In the present study, the potential correlation between high glucose and CTGF was investigated in cultured HUVSMCs. The major finding of this study is that high glucose up-regulates the expression of CTGF in HUVSMCs and knockdown of CTGF gene results in the inhibition of high glucose-induced VSMC proliferation and migration. These observations establish acritical role of CTGF in mediating high-glucose induced ECM accumulation in VSMC and suggest that inhibition of CTGF may be useful for preventing abnormal VSMC growth and migration in diabetic vessels.
CTGF was first identified as a 38-kDa cysteine-rich protein, which can be specifically induced by TGF-β. It is recently found that CTGF is expressed abundantly in atherosclerotic blood vessels, but only marginally in normal vascular tissues. CTGF is one of the key factors involved in the development of atherosclerotic lesions [13
]. To further assess the role of CTGF in diabetic cardiovascular complications, we examined whether CTGF was regulated by high glucose in VSMC. Our data show that exposure of HUVSMC to high glucose, but not iso-osmotic mannitol, leads to an increase of CTGF expression, and the induction of CTGF by high glucose is partly mediated via TGF-β pathway.
Some studies have showed that high glucose may mediate diabetic renal and macrovascular complications by stimulating ECM production [9
], and the increased ECM synthesis accounts mainly for intimal plaque formation in the atherosclerotic lesions in diabetic vessels, so the effect of blocking CTGF action on ECM expression was further examined in this study. By CTGF-specific siRNA, our results demonstrate that knockdown of CTGF expression prevents ECM production in VSMC, indicating that CTGF plays an important role in mediating ECM accumulation in VSMC in response to high glucose.
In addition to increased ECM deposition in VSMC, it has been recognized that VSMC proliferation within the vessel wall is another critical pathogenic feature in the development of atherosclerosis. Glucose metabolism has been implicated to play an important role in this cellular mechanism [1
]. Neointimal formation, the leading cause of restenosis, is also caused by proliferation of VSMCs. Patients with diabetes mellitus have higher restenosis rates after coronary angioplasty than non-diabetic patients. Enhanced proliferation of VSMC has also been demonstrated in diabetic experimental animal models [24
]. In addition, cultured VSMC cells grown in media with high glucose concentration (to mimic hyperglycemia of diabetes) have exhibited increased cell proliferation [23
] Several intracellular signals elicited by high glucose are responsible for VSMC cell proliferation, including increased expression of TGF-β receptor type II via PKC-β [28
], enhanced intracellular ROS production [29
], and suppressed apoptosis via upregulation of bcl-xl and bfl-1/A1 levels through PI-3K and ERK1/2 pathways in VSMCs [30
]. Our results suggest a role of CTGF in the HUVSMCs proliferation induced by high glucose.
The migration of VSMCs from the media into the neointima is important in the pathogenesis of atherosclerosis. This process is regulated by multiple factors, and it involves changes in the interaction between the ECM and intracellular signaling cascades that regulate cell movement [31
]. High glucose is one of the multiple factors that could increase VSMCs migration [29
]. CTGF over-expression can significantly increase the activity of MMP-2 in VSMC conditioned medium and increase the migration of VSMC [25
], which suggests a link between high glucose-induced VSMC migration and CTGF over-expression. MMP-2 is able to induce VSMC migration and proliferation in addition to ECM degradation, and it has also been shown to play an important role in atherosclerotic plaque formation and restenosis after vessel injury [33
]. Consistent with previous report [34
], our data demonstrate that CTGF-siRNA suppresses high glucose-induced HUVSMC migration via, at least partly, down-regulation of MMP-2.
Recently, RNA interference (RNAi) has reinvigorated the therapeutic prospects for inhibiting gene expression and promised many advantages over binding inhibitors, including high specificity. RNAi provides a new, reliable method to investigate gene function and has the potential for gene therapy. In mammalian cells, 21-or 22-nucleotide (nt) RNAs with 2-nt 3' overhangs (small inhibitory RNAs, siRNA) exhibit a RNAi effect [35
]. It is important to avoid homologous sequences within a target mRNA in a given protein family [35
]. One of the reported CTGF siRNA sequences targets the coding region 360–380 from the first nucleotide of the start codon of CTGF mRNA [36
], but it is located within one of the four conserved cysteine rich modular domains-the von Willebrand factor (vWF) domain (307–486 bp) in the CTGF mRNA [37
]. In order to construct a specific CTGF-siRNA, we searched for regions of low homology to other genes of the CCN family. With the help of some siRNA-design tools in the Internet, we designed five specific pairs of DNA templates coding siRNA against human CTGF-mRNA and reconstructed the plasmid pSilencer3.1-H1 siRNA-CTGF. However, we only observed one pair of the DNA templates coding the sequence (nucleotides 762–782) has significant effect (79%) to down-regulate the expression of basal and high glucose-induced CTGF expression in HUVSMCs. The reason why only one out of five pairs of siRNA shows specific gene knockdown is unclear. This problem remains to be one of the many challenges of therapeutic usage of siRNA [38
]. Down regulation of CTGF markedly reduces the synthesis of high glucose-induced ECM proteins such as collagen type I and fibronectin. Our results indicate that CTGF is involved in ECM accumulation under normal glucose condition, but also it is an important mediator in the ECM deposition induced by high glucose in VSMC. Antagonism of CTGF function could possibly attenuate progression of diabetic macrovascular complications.