Skip to main content

Advertisement

SpringerLink
Log in

The Ryanodine Receptor Contributes to the Lysophosphatidylcholine-Induced Mineralization in Valvular Interstitial Cells

  • Original Article
  • Published:
Cardiovascular Engineering and Technology Aims and scope Submit manuscript

Abstract

Purpose

Fibrocalcific aortic valve disease (CAVD) is caused by the deposition of calcific nodules in the aortic valve leaflets, resulting in progressive loss of function that ultimately requires surgical intervention. This process is actively mediated by the resident valvular interstitial cells (VICs), which, in response to oxidized lipids, transition from a quiescent to an osteoblast-like state. The purpose of this study was to examine if the ryanodine receptor, an intracellular calcium channel, could be therapeutically targeted to prevent this phenotypic conversion.

Methods

The expression of the ryanodine receptor in porcine aortic VICs was characterized by qRT-PCR and immunofluorescence. Next, the VICs were exposed to lysophosphatidylcholine, an oxidized lipid commonly found in low-density lipoprotein, while the activity of the ryanodine receptor was modulated with ryanodine. The cultures were analyzed for markers of cellular mineralization, alkaline phosphatase activity, proliferation, and apoptosis.

Results

Porcine aortic VICs predominantly express isoform 3 of the ryanodine receptors, and this protein mediates the cellular response to LPC. Exposure to LPC caused elevated intracellular calcium concentration in VICs, raised levels of alkaline phosphatase activity, and increased calcific nodule formation, but these changes were reversed when the activity of the ryanodine receptor was blocked.

Conclusions

Our findings suggest blocking the activity of the ryanodine receptor can attenuate the valvular mineralization caused by LPC. We conclude that oxidized lipids, such as LPC, play an important role in the development and progression of CAVD and that the ryanodine receptor is a promising target for pharmacological intervention.

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

Figure 1
Figure 2
Figure 3
Figure 4
Figure 5
Figure 6

Similar content being viewed by others

References

  1. Anderson, H. C. Matrix vesicles and calcification. Curr. Rheumatol. Rep. 5:222–226, 2003. https://doi.org/10.1007/s11926-003-0071-z.

    Article  Google Scholar 

  2. Balaoing, L. R., A. D. Post, A. Y. Lin, H. Tseng, J. L. Moake, and K. J. Grande-Allen. Laminin peptide-immobilized hydrogels modulate valve endothelial cell hemostatic regulation. PLoS ONE. 10:e0130749, 2015. https://doi.org/10.1371/journal.pone.0130749.

    Article  Google Scholar 

  3. Benjamin, E. J., M. J. Blaha, S. E. Chiuve, M. Cushman, S. R. Das, R. Deo, S. D. De Ferranti, J. Floyd, M. Fornage, C. Gillespie, C. R. Isasi, M. C. Jimenez, L. C. Jordan, S. E. Judd, D. Lackland, J. H. Lichtman, L. Lisabeth, S. Liu, C. T. Longenecker, R. H. MacKey, K. Matsushita, D. Mozaffarian, M. E. Mussolino, K. Nasir, R. W. Neumar, L. Palaniappan, D. K. Pandey, R. R. Thiagarajan, M. J. Reeves, M. Ritchey, C. J. Rodriguez, G. A. Roth, W. D. Rosamond, C. Sasson, A. Towfghi, C. W. Tsao, M. B. Turner, S. S. Virani, J. H. Voeks, J. Z. Willey, J. T. Wilkins, J. H. Y. Wu, H. M. Alger, S. S. Wong, and P. Muntner, Heart disease and stroke statistics—2017 update: a report from the American Heart Association. Circulation. 135:e146–e603, 2017. https://doi.org/10.1161/CIR.0000000000000485.

    Article  Google Scholar 

  4. Bowler, M. A., and W. D. Merryman. In vitro models of aortic valve calcification: solidifying a system. Cardiovasc. Pathol. 24:1–10, 2014. https://doi.org/10.1016/j.carpath.2014.08.003.

    Article  Google Scholar 

  5. Chen, J. H., and C. A. Simmons. Cell-matrix interactions in the pathobiology of calcific aortic valve disease: critical roles for matricellular, matricrine, and matrix mechanics cues. Circ. Res. 108:1510–1524, 2011. https://doi.org/10.1161/CIRCRESAHA.110.234237.

    Article  Google Scholar 

  6. Cheng, H., Q. Yao, R. Song, Y. Zhai, W. Wang, D. A. Fullerton, and X. Meng. Lysophosphatidylcholine activates the Akt pathway to upregulate extracellular matrix protein production in human aortic valve cells. J. Surg. Res. 213:243–250, 2017. https://doi.org/10.1016/j.jss.2017.02.028.

    Article  Google Scholar 

  7. Clapham, D. E. Calcium signaling. Cell. 131:1047–1058, 2007. https://doi.org/10.1016/j.cell.2007.11.028.

    Article  Google Scholar 

  8. Drzazga, A., A. Sowinska, A. Krzeminska, P. Rytczak, M. Koziolkiewicz, and E. Gendaszewska-Darmach. Lysophosphatidylcholine elicits intracellular calcium signaling in a GPR55-dependent manner. Biochem. Biophys. Res. Commun. 489:242–247, 2017. https://doi.org/10.1016/j.bbrc.2017.05.145.

    Article  Google Scholar 

  9. Fill, M., and J. A. Copello. Ryanodine receptor calcium release channels. Physiol. Rev. 82:893–922, 2002. https://doi.org/10.1152/physrev.00013.2002.

    Article  Google Scholar 

  10. Garg, A., S. V. Rao, G. Visveswaran, S. Agrawal, A. Sharma, L. Garg, I. Mahata, J. Garg, D. Singal, M. Cohen, and J. B. Kostis. Transcatheter aortic valve replacement versus surgical valve replacement in low-intermediate surgical risk patients: a systematic review and meta-analysis. J. Invasive Cardiol. 29:209–216, 2017.

    Google Scholar 

  11. Gomel, M. A., R. Lee, and K. J. Grande-Allen. Comparing the role of mechanical forces in vascular and valvular calcification progression. Front. Cardiovasc. Med. 5:197, 2019. https://doi.org/10.3389/fcvm.2018.00197.

    Article  Google Scholar 

  12. Gregory, C. A., W. G. Gunn, A. Peister, and D. J. Prockop. An Alizarin red-based assay of mineralization by adherent cells in culture: comparison with cetylpyridinium chloride extraction. Anal. Biochem. 329:77–84, 2004. https://doi.org/10.1016/j.ab.2004.02.002.

    Article  Google Scholar 

  13. Gu, X., and K. S. Masters. Role of the Rho pathway in regulating valvular interstitial cell phenotype and nodule formation. Am. J. Physiol. 2011. https://doi.org/10.1152/ajpheart.01178.2009.

    Article  Google Scholar 

  14. Hjortnaes, J., C. Goettsch, J. D. Hutcheson, G. Camci-Unal, L. Lax, K. Scherer, S. Body, F. J. Schoen, J. Kluin, A. Khademhosseini, and E. Aikawa. Simulation of early calcific aortic valve disease in a 3D platform: a role for myofibroblast differentiation. J. Mol. Cell. Cardiol. 94:13–20, 2016. https://doi.org/10.1016/j.yjmcc.2016.03.004.

    Article  Google Scholar 

  15. Hoffman, B. D., C. Grashoff, and M. A. Schwartz. Dynamic molecular processes mediate cellular mechanotransduction. Nature. 475:316–323, 2011. https://doi.org/10.1038/nature10316.

    Article  Google Scholar 

  16. Huk, D. J., B. F. Austin, T. E. Horne, R. B. Hinton, W. C. Ray, D. D. Heistad, and J. Lincoln. Valve endothelial cell-derived Tgfβ1 signaling promotes nuclear localization of Sox9 in interstitial cells associated with attenuated calcification significance. Arterioscler. Thromb. Vasc. Biol. 36:328–338, 2016. https://doi.org/10.1161/ATVBAHA.115.306091.

    Article  Google Scholar 

  17. Iannotti, J. P., S. Naidu, Y. Noguchi, R. M. Hunt, and C. T. Brighton. Growth plate matrix vesicle biogenesis. The role of intracellular calcium. Clin. Orthop. Relat. Res. 306:222–229, 1994.

    Google Scholar 

  18. Jeong, H., Y. H. Kim, Y. Lee, S. J. Jung, and S. B. Oh. TRPM2 contributes to LPC-induced intracellular Ca2+ influx and microglial activation. Biochem. Biophys. Res. Commun. 485:301–306, 2017. https://doi.org/10.1016/j.bbrc.2017.02.087.

    Article  Google Scholar 

  19. Jono, S., Y. Nishizawa, A. Shioi, and H. Morii. 1,25-Dihydroxyvitamin D3 increases in vitro vascular calcification by modulating secretion of endogenous parathyroid hormone related peptide. Circulation. 98:1302–1306, 1998. https://doi.org/10.1161/01.CIR.98.13.1302.

    Article  Google Scholar 

  20. Kim, K. M. Apoptosis and calcification. Scanning Microsc. 9:1137–1175, 1995.

    Google Scholar 

  21. Krause, T., M. U. Gerbershagen, M. Fiege, R. Weisshorn, and F. Wappler. Dantrolene—a review of its pharmacology, therapeutic use and new developments. Anaesthesia. 59:364–373, 2004. https://doi.org/10.1111/j.1365-2044.2004.03658.x.

    Article  Google Scholar 

  22. Li, X. H., and Y. J. Wu. Characteristics of lysophosphatidylcholine-induced Ca2+ response in human neuroblastoma SH-SY5Y cells. Life Sci. 80:886–892, 2007. https://doi.org/10.1016/j.lfs.2006.11.017.

    Article  Google Scholar 

  23. Li, C., S. Xu, and A. I. Gotlieb. The progression of calcific aortic valve disease through injury, cell dysfunction, and disruptive biologic and physical force feedback loops. Cardiovasc. Pathol. 22:1–8, 2013. https://doi.org/10.1016/j.carpath.2012.06.005.

    Article  Google Scholar 

  24. Liu, A. C., V. R. Joag, and A. I. Gotlieb. The emerging role of valve interstitial cell phenotypes in regulating heart valve pathobiology. Am. J. Pathol. 171:1407–1418, 2007. https://doi.org/10.2353/ajpath.2007.070251.

    Article  Google Scholar 

  25. Mahmut, A., M.-C. Boulanger, D. El Husseini, D. Fournier, R. Bouchareb, J.-P. Després, P. Pibarot, Y. Bossé, and P. Mathieu. Elevated expression of lipoprotein-associated phospholipase A2 in calcific aortic valve disease. J. Am. Coll. Cardiol. 63:460–469, 2014. https://doi.org/10.1016/j.jacc.2013.05.105.

    Article  Google Scholar 

  26. Massaeli, H., J. A. Austria, and G. N. Pierce. Lesions in ryanodine channels in smooth muscle cells exposed to oxidized low density lipoprotein. Arter. Thromb Vasc Biol. 20:328–334, 2000.

    Article  Google Scholar 

  27. Mathieu, P., and M. C. Boulanger. Basic mechanisms of calcific aortic valve disease. Can. J. Cardiol. 30:982–993, 2014. https://doi.org/10.1016/j.cjca.2014.03.029.

    Article  Google Scholar 

  28. Meissner, G. Ryanodine receptor/Ca2+ release channels and their regulation by endogenous effectors. Annu. Rev. Physiol. 56:485–508, 1994. https://doi.org/10.1146/annurev.ph.56.030194.002413.

    Article  Google Scholar 

  29. Mohty, D., P. Pibarot, J. P. Després, C. Côté, B. Arsenault, A. Cartier, P. Cosnay, C. Couture, and P. Mathieu. Association between plasma LDL particle size, valvular accumulation of oxidized LDL, and inflammation in patients with aortic stenosis. Arterioscler. Thromb. Vasc. Biol. 28:187–193, 2008. https://doi.org/10.1161/ATVBAHA.107.154989.

    Article  Google Scholar 

  30. Mosmann, T. Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays. J. Immunol. Methods. 65:55–63, 1983. https://doi.org/10.1016/0022-1759(83)90303-4.

    Article  Google Scholar 

  31. Nakamura, Y., M. Yasukochi, S. Kobayashi, K. Uehara, A. Honda, R. Inoue, I. Imanaga, and A. Uehara. Cell membrane-derived lysophosphatidylcholine activates cardiac ryanodine receptor channels. Pflugers Arch. 453:455–462, 2007. https://doi.org/10.1007/s00424-006-0141-y.

    Article  Google Scholar 

  32. Parisi, V., D. Leosco, G. Ferro, A. Bevilacqua, G. Pagano, C. de Lucia, P. Perrone Filardi, A. Caruso, G. Rengo, and N. Ferrara. The lipid theory in the pathogenesis of calcific aortic stenosis. Nutr. Metab. Cardiovasc. Dis. 25:519–525, 2015. https://doi.org/10.1016/j.numecd.2015.02.001.

    Article  Google Scholar 

  33. Puperi, D. S., R. W. O’Connell, Z. E. Punske, Y. Wu, J. L. West, and K. J. Grande-Allen. Hyaluronan hydrogels for a biomimetic spongiosa layer of tissue engineered heart valve scaffolds. Biomacromolecules. 17:1766–1775, 2016. https://doi.org/10.1021/acs.biomac.6b00180.

    Article  Google Scholar 

  34. Rajamannan, N. M., F. J. Evans, E. Aikawa, K. J. Grande-Allen, L. L. Demer, D. D. Heistad, C. A. Simmons, K. S. Masters, P. Mathieu, K. D. O’Brien, F. J. Schoen, D. A. Towler, A. P. Yoganathan, and C. M. Otto. Calcific aortic valve disease: not simply a degenerative process: a review and agenda for research from the National Heart and Lung and Blood Institute Aortic Stenosis Working Group. Executive summary: calcific aortic valve disease-2011 update. Circulation. 124:1783–1791, 2011. https://doi.org/10.1161/CIRCULATIONAHA.110.006767.

    Article  Google Scholar 

  35. Rodriguez, K. J., L. M. Piechura, and K. S. Masters. Regulation of valvular interstitial cell phenotype and function by hyaluronic acid in 2-D and 3-D culture environments. Matrix Biol. 30:70–82, 2011. https://doi.org/10.1016/j.matbio.2010.09.001.

    Article  Google Scholar 

  36. Rodriguez, K. J., L. M. Piechura, A. M. Porras, and K. S. Masters. Manipulation of valve composition to elucidate the role of collagen in aortic valve calcification. BMC Cardiovasc. Disord. 14:29, 2014. https://doi.org/10.1186/1471-2261-14-29.

    Article  Google Scholar 

  37. Song, R., Q. Zeng, L. Ao, J. A. Yu, J. C. Cleveland, K. S. Zhao, D. A. Fullerton, and X. Meng. Biglycan induces the expression of osteogenic factors in human aortic valve interstitial cells via toll-like receptor-2. Arterioscler. Thromb. Vasc. Biol. 32:2711–2720, 2012. https://doi.org/10.1161/ATVBAHA.112.300116.

    Article  Google Scholar 

  38. Stephens, E. H., J. L. Carroll, and K. J. Grande-Allen. The use of collagenase III for the isolation of porcine aortic valvular interstitial cells: rationale and optimization. J. Heart Valve Dis. 16:175–183, 2007.

    Google Scholar 

  39. Stephens, E. H., J. G. Saltarrelli, L. S. Baggett, I. Nandi, J. J. Kuo, A. R. Davis, E. A. Olmsted-Davis, M. J. Reardon, J. D. Morrisett, and K. J. Grande-Allen. Differential proteoglycan and hyaluronan distribution in calcified aortic valves. Cardiovasc. Pathol. 20:334–342, 2011. https://doi.org/10.1016/j.carpath.2010.10.002.

    Article  Google Scholar 

  40. Sutko, J. L., J. A. Airey, W. Welch, and L. Ruest. The pharmacology of ryanodine and related compounds. Pharmacol. Rev. 49:53–98, 1997.

    Google Scholar 

  41. Sylvester, P. W. Optimization of the tetrazolium dye (MTT) colorimetric assay for cellular growth and viability. Methods Mol. Biol. 716:157–168, 2011. https://doi.org/10.1007/978-1-61779-012-6_9.

    Article  Google Scholar 

  42. Thayer, P., K. Balachandran, S. Rathan, C. H. Yap, S. Arjunon, H. Jo, and A. P. Yoganathan. The effects of combined cyclic stretch and pressure on the aortic valve interstitial cell phenotype. Ann. Biomed. Eng. 39:1654–1667, 2011. https://doi.org/10.1007/s10439-011-0273-x.

    Article  Google Scholar 

  43. Tillman, T. S., and M. Cascio. Effects of membrane lipids on ion channel structure and function. Cell Biochem. Biophys. 38:161–190, 2003. https://doi.org/10.1385/cbb:38:2:161.

    Article  Google Scholar 

  44. Towler, D. A. Molecular and cellular aspects of calcific aortic valve disease. Circ. Res. 113:198–208, 2013. https://doi.org/10.1161/CIRCRESAHA.113.300155.

    Article  Google Scholar 

  45. Van Petegem, F. Ryanodine receptors: structure and function. J. Biol. Chem. 287:31624–31632, 2012. https://doi.org/10.1074/jbc.R112.349068.

    Article  Google Scholar 

  46. Vater, C., P. Kasten, and M. Stiehler. Culture media for the differentiation of mesenchymal stromal cells. Acta Biomater. 7:463–477, 2011. https://doi.org/10.1016/j.actbio.2010.07.037.

    Article  Google Scholar 

  47. Wang, H., M. W. Tibbitt, S. J. Langer, L. A. Leinwand, and K. S. Anseth. Hydrogels preserve native phenotypes of valvular fibroblasts through an elasticity-regulated PI3K/AKT pathway. Proc. Natl. Acad. Sci. U. S. A. 110:19336–19341, 2013. https://doi.org/10.1073/pnas.1306369110.

    Article  Google Scholar 

  48. Wiltz, D. C., R. I. Han, R. L. Wilson, A. Kumar, J. D. Morrisett, and K. J. Grande-Allen. Differential aortic and mitral valve interstitial cell mineralization and the induction of mineralization by lysophosphatidylcholine in vitro. Cardiovasc. Eng. Technol. 5:371–383, 2014. https://doi.org/10.1007/s13239-014-0197-3.

    Article  Google Scholar 

  49. Wu, Y., D. S. Puperi, K. J. Grande-Allen, and J. L. West. Ascorbic acid promotes extracellular matrix deposition while preserving valve interstitial cell quiescence within 3D hydrogel scaffolds. J. Tissue Eng. Regen. Med. 11:1963–1973, 2017. https://doi.org/10.1002/term.2093.

    Article  Google Scholar 

  50. Yip, C. Y. Y., J. H. Chen, R. Zhao, and C. A. Simmons. Calcification by valve interstitial cells is regulated by the stiffness of the extracellular matrix. Arterioscler. Thromb. Vasc. Biol. 29:936–942, 2009. https://doi.org/10.1161/ATVBAHA.108.182394.

    Article  Google Scholar 

  51. Zeng, Q., R. Song, D. A. Fullerton, L. Ao, Y. Zhai, S. Li, D. B. Ballak, J. C. Cleveland, T. B. Reece, T. A. McKinsey, D. Xu, C. A. Dinarello, and X. Meng. Interleukin-37 suppresses the osteogenic responses of human aortic valve interstitial cells in vitro and alleviates valve lesions in mice. Proc. Natl. Acad. Sci. 114:1631–1636, 2017. https://doi.org/10.1073/pnas.1619667114.

    Article  Google Scholar 

  52. Zhang, X., B. Xu, D. S. Puperi, A. L. Yonezawa, Y. Wu, H. Tseng, M. L. Cuchiara, J. L. West, and K. J. Grande-Allen. Integrating valve-inspired design features into poly(ethylene glycol) hydrogel scaffolds for heart valve tissue engineering. Acta Biomater. 14:11–21, 2015. https://doi.org/10.1016/j.actbio.2014.11.042.

    Article  Google Scholar 

Download references

Acknowledgments

This work was supported by the National Heart, Lung and Blood Institute [R21HL104377 to K.J.G.A. and T32HL007812 to J.D.M.] and the National Institute of Diabetes and Digestive and Kidney Disease [F30DK108541 to R.L.W.] of the National Institutes of Health. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health. The authors thank Jennifer P. Connell for her critical reading of the manuscript and Marci Kang and Vaidya Parthasarathy for their suggestions regarding research directions.

Conflict of interest

The authors have no conflicts of interest to declare.

Author information

Author notes

  1. Reid L. Wilson, Christopher B. Sylvester, and Dena C. Wiltz contributed equally to this work

Authors and Affiliations

  1. Department of Bioengineering, Rice University, 6100 Main St., MS 142, Houston, TX, 77005, USA

    Reid L. Wilson, Christopher B. Sylvester, Dena C. Wiltz, Aditya Kumar, Tahir H. Malik & K. Jane Grande-Allen

  2. Departments of Medicine and Biochemistry, Baylor College of Medicine, One Baylor Plaza, Houston, TX, 77030, USA

    Joel D. Morrisett

  3. Medical Scientist Training Program, Baylor College of Medicine, One Baylor Plaza, Houston, TX, 77030, USA

    Reid L. Wilson & Christopher B. Sylvester

Authors
  1. Reid L. Wilson
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Christopher B. Sylvester
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Dena C. Wiltz
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Aditya Kumar
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Tahir H. Malik
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Joel D. Morrisett
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. K. Jane Grande-Allen
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to K. Jane Grande-Allen.

Additional information

Associate Editor Craig Alexander Simmons oversaw the review of this article.

Publisher's Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Wilson, R.L., Sylvester, C.B., Wiltz, D.C. et al. The Ryanodine Receptor Contributes to the Lysophosphatidylcholine-Induced Mineralization in Valvular Interstitial Cells. Cardiovasc Eng Tech 11, 316–327 (2020). https://doi.org/10.1007/s13239-020-00463-1

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s13239-020-00463-1

Keywords

Search

Navigation