Korean J Physiol Pharmacol.  2013 Aug;17(4):307-314. 10.4196/kjpp.2013.17.4.307.

High Glucose Induces Connective Tissue Growth Factor Expression and Extracellular Matrix Accumulation in Rat Aorta Vascular Smooth Muscle Cells Via Extracellular Signal-Regulated Kinase 1/2

• Ha YM
• Lee DH
• Kim M
• Kang YJ

• KMID: 2285465
• DOI: http://doi.org/10.4196/kjpp.2013.17.4.307

Abstract

Connective tissue growth factor (CTGF) is a potent pro-fibrotic factor, which is implicated in fibrosis through extracellular matrix (ECM) induction in diabetic cardiovascular complications. It is an important downstream mediator in the fibrotic action of transforming growth factor beta (TGFbeta) and is potentially induced by hyperglycemia in human vascular smooth muscle cells (VSMCs). Therefore, the goal of this study is to identify the signaling pathways of CTGF effects on ECM accumulation and cell proliferation in VSMCs under hyperglycemia. We found that high glucose stimulated the levels of CTGF mRNA and protein and followed by VSMC proliferation and ECM components accumulation such as collagen type 1, collagen type 3 and fibronectin. By depleting endogenous CTGF we showed that CTGF is indispensable for the cell proliferation and ECM components accumulation in high glucose-stimulated VSMCs. In addition, pretreatment with the MEK1/2 specific inhibitors, PD98059 or U0126 potently inhibited the CTGF production and ECM components accumulation in high glucose-stimulated VSMCs. Furthermore, knockdown with ERK1/2 MAPK siRNA resulted in significantly down regulated of CTGF production, ECM components accumulation and cell proliferation in high glucose-stimulated VSMCs. Finally, ERK1/2 signaling regulated Egr-1 protein expression and treatment with recombinant CTGF reversed the Egr-1 expression in high glucose-induced VSMCs. It is conceivable that ERK1/2 MAPK signaling pathway plays an important role in regulating CTGF expression and suggests that blockade of CTGF through ERK1/2 MAPK signaling may be beneficial for therapeutic target of diabetic cardiovascular complication such as atherosclerosis.

Keyword

CTGF; Diabetic complication; Extracellular matrix; Hyperglycemia; VSMC

MeSH Terms

Animals
Aorta
Atherosclerosis
Butadienes
Cell Proliferation
Collagen
Connective Tissue
Connective Tissue Growth Factor
Diabetes Complications
Extracellular Matrix
Fibronectins
Fibrosis
Flavonoids
Glucose
Humans
Hyperglycemia
Muscle, Smooth, Vascular
Nitriles
Phosphotransferases
Rats
RNA, Messenger
RNA, Small Interfering
Transforming Growth Factor beta
Butadienes
Collagen
Connective Tissue Growth Factor
Fibronectins
Flavonoids
Glucose
Nitriles
Phosphotransferases
RNA, Messenger
RNA, Small Interfering
Transforming Growth Factor beta

Figure

• 

  Fig. 1 Effects of high glucose on VSMC proliferation. Cells were treated with glucose concentration (5.55, 11.1 and 22.2 mM) for 24 or 48 hrs. VSMC proliferation was measured (A) MTT assay (B) cell counting assay. Results are represented as the mean± S.E.M (n=4). *p<0.05 compared with 5.55 mM glucose, **p<0.01 compared with 5.55 mM glucose and ***p<0.001 compared with 5.55 mM glucose.

• 

  Fig. 2 High glucose increases CTGF, TGFβ1, and ECM components mRNA and protein expressions in VSMCs. Cells were treated with glucose concentration (5.55, 11.1 and 22.2 mM) for 24hrs or 48 hrs. After 24 hrs, CTGF, TGF-β1 (A) and ECM components mRNA (C) were measured by real-time PCR. After 24 or 48 hrs, CTGF, TGF-β1 (B) and ECM components protein expression (D) were detected by western blot. Results are represented as the mean±S.E.M (n=4). *p<0.05 compared with 5.55 mM glucose, **p<0.001 compared with 5.55 mM glucose.

• 

  Fig. 3 Effects of CTGF on high glucose-induced proliferation, ECM components mRNA and protein expression in VSMCs. Cells were transfected with CTGF siRNA and then treated with 5.55 or 22.2 mM of glucose. After treatment, CTGF protein expression measured by western blot (A) and cell proliferation measured by an MTT assay (B) Collagen type I, Collagen type III, and fibronectin mRNA levels measured by real-time PCR (C) and protein expression were measured by western blot (D). Results are represented as the mean±S.E.M (n=4). **p<0.01 compared 5.55 mM glucose, ***p<0.001 compared with 5.55 mM glucose, ##p<0.01 compared with 22.2 mM glucose, ###p<0.001 compared with 22.2 mM glucose.

• 

  Fig. 4 Effects of MAP Kinase inhibitors on high glucose-stimulated CTGF, ECM accumulation and proliferation in VSMCs. Cells were treated with PD98059 (25 µM), U0126 (2.5 µM), SB203580 (5 µM) and SP600125 (25 µM) and then treated with 5.55 or 22.2 mM of glucose. After treatment, CTGF protein expression measured by western blot (A, B) and cell proliferation measured by an MTT assay (C) Collagen type I, Collagen type III, and fibronectin protein expression were measured by western blot (D). Results are represented as the mean±S.E.M (n=4). *p<0.05 compared with 5.55 mM glucose, **p<0.001 compared with 5.55 mM glucose, #p<0.05 compared with 22.2 mM glucose.

• 

  Fig. 5 ERK1/2 siRNA transfection reduces high glucose-induced CTGF, ECM protein expression and cell proliferation in VSMCs. Cells were transfected with CTGF siRNA and then treated with 5.55 or 22.2 mM of glucose. After treatment, CTGF protein expression measured by western blot (A, B) and cell proliferation measured by an MTT assay (C) Collagen type I, Collagen type III, and fibronectin protein expression were measured by western blot (D). Results are represented as the mean±S.E.M (n=4). *p<0.05 compared with 5.55 mM glucose, **p<0.001 compared with 5.55 mM glucose, #p<0.05 compared with 22.2 mM glucose, ##p<0.001 compared with 22.2 mM glucose.

• 

  Fig. 6 CTGF regulated by Egr-1 through ERK1/2 MAPK signaling in high glucose-induced VSMCs. Treatment with high glucose of 22.2 mM, Egr-1 protein expression (A, B), pretreatment with PD98059 (25 µM), U0126 (2.5 µM) (C), transfection with Egr-1 or control siRNA and recombinant CTGF treatment (D) were measured by western blot. Pretreatment with PD98059 (25 µM), U0126 (2.5 µM) and CTGF treatment in high glucose of 22.2 mM analyzed by MTT assay. Results are represented as the mean±S.E.M (n=4). *p<0.01 compared with 5.55 mM glucose, #p<0.01 compared with 22.2 mM glucose, $p<0.01 compared with 22.2 mM glucose plus MEK1/2 inhibitor.

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