Shape-Specific Nanoceria Mitigate Oxidative Stress-Induced Calcification in Primary Human Valvular Interstitial Cell Culture.
Shape-Specific Nanoceria Mitigate Oxidative Stress-Induced Calcification in Primary Human Valvular Interstitial Cell Culture.
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DOI：
10.1007/s12195-017-0495-6
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发表时间：
2017-10
影响因子：
2.8
通讯作者：
Sant S
中科院分区：
文献类型：
--
作者：
Xue Y;St Hilaire C;Hortells L;Phillippi JA;Sant V;Sant S
缺乏有效的药物治疗使得瓣膜钙化成为瓣膜疾病和生物假体/机械瓣膜置换治疗患者的一个重要临床问题。瓣膜组织中活性氧（ROS）水平升高已被确定为瓣膜钙化的显著标志和驱动因素。然而，ros调节剂对瓣膜钙化的治疗价值仍然难以捉摸。我们假设ros调节形状特异性氧化铈纳米颗粒（CNPs）将抑制氧化应激诱导的瓣膜钙化。CNPs是一类自我再生的ros调节剂，在氧化微环境下可以在Ce3+和Ce4+之间切换。在这项工作中，我们利用两个患者来源的狭窄瓣膜间质细胞（hVIC）建立了氧化应激诱导的瓣膜钙化模型，并研究了形状特异性CNPs抑制hVIC钙化的治疗效果。从一名正常健康供体和两名主动脉瓣钙化患者身上获得人瓣膜间质细胞（hvic）。通过qRT-PCR检测hvcs表型（间充质细胞、肌成纤维细胞和成骨细胞）标志物表达和过氧化氢（H2O2）诱导氧化应激前后的抗氧化酶活性。通过水热或超声波法合成了四种形状特异性CNPs（球形、短棒状、长棒状和立方体），并通过alamarBlue®实验表征了它们在hvc中的生物相容性，通过DCFH-DA实验表征了它们清除ROS的能力。茜素红S染色和钙定量结果表明，H2O2和无机磷酸盐（Pi）可诱导体外hVIC钙化。评价CNPs对h2o2诱导的hVIC钙化的抑制作用。与正常hvic相比，从钙化瓣膜中分离的hvic表现出成骨细胞标志物表达升高和抗氧化酶活性降低。由于抗氧化酶活性受损，与正常hvic相比，急性h2o2诱导的氧化应激导致两种病变hvic的ROS水平和成骨细胞标志物表达升高。形状特异性CNPs表现出形状依赖的非生物ROS清除能力和良好的细胞相容性。杆状和球形CNPs通过降低细胞内ROS水平和成骨细胞标志物表达，以形状和剂量依赖的方式清除hvcs中h2o2诱导的氧化应激。此外，CNPs还增强了hvic中抗氧化酶的活性，以对抗氧化应激。立方体CNPs不是有效的ROS清除剂。在pi诱导的钙化模型中，H2O2的加入进一步增加了体外钙沉积，并呈时间依赖性。同时给药杆状CNPs与Pi和H2O2减轻了患病hvic的钙化。我们证明了钙化瓣膜衍生的hvic表现出抗氧化防御机制受损，并且比正常的hvic更容易受到氧化应激的影响。CNPs以形状依赖的方式清除h2o2诱导的hvic氧化应激。CNPs固有的活性氧清除能力及其诱导细胞抗氧化酶活性的能力可能赋予防止氧化应激加剧的钙化的保护作用。CNPs是治疗瓣膜钙化的有前途的抗氧化疗法，值得进一步研究。
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. 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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