Shape-Specific Nanoceria Mitigate Oxidative Stress-Induced Calcification in Primary Human Valvular Interstitial Cell Culture
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.
KeywordsNanoceria Reactive oxygen species (ROS) Valve calcification Patient-derived valvular interstitial cells (hVICs) Cerium oxide nanoparticle Nanoparticle shape
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.
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