Gee Terence W, Richards Jennifer M, Mahmut Ablajan, Butcher Jonathan T
Meinig School of Biomedical Engineering, Cornell University, Ithaca, NY, USA.
Biomaterials. 2021 Feb;269:120669. doi: 10.1016/j.biomaterials.2021.120669. Epub 2021 Jan 8.
Calcific aortic valve disease (CAVD) is an actively regulated degenerative disease process. Clinical lesions exhibit marked 3D complexity not represented in current in vitro systems. We here present a unique mechanically stressed 3D culture system that recapitulates valve interstitial cell (VIC) induced matrix calcification through myofibroblastic activation and osteoblastic differentiation. We test the hypothesis that valve endothelial (VEC) - interstitial collaborative interactions modulate the risk and complexity of calcific pathogenesis within mechanically stressed and pro-inflammatory environments.
Porcine aortic valve endothelial and interstitial cells (VEC and VIC) were seeded in a mechanically constrained collagen hydrogels alone or in co-culture configurations. Raised 3D VIC-filled lesions formed within 7 days when cultured in osteogenic media (OGM), and surprisingly exacerbated by endothelial coculture. We identified a spatially coordinated pro-endochondral vs. pro-osteogenic signaling program within the lesion. VEC underwent Endothelial-to-Mesenchymal Transformation (EndMT) and populated the lesion center. The spatial complexity of molecular and cellular signatures of this 3D in vitro CAVD system were consistent with human diseased aortic valve histology. SNAI1 was highly expressed in the VEC and subendothelial direct VIC corroborates with human CAVD lesions. Spatial distribution of Sox9 vs. Runx2 expression within the developed lesions (Sox9 peri-lesion vs. Runx2 predominantly within lesions) mirrored their expression in heavily calcified human aortic valves. Finally, we demonstrate the applicability of this platform for screening potential pharmacologic therapies through blocking the canonical NFκB pathway via BAY 11-7082.
Our results establish that VEC actively induce VIC pathological remodeling and calcification via EndMT and paracrine signaling. This mechanically constrained culture platform enables the interrogation of accelerated cell-mediated matrix remodeling behavior underpinned by this cellular feedback circuit. The high fidelity of this complex 3D model system to human CAVD mechanisms supports its use to test mechanisms of intercellular communication in valves and their pharmacological control.
钙化性主动脉瓣疾病（CAVD）是一个受主动调控的退行性疾病过程。临床病变呈现出显著的三维复杂性，这在当前的体外系统中并未得到体现。我们在此展示一种独特的机械应力三维培养系统，该系统通过肌成纤维细胞活化和成骨细胞分化来模拟瓣膜间质细胞（VIC）诱导的基质钙化。我们检验了这样一个假设，即在机械应力和促炎环境中，瓣膜内皮细胞（VEC）与间质细胞的协同相互作用会调节钙化发病机制的风险和复杂性。
将猪主动脉瓣内皮细胞和间质细胞（VEC和VIC）单独或以共培养形式接种到机械约束的胶原蛋白水凝胶中。在成骨培养基（OGM）中培养时，7天内形成了充满VIC的三维病变，令人惊讶的是，内皮细胞共培养使其加剧。我们在病变中确定了一个空间协调的软骨内成骨与成骨信号程序。VEC经历了内皮向间充质转化（EndMT）并聚集在病变中心。这个三维体外CAVD系统的分子和细胞特征的空间复杂性与人类病变主动脉瓣组织学一致。SNAI1在VEC和内皮下直接的VIC中高表达，并与人类CAVD病变相符。在发育中的病变内Sox9与Runx2表达的空间分布（Sox9在病变周围，Runx2主要在病变内）反映了它们在严重钙化的人类主动脉瓣中的表达。最后，我们通过BAY 11 - 7082阻断经典NFκB途径，证明了该平台在筛选潜在药物治疗方面的适用性。
我们的结果表明，VEC通过EndMT和旁分泌信号通路积极诱导VIC的病理重塑和钙化。这个机械约束的培养平台能够探究由这种细胞反馈回路支撑的加速细胞介导的基质重塑行为。这个复杂的三维模型系统对人类CAVD机制的高保真度支持其用于测试瓣膜中细胞间通讯的机制及其药理学控制。
