Abstract：
OBJECTIVE: Valve interstitial cells (VICs) undergo a transition to intermediate state cells before ultimately transforming into the osteogenic cell population, which is a pivotal cellular process in calcific aortic valve disease (CAVD). Herein, this study successfully delineated the stages of VIC osteogenic transformation and elucidated a novel key regulatory role of lumican (LUM) in this process.
METHODS: Single-cell RNA-sequencing (scRNA-seq) from nine human aortic valves was used to characterize the pathological switch process and identify key regulatory factors. The in vitro, ex vivo, in vivo, and double knockout mice were constructed to further unravel the calcification-promoting effect of LUM. Moreover, the multi-omic approaches were employed to analyse the molecular mechanism of LUM in CAVD.
RESULTS: ScRNA-seq successfully delineated the process of VIC pathological transformation and highlighted the significance of LUM as a novel molecule in this process. The pro-calcification role of LUM is confirmed on the in vitro, ex vivo, in vivo level, and ApoE-/-//LUM-/- double knockout mice. The LUM induces osteogenesis in VICs via activation of inflammatory pathways and augmentation of cellular glycolysis, resulting in the accumulation of lactate. Subsequent investigation has unveiled a novel LUM driving histone modification, lactylation, which plays a role in facilitating valve calcification. More importantly, this study has identified two specific sites of histone lactylation, namely, H3K14la and H3K9la, which have been found to facilitate the process of calcification. The confirmation of these modification sites\' association with the expression of calcific genes Runx2 and BMP2 has been achieved through ChIP-PCR analysis.
CONCLUSIONS: The study presents novel findings, being the first to establish the involvement of lumican in mediating H3 histone lactylation, thus facilitating the development of aortic valve calcification. Consequently, lumican would be a promising therapeutic target for intervention in the treatment of CAVD.
METHODS: Single-cell RNA-sequencing (scRNA-seq) from nine human aortic valves was used to characterize the pathological switch process and identify key regulatory factors. The in vitro, ex vivo, in vivo, and double knockout mice were constructed to further unravel the calcification-promoting effect of LUM. Moreover, the multi-omic approaches were employed to analyse the molecular mechanism of LUM in CAVD.
RESULTS: ScRNA-seq successfully delineated the process of VIC pathological transformation and highlighted the significance of LUM as a novel molecule in this process. The pro-calcification role of LUM is confirmed on the in vitro, ex vivo, in vivo level, and ApoE-/-//LUM-/- double knockout mice. The LUM induces osteogenesis in VICs via activation of inflammatory pathways and augmentation of cellular glycolysis, resulting in the accumulation of lactate. Subsequent investigation has unveiled a novel LUM driving histone modification, lactylation, which plays a role in facilitating valve calcification. More importantly, this study has identified two specific sites of histone lactylation, namely, H3K14la and H3K9la, which have been found to facilitate the process of calcification. The confirmation of these modification sites\' association with the expression of calcific genes Runx2 and BMP2 has been achieved through ChIP-PCR analysis.
CONCLUSIONS: The study presents novel findings, being the first to establish the involvement of lumican in mediating H3 histone lactylation, thus facilitating the development of aortic valve calcification. Consequently, lumican would be a promising therapeutic target for intervention in the treatment of CAVD.
摘要：
目的：瓣膜间质细胞（VIC）在最终转化为成骨细胞群体之前经历了向中间状态细胞的转变，这是钙化性主动脉瓣疾病（CAVD）的关键细胞过程。在这里，这项研究成功地描述了VIC成骨转化的阶段，并阐明了lumican（LUM）在此过程中的新的关键调节作用。
方法：使用来自九个人主动脉瓣的单细胞RNA测序（scRNA-seq）来表征病理转换过程并确定关键调节因子。在体外，离体,在体内，构建双基因敲除小鼠，进一步揭示LUM的钙化促进作用。此外，采用多组学方法分析LUM在CAVD中的分子机制。
结果：ScRNA-seq成功地描绘了VIC病理转化的过程，并强调了LUM作为该过程中的新型分子的重要性。LUM的促钙化作用在体外得到证实，离体,体内水平，和ApoE-/-//LUM-/-双敲除小鼠。LUM通过激活炎症途径和增强细胞糖酵解诱导VIC中的骨生成，导致乳酸的积累。随后的调查揭示了一种新颖的LUM驱动组蛋白修饰，乳酸化,在促进瓣膜钙化中起作用。更重要的是,这项研究确定了组蛋白乳酸化的两个特定位点，即,H3K14la和H3K9la,已发现促进钙化过程。通过ChIP-PCR分析已经实现了这些修饰位点与钙化基因Runx2和BMP2表达的相关性的确认。
结论：这项研究提出了新的发现，是第一个建立lumican参与介导H3组蛋白乳酸化的人，从而促进主动脉瓣钙化的发展。因此,lumican将是一个有希望的治疗目标，用于干预治疗CAVD。
方法：使用来自九个人主动脉瓣的单细胞RNA测序（scRNA-seq）来表征病理转换过程并确定关键调节因子。在体外，离体,在体内，构建双基因敲除小鼠，进一步揭示LUM的钙化促进作用。此外，采用多组学方法分析LUM在CAVD中的分子机制。
结果：ScRNA-seq成功地描绘了VIC病理转化的过程，并强调了LUM作为该过程中的新型分子的重要性。LUM的促钙化作用在体外得到证实，离体,体内水平，和ApoE-/-//LUM-/-双敲除小鼠。LUM通过激活炎症途径和增强细胞糖酵解诱导VIC中的骨生成，导致乳酸的积累。随后的调查揭示了一种新颖的LUM驱动组蛋白修饰，乳酸化,在促进瓣膜钙化中起作用。更重要的是,这项研究确定了组蛋白乳酸化的两个特定位点，即,H3K14la和H3K9la,已发现促进钙化过程。通过ChIP-PCR分析已经实现了这些修饰位点与钙化基因Runx2和BMP2表达的相关性的确认。
结论：这项研究提出了新的发现，是第一个建立lumican参与介导H3组蛋白乳酸化的人，从而促进主动脉瓣钙化的发展。因此,lumican将是一个有希望的治疗目标，用于干预治疗CAVD。
