Yuanyuan Ji

Angiotensin II Induces Inflammatory Response Partly Via Toll-Like Receptor 4-Dependent Signaling Pathway in Vascular Smooth Muscle Cells

Angiotensin (Ang II) plays an important role in atherosclerosis through proinflammatory effect. Toll-like receptor 4 (TLR4) may mediate inflammatory response. It is unknown whether TLR4 mediates the proinflammatory effect of Ang II. Thus, we observed the role and signaling pathway of TLR4 in Ang II-induced inflammation in rat vascular smooth muscle cells (VSMCs). Ang II and LPS stimulated TNF-α secretion and inhibited 6-keto-PGF1α production, upregulated MMP-9 and downregulated PPARγ and PPARαin rat VSMCs. Ang II also distinctly upregulated TLR4 expression in the cells. Pretreatment of the cells with anti-TLR4 antibody prior to Ang II stimulation significantly diminished the effects of Ang II. These suggest that Ang II stimulates VSMCs to produce inflammation through regulation of the proinflammatory and the antiinflammtory factors via TLR4-dependent mechanism. The further investigations showed that AT1 receptor antagonist losartan or ERK1/2 inhibitor PD098059 inhibited Ang II-induced TLR4 expression, TLR4 inhibitor prevented Ang II- induced IP-10 expression, anti-IP-10 antibody partly abolished Ang II- induced PKC increase, and PKC inhibitor chelerythrine suppressed Ang II- induced NF-κB expression. These demonstrate that TLR4-mediated proinflammatory effect of Ang II in VSMCs involves AT1/ERK1/2/TLR4/IP-10/ PKC/NF-κB pathway. Our results provide the evidence that Ang II induces inflammatory response involved in pathogenesis of atherosclerosis partly via TLR4- dependent signaling pathway in VSMCs.

PPARγ agonist, rosiglitazone, regulates angiotensin II-induced vascular inflammation through the TLR4-dependent signaling pathway

Atherosclerosis is increasingly recognized as a chronic inflammatory disease. Angiotensin II (Ang II) is a critical factor in inflammatory responses, so as to promote the pathogenesis of atherosclerosis. Toll-like receptor 4 (TLR4) activates signaling pathways leading to the expression of pro-inflammatory cytokines implicated in the etiology of atherosclerosis. Peroxisome proliferator-activated receptor γ (PPARγ) agonists are considered to be important in modulating vascular inflammation and atherosclerosis. Herein, we investigated the modulatory effects of rosiglitazone on Ang II-mediated inflammatory responses both in vivo and in vitro. We also examined whether TLR4-dependent signaling pathway was involved in the inhibitory effects of rosiglitazone on Ang II-induced pro-inflammatory responses in vascular smooth muscle cells (VSMCs). Male Sprague–Dawley rats received Ang II by subcutaneous infusion and/or rosiglitazone per os for 7 days. Systolic blood pressure rise in Ang II-infused rats was attenuated by rosiglitazone. Rosiglitazone also reduced Ang II-induced generation of pro-inflammatory mediators (TLR4, matrix metalloproteinase-9 and tumor necrosis factor-α), but enhanced production of anti-inflammatory mediators (PPARγ and 6-keto-PGF1α) both in vivo and in vitro. Furthermore, treatment of VSMCs with both the TLR4 inhibitor and TLR4 small-interfering RNA (siRNA) showed that the modulatory effects of rosiglitazone on Ang II-mediated inflammatory responses in VSMCs were related to TLR4. Treatment of the cells with rosiglitazone had little effect on Ang II receptors expression (AT1 and AT2), but downregulated AT1-dependent ERK1/2 activation. Then, treatment of VSMCs with TLR4 siRNA, interferon-gamma-inducible protein 10 (IP-10) siRNA and with the special protein kinase C (PKC) inhibitor further revealed that the signaling pathway (TLR4/IP-10/PKC/NF-κB) was involved in the inhibitory effects of rosiglitazone on Ang II-induced pro-inflammatory responses in VSMCs. In conclusion, TLR4 may be a drug target involved in the ameliorative effects of PPARγ agonist, rosiglitazone, on Ang II-mediated inflammatory responses in VSMCs. Moreover, rosiglitazone exerts its anti-inflammatory effect by interfering with the TLR4-dependent signaling pathway (ERK1/2/TLR4/IP-10/PKC/NF-κB) to prevent and treat atherosclerotic diseases.

PPARα activator fenofibrate modulates angiotensin II-induced inflammatory responses in vascular smooth muscle cells via the TLR4-dependent signaling pathway

Angiotensin II (Ang II) is a crucial contributor to inflammatory processes involved in development and progression of atherosclerotic lesion. Toll-like receptor 4 (TLR4) signaling responsible for the initiation of inflammation also participates in pathogenesis of atherosclerosis. The protective effect of peroxisome proliferator-activated receptor alpha (PPARalpha) activators on atherosclerosis may be due to their impact on vascular inflammation, plaque instability and thrombosis. However, mechanisms underlying the inhibitory effects of PPARalpha activators on Ang II-induced vascular inflammation and the TLR4-dependent signaling pathway involved in vascular smooth muscle cells (VSMCs) remain unclear. The present study demonstrated that PPARalpha activator fenofibrate decreased Ang II-induced generation of pro-inflammatory mediators such as TLR4, MMP-9 and TNF-alpha, but enhanced production of anti-inflammatory molecules like PPARalpha and 6-keto-PGF(1alpha) both in vivo and in vitro. Meanwhile, treatment of VSMCs with the TLR4 inhibitor or TLR4 siRNA showed that the inhibitory effects of fenofibrate on Ang II-induced inflammatory responses in VSMCs were dependent on TLR4. Furthermore, fenofibrate depressed Ang II-induced inflammatory responses in VSMCs by intervening the downstream effector molecules of the TLR4-dependent signaling pathway, including interferon-gamma inducible protein 10 (IP-10), protein kinases C (PKC) and nuclear factor kappaB (NF-kappaB). Thus, these findings provide the evidence for beneficial effects of PPARalpha activator fenofibrate to counter-regulate vascular inflammation induced by Ang II. More importantly, anti-inflammatory action of fenofibrate via interfering with the TLR4-dependent signaling pathway (TLR4/IP-10/PKC/NF-kappaB) works in concert to protect against atherosclerosis.

Angiotensin II Induces Angiogenic Factors Production Partly Via AT1/JAK2/STAT3/SOCS3 Signaling Pathway in MHCC97H Cells

Angiotensin II (Ang II) has been shown to function as a key role in neovascularization of hepatocellular carcinoma (HCC), but little is known its underlying mechanisms. The aim of this study was to explore the role of Janus kinase/signal transducer and activator of transcription (JAK/STAT) signaling pathway in Ang II-induced HCC angiogenic factors production. Herein, we found that Ang II upregulated angiogenic factors production such as vascular endothelial growth factor (VEGF), angiopoietin-2 (Ang-2) and Tie-2 in MHCC97H cells in a time- and concentration-dependent manner. And VEGF and Ang-2 caused a significant increase in angiogenic tube formation. Especially, Ang II-induced angiogenic tube formation was blunted by VEGF small interfering RNA (siRNA) and Ang-2 siRNA, respectively. The JAK2 inhibitor AG490 partly attenuated the effects of Ang II. Moreover, Ang II- induced JAK2 and STAT3 phosphorylation was significantly suppressed by losartan but not PD123319. Meanwhile, STAT3 phosphorylation and suppressor of cytokine signaling 3 (SOCS3) expression induced by Ang II were evidently impaired by AG490. More importantly, SOCS3 siRNA remarkably reinforced Ang II-induced VEGF, Ang-2 and Tie-2 generation in MHCC97H cells. Taken together, the present study demonstrates that Ang II induces angiogenic factors production partly via AT1/ JAK2/STAT3/SOCS3 signaling pathway in MHCC97H cells. These findings may provide important insights into the potential mechanism with respect to the AT1/ JAK2/ STAT3/SOCS3 signaling pathway associated with Ang II-induced angiogenesis in the pathogenesis of HCC.

Pro-inflammatory effect of fibrinogen and FDP on vascular smooth muscle cells by IL-6, TNF-α and iNOS

AIMS Atherosclerosis is a chronic inflammatory response of the arterial wall to multiple endothelial injuries. As one of the inflammatory markers, fibrinogen has been implicated in pathogenesis of atherosclerosis. But, it is not completely understood whether atherogenesis of fibrinogen is related to its pro-inflammatory effect on vascular smooth muscle cells (VSMCs). The purpose of the present study was to observe effects of fibrinogen and fibrin degradation products (FDP) on interleukin-6 (IL-6), tumor necrosis factor alpha (TNF-α), and inducible nitric oxide synthase (iNOS) generation in rat VSMCs. MAIN METHODS Rat VSMCs were cultured, and fibrinogen and FDP were used as stimulants for IL-6, TNF-α, and iNOS. IL-6 and TNF-α level in the supernatant were measured by ELISA, mRNA expression of IL-6, TNF-α, and iNOS were assayed with RT-PCR, and protein expression of iNOS was detected with western blot and immunocytochemistry. KEY FINDINGS Fibrinogen and FDP both significantly stimulated mRNA and protein expressions of IL-6, TNF-α and iNOS in VSMCs in time- and concentration-dependent ways. The pro-inflammatory potency of FDP is higher than fibrinogen, which seems to mean that smaller fragments of the protein have greater pro-inflammatory activity. Fibrinogen and FDP promote more protein expressions of IL-6 and TNF-α compared to iNOS, suggesting that fibrinogen and FDP produce a pro-inflammatory effect on VSMCs mainly by IL-6 and TNF-α. SIGNIFICANCE These findings are helpful to better understand pro-inflammatory effect of fibrinogen on VSMCs involved in atherogenesis, and imply a therapeutic strategy targeting hyperfibrinogenemia in atherosclerosis.

C-reactive protein triggers inflammatory responses partly via TLR4/IRF3/NF-κB signaling pathway in rat vascular smooth muscle cells.

AIMS C-reactive protein (CRP) plays an important role in the inflammatory process of atherosclerosis. Toll-like receptor 4 (TLR4) participates in atherogenesis by mediating the inflammatory responses. The aim of this experiment was to investigate the pro-inflammatory effects and mechanisms of CRP in rat vascular smooth muscle cells (VSMCs), especially focusing on the effects of CRP on IL-6 and peroxisome proliferator-activated receptor γ (PPARγ), and TLR4-dependent signal pathway. MAIN METHODS rat VSMCs were cultured, and CRP was used as a stimulant for IL-6 and peroxisome proliferator-activated receptor γ (PPARγ). IL-6 level in the culture supernatant was measured by ELISA, and mRNA and protein expressions were assayed by quantitative real-time PCR and western blot, respectively. RNA interference was used to assess the roles of TLR4 and interferon regulatory factor 3 (IRF3) in the pro-inflammatory signal pathway of CRP. KEY FINDINGS CRP stimulated IL-6 secretion, and inhibited mRNA and protein expression of PPARγ in VSMCs in a concentration-dependent manner. Additionally, CRP induced TLR4 expression, promoted nuclear translocation of NF-κB (p65), and augmented IκBα phosphorylation in VSMCs. Taken together, CRP induces the inflammatory responses through increasing IL-6 generation and reducing PPARγ expression in VSMCs, which is mediated by TLR4/IRF3/NF-κB signal pathway. SIGNIFICANCE CRP is able to stimulate IL-6 production and to inhibit PPARγ expression in VSMCs via MyD88-independent TLR4 signaling pathway (TLR4/IRF3/NF-κB). These provide the novel evidence for the pro-inflammatory action of CRP involved in atherogenesis.

PPARγ agonist rosiglitazone ameliorates LPS-induced inflammation in vascular smooth muscle cells via the TLR4/TRIF/IRF3/IP-10 signaling pathway

The activation of toll-like receptor 4 (TLR4) by lipopolysaccharide (LPS) can induce inflammation that are one of key etiological conditions for the development of many chronic inflammatory diseases including atherosclerosis and diabetes. Peroxisome proliferator-activated receptor γ (PPARγ) agonists play a crucial role in improving glucose and lipid homeostasis in the development of cardiovascular diseases. Evidence is growing that benefits of PPARγ agonists may also be derived from the anti-inflammatory and anti-atherosclerotic properties of these agents. However, the role of rosiglitazone in regulating LPS-induced vascular inflammation has yet to be fully elucidated. The current study demonstrated that rosiglitazone exerted a potent anti-inflammatory action via decreasing interleukin-18 (IL-18), tissue inhibitor of metalloproteinase-1 (TIMP-1), TLR4 and increasing PPARγ in LPS-induced VSMCs. Furthermore, treatment of VSMCs with the TLR4 blocker or TLR4 small-interfering RNA presented that the regulatory effects of rosiglitazone on LPS-mediated inflammation in VSMCs were dependent on TLR4. Interestingly, the results indicated that beneficial effects of rosiglitazone on LPS-induced inflammation in VSMCs were mediated via interference of TLR4 and its downstream signaling components including Toll-interleukin-1 (IL-1) receptor domain-containing adaptor inducing interferon-β (TRIF), interferon regulatory factor 3 (IRF3) and interferon-gamma inducible protein 10 (IP-10). In summary, PPARγ agonist rosiglitazone exerts anti-inflammatory property by antagonizing LPS-mediated inflammation in VSMCs. More importantly, the regulation of the TRIF-dependent TLR4 signaling pathway (TLR4/TRIF/ IRF3/IP-10) provides new insight to understand the mode of action of rosiglitazone for its anti-inflammatory effects.

The Prognostic Value of CXCR4 in Ovarian Cancer: A Meta-Analysis

Objective Recent reports have shown that C-X-C chemokine receptor 4 (CXCR4) is expressed in ovarian cancer and plays an important role in metastasis. However, the prognostic value of CXCR4 in ovarian cancer remains controversial and has not been emphasized. The aim of this study is to evaluate the prognostic significance of CXCR4 in ovarian cancer by performing a meta-analysis. Methods We systematically searched for studies evaluating the relationship between CXCR4 expression and the outcome of ovarian cancer patients. Only articles in which CXCR4 expression was detected by immunohistochemical staining were included. Hazard ratios (HRs) and relative risk (RR) with 95% confidence intervals (CIs) were pooled as effect size (ES) across studies for overall survival (OS) and progression-free survival (PFS). Results A total of 729 patients from 7 studies (6 articles) were included in this meta-analysis. Our results showed that high CXCR4 expression was significantly associated with poor prognosis in terms of OS (ES, 2.81; 95% CI, 1.16–6.80; p = 0.022) and PFS (ES, 8.48; 95% CI, 2.13–33.70; p = 0.002) in ovarian cancer patients. The association between high CXCR4 expression and poor ovarian cancer prognosis in OS was also statistically significant in subgroups of Asian and III-IV patients constituting 70%. Conclusions The present meta-analysis indicated that high CXCR4 expression was associated with poor prognosis in ovarian cancer. More studies, especially larger scale and well-matched researches, are warranted to clarify the prognostic effect of CXCR4 on the outcome of ovarian cancer.

Toll-Like Receptor 4 Signaling Mediates Inflammatory Activation Induced by C-Reactive Protein in Vascular Smooth Muscle Cells

C-reactive protein (CRP) is considered to induce the inflammatory response during atherosclerosis. Toll-like receptor 4 (TLR4)-mediated inflammatory signaling has been shown to facilitate atherogenesis. It remains thoroughly unknown whether TLR4 mediates the proinflammatory effect of CRP. Thus, the study was designed to explore TLR4-related mechanism of proinflammatory effect of CRP in rat vascular smooth muscle cells (VSMCs). CRP increased mRNA levels and protein expressions of vascular endothelial growth factor A (VEGF-A) and inducible nitric oxide synthase (iNOS) in VSMCs, and enhanced NO secretion in the medium. But, CRP hindered nuclear translocation of glucocorticoid receptor (GR) and decreased mRNA level and protein phosphorylation of GR in VSMCs. TLR4 small-interfering RNA (siRNA) significantly reversed the effects of CRP. These suggest that CRP is able to induce inflammatory responses via TLR4 in VSMCs. More importantly, our data provide novel evidence that CRP exerts a proinflammatory action via TLR4-dependent signaling pathway (AT1R-p38 MAPK-TLR4-PKCα) in VSMCs.

C-Reactive Protein Induces TNF-α Secretion by p38 MAPK–TLR4 Signal Pathway in Rat Vascular Smooth Muscle Cells

Atherosclerosis is a chronic inflammatory disease. C-reactive protein (CRP) not only is an inflammatory marker but also regulates the expressions of other inflammatory cytokines associated with the pathogenesis of atherosclerosis. Toll-like receptor 4 (TLR4) also contributes to atherogenesis via transducting inflammatory signals. Herein, our studies focused on characterizing the effect of CRP on tumor necrosis factor α (TNF-α) production and TLR4-related molecular mechanisms in rat vascular smooth muscle cells (VSMCs). The results showed that CRP stimulated VSMCs to secrete TNF-α and enhanced TLR4 expression in a time-concentration-dependent manner. TLR4 knockdown significantly inhibited CRP-induced TNF-α generation, and p38 mitogen-activated protein kinase (MAPK) blocker SB203580 depressed TLR4 expression and TNF-α production initiated by CRP in VSMCs. The data demonstrate that CRP triggers an inflammatory response in rat VSMCs by inducing TNF-α secretion, which is mediated by p38 MAPK–TLR4 signaling pathway.