Table_1_Integrated proteomic and metabolomic profile analyses of cardiac valves revealed molecular mechanisms and targets in calcific aortic valve disease.DOCX

BackgroundThis study aimed to define changes in the metabolic and protein profiles of patients with calcific aortic valve disease (CAVD).
Methods and resultsWe analyzed cardiac valve samples of patients with and without (control) CAVD (n = 24 per group) using untargeted metabolomics and tandem mass tag-based quantitative proteomics. Significantly different metabolites and proteins between the CAVD and control groups were screened; then, functional enrichment was analyzed. We analyzed co-expressed differential metabolites and proteins, and constructed a metabolite-protein-pathway network. The expression of key proteins was validated using western blotting. Differential analysis identified 229 metabolites in CAVD among which, 2-aminophenol, hydroxykynurenine, erythritol, carnosine, and choline were the top five. Proteomic analysis identified 549 differentially expressed proteins in CAVD, most of which were localized in the nuclear, cytoplasmic, extracellular, and plasma membranes. Levels of selenium binding protein 1 (SELENBP1) positively correlated with multiple metabolites. Adenosine triphosphate-binding cassette transporters, starch and sucrose metabolism, hypoxia-inducible factor 1 (HIF-1) signaling, and purine metabolism were key pathways in the network. Ectonucleotide pyrophosphatase/phosphodiesterase 1 (ENPP1), calcium2+/calmodulin-dependent protein kinase II delta (CAMK2D), and ATP binding cassette subfamily a member 8 (ABCA8) were identified as hub proteins in the metabolite-protein-pathway network as they interacted with ADP, glucose 6-phosphate, choline, and other proteins. Western blotting confirmed that ENPP1 was upregulated, whereas ABCA8 and CAMK2D were downregulated in CAVD samples.
ConclusionThe metabolic and protein profiles of cardiac valves from patients with CAVD significantly changed. The present findings provide a holistic view of the molecular mechanisms underlying CAVD that may lead to the development of novel diagnostic biomarkers and therapeutic targets to treat CAVD.

研究背景
本研究旨在明确钙化性主动脉瓣疾病（calcific aortic valve disease, CAVD）患者的代谢组谱与蛋白质组谱变化。

方法与结果
本研究采用非靶向代谢组学（untargeted metabolomics）与基于串联质量标签（tandem mass tag）的定量蛋白质组学技术，对伴与不伴钙化性主动脉瓣疾病的对照组患者的心脏瓣膜样本进行分析，每组样本量为24例。研究团队首先筛选出钙化性主动脉瓣疾病组与对照组间存在显著差异的代谢物与蛋白质，随后开展功能富集分析；对共表达的差异代谢物与蛋白质进行关联分析，并构建代谢物-蛋白质-通路调控网络；最后采用蛋白质印迹法（western blotting）验证关键蛋白质的表达水平。
差异分析共鉴定出钙化性主动脉瓣疾病组中的229种差异代谢物，其中排名前五的分别为2-氨基苯酚（2-aminophenol）、羟基犬尿氨酸（hydroxykynurenine）、赤藓糖醇（erythritol）、肌肽（carnosine）与胆碱（choline）。蛋白质组学分析共鉴定出549种钙化性主动脉瓣疾病组的差异表达蛋白质，其中大多数定位于细胞核、细胞质、细胞外区域与质膜。硒结合蛋白1（selenium binding protein 1, SELENBP1）的表达水平与多种代谢物呈正相关。腺苷三磷酸结合盒转运体（adenosine triphosphate-binding cassette transporters）、淀粉与蔗糖代谢、缺氧诱导因子1（hypoxia-inducible factor 1, HIF-1）信号通路以及嘌呤代谢是该调控网络中的关键通路。外核苷酸焦磷酸酶/磷酸二酯酶1（ectonucleotide pyrophosphatase/phosphodiesterase 1, ENPP1）、钙/钙调蛋白依赖性蛋白激酶IIδ（calcium2+/calmodulin-dependent protein kinase II delta, CAMK2D）以及ATP结合盒亚家族A成员8（ATP binding cassette subfamily a member 8, ABCA8）被鉴定为该调控网络中的核心蛋白质，它们可与腺苷二磷酸（ADP）、葡萄糖-6-磷酸（glucose 6-phosphate）、胆碱以及其他蛋白质发生相互作用。蛋白质印迹实验证实，在钙化性主动脉瓣疾病患者的瓣膜样本中，ENPP1表达上调，而ABCA8与CAMK2D表达下调。

研究结论
钙化性主动脉瓣疾病患者心脏瓣膜的代谢组谱与蛋白质组谱发生了显著改变。本研究结果全面揭示了钙化性主动脉瓣疾病潜在的分子机制，有望为开发用于治疗钙化性主动脉瓣疾病的新型诊断生物标志物与治疗靶点提供理论依据。