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.
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.
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.
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.
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We acknowledge funding support from the School of Pharmacy, University of Pittsburgh (SS) and HL117917, NHLBI (CSH). YX acknowledges Graduate Student Research Scholarship from the School of Pharmacy, University of Pittsburgh. We thank Dr. Thomas Gleason, Center for Thoracic Aortic Disease, University of Pittsburgh for providing us with valve cusp tissue to collect the valve cells and Jennifer Hill for isolating valve cells. We thank Dr. Donna Stolz, Center for Biologic Imaging, University of Pittsburgh for access to TEM facility and Akhil Patel, School of Pharmacy, University of Pittsburgh for acquiring the TEM images. We thank Dr. Paul Johnston, School of Pharmacy, University of Pittsburgh for access to the spectrophotometer.
Conflict of interest
Shilpa Sant has an invention disclosure filed as “shape-specific CNPs as ROS and immune-modulating agents”. Yingfei Xue, Cynthia St. Hilaire, Luis Hortells, Julie A. Phillippi, and Vinayak Sant declare that they have no conflicts of interest.
All human subjects researches were carried out in accordance with the ethical standards approved by the University of Pittsburgh Institutional Review Board and with the 1964 Helsinki declaration and its later amendments or comparable ethical standards. No animal studies were carried out by the authors for this article.
Shilpa Sant, PhD, is an Assistant Professor at University of Pittsburgh in the Department of Pharmaceutical Sciences with secondary appointment in Bioengineering. She is also a member faculty at the McGowan Institute for Regenerative Medicine and UPMC Hillman Cancer Center. Dr. Sant received a PhD in Pharmaceutical Technology from University of Montreal, Canada; MS in Pharmacology and B.Pharm. in Pharmaceutical Sciences from University of Mumbai. Before joining Pitt, she was a Ruth Kirschstein fellow with Drs. Ali Khademhosseini and Richard Maas at the Wyss Institute for Biologically Inspired Engineering and the Center for Bioengineering at Brigham and Women’s Hospital. Her major research interests include bioinspired approaches for regenerative therapies and development of biomimetic microenvironments for in vitro three-dimensional disease progression models. Her lab uses interdisciplinary approaches to study role of microenvironments on disease progression in the same cells without any genetic manipulations or artificial culture conditions. Dr. Sant has contributed more than 50 papers, book chapters and patents, including reports in Cancer Research, Advanced Materials, Journal of Controlled Release, and Advanced Drug Delivery Reviews. Her work is highlighted on PNAS journal club, MaterialsToday news, and Women in Nanoscience blog. She has also edited a book entitled “Nanomaterials in Tissue Engineering: Fabrication and Applications” and a journal issue “Stem Cells: Microenvironment, Micro/Nanotechnology, and Application”, in Stem Cells International. Dr. Sant’s exemplary achievements in research have been recognized by prestigious fellowships: Ruth L. Kirschstein National Research Service Award (NIH, USA), Post-doctoral Fellowship (Le Fonds de Recherche du Quebec Nature et Technologies (FRQNT), Canada), Post-graduate Scholarship (Natural Sciences and Engineering Research Council of Canada (NSERC), Canada). She has also received several awards including “2016 CMBE-BMES Rising Star Early Career Faculty Award” “2013 CMBE-BMES Rising Star/Fellow Award”, “2010 Society For Biomaterials – STAR Award” to name a few. She serves as a reviewer on several NIH and NSF grant review panels. She also serves as an Associate Editor for IEEE Transactions on NanoBioScience, and editorial board member for Scientific Reports and In Silico Pharmacology.
This article is part of the 2017 CMBE Young Innovators special issue.
Associate Editor Alyssa Panitch oversaw the review of this article.
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Xue, Y., St. Hilaire, C., Hortells, L. et al. Shape-Specific Nanoceria Mitigate Oxidative Stress-Induced Calcification in Primary Human Valvular Interstitial Cell Culture. Cel. Mol. Bioeng. 10, 483–500 (2017). https://doi.org/10.1007/s12195-017-0495-6
- Reactive oxygen species (ROS)
- Valve calcification
- Patient-derived valvular interstitial cells (hVICs)
- Cerium oxide nanoparticle
- Nanoparticle shape