TGF-β1and PPARα Agonist Cooperatively Inhibit Vascular Smooth Muscle Cell Proliferation

Objective: Vascular smooth muscle cells (VSMCs) can synthesize andsecrete many extracellular matrix components and a variety of cytokines, andcan response to different stimulations through cytokine and growth factorreceptors on cell surface, thus altering the biological behavior of VSMCs.Krüppel like factor4(KLF4) is a class of zinc finger transcription factor, andthere are three consecutive C₂H₂zinc-figure structure in the carboxy-terminal,which binds to specific DNA sequences directly and functions as atranscriptional regulator.Peroxisome proliferator-activated receptors (PPARs) regulate geneexpression as transcription factors, involved in the regulation of celldifferentiation, development, metabolism and tumorigenesis. PPARs havethree types of α, β/δ, γ, and PPARα is widely expressed in epithelial cell (EC)and VSMC to play the role of anti-inflammatory and anti-atherogenic sclerosis,etc. Fenofibrate, a PPARα agonist, has been widely used in atherosclerosis,myocardial infarction and other cardiovascular diseases as a clinical drug.Transforming growth factor β (TGF-β) family members are factors withmultiple functions, binding to specific type I and type II serine-threoninekinase receptors, and play a role in regulation of VSMC proliferation,differentiation, and apoptosis through a variety of signaling pathways.The present experiment demonstrated that TGF-β1and PPARα agonistFenofibrate can significantly inhibit VSMC proliferation. On the basis of thisfinding, we further explored the molecular mechanisms whereby TGF-β1andPPARα cooperatively inhibit VSMC proliferation.Methods: VSMCs were isolated from the thoracic aorta ofSprague-Dawley rats. Cell passages3⁵were used in all experiments. Theexpression of KLF4, SM22α, SM-α-actin, PCNA, and cyclin E was detected by Western blotting. Flow cytometric analysis was used to reveal thedistribution of VSMCs in various phases of the cell cycle. Cell counting andBrdU incorporation experiments were done to examine cell proliferation.Wound healing and Boyden chamber assays were performed to observe cellmigration.Results:1TGF-β1and PPARα agonist Fenofibrate cooperatively promote KLF4expressionWestern blot analysis showed that the protein level of KLF4increasedsignificantly when VSMCs were treated with TGF-β1and Fenofibrate,respectively, and further increased when VSMCs were treated with both ofthem. In contrast, GW6471, a PPARα-specific inhibitor, could inhibitTGF-β1-induced expression of KLF4. These studies demonstrated thatTGF-β1and PPARα signaling pathways could coordinately promote theexpression of KLF4.2TGF-β1and PPARα agonist Fenofibrate cooperatively induce VSMCdifferentiationFenofibrate could significantly increase TGF-β1-induced expression ofVSMC markers SM22α and SM-α-actin. To further demonstrate thatFenofibrate and TGF-β1can cooperatively promote VSMC differentiation,VSMCs were treated with GW6471, a PPARα inhibitor. The results showedthat TGF-β1-induced expression of SM22α and SM-α-actin was inhibited byGW6471. This further indicated that TGF-β1and PPARα signaling worktogether to promote the differentiation of VSMCs.3TGF-β1and Fenofibrate cooperatively inhibit VSMC proliferationProliferation of VSMCs plays a key role in the pathogenesis of a varietyof proliferative vascular diseases, such as atherosclerosis, restenosis andhypertension. In order to observe the effect of TGF-β1and Fenofibrate onVSMC proliferation, Western blot, cell counting and BrdU incorporationassays were performed. Western blot analysis showed that TGF-β1inhibitedthe expression of PCNA (proliferating cell nuclear antigen), a VSMC proliferation marker. A further reduction in PCNA level was observed whenthe cells were treated with TGF-β1and Fenofibrate. This result indicated thatTGF-β1and Fenofibrate cooperatively inhibit VSMC proliferation. Cellcounting and BrdU incorporation assays obtained the same results.4Inhibition of VSMC proliferation by TGF-β1and Fenofibrate isrelated to suppression of the expression of cyclin ETo further investigate the mechanism by which TGF-β1and Fenofibrateinhibit VSMC proliferation, we detected the expression of cell cycle-relatedproteins. Western blot analysis showed that Fenofibrate significantly promotedTGF-β1-induced inhibition of cyclin E expression.G1-S switch is a key point in cell proliferation, so we examined thealteration of VSMC cell cycle by flow cytometry (FCM). Cell cycle analysisshowed that the G0/G1cell population markedly increased after TGF-β1treatment, and Fenofibrate enhanced this increase induced by TGF-β1. Inaddition, there was a reduction in cell proportion in S phase and G2/M phaseafter TGF-β1treatment, and this reduction was also enhanced by Fenofibratetreatment. These results indicated that TGF-β1and Fenofibrate cooperativelyinhibit the expression of cyclin E and cell cycle progression, subsequentlyleading to cell proliferation inhibition.5TGF-β1and Fenofibrate cooperatively inhibit VSMC migrationVSMC migration is closely related to cell proliferation. To observe theeffect of TGF-β1and Fenofibrate on VSMC migration, wound healingexperiment and Boyden chamber analysis were performed. The data showedthat TGF-β1or Fenofibrate treatment decreased the mobility of VSMCs and afurther decrease was observed when the cells were treated with both TGF-β1and Fenofibrate.Conclusions:1TGF-β1and PPARα agonist Fenofibrate cooperatively induce VSMCdifferentiation.2TGF-β1and Fenofibrate inhibit VSMC proliferation by suppressing theexpression of cyclin E and cell cycle progression. 3TGF-β1and Fenofibrate cooperatively inhibit VSMC migration.4TGF-β1and Fenofibrate cooperatively inhibit VSMC proliferation andinduce VSMC differentiation by promoting KLF4expression.