Pei-pei Lu

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.

Fibrinogen, fibrin, and FDP induce C-reactive protein generation in rat vascular smooth muscle cells: Pro-inflammatory effect on atherosclerosis

Atherosclerosis is a chronic inflammatory disease in the vessel. As an inflammatory cytokine, C-reactive protein (CRP) participates in the pathogenesis of atherosclerosis through multiple bioactivities. It has been widely accepted that hyperfibrinogenemia is associated with the formation and progression of atherosclerosis. But, it is unknown whether fibrinogen exerts a pro-inflammatory effect on vascular smooth muscle cells (VSMCs). The purpose of the present study was to observe the effect of fibrinogen, fibrin, and fibrin degradation products (FDP) on CRP generation in VSMCs. CRP mRNA expression was identified with the reverse transcription polymerase chain reaction. CRP level in the supernatant of VSMCs was measured with the enzyme-linked immunosorbent assay. CRP expression in VSMCs was examined with the immunocytochemical method. The results showed that fibrinogen, fibrin, and FDP all induced CRP production in VSMCs both in mRNA level and in protein level in a time- and concentration-dependent manner. The potency is FDP>fibrin>fibrinogen, which seems to mean that their pro-inflammatory activity decreases with increase of molecular weight of these three proteins. The finding provides a new mechanism for atherogenic effect of fibrin(ogen) and FDP, and emphasizes the importance of therapy of 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.

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.

Pravastatin inhibits fibrinogen‑ and FDP‑induced inflammatory response via reducing the production of IL‑6, TNF‑α and iNOS in vascular smooth muscle cells

Atherosclerosis is a chronic inflammatory response of the arterial wall to pro‑atherosclerotic factors. As an inflammatory marker, fibrinogen directly participates in the pathogenesis of atherosclerosis. Our previous study demonstrated that fibrinogen and fibrin degradation products (FDP) produce a pro‑inflammatory effect on vascular smooth muscle cells (VSMCs) through inducing the production of interleukin‑6 (IL‑6), tumor necrosis factor‑α (TNF‑α) and inducible nitric oxide synthase (iNOS). In the present study, the effects of pravastatin on fibrinogen‑ and FDP‑induced expression of IL‑6, TNF‑α and iNOS were observed in VSMCs. The results showed that pravastatin dose‑dependently inhibited fibrinogen‑ and FDP‑stimulated expression of IL‑6, TNF‑α and iNOS in VSMCs at the mRNA and protein level. The maximal inhibition of protein expression of IL‑6, TNF‑α and iNOS was 46.9, 42.7 and 49.2% in fibrinogen‑stimulated VSMCs, and 50.2, 49.8 and 53.6% in FDP‑stimulated VSMCs, respectively. This suggests that pravastatin has the ability to relieve vascular inflammation via inhibiting the generation of IL‑6, TNF‑α and iNOS. The results of the present study may aid in further explaining the beneficial effects of pravastatin on atherosclerosis and related cardiovascular diseases. In addition, they suggest that application of pravastatin may be beneficial for prevention of atherosclerosis formation in hyperfibrinogenemia.

Rosiglitazone regulates c-reactive protein-induced inflammatory responses via glucocorticoid receptor-mediated inhibition of p38 mitogen-activated protein kinase-toll-like receptor 4 signal pathway in vascular smooth muscle cells.

C-reactive protein (CRP) activates toll-like receptor 4 (TLR4) to initiate inflammatory response involved in the pathogenesis of atherosclerosis through mitogen-activated protein kinase (MAPK) signal pathways. Rosiglitazone, a synthetic peroxisome proliferator-activated receptor γ (PPARγ) agonist, is considered to be an important inhibitor of the inflammatory response. The present study was to explore the effect of rosiglitazone on the CRP-induced inflammatory responses and the related signal pathway in vascular smooth muscle cells (VSMCs). The results showed that rosiglitazone reduced the expressions of proinflammatory cytokines, such as vascular endothelial growth factor-A and inducible nitric oxide synthase, and enhanced the expression or activation of anti-inflammatory transcription factors including PPARγ and glucocorticoid receptor (GR) in VSMCs in response to CRP. The further investigations indicated that rosiglitazone inhibited CRP-induced TLR4 expression and p38 MAPK phosphorylation in VSMCs, and TLR4 knockdown potentiated the inhibitory effects of rosiglitazone on vascular endothelial growth factor-A and inducible nitric oxide synthase expressions. In addition, GR antagonist RU486 but not PPARγ inhibitor GW9662 remarkably weakened the inhibitory effects of rosiglitazone on CRP-induced TLR4 expression and p38 phosphorylation in VSMCs. But GW9662 did not affect rosiglitazone-induced GR phosphorylation. These suggest that rosiglitazone exerts its anti-inflammatory effect through activating GR and subsequently inhibiting p38 MAPK-TLR4 signaling pathway in CRP-stimulated VSMCs.

Effects and mechanisms of the functional parts of Dahuang Zhechong Pill (大黄虫丸) containing serum on platelet-derived growth factor-stimulated proliferation of vascular smooth muscle cells

ObjectiveTo investigate and compare the effects and mechanisms of three functional parts of Dahuang Zhechong Pill (大黄虫丸, DHZCP), including drugs with the function of removing blood stasis and promoting blood circulation (FP-I), drugs with the function of expelling heat and moistening dryness (FP-II), and drugs with the function of nourishing yin and replenishing blood (FP-III) of DHZCP, on platelet-derived growth factor (PDGF)-stimulated vascular smooth muscle cells (VSMCs) proliferation with the method of serum pharmacology.MethodsVSMCs proliferation of rat was assayed by measuring the cell viability with the 3-(4, 5-dimethylthiazol-2yl)-2, 5-diphenyltetrazolium bromide (MTT) method. DNA synthesis in VSMCs was examined by detecting 5′-bromo-2′-deoxyuridine incorporation with the immunocytochemical method. Cycle of VSMCs was evaluated with flow cytometry. Expression of cyclin D1, p27, PKCα, and phosphorylated extracellular signal regulated kinase 1/2 (ERK1/2) was quantified by the Western blotting method.ResultsThe FP-I and FP-III containing serum was capable of inhibiting PDGF-stimulated proliferation and DNA synthesis of VSMCs, arrested VSMCs in G phase, downregulated cyclin D1, and upregulated p27 expression (P <0.01 or P <0.05). The FP-I and FP-III containing serum also inhibited the PDGF-induced phosphorylation of tyrosine of ERK1/2 and PKCα expression (P <0.01 or P <0.05).ConclusionsFP-I and FP-III of DHZCP are able to inhibit VSMCs proliferation via interrupting PKCα-ERK1/2 signaling, modulating the expression of cell cycle proteins to result in arresting the cells in G phase. The inhibitory effect is mainly related to the function of removing blood stasis and promoting blood circulation, slightly to the function of nourishing yin and replenishing blood, but not to the function of expelling heat and moistening dryness.

Dahuang Zhechong Pill (大黄虫丸) containing serum inhibited platelet-derived growth factor-stimulated vascular smooth muscle cells proliferation by inducing G1 arrest partly via suppressing protein kinase C α-Extracellular regulated kinase 1/2 signaling pathway

ObjectiveTo investigate effects of Dahuang Zhechong Pill (大黄{ie371-1}虫丸, DHZCP) on the cell cycle and the related signal pathways in vascular smooth muscle cells (VSMCs) stimulated by platelet-derived growth factor (PDGF) with the method of serum pharmacology.MethodsDNA synthesis in VSMCs was examined by detecting 5′-bromo-2′-deoxyuridine incorporation with the immunocytochemical method. The cycle of VSMCs was evaluated with flow cytometry. Expressions of cyclin D1, p27, protein kinase Cα (PKCα), and phosphorylated extracellular signal regulated kinase 1/2 (ERK1/2) were quantified by Western blot method.ResultsDHZCP containing serum significantly inhibited DNA synthesis of PDGF-stimulated VSMCs, arrested the cells in G G1 phase, modulated the protein expressions of cyclin D D1 and p27, and suppressed the activation of PKCα and ERK1/2.ConclusionDHZCP containing serum inhibits VSMCs proliferation via modulating the expressions of cell cycle proteins to arrest the cell in G G1 phase, which is attributed to, at least in part, suppressing PKCα-ERK1/2 signaling in VSMCs.