Cellular and Molecular Bioengineering

, Volume 10, Issue 5, pp 483–500 | Cite as

Shape-Specific Nanoceria Mitigate Oxidative Stress-Induced Calcification in Primary Human Valvular Interstitial Cell Culture

  • Yingfei Xue
  • Cynthia St. Hilaire
  • Luis Hortells
  • Julie A. Phillippi
  • Vinayak Sant
  • Shilpa Sant
Article

Abstract

Introduction

Lack of effective pharmacological treatment makes valvular calcification a significant clinical problem in patients with valvular disease and bioprosthetic/mechanical valve replacement therapies. Elevated levels of reactive oxygen species (ROS) in valve tissue have been identified as a prominent hallmark and driving factor for valvular calcification. However, the therapeutic value of ROS-modulating agents for valvular calcification remains elusive. We hypothesized that ROS-modulating shape-specific cerium oxide nanoparticles (CNPs) will inhibit oxidative stress-induced valvular calcification. CNPs are a class of self-regenerative ROS-modulating agents, which can switch between Ce3+ and Ce4+ in response to oxidative microenvironment. In this work, we developed oxidative stress-induced valve calcification model using two patient-derived stenotic valve interstitial cells (hVICs) and investigated the therapeutic effect of shape-specific CNPs to inhibit hVIC calcification.

Methods

Human valvular interstitial cells (hVICs) were obtained from a normal healthy donor and two patients with calcified aortic valves. hVICs were characterized for their phenotypic (mesenchymal, myofibroblast and osteoblast) marker expression by qRT-PCR and antioxidant enzymes activity before and after exposure to hydrogen peroxide (H2O2)-induced oxidative stress. Four shape-specific CNPs (sphere, short rod, long rod, and cube) were synthesized via hydrothermal or ultra-sonication method and characterized for their biocompatibility in hVICs by alamarBlue® assay, and ROS scavenging ability by DCFH-DA assay. H2O2 and inorganic phosphate (Pi) were co-administrated to induce hVIC calcification in vitro as demonstrated by Alizarin Red S staining and calcium quantification. The effect of CNPs on inhibiting H2O2-induced hVIC calcification was evaluated.

Results

hVICs isolated from calcified valves exhibited elevated osteoblast marker expression and decreased antioxidant enzyme activities compared to the normal hVICs. Due to the impaired antioxidant enzyme activities, acute H2O2-induced oxidative stress resulted in higher ROS levels and osteoblast marker expression in both diseased hVICs when compared to the normal hVICs. Shape-specific CNPs exhibited shape-dependent abiotic ROS scavenging ability, and excellent cytocompatibility. Rod and sphere CNPs scavenged H2O2-induced oxidative stress in hVICs in a shape- and dose-dependent manner by lowering intracellular ROS levels and osteoblast marker expression. Further, CNPs also enhanced activity of antioxidant enzymes in hVICs to combat oxidative stress. Cube CNPs were not effective ROS scavengers. The addition of H2O2 in the Pi-induced calcification model further increased calcium deposition in vitro in a time-dependent manner. Co-administration of rod CNPs with Pi and H2O2 mitigated calcification in the diseased hVICs.

Conclusions

We demonstrated that hVICs derived from calcified valves exhibited impaired antioxidant defense mechanisms and were more susceptible to oxidative stress than normal hVICs. CNPs scavenged H2O2-induced oxidative stress in hVICs in a shape-dependent manner. The intrinsic ROS scavenging ability of CNPs and their ability to induce cellular antioxidant enzyme activities may confer protection from oxidative stress-exacerbated calcification. CNPs represent promising antioxidant therapy for treating valvular calcification and deserve further investigation.

Keywords

Nanoceria Reactive oxygen species (ROS) Valve calcification Patient-derived valvular interstitial cells (hVICs) Cerium oxide nanoparticle Nanoparticle shape 

Supplementary material

12195_2017_495_MOESM1_ESM.docx (1.7 mb)
Supplementary material 1 (DOCX 1727 kb)

References

  1. 1.
    Mozaffarian, D., E. J. Benjamin, A. S. Go, D. K. Arnett, M. J. Blaha, M. Cushman, S. R. Das, S. de Ferranti, J.-P. Després, H. J. Fullerton, V. J. Howard, M. D. Huffman, C. R. Isasi, M. C. Jiménez, S. E. Judd, B. M. Kissela, J. H. Lichtman, L. D. Lisabeth, S. Liu, R. H. Mackey, D. J. Magid, D. K. McGuire, E. R. Mohler, C. S. Moy, P. Muntner, M. E. Mussolino, K. Nasir, R. W. Neumar, G. Nichol, L. Palaniappan, D. K. Pandey, M. J. Reeves, C. J. Rodriguez, W. Rosamond, P. D. Sorlie, J. Stein, A. Towfighi, T. N. Turan, S. S. Virani, D. Woo, R. W. Yeh, and M. B. Turner. Executive summary: heart disease and stroke statistics—2016 update. Circulation 133:447–454, 2016.CrossRefGoogle Scholar
  2. 2.
    Otto, C. M. Calcific aortic stenosis—time to look more closely at the valve. N. Engl. J. Med. 359:1395–1398, 2008.CrossRefGoogle Scholar
  3. 3.
    Schoen, F. J., and R. J. Levy. Calcification of tissue heart valve substitutes: progress toward understanding and prevention. Ann. Thorac. Surg. 79:1072–1080, 2005.CrossRefGoogle Scholar
  4. 4.
    Xue, Y., V. Sant, J. Phillippi, and S. Sant. Biodegradable and biomimetic elastomeric scaffolds for tissue-engineered heart valves. Acta Biomater. 48:2–19, 2017.CrossRefGoogle Scholar
  5. 5.
    Hutcheson, J. D., E. Aikawa, and W. D. Merryman. Potential drug targets for calcific aortic valve disease. Nat. Rev. Cardiol. 11:218–231, 2014.CrossRefGoogle Scholar
  6. 6.
    Mathieu, P., and M.-C. Boulanger. Basic mechanisms of calcific aortic valve disease. Can. J. Cardiol. 30:982–993, 2014.CrossRefGoogle Scholar
  7. 7.
    Sverdlov, A. L., D. T. Ngo, M. J. Chapman, O. A. Ali, Y. Y. Chirkov, and J. D. Horowitz. Pathogenesis of aortic stenosis: not just a matter of wear and tear. Am. J. Cardiovasc. Dis. 1:185–199, 2011.Google Scholar
  8. 8.
    Butcher, J. T., G. J. Mahler, and L. A. Hockaday. Aortic valve disease and treatment: the need for naturally engineered solutions. Adv. Drug Deliv. Rev. 63:242–268, 2011.CrossRefGoogle Scholar
  9. 9.
    Heistad, D. D., Y. Wakisaka, J. Miller, Y. Chu, and R. Pena-Silva. Novel aspects of oxidative stress in cardiovascular diseases. Circ. J. 73:201–207, 2009.CrossRefGoogle Scholar
  10. 10.
    Miller, J. D., R. M. Weiss, and D. D. Heistad. Calcific aortic valve stenosis: methods, models, and mechanisms. Circ. Res. 108:1392–1412, 2011.CrossRefGoogle Scholar
  11. 11.
    Yip, C. Y. Y., and C. A. Simmons. The aortic valve microenvironment and its role in calcific aortic valve disease. Cardiovasc. Pathol. 20:177–182, 2011.CrossRefGoogle Scholar
  12. 12.
    Schoen, F. J. Mechanisms of function and disease of natural and replacement heart valves. Annu. Rev. Pathol.: Mech. Dis. 7:161–183, 2012.CrossRefGoogle Scholar
  13. 13.
    Hutcheson, J. D., M. C. Blaser, and E. Aikawa. Giving calcification its due: recognition of a diverse disease. Circ. Res. 120:270–273, 2017.CrossRefGoogle Scholar
  14. 14.
    Miller, J. D., Y. Chu, R. M. Brooks, W. E. Richenbacher, R. Peña-Silva, and D. D. Heistad. Dysregulation of antioxidant mechanisms contributes to increased oxidative stress in calcific aortic valvular stenosis in humans. J. Am. Coll. Cardiol. 52:843–850, 2008.CrossRefGoogle Scholar
  15. 15.
    Miller, J. D., R. M. Weiss, K. M. Serrano, R. M. Brooks, C. J. Berry, K. Zimmerman, S. G. Young, and D. D. Heistad. Lowering plasma cholesterol levels halts progression of aortic valve disease in mice. Circulation 119:2693–2701, 2009.CrossRefGoogle Scholar
  16. 16.
    Fernández Esmerats, J., J. Heath, and H. Jo. Shear-sensitive genes in aortic valve endothelium. Antioxid. Redox Signal. 25:401–414, 2016.CrossRefGoogle Scholar
  17. 17.
    Bostrom, K. I., N. M. Rajamannan, and D. A. Towler. The regulation of valvular and vascular sclerosis by osteogenic morphogens. Circ. Res. 109:564–577, 2011.CrossRefGoogle Scholar
  18. 18.
    Branchetti, E., R. Sainger, P. Poggio, J. B. Grau, J. Patterson-Fortin, J. E. Bavaria, M. Chorny, E. Lai, R. C. Gorman, R. J. Levy, and G. Ferrari. Antioxidant enzymes reduce DNA damage and early activation of valvular interstitial cells in aortic valve sclerosis. Arterioscler. Thromb. Vasc. Biol. 33:e66–e74, 2012.CrossRefGoogle Scholar
  19. 19.
    Bowler, M. A., and W. D. Merryman. in vitro models of aortic valve calcification: solidifying a system. Cardiovasc. Pathol. 24:1–10, 2015.CrossRefGoogle Scholar
  20. 20.
    Aikawa, E., K. L. Cloyd, I. El-Hamamsy, S. Boonrungsiman, M. Hedegaard, E. Gentleman, P. Sarathchandra, F. Colazzo, M. M. Gentleman, M. H. Yacoub, A. H. Chester, and M. M. Stevens. Characterization of porcine aortic valvular interstitial cell ‘calcified’ nodules. PLoS ONE 7:e48154, 2012.CrossRefGoogle Scholar
  21. 21.
    Mulholland, D. L., and A. I. Gotlieb. Cell biology of valvular interstitial cells. Can. J. Cardiol. 12:231–236, 1996.Google Scholar
  22. 22.
    Liberman, M., E. Bassi, M. K. Martinatti, F. C. Lario, J. Wosniak, P. M. A. Pomerantzeff, and F. R. M. Laurindo. Oxidant generation predominates around calcifying foci and enhances progression of aortic valve calcification. Arterioscler. Thromb. Vasc. Biol. 28:463–470, 2007.CrossRefGoogle Scholar
  23. 23.
    Shao, J. S. Molecular mechanisms of vascular calcification: lessons learned from the aorta. Arterioscler. Thromb. Vasc. Biol. 26:1423–1430, 2006.CrossRefGoogle Scholar
  24. 24.
    Griendling, K. K. Oxidative stress and cardiovascular injury: part II: animal and human studies. Circulation 108:2034–2040, 2003.CrossRefGoogle Scholar
  25. 25.
    Kobayashi, S., N. Inoue, H. Azumi, T. Seno, K. Hirata, S. Kawashima, Y. Hayashi, H. Itoh, H. Yokozaki, and M. Yokoyama. Expressional changes of the vascular antioxidant system in atherosclerotic coronary arteries. J. Atheroscler. Thromb. 9:184–190, 2002.CrossRefGoogle Scholar
  26. 26.
    Kim, K. M. Calcification of matrix vesicles in human aortic valve and aortic media. Fed. Proc. 35:156–162, 1976.Google Scholar
  27. 27.
    Walkey, C., S. Das, S. Seal, J. Erlichman, K. Heckman, L. Ghibelli, E. Traversa, J. F. McGinnis, and W. T. Self. Catalytic properties and biomedical applications of cerium oxide nanoparticles. Environ. Sci.: Nano 2:33–53, 2015.Google Scholar
  28. 28.
    Nelson, B., M. Johnson, M. Walker, K. Riley, and C. Sims. Antioxidant cerium oxide nanoparticles in biology and medicine. Antioxidants 5:15, 2016.CrossRefGoogle Scholar
  29. 29.
    Rzigalinski, B. A., C. S. Carfagna, and M. Ehrich. Cerium oxide nanoparticles in neuroprotection and considerations for efficacy and safety. WIREs Nanomed. Nanobiotechnol. 9:e1444, 2017. doi:10.1002/wnan.1444.CrossRefGoogle Scholar
  30. 30.
    Xue, Y., S. R. Balmuri, A. Patel, V. Sant, and S. Sant. Synthesis, physico-chemical characterization, and antioxidant effect of PEGylated cerium oxide nanoparticles. Drug Deliv. Transl. Res. 2017. doi:10.1007/s13346-017-0396-1.
  31. 31.
    Niu, J., K. Wang, and P. E. Kolattukudy. Cerium oxide nanoparticles inhibits oxidative stress and nuclear factor- B activation in H9c2 cardiomyocytes exposed to cigarette smoke extract. J. Pharmacol. Exp. Ther. 338:53–61, 2011.CrossRefGoogle Scholar
  32. 32.
    Pagliari, F., C. Mandoli, G. Forte, E. Magnani, S. Pagliari, G. Nardone, S. Licoccia, M. Minieri, P. Di Nardo, and E. Traversa. Cerium oxide nanoparticles protect cardiac progenitor cells from oxidative stress. ACS Nano 6:3767–3775, 2012.CrossRefGoogle Scholar
  33. 33.
    Kolli, M. B., N. D. P. K. Manne, R. Para, S. K. Nalabotu, G. Nandyala, T. Shokuhfar, K. He, A. Hamlekhan, J. Y. Ma, P. S. Wehner, L. Dornon, R. Arvapalli, K. M. Rice, and E. R. Blough. Cerium oxide nanoparticles attenuate monocrotaline induced right ventricular hypertrophy following pulmonary arterial hypertension. Biomaterials 35:9951–9962, 2014.CrossRefGoogle Scholar
  34. 34.
    Niu, J., A. Azfer, L. Rogers, X. Wang, and P. Kolattukudy. Cardioprotective effects of cerium oxide nanoparticles in a transgenic murine model of cardiomyopathy. Cardiovasc. Res. 73:549–559, 2007.CrossRefGoogle Scholar
  35. 35.
    Mai, H.-X., L.-D. Sun, Y.-W. Zhang, R. Si, W. Feng, H.-P. Zhang, H.-C. Liu, and C.-H. Yan. Shape-selective synthesis and oxygen storage behavior of ceria nanopolyhedra, nanorods, and nanocubes. J. Phys. Chem. B 109:24380–24385, 2005.CrossRefGoogle Scholar
  36. 36.
    Li, Y., M. Kröger, and W. K. Liu. Shape effect in cellular uptake of PEGylated nanoparticles: comparison between sphere, rod, cube and disk. Nanoscale 7:16631–16646, 2015.CrossRefGoogle Scholar
  37. 37.
    Jo, D. H., J. H. Kim, T. G. Lee, and J. H. Kim. Size, surface charge, and shape determine therapeutic effects of nanoparticles on brain and retinal diseases. Nanomed.: Nanotechnol. Biol. Med. 11:1603–1611, 2015.CrossRefGoogle Scholar
  38. 38.
    Gould, R. A., and J. T. Butcher. Isolation of valvular endothelial cells. J. Vis. Exp. 23:12, 2010.Google Scholar
  39. 39.
    Zhang, D., H. Fu, L. Shi, C. Pan, Q. Li, Y. Chu, and W. Yu. Synthesis of CeO2 nanorods via ultrasonication assisted by polyethylene glycol. Inorg. Chem. 46:2446–2451, 2007.CrossRefGoogle Scholar
  40. 40.
    Gaharwar, A. K., S. M. Mihaila, A. Swami, A. Patel, S. Sant, R. L. Reis, A. P. Marques, M. E. Gomes, and A. Khademhosseini. Bioactive silicate nanoplatelets for osteogenic differentiation of human mesenchymal stem cells. Adv. Mater. 25:3329–3336, 2013.CrossRefGoogle Scholar
  41. 41.
    Cox, R. F., A. Hernandez-Santana, S. Ramdass, G. McMahon, J. H. Harmey, and M. P. Morgan. Microcalcifications in breast cancer: novel insights into the molecular mechanism and functional consequence of mammary mineralisation. Br. J. Cancer 106:525–537, 2012.CrossRefGoogle Scholar
  42. 42.
    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.CrossRefGoogle Scholar
  43. 43.
    Pesce, M., N. Latif, A. Quillon, P. Sarathchandra, A. McCormack, A. Lozanoski, M. H. Yacoub, and A. H. Chester. Modulation of human valve interstitial cell phenotype and function using a fibroblast growth factor 2 formulation. PLoS ONE 10:e0127844, 2015.CrossRefGoogle Scholar
  44. 44.
    Monzack, E. L., and K. S. Masters. Can valvular interstitial cells become true osteoblasts? A side-by-side comparison. J. Heart Valve Dis. 20:449–463, 2011.Google Scholar
  45. 45.
    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.CrossRefGoogle Scholar
  46. 46.
    MatÉs, J. M., C. Pérez-Gómez, and I. N. De Castro. Antioxidant enzymes and human diseases. Clin. Biochem. 32:595–603, 1999.CrossRefGoogle Scholar
  47. 47.
    Gough, D. R., and T. G. Cotter. Hydrogen peroxide: a Jekyll and Hyde signalling molecule. Cell Death Dis. 2:e213, 2011.CrossRefGoogle Scholar
  48. 48.
    Byon, C. H., A. Javed, Q. Dai, J. C. Kappes, T. L. Clemens, V. M. Darley-Usmar, J. M. McDonald, and Y. Chen. Oxidative stress induces vascular calcification through modulation of the osteogenic transcription factor Runx2 by AKT signaling. J. Biol. Chem. 283:15319–15327, 2008.CrossRefGoogle Scholar
  49. 49.
    Lai, C.-F., J.-S. Shao, A. Behrmann, K. Krchma, S.-L. Cheng, and D. A. Towler. TNFR1-activated reactive oxidative species signals up-regulate osteogenic Msx2 programs in aortic myofibroblasts. Endocrinology 153:3897–3910, 2012.CrossRefGoogle Scholar
  50. 50.
    Pulido-Reyes, G., I. Rodea-Palomares, S. Das, T. S. Sakthivel, F. Leganes, R. Rosal, S. Seal, and F. Fernández-Piñas. Untangling the biological effects of cerium oxide nanoparticles: the role of surface valence states. Sci. Rep. 5:15613, 2015.CrossRefGoogle Scholar
  51. 51.
    Sakthivel, T., S. Das, A. Kumar, D. L. Reid, A. Gupta, D. C. Sayle, and S. Seal. Morphological phase diagram of biocatalytically active ceria nanostructures as a function of processing variables and their properties. ChemPlusChem 78:1446–1455, 2013.CrossRefGoogle Scholar
  52. 52.
    Pirmohamed, T., J. M. Dowding, S. Singh, B. Wasserman, E. Heckert, A. S. Karakoti, J. E. S. King, S. Seal, and W. T. Self. Nanoceria exhibit redox state-dependent catalase mimetic activity. Chem. Commun. 46:2736, 2010.CrossRefGoogle Scholar
  53. 53.
    Popov, A. L., N. R. Popova, I. I. Selezneva, A. Y. Akkizov, and V. K. Ivanov. Cerium oxide nanoparticles stimulate proliferation of primary mouse embryonic fibroblasts in vitro. Mater. Sci. Eng.: C 68:406–413, 2016.CrossRefGoogle Scholar
  54. 54.
    Giachelli, C. M. The emerging role of phosphate in vascular calcification. Kidney Int. 75:890–897, 2009.CrossRefGoogle Scholar
  55. 55.
    Mazière, C., V. Savitsky, A. Galmiche, C. Gomila, Z. Massy, and J.-C. Mazière. Oxidized low density lipoprotein inhibits phosphate signaling and phosphate-induced mineralization in osteoblasts. Involvement of oxidative stress. Biochim. Biophys. Acta (BBA)—Mol. Basis Dis. 1802:1013–1019, 2010.CrossRefGoogle Scholar
  56. 56.
    Zhang, Q., K. Ge, H. Ren, C. Zhang, and J. Zhang. Effects of cerium oxide nanoparticles on the proliferation, osteogenic differentiation and adipogenic differentiation of primary mouse bone marrow stromal cells in vitro. J. Nanosci. Nanotechnol. 15:6444–6451, 2015.CrossRefGoogle Scholar
  57. 57.
    Mason, D., Y.-Z. Chen, H. V. Krishnan, and S. Sant. Cardiac gene therapy: recent advances and future directions. J. Control. Release 215:101–111, 2015.CrossRefGoogle Scholar

Copyright information

© Biomedical Engineering Society 2017

Authors and Affiliations

  1. 1.Department of Pharmaceutical SciencesUniversity of Pittsburgh School of PharmacyPittsburghUSA
  2. 2.Department of Medicine, Division of Cardiology & Vascular Medicine InstituteUniversity of PittsburghPittsburghUSA
  3. 3.Department of BioengineeringUniversity of Pittsburgh Swanson School of EngineeringPittsburghUSA
  4. 4.Department of Cardiothoracic SurgeryUniversity of PittsburghPittsburghUSA
  5. 5.McGowan Institute for Regenerative MedicineUniversity of PittsburghPittsburghUSA
  6. 6.PittsburghUSA

Personalised recommendations