Evaluation of canine 2D cell cultures as models of myxomatous mitral valve degeneration

The utility of cells cultured from the mitral valve as models of myxomatous diseases needs to be properly validated. In this study valve interstitial cells (VICs) and valve endothelial cells (VECs) were cultured from normal and diseased canine mitral valves in 2% (v/v) or 10% FBS media, in the presence of TGFβ1, 2 and 3, the TGFβ RI kinase inhibitor SB431542 and TGFβ neutralising antibodies, 5HT and the 5HT2RB antagonist LY272015. Cultures were examined by morphology, transcriptomic profiling, protein expression of the cell specific markers αSMA and SM22α (VICs), and CD31 (VECs), deposition of proteoglycans (PG), the PG versican, and the TGFβs themselves. VECs derived from normal valves were CD31+/αSMA-, but those from diseased valves were αSMA+, indicating endothelial-to-mesenchymal (EndoMT) transition had occurred. The TGFβs induced EndoMT in normal VECs, and this was abolished by SB431542, with significant changes in αSMA, CD31 and HAS2 expression (PACTA2 (αSMA), 5HTR2B, TAGLN (SM22α) and MYH10 (SMemb), gene ontology terms and canonical signalling pathways. Normal and diseased VICs and normal VECs from canine mitral valves can be successfully grown in culture with retention of phenotype, which can be manipulated using TGFβ1 and the TGFβ RI kinase inhibitor SB431542. This optimized cell system can now be used to model MMVD to elucidate disease mechanisms and identify key regulators of disease progression.

以二尖瓣来源的细胞作为黏液瘤样疾病模型的实用性仍需得到充分验证。本研究中，我们在体积分数2%或10%胎牛血清（Fetal Bovine Serum, FBS）培养基中，添加转化生长因子β1、2、3（Transforming Growth Factor β, TGFβ）、TGFβ I型受体激酶抑制剂SB431542、TGFβ中和抗体、5-羟色胺（5-Hydroxytryptamine, 5HT）以及5HT2B受体拮抗剂LY272015，对来自正常和病变犬二尖瓣的瓣膜间质细胞（Valve Interstitial Cells, VICs）与瓣膜内皮细胞（Valve Endothelial Cells, VECs）进行体外培养。通过形态学观察、转录组分析、细胞特异性标志物的蛋白表达检测（瓣膜间质细胞标志物α平滑肌肌动蛋白（αSMA）与SM22α，瓣膜内皮细胞标志物CD31）、蛋白聚糖（Proteoglycans, PG）及其核心蛋白versican的沉积情况，以及TGFβ自身的表达水平对培养细胞进行全面鉴定。结果显示，源自正常二尖瓣的VECs呈CD31+/αSMA-表型，而病变二尖瓣来源的VECs则为αSMA+表型，表明已发生内皮细胞向间充质细胞转化（Endothelial-to-Mesenchymal Transition, EndoMT）。TGFβ可诱导正常VECs发生EndoMT，该效应可被SB431542完全阻断，且伴随αSMA、CD31及透明质酸合成酶2（HAS2）、PACTA2（即αSMA）、5HTR2B、TAGLN（SM22α）、MYH10（SMemb）的表达出现显著变化，同时涉及特定基因本体（Gene Ontology, GO）术语及经典信号通路的异常调控。本研究成功实现了犬二尖瓣来源的正常与病变VICs、正常VECs的体外培养，且可保留其固有细胞表型；通过TGFβ1及TGFβ I型受体激酶抑制剂SB431542可对该表型进行精准操控。该优化后的细胞培养体系可用于模拟犬二尖瓣黏液瘤样疾病（Myxomatous Mitral Valve Disease, MMVD），以阐明疾病发病机制并筛选疾病进展的关键调控因子。