Phadwal K, Tang Q, Kurian D, Tan X, Cawthorn W P, MacRae V E
The Roslin Institute and Royal (Dick) School of Veterinary Studies, University of Edinburgh, Midlothian, United Kingdom.
University/BHF Centre for Cardiovascular Science, The Queen's Medical Research Institute, Edinburgh Bioquarter, University of Edinburgh, Edinburgh, United Kingdom.
Front Cardiovasc Med. 2025 Jul 21;12:1586775. doi: 10.3389/fcvm.2025.1586775. eCollection 2025.
Calcific aortic valve disease (CAVD) is a common and progressive valvular heart disease characterised by the pathological calcification of valve interstitial cells (VICs). Current clinical treatments, such as surgical valve replacement and transcatheter valve implantation, are invasive and do not target the underlying molecular mechanisms of calcification. Emerging evidence suggests that metabolic interventions may modulate cellular calcification processes. In this study, we investigated the potential of nutrient restriction (NR) as a non-invasive strategy to mitigate VIC calcification, with a particular focus on the role of the ubiquitin-proteasome system (UPS).
Primary rat valvular interstitial cells (RVICs) were cultured and subjected to in vitro calcification using calcium- and phosphate-enriched media. Nutrient restriction was induced by incubating cells in Hank's Balanced Salt Solution (HBSS). Calcification was assessed by quantifying calcium deposition and osteogenic marker expression. To explore the underlying molecular changes, a stable isotope labelling by amino acids in cell culture (SILAC)-based proteomic analysis was performed. The role of the UPS was further examined using pharmacological inhibition with MG132 and siRNA-mediated knockdown of key UPS components, including Cullin-2 (Cul2) and Ubiquitin-conjugating enzyme E2 H (Ube2H).
Nutrient restriction significantly downregulated the expression of osteogenic markers and reduced calcium deposition in RVICs. SILAC-based proteomics revealed the upregulation of multiple components of the UPS in nutrient-restricted cells. Notably, Cul2 and Ube2H were identified as potential key mediators of the anti-calcification effects observed. Inhibition of the proteasome with MG132 exacerbated calcification, while knockdown of Cul2 using siRNA increased osteogenic marker expression and calcium deposition, indicating the essential role of Cul2 in modulating VIC calcification under nutrient-restricted conditions.
This study demonstrates that nutrient restriction effectively attenuates VIC calcification through the modulation of the ubiquitin-proteasome system. The protective role of UPS components, particularly Cul2 and Ube2H, suggests that targeting this pathway could represent a novel therapeutic approach for the management of CAVD. These findings also raise the possibility of employing dietary or metabolic interventions as non-invasive strategies to prevent or delay valve calcification.
钙化性主动脉瓣疾病（CAVD）是一种常见的进行性心脏瓣膜疾病，其特征为瓣膜间质细胞（VICs）发生病理性钙化。目前的临床治疗方法，如外科瓣膜置换术和经导管瓣膜植入术，具有侵入性，且未针对钙化的潜在分子机制。新出现的证据表明，代谢干预可能调节细胞钙化过程。在本研究中，我们研究了营养限制（NR）作为一种减轻VIC钙化的非侵入性策略的潜力，特别关注泛素-蛋白酶体系统（UPS）的作用。
培养原代大鼠瓣膜间质细胞（RVICs），并使用富含钙和磷的培养基使其进行体外钙化。通过在汉克平衡盐溶液（HBSS）中培养细胞来诱导营养限制。通过定量钙沉积和成骨标志物表达来评估钙化情况。为了探究潜在的分子变化，进行了基于细胞培养中氨基酸稳定同位素标记（SILAC）的蛋白质组学分析。使用MG132进行药理学抑制以及用小干扰RNA（siRNA）介导关键UPS成分（包括Cullin-2（Cul2）和泛素结合酶E2 H（Ube2H））的敲低，进一步研究UPS的作用。
营养限制显著下调了RVICs中成骨标志物的表达，并减少了钙沉积。基于SILAC的蛋白质组学揭示了营养限制细胞中UPS多个成分的上调。值得注意的是，Cul2和Ube2H被确定为观察到的抗钙化作用的潜在关键介质。用MG132抑制蛋白酶体加剧了钙化，而使用siRNA敲低Cul2增加了成骨标志物表达和钙沉积，表明Cul2在营养限制条件下调节VIC钙化中起重要作用。
本研究表明，营养限制通过调节泛素-蛋白酶体系统有效地减轻了VIC钙化。UPS成分，特别是Cul2和Ube2H的保护作用表明，靶向该途径可能代表一种治疗CAVD的新方法。这些发现还增加了采用饮食或代谢干预作为预防或延缓瓣膜钙化的非侵入性策略的可能性。
