Department of Cardiology, Cardiovascular Key Laboratory of Zhejiang Province, The Second Affiliated Hospital, Zhejiang University School of Medicine, Hangzhou, Zhejiang Province, 310009, People's Republic of China.
The Affiliated Cardiovascular Hospital of Qingdao University, Qingdao, Shandong Province, 266071, People's Republic of China.
Int J Nanomedicine. 2021 May 27;16:3633-3648. doi: 10.2147/IJN.S303714. eCollection 2021.
The deposition of hydroxyapatite (HAp) crystals plays an important role in the development of vascular calcification (VC). This study aimed to demonstrate the effects of nanosized HAp (nHAp) on vascular smooth muscle cells (VSMCs) and VC progression.
Transmission electron microscopy (TEM) was used to examine cellular uptake of nHAp. Cell viability was determined using CCK-8 assay kit. Mitochondrial impairment and reactive oxygen species were detected by TEM and fluorescence dye staining, respectively. Cell apoptosis was detected by Western blot analysis and Annexin V staining. Mouse model of VC was built via applying nHAp on the surface of abdominal aorta. Calcification was visualized by Alizarin red and von Kossa staining.
We found that nHAp could promote osteogenic transformation of VSMCs by elevating expression of runt-related factor 2 (Runx2), osteopontin (OPN) and alkaline phosphatase (ALP), impairing function and morphology of mitochondria and inducing apoptosis of VSMCs. More phosphorylation of c-Jun N-terminal protein kinase/c-JUN (JNK/c-JUN) in VSMCs was detected after mixing nHAp with VSMCs. HAp-induced osteogenic transformation of VSMCs was blocked by JNK inhibitor SP600125, resulted in decreased ALP activity, less Runx2 and OPN expressions. SP600125 also inhibited apoptosis of VSMCs. Application of nHAp to outside of aorta induced osteogenic transformation and apoptosis of VSMCs, and significant deposition of calcium on the vessel walls of mice, which can be effectively attenuated by SP600125.
JNK/c-JUN signaling pathway is critical for nHAp-induced calcification, which could be a potential therapeutic target for controlling the progression of VC.
羟基磷灰石（HAp）晶体的沉积在血管钙化（VC）的发展中起着重要作用。本研究旨在证明纳米 HAp（nHAp）对血管平滑肌细胞（VSMCs）和 VC 进展的影响。
透射电子显微镜（TEM）用于观察 nHAp 的细胞摄取。使用 CCK-8 测定试剂盒测定细胞活力。通过 TEM 和荧光染料染色分别检测线粒体损伤和活性氧。通过 Western blot 分析和 Annexin V 染色检测细胞凋亡。通过将 nHAp 施加到腹主动脉表面在小鼠模型中构建 VC。通过茜素红和 von Kossa 染色可视化钙化。
我们发现 nHAp 通过提高 runt 相关转录因子 2（Runx2）、骨桥蛋白（OPN）和碱性磷酸酶（ALP）的表达，损害线粒体的功能和形态，并诱导 VSMCs 凋亡，从而促进 VSMCs 的成骨转化。在 nHAp 与 VSMCs 混合后，VSMCs 中检测到更多的 c-Jun N-末端蛋白激酶/c-JUN（JNK/c-JUN）磷酸化。JNK 抑制剂 SP600125 阻断了 HAp 诱导的 VSMCs 成骨转化，导致 ALP 活性降低、Runx2 和 OPN 表达减少。SP600125 还抑制了 VSMCs 的凋亡。将 nHAp 施加到主动脉外会引起 VSMCs 的成骨转化和凋亡，并在小鼠的血管壁上沉积大量钙，SP600125 可有效抑制钙沉积。
JNK/c-JUN 信号通路对 nHAp 诱导的钙化至关重要，这可能是控制 VC 进展的潜在治疗靶点。
