylation End Products, Advanced
Phosphotransferases
RNA, Messenger

Figure

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  Fig. 1 Fluvastatin inhibits AGE-induced CTGF expression in VSMCs. Cells were treated with AGE 10 µg/ml for 0, 6, 12, 18, 24 h. CTGF mRNA level was determined by qRT-PCR analysis (A) CTGF protein level was determined by Western blot (C). Cells were treated with 2 or 5 µM fluvastatin for 1 h before incubation with AGEs for 24 h. CTGF mRNA level was determined by qRT-PCR analysis (B) and CTGF protein level was determined by Western blot (D). Data are representative of three independent experiments with similar results *p<0.05 **p<0.01 vs. untreated cells, #p<.05 ##p<0.01 vs. AGE-treated cells.

• 

  Fig. 2 AGEs induce CTGF expression in VSMCs via ERK/JNK pathways. Cells were treated with 10 µg/ml AGEs for 0, 5, 10, 15, 30, 60 min, protein level were determined by western blotting (A). The cells were pretreated with PD98059 (1 µM), U0126 (1 µM), SB203580 (10 µM) and SP600125 (25 µM) for 1 h and then incubated with AGE (10 µg/ml) for 24 h, CTGF protein level were determined by western blotting (B), cell proliferation was determined by MTT assay (C). The cells were pretreated with fluvastatin for 1 h and then incubated with AGE (10 µg/ml) for 30 min, MAPK protein level were determined by western blotting (D). Data are representative of three independent experiments with similar results **p<0.01 vs. untreated cells, #p<0.05, ##p<0.01 vs. AGE-treated cells.

• 

  Fig. 3 AGEs induce CTGF expression in VSMCs via Egr-1 pathways. Cells were pretreated with PD98059 (1 µM), U0126 (1 µM), SB203580 (10 µM) and SP600125 (25 µM) for 1 h and then incubated with AGE (10 µg/ml) for 1 h, Egr-1 protein expression was determined by western blotting (A). The cells were transfected with Egr-1 siRNA and then treated with AGE (10 µg/ml) for 24 h. proliferation determined by MTT assay (B) and Egr-1 and CTGF protein level were determined by western blotting (C, D). Data are representative of three independent experiments with similar results **p<0.01 vs. untreated cells, ##p<0.01 vs. AGE-treated cells.

• 

  Fig. 4 Fluvastatin inhibits AGE-induced VSMC proliferation, migration and ECM production via inhibiting CTGF. Cells were pretreated with Fluvastatin in the presence or absence of recombinant CTGF (100 nM) for 1 h and then exposed to AGE (10 µg/ml) for 24 h, cell proliferation was determined by MTT assay (A), mRNA level of fibronection, collagan1, collagan3 were determined by qRT-PCR analysis (B–D). After scratching, serum-starved VSMCs were pretreated with fluvastatin in the presence or absence of recombinant CTGF (100 nM) for 1 h and then exposed to AGE (10 µg/ml) for 36 h (E). Wound area was visualized by a phase contrast microscope. Data represent mean±SD from three independent repeats (n=4). **p<0.01 vs. untreated cells, #p<0.05 ##p<0.01 vs. AGE-treated cells, $p<0.05 $$p<0.01 vs. AGE+fluvastatin.

• 

  Fig. 5 Fluvastatin increases cell cycle arrest through inhibition of CTGF in AGE-induced VSMCs. Cells were seeds onto 6-well plates at a density of 1×104 cells/mL, pretreated with fluvastatun (5 µM) in the presence or absence of recombinant CTGF (100 nM) for 1 h and then exposed to AGE (10 µg/ml) for 36 h. Cell cycle was analyzed by FACS analysis (A, B). Expression of cell cycle regulating genes was determined by western blotting (C). Data represent mean±SD from three independent repeats (n=4). **p<0.001 vs. untreated cells, ##p<0.001 vs. AGE-treated cells, $$p<0.001 vs. AGE+fluvastatin.

• 

  Fig. 6 Schematics of the fluvastatin inhibits AGE-induced gene expression in VSMCs. AGEs stimulation of vascular smooth muscle cells leads to an increase in MEK MAP kinase, ERK1/2, which regulates the transcription factor, Egr-1 gene. Subsequently, the increased expression of Egr-1 leads to elevated levels of CTGF protein, which promote the proliferation, migration and ECM accumulation of vascular smooth muscle cells. Fluvastatin inhibits MEK MAP kinase, which downregulated the transcription of Egr-1. Subsequently, it leads to inhibited levels of CTGF protein, which downregulated the proliferation, migration and ECM accumulation in VSMCs.

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