TLR4 Signaling Mediates Inflammatory Responses Induced By CRP In Rat VSMCs And Inhibitory Effects Of Rosiglitazone

Background:Although it is known that atherosclerosis is a chronic inflammatory and immunity-related disease associated with ischemic cardiocerebrovascular diseases, the pathologenesis of atherosclerosis remains poorly understood. Toll-like receptors (TLRs), as important mediators of innate immunity, trigger the inflammatory response by activation of pro-inflammatory transcription factors such as NF-κB and AP-1. Especially, TLR4-mediated inflammatory signaling has been shown to facilitate atherogenesis.C-reactive protein (CRP), the prototypic marker of inflammation, has been confirmed in numerous studies to predict cardiovascular events. In addition to being a powerful risk factor, much data suggest that CRP exerts direct pro-inflammatory and pro-atherogenic effects on endothelial cells, vascular smooth muscle cells (VSMCs) and monocytes through increasing expressions of adhesion molecules, cytokines, chemokines and growth factors in vitro. Moreover, CRP deposits in the lesions of human and experimental atherosclerosis, and colocalizes with the terminal complement complex and foam cells. On the basis of its pleiotropic roles in cardiovascular diseases, CRP may contribute to development of atherosclerosis and the subsequent cardiovascular events by inducing inflammatory responses. But, whether the pro-inflammatory effect of CRP involves TLR4-dependent signaling pathway is still unknown.Peroxisome proliferator-activated receptors (PPARs) are ligand-activated transcription factors belonging to the nuclear receptor family. PPARs participate in regulating lipid and lipoprotein metabolism and glucose homeostasis and influencing cellular proliferation, differentiation and apoptosis. Thiazolidinediones (TZDs), the synthetic PPARγagonists, not only improve insulin resistance in patients with type II diabetes but also exert a broad spectrum of anti-inflammatory and anti-atherogenic effects in vitro and in animal models of atherosclerosis, including inhibition of expressions of matrix metalloproteinases, iNOS and several interleukins. Although the ability of TZDs to antagonize inflammatory responses is linked to anti-atherogenic actions, the exact mechanisms of rosiglitazone against CRP-mediated inflammatory responses remain to be established. Objective:1. To probe TLR4-dependent signaling pathway of pro-inflammatory effect of CRP in VSMCs.2. To explore the novel anti-inflammatory effects and mechanisms of rosiglitazone in CRP-stimulated VSMCs.Protocol and methods:1. Study on expressions of TLR4 and inflammatory cytokines in CRP-stimulated VSMCs Rat VSMCs were cultured and used in all the experiments. VSMCs were incubated with CRP (10, 25, 50μg/ml) or LPS (100 ng/ml) for 9 h or 30 min (for GR protein). Then, TLR4 and GR expressions in VSMCs were identified with the immunocytofluorescence staining followed by fluorescent microscope observation. mRNA and protein expressions of VEGF-A, iNOS, TLR4, PPARγand GR were analyzed by quantitative real-time PCR and western blot, respectively.2. Study on TLR4-dependent signaling pathway for CRP-induced inflammation in VSMCs VSMCs were transiently transfected with 100 nM TLR4 small-interfering RNA (siRNA) or negative control siRNA (NC siRNA) using DharmaFECT 2 transfection reagents for 48 h, and subsequently stimulated with 25μg/ml CRP for 9 h or 30 min (for GR protein). mRNA and protein levels of VEGF-A, iNOS, PPARγand GR were analyzed by the same methods as above.VSMCs were stimulated with CRP (25μg/ml) for 9 h after pretreatment for 30 min with AT1R antagonist losartan, AT2R blocker PD123319, JNK1/2 inhibitor SP600125, p38 MAPK inhibitor SB203580 and ERK1/2 inhibitor PD098059 alone or in the different combinations. Then, TLR4 expression was determined by the same methods as above.VSMCs were stimulated with CRP (25μg/ml) for 9 h after pretreatment for 30 min with AT1R antagonist losartan, JNK1/2 inhibitor SP600125, p38 MAPK inhibitor SB203580 or ERK1/2 inhibitor PD098059. Then, protein expressions of VEGF-A and iNOS were determined by western blot.In another experiment, VSMCs were stimulated with CRP (25μg/ml) for 30 min after pretreated for 30 min with losartan and then, p38 expression was measured by western blot.To probe role of TLR4 in CRP-induced PKCαexpression, the cells were exposed to CRP for another 9 h after transiently transfected with TLR4 siRNA (100 nM) or NC siRNA for 48 h and then, mRNA and protein expression of PKCαwas measured by the same methods as above.VSMCs were stimulated with CRP (25μg/ml) for 9 h after pretreatment for 1 h with PKCα inhibitor G? 6976. Then, protein expressions of VEGF-A and iNOS were determined by western blot.To further explore role of PKCαin CRP-induced IRF3 expression, VSMCs were stimulated with CRP for 9 h (for mRNA) or 30 min (for protein) after transfected with PKCαsiRNA or NC siRNA for 48 h and then, mRNA and protein expression of IRF3 was assessed by the same methods as above.After the transfected cells with IRF3 siRNA or NC siRNA for 48 h were stimulated by CRP for 9 h or 30 min, NF-κB p65 and IκBαexpressions were investigated by western blot.3. Study on anti-inflammatory effects of rosiglitazone in CRP-stimulated VSMCs VSMCs were treated for 2 h with the different concentrations of rosiglitazone (5, 10 and 20μM) before exposure of the cells to CRP (25μg/ml) for 9 h or 30 min (for GR protein). Then, TLR4 and GR expressions in VSMCs were identified with the immunocytofluorescence staining followed by fluorescent microscope observation. mRNA and protein levels of VEGF-A, iNOS, TLR4, PPARγand GR were analyzed by quantitative real-time PCR and western blot, respectively.4. Study on the inhibitory effects of rosiglitazone on the TLR4-dependent signaling pathway of CRP-induced inflammation in VSMCsAfter application of TLR4 siRNA or NC siRNA for 48 h, VSMCs were exposed to rosiglitazone (10μM) for another 2 h, and subsequently stimulated with CRP (25μg/ml) for 9 h. Then, mRNA and protein expressions of VEGF-A and iNOS were analyzed by the same methods as above.The cells were treated with rosiglitazone (10μM) for 2 h after pretreatment of the cells with p38 inhibitor SB203580 for 30 min, PPARγinhibitor GW9662 for 1h or GR inhibitor RU486 for 1 h and then, stimulated by CRP (25μg/ml) for another 9 h. Finally, TLR4 protein expression was determined by western blot.The cells were incubated with rosiglitazone (10μM) for 2 h after pretreated for 1h with GW9662 or/and RU486. Then, the cells were stimulated by CRP (25μg/ml) for another 30 min, and p38 expression was measured using western blot.The cells were incubated with rosiglitazone (10μM) for 2 h after pretreated for 1h with GW9662 and then, stimulated by CRP (25μg/ml) for another 30 min. Finally, GR expression was assessed with western blot.Results:1. Effects of CRP on the expressions of TLR4 and inflammatory cytokines in VSMCs The results showed that CRP concentration-dependently enhanced VEGF-A, iNOS and TLR4 expressions, downregulated expressions of PPARγand GR in mRNA and protein levels. In addition, CRP hindered GR nuclear translocation in VSMCs.2. TLR4 signaling pathway of CRP-induced inflammatory response in VSMCs The results indicated that CRP increased VEGF-A and iNOS expressions, and decreased PPARγand GR expressions in VSMCs. Knockdown of TLR4 with special siRNA partly reversed the effects of CRP. These demonstrate that CRP regulates VEGF-A, iNOS, PPARγand GR expressions through TLR4.CRP-induced expression of TLR4 was inhibited by losartan, SP600125, SB203580 or PD098059 but not PD123319, and the inhibitory effect was more obvious when the cells were pretreated with the combination of losartan and SP600125 or SB203580 or PD098059. These confirm that CRP-induced TLR4 expression in VSMCs involves AT1R and MAPK.CRP-induced VEGF-A and iNOS expressions were apparently depressed by pretreating the cells with losartan or SB203580. And the inhibitory effect was more obvious when the cells were pretreated with the combination of losartan and SB203580. Furthermore, we found that losartan remarkably inhibited p38 phosphorylation induced by CRP in VSMCs. Although PD123319, PD98059 and SP600125 produced an inhibitory trend to VEGF-A and iNOS expressions in CRP-stimulated VSMCs, there was no significant effect in statistics. These suggest that CRP induces VEGF-A and iNOS up-regulations in VSMCs via AT1R and p38.The results also showed that TLR4 siRNA significantly blunted CRP-induced mRNA and protein expression of PKCαin VSMCs, and PKCαinhibitor G? 6976 inhibited protein expressions of VEGF-A and iNOS in VSMCs. Further experiments found that CRP-induced expression of IRF3 was substantially prevented by PKCαsiRNA. CRP significantly enhanced NF-κB p65 nuclear translocation and IκBαphosphorylation in VSMCs, and IRF3 siRNA abolished the effects.3. Inhibitory effects of rosiglitazone on CRP-stimulated expressions of TLR4 and inflammatory cytokines in VSMCsPretreatment of the cells with rosiglitazone concentration-dependently inhibited expressions of VEGF-A, iNOS and TLR4, but elevated expressions of PPARγand GR in mRNA and protein levels in CRP-stimulated VSMCs. Additionally, rosiglitazone reversed CRP-hindered GR nuclear translocation in VSMCs.4. Inhibitory effects of rosiglitazone on TLR4 signaling pathway of CRP-induced inflammation in VSMCsStimulation of the cells with CRP resulted in VEGF-A and iNOS elevation, whereas TLR4 siRNA and rosiglitazone partially reversed its effects in VSMCs. Moreover, treatment of the cells with combination of the TLR4 siRNA and rosiglitazone synergistically reversed CRP-induced changes of VEGF-A and iNOS.Our experiment also found that TLR4 siRNA enhanced PPARγand GR protein expressions in CRP-stimulated VSMCs. Rosiglitazone also produced the similar effects on protein expressions of PPARγand GR to TLR4 siRNA. Treatment of the cells with rosiglitazone further reversed CRP-induced changes of PPARγand GR in TLR4 siRNA-transfected cells.SB203580 or rosiglitazone alone inhibited CRP-induced TLR4 expression in VSMCs. Furthermore, the combination of SB203580 and rosiglitazone exhibited an additional inhibitory effect on TLR4 expression in CRP-stimulated VSMCs. Additionally, p38 phosphorylation under CRP stimulation was impaired by rosiglitazone. These indicate that blockade of p38 is involved in the inhibitory effect of rosiglitazone on TLR4 expression in CRP-stimulated VSMCs.RU486 but not GW9662 remarkably blunted rosiglitazone-attenuated p38 phosphorylation and subsequent TLR4 expression in CRP-stimulated VSMCs. PPARγinhibitor GW9662 did not affect GR phosphorylation in response to rosiglitazone in CRP-treated VSMCs. Therefore, the negative regulation of rosiglitazone on p38—TLR4 signal pathway directly depends on GR in CRP-stimulated VSMCs.Conclusions:1. CRP enhances generation of pro-inflammatory mediators such as TLR4, VEGF-A and iNOS, but reduces expression of anti-inflammatory molecules like PPARγand GR in VSMCs.2. CRP induces inflammatory response via TLR4-dependent signaling pathway (AT1—p38—TLR4—PKCα—IRF3—NF-κB) in VSMCs. This finding may contribute to elucidate the novel pro-inflammatory and pro-atherosclerotic mechanisms of CRP.3. Rosiglitazone decreased CRP-induced generations of pro-inflammatory mediators such as TLR4, VEGF-A and iNOS, but increased the expressions of anti-inflammatory molecules like PPARγand GR in VSMCs.4. Rosiglitazone exerts an anti-inflammatory effect by activating GR and subquently inhibiting p38—TLR4 signaling pathway in CRP-stimulated VSMCs. These findings provide new mechanisms to beneficial effects of rosiglitazone in the prevention and treatment of cardiovascular diseases including atherosclerosis.