Bilical vein smooth muscle cells (HUVSMC) have been characterized as a model for investigation of VSMC functions [19]. Therefore, HUVSMCs were employed as a model to study the effects of high glucose on the expression of CTGF and also other ECM genes by RNA interference and neutralization antibody within this paper. Our data demonstrate that high-glucose-stimulated VSMC development and migration, at the same time as the high-glucose-induced ECM elements deposition in VSMCs were attenuated by CTGF inhibition, which recommended that therapies targeting CTGF could possibly be helpful in preventing intimal hyperplasia in the atherosclerotic lesions in diabetic macrovascular complications.ResultsEffect of higher glucose on CTGF expression in HUVSMCs To decide whether or not higher glucose modulates the expression of CTGF mRNA, HUVSMCs were treated with 25 mmol/L D-glucose, and total RNA was isolated at a variety of instances from six to 48 hours. Real-time quantitative RT-PCR revealed that higher glucose swiftly induced the expression of CTGF above basal levels 6 hours soon after remedy. The induction of CTGF expression was peaked at 12 hours just after treatment, then declined to close to baseline by 24 hours (Figure 1a). To exclude the possibility that high-glucose-induced CTGF expression was triggered by increased osmolarity, we tested the impact of 25 mmol/L mannitol on CTGF mRNA expression. Compared with cells inside the standard glucose medium, there was no considerable stimulatory effect on CTGF expression in HUVSMC cells incubated for 24 hours in regular glucose media containing 25 mmol/L mannitol, confirming the specificity in the high glucose response in stimulating the CTGF expression in HUVSMCs (Figure 1a).Below serum-starvation condition, growth-arrested HUVSMCs expressed low level of CTGF protein, as shown by Western blot as a band of 38 Kda. Total cellular CTGF protein levels began to enhance after treated with high glucose for 12 hours, and peaked at 24 hours post-treatment. The elevated CTGF level lasted up to 48 hours right after therapy (Figure 1b). The expression of CTGF protein was also analyzed by immunocytochemistry, which showed that growth-arrested HUVSMCs presented a slight CTGF staining, and treatment with higher glucose for 24 hours significantly enhanced cytoplasmic CTGF αLβ2 medchemexpress staining (Figure 1c). These information suggest that high glucose induces each CTGF mRNA and protein production in HUVSMCs. TGF- has been identified as a potent inducer of CTGF expression and it is also a really critical regulator of ECM in unique cell types [20,21]. Our outcomes showed that TGF- treatment (ten ng/mL) also induced CTGF expression within the HUVSMCs. Induction of CTGF by higher glucose may perhaps happen indirectly, mediated by the action of TGF-. To test this hypothesis, we examined the effect of a neutralization antibody of TGF- (10 g/mL, R D Systems, USA) on high glucose-induced CTGF expression. We observed that the blockade of TGF- by a neutralization antibody against active TGF- partly decreased high glucoseinduced CTGF gene and protein production (Figure 2a and 2b). This partial inhibition suggests that endogenous TGF- synthesis is, at the least partly, involved in high glucose-induced CTGF production.Function of CTGF in high glucose-induced ECM accumulation in HUVSMCs Earlier research have showed that high glucose elevated ECM accumulation in cultured smooth muscle Histone Methyltransferase Storage & Stability cellsPage two of(web page quantity not for citation purposes)BMC Cell Biology 2007, eight:http://www.biomedcentral.com/1471-2121/8/Figure 1 High glucose increases CTGF mRNA express.