Heuschkel Marina A, Babler Anne, Heyn Jonas, van der Vorst Emiel P C, Steenman Marja, Gesper Maren, Kappel Ben A, Magne David, Gouëffic Yann, Kramann Rafael, Jahnen-Dechent Willi, Marx Nikolaus, Quillard Thibaut, Goettsch Claudia
Department of Internal Medicine I-Cardiology, Medical Faculty, RWTH Aachen University, Aachen, Germany.
Institute of Experimental Medicine and Systems Biology, University Hospital, RWTH Aachen, Aachen, Germany.
Front Cardiovasc Med. 2022 Sep 20;9:959457. doi: 10.3389/fcvm.2022.959457. eCollection 2022.
Vascular calcification (VC) is a major risk factor for cardiovascular morbidity and mortality. Depending on the location of mineral deposition within the arterial wall, VC is classified as intimal and medial calcification. Using mineralization assays, we developed protocols triggering both types of calcification in vascular smooth muscle cells (SMCs) following diverging molecular pathways.
Human coronary artery SMCs were cultured in osteogenic medium (OM) or high calcium phosphate medium (CaP) to induce a mineralized extracellular matrix. OM induces osteoblast-like differentiation of SMCs-a key process in intimal calcification during atherosclerotic plaque remodeling. CaP mimics hyperphosphatemia, associated with chronic kidney disease-a risk factor for medial calcification. Transcriptomic analysis revealed distinct gene expression profiles of OM and CaP-calcifying SMCs. OM and CaP-treated SMCs shared 107 differentially regulated genes related to SMC contraction and metabolism. Real-time extracellular efflux analysis demonstrated decreased mitochondrial respiration and glycolysis in CaP-treated SMCs compared to increased mitochondrial respiration without altered glycolysis in OM-treated SMCs. Subsequent kinome and drug repurposing analysis (Connectivity Map) suggested a distinct role of protein kinase C (PKC). validation experiments demonstrated that the PKC activators prostratin and ingenol reduced calcification triggered by OM and promoted calcification triggered by CaP.
Our direct comparison results of two calcification models strengthen previous observations of distinct intracellular mechanisms that trigger OM and CaP-induced SMC calcification . We found a differential role of PKC in OM and CaP-calcified SMCs providing new potential cellular and molecular targets for pharmacological intervention in VC. Our data suggest that the field should limit the generalization of results found in studies using different calcification protocols.
血管钙化（VC）是心血管疾病发病和死亡的主要危险因素。根据矿物质在动脉壁内沉积的位置，VC可分为内膜钙化和中膜钙化。通过矿化分析，我们开发了在不同分子途径后触发血管平滑肌细胞（SMC）两种类型钙化的方案。
将人冠状动脉SMC在成骨培养基（OM）或高钙磷培养基（CaP）中培养以诱导矿化细胞外基质。OM诱导SMC向成骨细胞样分化，这是动脉粥样硬化斑块重塑过程中内膜钙化的关键过程。CaP模拟与慢性肾病相关的高磷血症，慢性肾病是中膜钙化的危险因素。转录组分析揭示了OM和CaP诱导钙化的SMC的不同基因表达谱。OM和CaP处理的SMC共有107个与SMC收缩和代谢相关的差异调节基因。实时细胞外流出分析表明，与OM处理的SMC中线粒体呼吸增加而糖酵解未改变相比，CaP处理的SMC中线粒体呼吸和糖酵解均降低。随后的激酶组和药物再利用分析（连接图谱）表明蛋白激酶C（PKC）具有独特作用。验证实验表明，PKC激活剂prostratin和ingenol可减少OM触发的钙化并促进CaP触发的钙化。
我们对两种钙化模型的直接比较结果强化了先前关于触发OM和CaP诱导的SMC钙化的不同细胞内机制的观察。我们发现PKC在OM和CaP钙化的SMC中具有不同作用，为VC的药物干预提供了新的潜在细胞和分子靶点。我们的数据表明，该领域应限制在使用不同钙化方案的研究中所发现结果的推广。
