Skip to main content

Advertisement

SpringerLink
Log in

Lipopolysaccharide Activates Toll-Like Receptor 4 and Prevents Cardiac Fibroblast-to-Myofibroblast Differentiation

  • Published:
Cardiovascular Toxicology Aims and scope Submit manuscript

Abstract

Bacterial lipopolysaccharide (LPS) is a known ligand of Toll-like receptor 4 (TLR4) which is expressed in cardiac fibroblasts (CF). Differentiation of CF to cardiac myofibroblasts (CMF) is induced by transforming growth factor-β1 (TGF-β1), increasing alpha-smooth muscle actin (α-SMA) expression. In endothelial cells, an antagonist effect between LPS-induced signaling and canonical TGF-β1 signaling was described; however, it has not been studied whether in CF and CMF the expression of α-SMA induced by TGF-β1 is antagonized by LPS and the mechanism involved. In adult rat CF and CMF, α-SMA, ERK1/2, Akt, NF-κβ, Smad3, and Smad7 protein levels were determined by western blot, TGF-β isoforms by ELISA, and α-SMA stress fibers by immunocytochemistry. CF and CMF secrete the three TGF-β isoforms, and the secretion levels of TGF-β2 was affected by LPS treatment. In CF, LPS treatment decreased the protein levels of α-SMA, and this effect was prevented by TAK-242 (TLR4 inhibitor) and LY294002 (Akt inhibitor), but not by BAY 11-7082 (NF-κβ inhibitor) and PD98059 (ERK1/2 inhibitor). TGF-β1 increased α-SMA protein levels in CF, and LPS prevented partially this effect. In addition, in CMF α-SMA protein levels were decreased by LPS treatment, which was abolished by TAK-242. Finally, in CF LPS decreased the p-Smad3 phosphorylation and increased the Smad7 protein levels. LPS treatment prevents the CF-to-CMF differentiation and reverses the CMF phenotype induced by TGF-β1, through decreasing p-Smad3 and increasing Smad7 protein levels.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5
Fig. 6
Fig. 7

Similar content being viewed by others

References

  1. Abdalla, M., Goc, A., Segar, L., & Somanath, P. (2013). Response factor differentiation via myocardin and serum expression and myofibroblast Akt1 mediates alpha-smooth muscle actin. Journal of Biological Chemistry, 288(46), 33483–33493. PubMed PMID: 24106278. PubMed Central PMCID: PMC3829193.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  2. Ayache, N., Boumediene, K., Mathy-Hartert, M., Reginster, J. Y., Henrotin, Y., & Pujol, J. P. (2002). Expression of TGF-betas and their receptors is differentially modulated by reactive oxygen species and nitric oxide in human articular chondrocytes. Osteoarthritis Cartilage, 10, 344–352. PubMed PMID: 12027535.

    Article  CAS  PubMed  Google Scholar 

  3. Bhattacharyya, S., Kelley, K., Melichian, D. S., Tamaki, Z., Fang, F., Su, Y., et al. (2013). Toll-like receptor 4 signaling augments transforming growth factor-β responses: a novel mechanism for maintaining and amplifying fibrosis in scleroderma. American Journal of Pathology, 182, 192–205.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  4. Briest, W., Homagk, L., Rassler, B., Ziegelhöffer-Mihalovicová, B., Meier, H., Tannapfel, A., et al. (2004). Norepinephrine-induced changes in cardiac transforming growth factor-beta isoform expression pattern of female and male rats. Hypertension, 44(4), 410–418. PubMed PMID: 15326086.

    Article  CAS  PubMed  Google Scholar 

  5. Brown, R. D., Ambler, S. K., Mitchell, M. D., & Long, C. S. (2005). The cardiac fibroblast: therapeutic target in myocardial remodeling and failure. Annual Review of Pharmacology and Toxicology, 45, 657–687. PubMed PMID: 15822192.

    Article  CAS  PubMed  Google Scholar 

  6. Chen, T., Guo, J., Han, C., Yang, M., & Cao, X. (2009). Heat shock protein 70, released from heat-stressed tumor cells, initiates antitumor immunity by inducing tumor cell chemokine production and activating dendritic cells via TLR4 pathway. Journal of Immunology, 182(3), 1449–1459. PubMed PMID: 19155492.

    Article  CAS  Google Scholar 

  7. Conery, A. R., Cao, Y., Thompson, E. A., Townsend, C. M., Jr., Ko, T. C., & Luo, K. (2004). Akt interacts directly with Smad3 to regulate the sensitivity to TGF-beta induced apoptosis. Nature Cell Biology, 6(4), 366–372. PubMed PMID: 15104092.

    Article  CAS  PubMed  Google Scholar 

  8. Copaja, M., Venegas, D., Aránguiz, P., Canales, J., Vivar, R., Catalán, M., et al. (2011). Simvastatin induces apoptosis by a Rho-dependent mechanism in cultured cardiac fibroblasts and myofibroblasts. Toxicology and Applied Pharmacology, 255(1), 57–64. PubMed PMID: 21651924.

    Article  CAS  PubMed  Google Scholar 

  9. Dabiri, G., Campaner, A., Morgan, J. R., & Van De Water, L. (2006). A TGF-beta1-dependent autocrine loop regulates the structure of focal adhesions in hypertrophic scar fibroblasts. Journal of Investigative Dermatology, 126(5), 963–970. PubMed PMID: 16498397.

    Article  CAS  PubMed  Google Scholar 

  10. Deten, A., Hölzl, A., Leicht, M., Barth, W., & Zimmer, H. G. (2001). Changes in extracellular matrix and in transforming growth factor beta isoforms after coronary artery ligation in rats. Journal of Molecular and Cellular Cardiology, 33, 1191–1207. PubMed PMID: 11444923.

    Article  CAS  PubMed  Google Scholar 

  11. Díaz-Araya, G., Vivar, R., Humeres, C., Boza, P., Bolívar, S., & Muñoz, C. (2015). Cardiac fibroblasts as sentinel cells in cardiac tissue: Receptors, signaling pathways and cellular functions. Pharmacological Research, 101, 30–40. PubMed PMID: 26151416.

    Article  PubMed  Google Scholar 

  12. Dobaczewski, M., Bujak, M., Li, N., Gonzalez-Quesada, C., Mendoza, L. H., Wang, X. F., et al. (2010). Smad3 signaling critically regulates fibroblast phenotype and function in healing myocardial infarction. Circulation Research, 107, 418–428. PubMed PMID: 20522804. PubMed Central PMCID: PMC2917472.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  13. Dobaczewski, M., Gonzalez-Quesada, C., & Frangogiannis, N. G. (2010). The extracellular matrix as a modulator of the inflammatory and reparative response following myocardial infarction. Journal of Molecular and Cellular Cardiology, 48, 504–511. PubMed PMID: 19631653. PubMed Central PMCID: PMC2824059.

    Article  CAS  PubMed  Google Scholar 

  14. Dong, R. Q., Wang, Z. F., Zhao, C., Gu, H. R., Hu, Z. W., Xie, J., et al. (2015). Toll-like receptor 4 knockout protects against isoproterenol-induced cardiac fibrosis: The role of autophagy. Journal of Cardiovascular Pharmacology and Therapeutics, 20(1), 84–92. PubMed PMID: 24950765.

    Article  CAS  PubMed  Google Scholar 

  15. Ebisawa, T., Fukuchi, M., Murakami, G., Chiba, T., Tanaka, K., Imamura, T., et al. (2001). Smurf1 interacts with transforming growth factor-β type I receptor through Smad7 and induces receptor degradation. Journal of Biological Chemistry, 276, 12477–12480. PubMed PMID: 11278251.

    Article  CAS  PubMed  Google Scholar 

  16. Fan, D., Takawale, A., Lee, J., & Kassiri, Z. (2012). Cardiac fibroblasts, fibrosis and extracellular matrix remodeling in heart disease. Fibrogenesis Tissue Repair, 5(1), 15. PubMed PMID: 22943504. PubMed Central PMCID: PMC3464725.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  17. Fang, H., Ang, B., Xu, X., Huang, X., Wu, Y., Sun, Y., et al. (2014). TLR4 is essential for dendritic cell activation and anti-tumor T-cell response enhancement by DAMPs released from chemically stressed cancer cells. Cellular and Molecular Immunology, 11(2), 150–159. PubMed PMID: 24362470. PubMed Central PMCID: PMC4003380.

    Article  CAS  PubMed  Google Scholar 

  18. Goodall, K. J., Poon, I. K., Phipps, S., & Hulett, M. D. (2014). Soluble heparan sulfate fragments generated by heparanase trigger the release of pro-inflammatory cytokines through TLR-4. PLoS One, 9(10), e109596.

    Article  PubMed  PubMed Central  Google Scholar 

  19. Gronroos, E., Hellman, U., Heldin, C. H., & Ericsson, J. (2002). Control of Smad7 stability by competition between acetylation and ubiquitination. Molecular Cell, 10, 483–493. PubMed PMID: 12408818.

    Article  CAS  PubMed  Google Scholar 

  20. Hayashi, H., Abdollah, S., Qiu, Y., Cai, J., Xu, Y. Y., Grinnell, B. W., et al. (1997). The MAD-related protein Smad7 associates with the TGF-β receptor and functions as an antagonist of TGF-β signaling. Cell, 89, 1165–1173. PubMed PMID: 9215638.

    Article  CAS  PubMed  Google Scholar 

  21. He, Z., Gao, Y., Deng, Y., Li, W., Chen, Y., Xing, S., et al. (2012). Lipopolysaccharide induces lung fibroblast proliferation through Toll-like receptor 4 signaling and the phosphoinositide3-kinase-Akt pathway. PLoS ONE, 7(4), e35926. PubMed PMID: 22563417. PubMed Central PMCID: PMC3338545..

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  22. Kavsak, P., Rasmussen, R. K., Causing, C. G., Bonni, S., Zhu, H., Thomsen, G. H., et al. (2000). Smad7 binds to Smurf2 to form an E3 ubiquitin ligase that targets the TGF-β receptor for degradation. Molecular Cell, 6, 1365–1375. PubMed PMID: 11163210.

    Article  CAS  PubMed  Google Scholar 

  23. Kume, S., Haneda, M., Kanasaki, K., Sugimoto, T., Araki, S., Isshiki, K., et al. (2007). SIRT1 inhibits transforming growth factor β-induced apoptosis in glomerular mesangial cells via Smad7 deacetylation. Journal of Biological Chemistry, 282, 151–158. PubMed PMID: 17098745.

    Article  CAS  PubMed  Google Scholar 

  24. Lew, W. Y., Bayna, E., Dalle Molle, E., Contu, R., Condorelli, G., & Tang, T. (2014). Myocardial fibrosis induced by exposure to subclinical lipopolysaccharide is associated with decreased miR-29c and enhanced NOX2 expression in mice. PLoS ONE, 9(9), e107556R.D.

    Article  Google Scholar 

  25. Li, P., Wang, D., Lucas, J., Oparil, S., Xing, D., Cao, X., et al. (2008). Atrial natriuretic peptide inhibits transforming growth factor beta-induced Smad signaling and myofibroblast transformation in mouse cardiac fibroblasts. Circulation Research, 102, 185–192. PubMed PMID: 17991884.

    Article  CAS  PubMed  Google Scholar 

  26. Yu, L., Wang, L., & Chen, S. (2010). Endogenous toll-like receptor ligands and their biological significance. Journal of Cellular Molecular Medicine, 14(11), 2592–2603. PMCID: PMC4373479.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  27. Massagué, J. (1990). The transforming growth factor-beta family. Annual Review of Cell Biology, 6, 597–641. PubMed PMID: 2177343.

    Article  PubMed  Google Scholar 

  28. Okamura, Y., Watari, M., Jerud, E. S., Young, D. W., Ishizaka, S. T., Rose, J., et al. (2001). The extra domain A of fibronectin activates Toll-like receptor 4. Journal of Biological Chemistry, 276(13), 10229–10233. PubMed PMID: 11150311.

    Article  CAS  PubMed  Google Scholar 

  29. Peri, F., & Calabrese, V. (2014). Toll-like receptor 4 (TLR4) modulation by synthetic and natural compounds: an update. Journal of Medicinal Chemistry, 57(9), 3612–3622. PubMed PMID: 24188011. PubMed Central PMCID: PMC4040398.

    Article  CAS  PubMed  Google Scholar 

  30. Tavener, S. A., & Kubes, P. (2005). Is there a role for cardiomyocyte toll-like receptor 4 in endotoxemia? Trends in Cardiovascular Medicine, 15(5), 153–157. PubMed PMID: 16165010.

    Article  CAS  PubMed  Google Scholar 

  31. Remy, I., Montmarquette, A., & Michnick, S. W. (2004). PKB/Akt modulates TGF-beta signaling through a direct interaction with Smad3. Nature Cell Biology, 6(4), 358–365. PubMed PMID: 15048128.

    Article  CAS  PubMed  Google Scholar 

  32. Sandbo, N., Taurin, S., Yau, D. M., Kregel, S., Mitchell, R., & Dulin, N. O. (2007). Downregulation of smooth muscle alpha-actin expression by bacterial lipopolysaccharide. Cardiovascular Research, 74(2), 262–269. PubMed PMID: 17303098.

    Article  CAS  PubMed  Google Scholar 

  33. Serini, G., & Gabbiani, G. (1996). Modulation of alpha-smooth muscle actin expression in fibroblasts by transforming growth factor-beta isoforms: An in vivo and in vitro study. Wound Repair Regen, 4, 278–287. PubMed PMID: 17177825.

    Article  CAS  PubMed  Google Scholar 

  34. Stawowy, P., Goetze, S., Margeta, C., Fleck, E., & Graf, K. (2003). LPS regulate ERK1/2-dependent signaling in cardiac fibroblasts via PKC-mediated MKP-1 induction. Biochemical and Biophysical Research Communications, 303, 74–80. PubMed PMID: 12646169.

    Article  CAS  PubMed  Google Scholar 

  35. Sugiura, H., Ichikawa, T., Koarai, A., Yanagisawa, S., Minakata, Y., Matsunaga, K., et al. (2009). Activation of Toll-like receptor 3 augments myofibroblast differentiation. American Journal of Respiratory Cell and Molecular Biology, 40(6), 654–662. PubMed PMID: 18988918.

    Article  CAS  PubMed  Google Scholar 

  36. Tiede, K., Stöter, K., Petrik, C., Chen, W. B., Ungefroren, H., Kruse, M. L., et al. (2003). Angiotensin II AT (1)-receptor induces biglycan in neonatal cardiac fibroblasts via autocrine release of TGF-β in vitro. Cardiovascular Research, 60(3), 538–546.

    Article  CAS  PubMed  Google Scholar 

  37. Timmers, L., Sluijter, J. P., van Keulen, J. K., Hoefer, I. E., Nederhoff, M. G., Goumans, M. J., et al. (2008). Toll-like receptor 4 mediates maladaptive left ventricular remodeling and impairs cardiac function after myocardial infarction. Circulation Research, 102(2), 257–264. PubMed PMID: 18007026.

    Article  CAS  PubMed  Google Scholar 

  38. Tomasek, J., McRae, J., Owens, G., & Haaksma, C. (2005). Regulation of α-Smooth Muscle Actin Expression in Granulation Tissue Myofibroblasts Is Dependent on the Intronic CArG Element and the Transforming Growth Factor-β1 Control Element. American Journal of Pathology, 166(5), 1343–1351. PubMed PMID: 15855636. PubMed Central PMCID: PMC1606390.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  39. Tomita, K., Takashina, M., Mizuno, N., Sakata, K., Hattori, K., Imura, J., et al. (2015). Cardiac fibroblasts: Contributory role in septic cardiac dysfunction. Journal of Surgical Research, 193(2), 874–887.

    Article  CAS  PubMed  Google Scholar 

  40. Turner, N. A., & Porter, K. E. (2013). Function and fate of myofibroblasts after myocardial infarction. Fibrogenesis Tissue Repair, 6(1), 5. PubMed PMID: 23448358. PubMed Central PMCID: PMC3599637.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  41. Uematsu, S., & Akira, S. (2008). Toll-like receptors and innate inmunity. Handbook of Experimental Pharmacology, 183, 1–21. PubMed PMID: 18071652.

    Article  CAS  Google Scholar 

  42. Vivar, R., Humeres, C., Varela, M., Ayala, P., Guzmán, N., Olmedo, I., et al. (2011). Cardiac fibroblast death by ischemia/reperfusion is partially inhibited by IGF-1 through both PI3 K/Akt and MEK-ERK pathways. Experimental and Molecular Pathology, 93(1), 1–7. PubMed PMID: 22537549.

    Article  Google Scholar 

  43. Verstrepen, L., Bekaert, T., Chau, T. L., Tavernier, J., Chariot, A., & Beyaert, R. (2008). TLR-4, IL-1R and TNF-R signaling to NF-kappaβ: Variations on a common theme. Cellular and Molecular Life Sciences, 65, 2964–2978. PubMed PMID: 18535784.

    Article  CAS  PubMed  Google Scholar 

  44. Webber, J., Meran, S., Steadman, R., & Phillips, A. (2009). Hyaluronan orchestrates transforming growth factor-beta1-dependent maintenance of myofibroblast phenotype. Journal of Biological Chemistry, 284(14), 9083–9092. PubMed PMID: 19193641. PubMed Central PMCID: PMC2666557.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  45. Weber, K. T. (2004). Fibrosis in hypertensive heart disease: Focus on cardiac fibroblasts. Journal of Hypertension, 22(1), 47–50. PubMed PMID: 15106793.

    Article  CAS  PubMed  Google Scholar 

  46. Yan, X., Lin, Z., Chen, F., Zhao, X., Chen, H., Ning, Y., et al. (2009). Human BAMBI cooperates with Smad7 to inhibit transforming growth factor-β signaling. Journal of Biological Chemistry, 284, 30097–30104. PubMed PMID: 19758997; PubMed Central PMCID: PMC2781564.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  47. Yoshida, K., & Matsuzaki, K. (2012). Differential regulation of TGF-β/Smad signaling in hepatic stellate cells between acute and chronic liver injuries. Front Physiol, 3, 53. PubMed PMID: 22457652. PubMed Central PMCID: PMC3307138.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  48. Zhang, S., Fei, T., Zhang, L., Zhang, R., Chen, F., Ning, Y., et al. (2007). Smad7 antagonizes transforming growth factor-β signaling in the nucleus by interfering with functional Smad-DNA complex formation. Molecular and Cellular Biology, 27, 4488–4499.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  49. Zeuke, S., Ulmer, A. J., Kusumoto, S., Katus, H. A., & Heine, H. (2002). TLR4-mediated inflammatory activation of human coronary artery endothelial cells by LPS. Cardiovascular Research, 56(1), 126–134. PubMed PMID: 12237173.

    Article  CAS  PubMed  Google Scholar 

Download references

Acknowledgements

This work was supported by FONDECYT (Grant 1130300 to G. Díaz-Araya) and CONICYT (Grant 63130233 to S Bolivar).

Author information

Authors and Affiliations

  1. Advanced Center for Chronic Diseases (ACCDiS), Faculty of Chemical and Pharmaceutical Sciences and Faculty of Medicine, University of Chile, 8380492, Santiago, Chile

    Roxana Santana, Rodolfo Landaeta, Pía Boza, Claudio Humeres, Raúl Vivar, Claudia Muñoz, Viviana Pardo, Samuel Fernandez, Renatto Anfossi & Guillermo Diaz-Araya

  2. Department of Pharmacological and Toxicological Chemistry, Faculty of Chemical and Pharmaceutical Sciences, University of Chile, Santiago, Chile

    Samir Bolívar, Roxana Santana, Rodolfo Landaeta, Pía Boza, Claudio Humeres, Raúl Vivar, Claudia Muñoz, Viviana Pardo, Samuel Fernandez, Renatto Anfossi & Guillermo Diaz-Araya

  3. Center of Medical Investigations, Catholic University of Chile, Santiago, Chile

    Pedro Ayala

  4. Faculty of Chemistry and Pharmacy, Atlantic University, Barranquilla, Colombia

    Samir Bolívar

Authors
  1. Samir Bolívar
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Roxana Santana
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Pedro Ayala
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Rodolfo Landaeta
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Pía Boza
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Claudio Humeres
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Raúl Vivar
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Claudia Muñoz
    View author publications

    You can also search for this author in PubMed Google Scholar

  9. Viviana Pardo
    View author publications

    You can also search for this author in PubMed Google Scholar

  10. Samuel Fernandez
    View author publications

    You can also search for this author in PubMed Google Scholar

  11. Renatto Anfossi
    View author publications

    You can also search for this author in PubMed Google Scholar

  12. Guillermo Diaz-Araya
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Guillermo Diaz-Araya.

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Bolívar, S., Santana, R., Ayala, P. et al. Lipopolysaccharide Activates Toll-Like Receptor 4 and Prevents Cardiac Fibroblast-to-Myofibroblast Differentiation. Cardiovasc Toxicol 17, 458–470 (2017). https://doi.org/10.1007/s12012-017-9404-4

Download citation

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s12012-017-9404-4

Keywords

Search

Navigation