Research Of The Effects And Mechanisms Of Connective Tissue Growth Factor On Calcification Of Vascular Smooth Muscle Cells In Vitro

Vascular calcification is an active and cell-regulated process resemble to skeletal mineralization and is prevalent in patients with atherosclerosis, aging, end-stage renal failure, uremia, and type â…¡ diabetes. Osteogenic differentiation of vascular smooth muscle cells (VSMCs) is essential in the development of vascular calcification. Connective tissue growth factor (CTGF) plays an important role in the pathogenesis of atherosclerosis. However, the effect of CTGF on the phenotypic transformation of VSMCs and the calcification of VSMCs have not been investigated. In the present study we defined the effect of CTGF in VSMCs transdifferentiation, VSMCs calcification and the molecular signaling by using an in vitro calcification model.Methods:VSMCs used in the present studies were identified by immunohistochmistry. Cells were divided into two groups:CTGF treated group (50ng/ml) and the control group. Mineralized matrix staining was performed with0.1%Alizarin red, the cellular mineral deposition was measured by testing the concentration of calcium. Real-time PCR and Western blot were used to examine the expressions of bone markers including Cbfa-1/Runx-2, ALP, OC, OPG and the expressions of MAPKs, including c-jun N-terminal Kinase (JNK), p38and ERK1/2. The ERK-specific inhibitor PD98059was used to blocked the activation of ERK.Results:VSMCs were obtained from the thoracic aortas of mouse, and the expression of smooth muscle specific a-actin antibody (a-SMA) was positive.After culture with CTGF for14days, the expression of bone markers including Cbfa-1/Runx-2, ALP, OC and OPG increased when compared with the control group, which were in a dose-dependent manner. The calcium deposition and calcium content was also increased in VSMCs with CTGF. Western blot analysis revealed that CTGF activated ERK and the peak activation of ERK occurred at30min. However, CTGF had no effect on the activation of JNK and p-38. Furthermore, a ERK-specific inhibitor, PD98059, significantly suppressed the effect of CTGF on VSMCs calcification and phenotypic marker expression.Conclusions:Taken together, our results reveal that CTGF enhances in vitro VSMCs calcification by inducing osteogenic differentiation of VSMCs, and this effect might be activated via the ERK pathway.