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

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 microen-vironment. 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 th...

Semicarbazide-Sensitive Amine Oxidase Increases in Calcific Aortic Valve Stenosis and Contributes to Valvular Interstitial Cell Calcification

“…Valvular oxidative stress predominates around calcifying foci and enhances progression of CAVS. Acute H 2 O 2 -induced oxidative stress and the resulting higher reactive oxygen species (ROS) levels induce osteoblastic differentiation of human valvular interstitial cells (VIC), which are the main structural cells of the aortic valve [3]. These processes highly resemble those observed in atherosclerosis, in which, for example, vascular peroxidase 1, an enzyme generating H 2 O 2 , has been implicated in Ox-LDL-induced calcification of vascular smooth muscle cells [4].…”

“…The addition of H 2 O 2 in the Pi-induced calcification model further increased calcium deposition in vitro, and VIC derived from calcified valves were more susceptible to oxidative stress compared with VIC from healthy valves [3]. Oxidative stress also induces the expression of Runx2, a transcriptional factor of osteoblasts [27], expressed by VIC under osteogenic conditions [31].…”

Valve leaflet‐inspired elastomeric scaffolds with tunable and anisotropic mechanical properties

“…De‐identified human valvular tissues were collected from the University of Pittsburgh Medical Center (UPMC) as per the protocols approved by the University of Pittsburgh Institutional Review Board, PRO16050264 (St. Hilaire) and PRO0702120 with informed consent (Dr. Gleason). The hVICs were isolated from the donor tissue as per our previously reported protocol . The hVICs were then propagated and cultured using Dulbecco's Modified Eagle Medium (DMEM) supplemented with 10 % fetal bovine serum according to the established protocol .…”

“…The hVICs were isolated from the donor tissue as per our previously reported protocol . The hVICs were then propagated and cultured using Dulbecco's Modified Eagle Medium (DMEM) supplemented with 10 % fetal bovine serum according to the established protocol . All experiments were carried out by using cells between passages 4 and 8.…”

Nanoceria and Its Biomedical Relevance

“…Nanoceria and manganese ferrite nanoparticles anchored to mesoporus silica nanoparticles showed synergistic effects by scavenging ROS and promoting recruitment of anti-inflammatory macrophages in joints of rat RA model. 12 Nanoceria also showed therapeutic potential by preventing valvular calcification mediated by ROS related damage, 13 cardio-protective effects by preventing myocardial remodelling, 14 and attenuating ischemia reperfusion induced hepatic injury in rats. 15 Antioxidant and anti-inflammatory properties of nanoceria were found beneficial in cisplatin induced nephrotoxicity.…”