TLR4-mediated Proinflammatory Signaling Pathway Of Ang Ⅱ And Inhibitory Effects Of PPARα/γ Agonists

Background:Atherosclerosis is the main pathological basis of ischemic cardio-cerebral vascular diseases, and a chronic inflammatory and immunity-related disease. So far, pathogenesis of atherosclerosis is unclear. Toll-like receptor (TLR) family has been identified as a major component of pathogen-associated molecular-pattern-recognition molecules. Among members of TLR family, TLR4 is a new central signaling receptor providing an important link between immune response, inflammatory response and lipid metabolism. Activation of TLR4 can initiate inflammatory response to contribute to development of atherosclerosis.Angiotensin II (Ang II) is a main peptide hormone of rennin-angiotensin system, and plays an important role in pathogenesis of cardiovascular diseases including atherosclerosis, myocardial infarction and hypertension. Ang II is able to stimulate vascular cells to produce cytokines and growth factors via receptor-mediated way and subsequent activation of multiple intracellular signaling pathways, which participate in vascular inflammatory responses and cardiovascular disorders. Our previous study demonstrates that Ang II induces secretion of C-reactive protein (CRP) and IL-6 to produce inflammation in VSMCs via activating Ang II type 1 receptor (AT1) and subsequent extracellular signal-regulated kinase 1/2 (ERK1/2) pathway. But, whether the proinflammatory effect of Ang II involves TLR4 signaling is unknown.The peroxisome proliferator-activated receptors(PPARs)are members of the nuclear receptor superfamily of transcription factors that control the expressions of a large scope of genes. PPAR family plays a crucial role, not only in improving glucose and lipid homeostasis, but also in inhibiting vascular inflammation, oxidative stress and atherosclerosis. PPARαagonist fenofibrate and PPARγagonist rosiglitazone have recently been shown to possess anti-inflammatory and anti-arteriosclerotic properties by lowering levels of CRP, IL-6 and MCP-1 and inhibiting VCAM-1 and E-selectin expressions and TNF-αproduction. Although PPARαand PPARγagonists exert anti-inflammatory and anti-arteriosclerotic actions in cardiovascular system, the exact mechanisms of fenofibrate and rosiglitazone against Ang II-mediated inflammatory responses remain to be established. Objective:1. To probe TLR4-dependent signaling pathway for pro-inflammatory effect of Ang II.2. To explore a novel anti-inflammatory and anti-atherosclerotic mechanisms of PPARs agonists.Methods:1. Animal experimentMale Sprague-Dawley rats were subcutaneously infused with Ang II at a dose of 150 ng/kg/min for 7 days by Alzet osmotic minipumps. Fenofibrate ( 150 mg/kg ) and rosiglitazone(5 and 10 mg/kg)were intragastrically administered to rats for 7 days, starting from the day of Ang II infusion. Serum TNF-αand plasma 6-keto prostaglandin F1α(6-keto-PGF1α)levels were examined by the radioimmunity assay. One portion of the thoracic aorta was used for analysis of protein and mRNA expressions of TLR4, matrix metalloproteinase-9(MMP-9), PPARαand PPARγ, and other portion of the thoracic aorta was prepared for the immunohistochemical evaluation.2. Cell culture and treatmentsRat VSMCs were cultured and used in all the experiments. VSMCs were incubated with Ang II or LPS for the indicated time, or the cells were pretreated for 1 h with fenofibrate or rosiglitazone before exposure to Ang II stimulation. Then,the conditioned media were collected for determining TNF-αand 6-keto-PGF1αconcentrations with ELISA. TLR4 and MMP-9 expressions in VSMCs were identified with the immunocytofluorescence staining followed by confocal microscope observation. Protein and mRNA expressions of TLR4, MMP-9, PPARαand PPARγwere analyzed by western blot, RT-PCR and quantitative real-time PCR, respectively.3. Study on TLR4-dependent signaling pathway for Ang II-induced inflammation in VSMCsVSMCs were pretreated with anti-TLR4 antibody for 1 h and subsequently stimulated with Ang II for 24 h. TNF-αand 6-keto-PGF1αlevels in the supernatant were measured with ELISA, and protein and mRNA expressions of MMP-9, PPARαand PPARγwere analyzed by the same methods as above.To certify AT receptor subtypes and ERK1/2 signal involved in Ang II -induced TLR4 activation in VSMCs, the cells were stimulated with Ang II after pretreated with AT1 receptor antagonist losartan, AT2 receptor antagonist PD123319 and ERK1/2 inhibitor PD098059 alone or in the different combinations. Then, protein and mRNA expressions of TLR4 were analyzed by the same methods as above. To probe role of TLR4 in Ang II-induced IP-10 expression, VSMCs were preincubated with anti-TLR4 antibody prior to Ang II stimulation, and protein and mRNA expressions of IP-10 were analyzed by the same methods as above.To further explore role of IP-10 in Ang II-induced PKC expression, VSMCs were pretreated with anti-IP-10 antibody and subsequently stimulated with Ang II. Then, protein and mRNA expressions of PKC were analyzed by the same methods as above. Finally, VSMCs were preincubated with a specific PKC inhibitor chelerythrine prior to Ang II stimulation, and protein expression of NF-κBp65 was analyzed by western blot to investigate role of PKC in Ang II-induced NF-κB expression.4. Small-interfering RNAVSMCs were transiently transfected with TLR4 small-interfering RNA (siRNA), IP-10 siRNA or negative control siRNA (NC siRNA) using DharmaFECT 2 transfection reagents. After application of NC siRNA or TLR4 siRNA, VSMCs were exposed to fenofibrate or rosiglitazone, and subsequently stimulated with Ang II. TNF-αand 6-keto-PGF1αconcentrations were determined by ELISA, and mRNA levels of MMP-9, PPARαand PPARγwere analyzed by the same methods as above.5. Study on inhibitory effects of fenofibrate and rosiglitazone on the TLR4-dependentsignaling pathway for Ang II-induced inflammation in VSMCs. VSMCs were pretreated with anti-TLR4 antiobody prior to incubation with fenofibrate or rosiglitazone, and subsequently stimulated with Ang II or LPS. Then, TNF-αand 6-keto-PGF1αlevels in the supernatant were measured with ELISA, and protein expressions of MMP-9, PPARαand PPARγwere analyzed by western blot.To clarify whether blockade of the AT1/ERK1/2 signaling was involved in the inhibitory effect of fenofibrate and rosiglitazone on Ang II-induced TLR4 expression in VSMCs, VSMCs were subjected to losartan, PD123319 and PD098059, followed by treatment of fenofibrate or rosiglitazone before stimulation with Ang II. Then, protein expressions of TLR4 and AT1 and ERK1/2 phosphorylation were analyzed by western blot.Furthermore, after application of TLR4 siRNA or IP-10 siRNA, VSMCs were exposed to fenofibrate or rosiglitazone, and subsequently stimulated with Ang II. IP-10 or PKC protein expression was also detected by western blot.Finally, we observed whether the blockade of PKC was required for the inhibitory effect of fenofibrate and rosiglitazone on Ang II-induced protein expression of NF-κB. VSMCs were pretreated with chelerythrine prior to treatment with fenofibrate or rosiglitazone, and subsequently stimulated with Ang II or a specific PKC activator PMA. Then, NF-κB p65 protein expression was determined by western blot. Results:1. Effects of Ang II on TLR4 expression and production of inflammatory cytokines in VSMCs and inhibitory effects of PPARα/γagonists in vivo The results showed that Ang II increased serum TNF-αand decreased plasma 6-keto-PGF1αcompared with control. In fenofibrate and rosiglitazone treated groups, there was a evident decrease in TNF-αlevel and increase in 6-keto-PGF1αsecretion compared with Ang II-treated group.The immunohistochemical staining found that there existed a stronger TLR4 expression on the luminal surface of thoracic aorta and MMP-9 expression on the vascular media in Ang II-treated group, whereas fenofibrate and rosiglitazone obviously attenuated TLR4 and MMP-9 expressions. Identification of smooth muscle cells with the antibody toα-actin showed that TLR4 and MMP-9 were expressed predominantly in smooth muscle cells. Meanwhile, protein and mRNA expressions of TLR4 and MMP-9 in the thoracic aorta were also significantly enhanced, and protein and mRNA expressions of PPARαand PPARγwere dramatically reduced in Ang II- treated group. Fenofibrate down-regulated protein and mRNA expressions of TLR4 and MMP-9, up-regulated PPARαexpression and but, had little effect on PPARγexpression in the thoracic aorta of Ang II-infused rats. Rosiglitazone down-regulated protein and mRNA expressions of TLR4 and MMP-9, up-regulated PPARγexpression and but, showed little effect on PPARαexpression in the thoracic aorta of Ang II-infused rats.2. Effects of Ang II on TLR4 expression and production of inflammatory cytokines in VSMCs and inhibitory effects of PPARα/γagonists in vitro Incubation of the cells with Ang II or LPS increased TNF-α, but decreased 6-keto-PGF1αin time- and concentration-dependent manners. However, pretreatment of the cells with fenofibrate and rosiglitazone concentration-dependently inhibited TNF-αproduction and promoted 6-keto-PGF1αsecretion in Ang II-stimulated VSMCs.Ang II and LPS elicited TLR4 and MMP-9 expressions in VSMCs identified by the immunocytofluorescence in a concentration-dependent manner, whereas fenofibrate and rosiglitazone attenuated Ang II-induced TLR4 and MMP-9 expressions in VSMCs.Ang II and LPS also up-regulated protein and mRNA expressions of TLR4 and MMP-9, and down-regulated protein and mRNA expressions of PPARαand PPARγin VSMCs in a concentration-dependent manner. Similarly, fenofibrate and rosiglitazone concentration- dependently suppressed protein and mRNA expressions of TLR4 and MMP-9, and elevated protein and mRNA expressions of PPARαor PPARγ. But, fenofibrate had little effect on protein and mRNA expression of PPARγ, and rosiglitazone did not affect protein and mRNA expression of PPARαin Ang II-stimulated VSMCs. 3. TLR4 signaling pathway of Ang II-induced inflammation in VSMCs The results indicated that Ang II increased TNF-αand reduced 6-keto-PGF1αin the supernatant of VSMCs. Blocking TLR4 with anti-TLR4 antibody reversed Ang II-induced changes of TNF-αand 6-keto-PGF1α, inhibited Ang II-induced protein and mRNA expression of MMP-9, and up-regulated Ang II-induced protein and mRNA expressions of PPARγand PPARαin VSMCs.Ang II-induced protein and mRNA expression of TLR4 were significantly depressed by pretreatment of the cells with losartan or PD098059, but not PD123319, and the inhibitory effect was more obvious when the cells were pretreated with the combination of losartan, PD123319 and PD098059.The results also showed that anti-TLR4 antibody partially but significantly blunted Ang II- induced protein and mRNA expression of IP-10 in VSMCs. Ang II-induced protein and mRNA expression of PKC was substantially prevented by anti-IP-10 antibody, and chelerythrine significantly suppressed the total NF-κBp65 protein expression.4. Inhibitory effects of PPARα/γagonists on TLR4 signaling pathway of Ang II-induced inflammation in VSMCsStimulation of the cells with Ang II or LPS resulted in TNF-αelevation and 6-keto-PGF1αreduction, whereas the TLR4 inhibitor, fenofibrate and rosiglitazone partially reversed the Ang II- and LPS-induced effects in VSMCs. Moreover, treatment of the cells with combination of the TLR4 blocker and fenofibrate or rosiglitazone synergistically reversed Ang II-induced changes of TNF-αand 6-keto-PGF1α.The experiment also found that TLR4 inhibitor reduced MMP-9 protein expression and enhanced PPARγand PPARαprotein expressions in Ang II- and LPS-stimulated VSMCs. Fenofibrate and rosiglitazone also produced the similar effects on protein expressions of MMP-9 and PPARαor PPARγto TLR4 inhibitor and but, had little effect on PPARγor PPARαprotein expression. Treatment of the cells with TLR4 blocker and fenofibrate or rosiglitazone also synergistically reversed Ang II-induced changes of MMP-9 and PPARαor PPARγprotein expressions.TLR4 siRNA decreased Ang II-induced TNF-αproduction and increased 6-keto-PGF1αrelease. Fenofibrate and rosiglitazone also reduced Ang II-induced TNF-αproduction and enhanced Ang II-decreased 6-keto-PGF1αrelease in negative control. But, the effects were nearly abolished in TLR4 siRNA control. In addition, fenofibrate and rosiglitazone did not change basal TNF-αproduction, and but significantly increased 6-keto-PGF1αlevel in the unstimulated VSMCs. The similar results to TNF-αwere achieved for mRNA expression of MMP-9. Furthermore, TLR4 siRNA also abolished Ang II-induced decrease of mRNA expressions of PPARγand PPARα. Fenofibrate or rosiglitazone regulated PPARαor PPARγmRNA levels in negative control both in the normal and Ang II-stimulated VSMCs, and but TLR4 siRNA did not change the effect of fenofibrate or rosiglitazone on PPARαor PPARγ. Fenofibrate or rosiglitazone also did not produce significant effect on mRNA levels of PPARγor PPARαin negative control and in TLR4 siRNA control .The another results revealed that losartan and PD098059 inhibited Ang II-induced TLR4 protein expression in VSMCs, whereas PD123319 had no effect. Although fenofibrate alone inhibited Ang II-induced TLR4 protein expression in VSMCs, it did not have any additional effects to AT1, AT2 and ERK1/2 blockades. Rosiglitazone alone also inhibited Ang II-induced TLR4 protein expression in VSMCs, and did not have any additional effects to AT1 or AT2 blockade. But, rosiglitazone produced an additional effect to PD098059 blockade. Furthermore, Ang II-induced ERK1/2 phosphorylation was slightly impaired by fenofibrate and remarkably inhibited by rosiglitazone. Meanwhile, treatment of VSMCs with Ang II significantly down-regulated AT1 protein expression. But, fenofibrate and rosiglitazone did not produce an additional inhibitory effect on AT1 expression in Ang II-treated cells.The results also indicated that TLR4 siRNA reduced Ang II-induced IP-10 protein expression. Fenofibrate and rosiglitazone obviously depressed Ang II-induced IP-10 protein expression in negative control, and but did not further decreased Ang II-induced IP-10 protein expression in TLR4 siRNA control. IP-10 siRNA diminished Ang II-induced PKC protein expression. Although fenofibrate and rosiglitazone repressed Ang II-induced PKC protein expression, IP-10 siRNA abolished the inhibitory effects. Besides, fenofibrate and rosiglitazone significantly suppressed Ang II- and PMA-induced NF-κBp65 protein expression in VSMCs, and potentiated the inhibitory effect of the specific PKC inhibitor.Taken together, all results in the present study suggest that fenofibrate and rosiglitazone exert theirs inhibitory actions on Ang II-induced inflammatory responses in VSMCs via interfering with the TLR4-dependent signaling pathway.Conclusions:1)Ang II enhances generation of pro-inflammatory mediators such as TLR4, MMP-9 and TNF-α, but reduces production of anti-inflammatory molecules like PPARα, PPARγand 6-keto-PGF1αboth in vivo and in vitro.2)Ang II induces inflammatory response involved in pathogenesis of atherosclerosis via TLR4-dependent signaling pathway (AT1→ERK1/2→TLR4→IP-10→PKC→NF-κB) in VSMCs. This finding may contribute to elucidate the novel pro-inflammatory and pro-atherosclerotic mechanisms of Ang II. 3)Fenofibrate and rosiglitazone decreased Ang II-induced generation of pro-inflammatory mediators such as TLR4, MMP-9 and TNF-α, but increased production of anti-inflammatory molecules like PPARαor PPARγand 6-keto-PGF1αboth in vivo and in vitro.4 ) Anti-inflammatory action of PPARαagonist fenofibrate via interfering with the TLR4-dependent signaling pathway (TLR4/IP-10/ PKC/NF-κB) works in concert to protect against atherosclerosis. Meanwhile, PPARγagonist rosiglitazone exerts its anti-inflammatory effect via interfering with the TLR4-dependent signaling pathway (ERK1/2/TLR4/IP-10/ PKC/NF-κB). These findings provide new mechanisms to beneficial effects of fenofibrate and rosiglitazone in the prevention and treatment of cardiovascular diseases including atherosclerosis.