Positive Feedback Regulation of Proliferation in Vascular Smooth Muscle Cells Stimulated by Lipopolysaccharide Is Mediated through the TLR 4/Rac1/Akt Pathway

Jiang, Dehua; Li, Dongye; Cao, Lijuan; Wang, Lele; Zhu, Shasha; Xu, Tongda; Wang, Cheng; Pan, Defeng

doi:10.1371/journal.pone.0092398

Cited by 27 publications

(35 citation statements)

References 46 publications

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“…Images of cells dyed with Apollo 567 were captured with a ‘red’ filter, while Hoechst 33342-stained cell images were captured with a ‘blue’ filter. The percentage of EdU-positive cells was calculated using Image-Pro Plus version 6.0 for Windows (Media Cybernetics, Inc, USA) according to the manufacture’s protocol as previously described [15]. …”

Section: Methodsmentioning

confidence: 99%

“…cDNA samples were obtained using Script M-MLV (Tiangen Biochemical Technology, China). qRT-PCR was performed using 2× Super Real PreMix (SYBR Green, Tiangen) and 50× ROX Reference Dye on an ABI 7500 QPCR System (Applied Biosystems, USA) as previously described [15]. The results were normalized to the expression levels of β-actin prior to further analysis.…”

Section: Methodsmentioning

confidence: 99%

“…Our previous study revealed that LPS contributes to VSMCs proliferation via the TLR4/Rac1/Akt signalling pathway [15]. In the current study, we use small interfering RNA (siRNA) to provide further evidence for the involvement of the TLR4/Rac1/Akt signalling pathway in LPS-mediated VSMCs proliferation.…”

Section: Introductionmentioning

confidence: 92%

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LPS Promotes Vascular Smooth Muscle Cells Proliferation Through the TLR4/Rac1/Akt Signalling Pathway

Yin

Jiang²,

et al. 2017

Cell Physiol Biochem

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Background/Aims: Lipopolysaccharide (LPS) is a potent activator of vascular smooth muscle cells (VSMCs) proliferation, but the underlying mechanism remains unknown. In this study, we knocked down Toll-like receptor 4 (TLR4) and Ras-related C3 botulinum toxin substrate 1 (Rac1) expression using small interfering RNA (siRNA) in order to investigate the effects and possible mechanisms of LPS-induced VSMCs proliferation. Methods: VSMCs proliferation was monitored by 5-ethynyl-2’-deoxyuridine staining, and Rac1 activity was measured via Glutathione S-transferase pull-down assay. mRNAs encoding proliferating cell nuclear antigen (PCNA), smooth muscle 22α (SM22α), myosin heavy chain (MYH) and transient receptor potential channel 1 (TRPC1) were detected by qRT-PCR. The expression of total Akt, p-Akt (308), p-Akt (473), SM22α, MYH and TRPC1 protein was analysed by Western blot. Results: Treatment with TLR4 siRNA (siTLR4) or Rac1 siRNA (siRac1) significantly decreased LPS-induced VSMCs proliferation. Moreover, LPS-induced activation of Rac1 through TLR4 was observed. Western blot analysis revealed that transfection with siTLR4 or siRac1 inhibited LPS-induced Akt phosphorylation. We discovered that LPS stimulated VSMCs proliferation via phenotypic modulation and that this effect was partially inhibited by pre-treatment with siTLR4 or siRac1. Further, TLR4 and Rac1 are involved in LPS-induced activation of TRPC1. Conclusion: This study suggests that LPS exerts an effect on VSMCs proliferation and that the TLR4/Rac1/Akt signalling pathway mediates this effect.

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Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

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LPS Promotes Vascular Smooth Muscle Cells Proliferation Through the TLR4/Rac1/Akt Signalling Pathway

Yin

Jiang²,

et al. 2017

Cell Physiol Biochem

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“…To investigate the effect of IRAK1/4 inhibition on the TLR4/NF B signaling pathway in VSMCs, LPS was employed, which can upregulate TLR4 expression and promote an inflammatory response in VSMCs 5) . In our previous study, VSMCs were stimulated with LPS from 0 to 100 g/mL for 24 h, and we observed that VSMC proliferation was stimulated by LPS at 10 g/mL 24) . The cultures were divided into four groups: control (no drug treatment), LPS, LPS IRAK1/4 inhibitor, and IRAK1/4 inhibitor.…”

Section: In Vitro Studiesmentioning

confidence: 80%

“…seeded in 6-well plates and stimulated with LPS (10 g/mL) in the presence or absence of the IRAK1/4 inhibitor (1 M) for 24 h. VSMC proliferation was determined by counting the cell number. As described in our previous in vitro study, each well was incubated with serum-free DMEM supplemented with 500 L EdU (50 M) for 2 h. The EdU staining was conducted according to the manufacturer's protocol as previously described 24) . The proliferative VSMCs were represented by the EdU-positive cells.…”

Section: Tlr Signaling Pathway Is Involved In Neointimal Formationmentioning

confidence: 99%

Interleukin-1 Receptor-Associated Kinase 1/4 as a Novel Target for Inhibiting Neointimal Formation After Carotid Balloon Injury

Bai

Zhou

et al. 2015

JAT

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Aim:Interleukin-1 receptor-associated kinase 1 (IRAK1) and IRAK4 play essential roles in the induction of inflammatory gene products. We aimed to investigate the effect of the inhibition of IRAK1 and IRAK4 kinase activities on neointimal formation in rats with carotid artery balloon injuries using the IRAK1/4 inhibitor N-(2-Morpholinylethyl)-2-(3-nitrobenzoylamido)-benzimidazole, a cell-permeable benzimidazole compound. Methods: Wistar rats and vascular smooth muscle cells (VSMCs) isolated from the thoracic aortas were used. Toll-like receptor 4 (TLR4)-mediated nuclear factor kappa B (NF B) signaling pathway was revealed by microarrays analysis. In addition, the differential expression of the TLR4 pathway genes, including TLR4, IRAK1, I B , and interleukin-1 (IL-1 ), was confirmed by quantitative real-time polymerase chain reaction. Immunohistochemical staining, elastic-van Gieson and Masson staining, 5-ethynyl-2´-deoxyuridine staining, enzyme-linked immunosorbent assay, transwell migration assay and western blotting were also contributed for relevant detection. Results:The expression of TLR4 protein gradually increased at days 1, 3, 7, and 21 after balloon injury compared with the uninjured group. The dual inhibition of IRAK1 and IRAK4 attenuated neointimal formation and fibrotic remodeling after injury in vivo and suppressed VSMC proliferation and migration in vitro. The production of mediators such as tumor necrosis factor-and IL-1 in injured arteries were also reduced by the inhibition of IRAK1 and IRAK4. The expression of NF B p65-and F4/80-positive cells in inhibitor rats were fewer than those in control rats at day 7, while IRAK1 expression was markedly higher at day 3 in inhibitor rats. Furthermore, western blotting analysis revealed that the IRAK1/4 inhibitor suppressed the IRAK1 and IRAK4 kinase activities and the activation of the TLR4-mediated NF B pathway in vivo and in vitro. Conclusions: This study suggested that IRAK1/4 could serve as a potential therapeutic target to suppress neointimal formation in carotid arteries after balloon injury through the TLR4/NF B signaling pathway. IntroductionRestenosis is a healing response of the arterial wall to mechanical injury after percutaneous coronary intervention (PCI). Vascular smooth muscle cell (VSMC) proliferation and migration are critical to 1318 tion of IRAK1 and IRAK4 kinase activities would be predicted to leave some degree of host defense intact while still reducing inflammatory responses 18) . A novel cell-permeable benzimidazole compound can act as a potent and selective inhibitor of IRAK1 and IRAK4 and has been tested in plasmacytoid dendritic cells from systemic lupus erythematosus and rheumatoid arthritis patients 19) , human primary small airway epithelial cells in chronic obstructive pulmonary disease 20) , and rat brain hypoxia/ischemiainduced neuronal injury 21) ; however, it has not yet been employed in vascular research. To the best of our knowledge, there are no published studies on the role of IRAK1 and IRAK4 kinase activities in...

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Effects of LPS on the accumulation of lipid droplets, proliferation, and steroidogenesis in goat luteinized granulosa cells

Zhang

Wang

et al. 2019

J Biochem & Molecular Tox

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Lipopolysaccharide (LPS) can cause ovarian dysfunction and infertility in mammals. The purpose of this study was to investigate the effects of LPS on the accumulation of lipid droplets (LDs), proliferation, and steroidogenesis in goat luteinized granulosa cells (LGCs). GCs isolated from the ovarian follicles were spontaneously luteinized under media with fetal bovine serum, resulting in increased progesterone and shifted shape from spherical to star with multiple prolongations. Then, LGCs were treated with LPS (0-10 μg/mL) for 0-48 hours. Oil Red O staining was performed to observe LDs accumulation and commercial kit was applied to detect intracellular triglyceride (TG) content. The cell proliferation were detected by cell counting kit-8. Expressions of cellcycle-related genes were determined by real-time polymerase chain reaction. Estradiol (E 2 ) and progesterone (P 4 ) from cell supernatants were determined by enzyme-linked immunosorbent assay, and expressions of STAR, P450scc, 3β-hydroxysteroid dehydrogenase (3β-HSD) and CYP19A1 were detected by Western blot. Results showed that LPS treatment significantly increased LDs accumulation after 24 hours, and 5 μg/mL LPS increased TG content (P < 0.05). LPS treatment for 24 hours stimulated the LGCs activities (P<0.05), which was confirmed by the increases in the expressions of proliferating cell nuclear antigen (PCNA), cyclinB1 and cyclinD1, while 48 hours treatment had no effect. LPS treatment suppressed E 2 and P 4 output of LGCs (P < 0.05). Western blot results showed that 10 μg/mL LPS decreased the protein expression of 3β-HSD in LGCs (P < 0.05). In conclusion, LPS increased LDs accumulation and cell proliferation, and LPS-mediated P 4 reduction could be attributed to the decreased 3β-HSD protein expression, which provide new information for the regulation of ovarian function in goats. HIGHLIGHTSLPS increased the content of lipid droplet in goat luteinized granulosa cells. LPS promoted the proliferation of goat luteinized granulosa cells after treatment for 24 hours. LPS suppressed P4 and E2 output which can be attributed to the decreased 3β-HSD protein expression. K E Y W O R D S cell proliferation, lipid droplet, lipopolysaccharide, luteinized granulosa cells, steroidogenesis J Biochem Mol Toxicol. 2019;33:e22329.wileyonlinelibrary.com/journal/jbt

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Positive Feedback Regulation of Proliferation in Vascular Smooth Muscle Cells Stimulated by Lipopolysaccharide Is Mediated through the TLR 4/Rac1/Akt Pathway

Cited by 27 publications

References 46 publications

LPS Promotes Vascular Smooth Muscle Cells Proliferation Through the TLR4/Rac1/Akt Signalling Pathway

LPS Promotes Vascular Smooth Muscle Cells Proliferation Through the TLR4/Rac1/Akt Signalling Pathway

Interleukin-1 Receptor-Associated Kinase 1/4 as a Novel Target for Inhibiting Neointimal Formation After Carotid Balloon Injury

Effects of LPS on the accumulation of lipid droplets, proliferation, and steroidogenesis in goat luteinized granulosa cells

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