Goto Shinji, Rogers Maximillian A, Blaser Mark C, Higashi Hideyuki, Lee Lang H, Schlotter Florian, Body Simon C, Aikawa Masanori, Singh Sasha A, Aikawa Elena
Center for Interdisciplinary Cardiovascular Sciences, Cardiovascular Medicine, Brigham and Women's Hospital, Harvard Medical School, Boston, MA, United States.
Department of Anesthesia, Critical Care and Pain Medicine, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, MA, United States.
Front Cardiovasc Med. 2019 Apr 16;6:49. doi: 10.3389/fcvm.2019.00049. eCollection 2019.
Aortic valvular interstitial cells (VICs) isolated from patients undergoing valve replacement are commonly used as models of calcific aortic valve disease (CAVD). Standardization of VIC calcification, however, has not been implemented, which impairs comparison of results from different studies. We hypothesized that different culture methods impact the calcification phenotype of human VICs. We sought to identify the key parameters impacting calcification in primary human VICs to standardize CAVD research. Here we report that in calcification media containing organic phosphate, termed osteogenic media (OM), primary human VICs exhibited a passage-dependent decrease in calcification potential, which was not observed in calcification media containing inorganic phosphate, termed pro-calcifying media (PM). We used Alizarin red staining to compare the calcification potential of VICs cultured in OM and PM between the first and fourth passages after cell isolation from human CAVD tissues. Human VICs showed consistent Alizarin red stain when cultured with PM in a passage-independent manner. VICs cultured in OM did not exhibit consistent calcification potential between donors in early passages and consistently lacked positive Alizarin red stain in late passages. We performed whole cell, cytoplasmic and nuclear fractionation proteomics to identify factors regulating VIC passage-dependent calcification in OM. Proteomics cluster analysis identified tissue non-specific alkaline phosphatase (TNAP) as a regulator of passage-dependent calcification in OM. We verified an association of TNAP activity with calcification potential in VICs cultured in OM, but not in PM in which VICs calcified independent of TNAP activity. This study demonstrates that media culture conditions and cell passage impact the calcification potential of primary human VICs and should be taken into consideration in cell culture models of CAVD. Our results help standardize CAVD modeling as part of a greater effort to identify disease driving mechanisms and therapeutics for this unmet medical need.
从接受瓣膜置换手术的患者中分离出的主动脉瓣膜间质细胞（VICs）通常被用作钙化性主动脉瓣疾病（CAVD）的模型。然而，VIC钙化的标准化尚未实现，这影响了不同研究结果的比较。我们假设不同的培养方法会影响人VICs的钙化表型。我们试图确定影响原代人VICs钙化的关键参数，以规范CAVD研究。在此我们报告，在含有有机磷酸盐的钙化培养基（称为成骨培养基，OM）中，原代人VICs的钙化潜能呈现传代依赖性下降，而在含有无机磷酸盐的钙化培养基（称为促钙化培养基，PM）中未观察到这种情况。我们使用茜素红染色来比较从人CAVD组织分离细胞后，第一代和第四代在OM和PM中培养的VICs的钙化潜能。人VICs在与PM一起培养时，茜素红染色呈现传代无关的一致性。在OM中培养的VICs在早期传代时供体之间的钙化潜能不一致，在后期传代时始终缺乏阳性茜素红染色。我们进行了全细胞、细胞质和细胞核分级蛋白质组学分析，以确定调节OM中VIC传代依赖性钙化的因素。蛋白质组学聚类分析确定组织非特异性碱性磷酸酶（TNAP）是OM中传代依赖性钙化的调节因子。我们验证了TNAP活性与在OM中培养的VICs的钙化潜能相关，但在VICs钙化与TNAP活性无关的PM中则不然。本研究表明，培养基培养条件和细胞传代会影响原代人VICs的钙化潜能，在CAVD的细胞培养模型中应予以考虑。我们的结果有助于规范CAVD建模，作为识别这种未满足医疗需求的疾病驱动机制和治疗方法的更大努力的一部分。
Front Cardiovasc Med. 2019-4-16
Front Cardiovasc Med. 2021-8-30
Front Cardiovasc Med. 2023-6-20
Am J Physiol Heart Circ Physiol. 2020-9-28
Eur Heart J. 2025-2-14
Bioengineering (Basel). 2024-9-24
Biophys Rev (Melville). 2021-12-10
Cell Regen. 2024-3-1
Circ Genom Precis Med. 2019-10-15
J Am Coll Cardiol. 2019-1-29
J Proteome Res. 2018-10-29
Atherosclerosis. 2018-8-25
Nanomaterials (Basel). 2018-5-3
Proc Natl Acad Sci U S A. 2017-12-27
Zotero 插件
