Department of Chemical and Biochemical Engineering, University of Western Ontario, London, ON, N6A 5C1, Canada.
Division of Cardiology, Keenan Research Centre for Biomedical Science and Li Ka Shing Knowledge Institute of St. Michael's Hospital, Toronto, ON, M5B 1W8, Canada.
Sci Rep. 2020 Mar 11;10(1):4466. doi: 10.1038/s41598-020-61292-9.
Intraflagellar transport protein 88 (Ift88) is required for ciliogenesis and shear stress-induced dissolution of cilia in embryonic endothelial cells coincides with endothelial-to-mesenchymal transition (EndMT) in the developing heart. EndMT is also suggested to underlie heart and lung fibrosis, however, the mechanism linking endothelial Ift88, its effect on EndMT and organ fibrosis remains mainly unexplored. We silenced Ift88 in endothelial cells (ECs) in vitro and generated endothelial cell-specific Ift88-knockout mice (Ift88) in vivo to evaluate EndMT and its contribution towards organ fibrosis, respectively. Ift88-silencing in ECs led to mesenchymal cells-like changes in endothelial cells. The expression level of the endothelial markers (CD31, Tie-2 and VE-cadherin) were significantly reduced with a concomitant increase in the expression level of mesenchymal markers (αSMA, N-Cadherin and FSP-1) in Ift88-silenced ECs. Increased EndMT was associated with increased expression of profibrotic Collagen I expression and increased proliferation in Ift88-silenced ECs. Loss of Ift88 in ECs was further associated with increased expression of Sonic Hedgehog signaling effectors. In vivo, endothelial cells isolated from the heart and lung of Ift88 mice demonstrated loss of Ift88 expression in the endothelium. The Ift88 mice were born in expected Mendelian ratios without any adverse cardiac phenotypes at baseline. Cardiac and pulmonary endothelial cells isolated from the Ift88 mice demonstrated signs of EndMT and bleomycin treatment exacerbated pulmonary fibrosis in Ift88 mice. Pressure overload stress in the form of aortic banding did not reveal a significant difference in cardiac fibrosis between Ift88 mice and control mice. Our findings demonstrate a novel association between endothelial cilia with EndMT and cell proliferation and also show that loss of endothelial cilia-associated increase in EndMT contributes specifically towards pulmonary fibrosis.
内纤毛运输蛋白 88（Ift88）是纤毛发生所必需的，并且胚胎内皮细胞中剪切力诱导的纤毛溶解与心脏发育中的内皮细胞向间充质转化（EndMT）同时发生。EndMT 也被认为是心脏和肺纤维化的基础，但内皮细胞 Ift88 与之相关的机制、它对 EndMT 和器官纤维化的影响在很大程度上仍未得到探索。我们在体外沉默内皮细胞（ECs）中的 Ift88，并在体内生成内皮细胞特异性 Ift88 敲除小鼠（Ift88），分别评估 EndMT 及其对器官纤维化的贡献。ECs 中的 Ift88 沉默导致内皮细胞发生间充质样变化。内皮标志物（CD31、Tie-2 和 VE-cadherin）的表达水平显著降低，而间充质标志物（αSMA、N-Cadherin 和 FSP-1）的表达水平则显著升高。在 Ift88 沉默的 ECs 中，EndMT 的增加与致纤维化 Collagen I 表达的增加和增殖的增加相关。ECs 中 Ift88 的缺失与 Sonic Hedgehog 信号效应物的表达增加进一步相关。在体内，从 Ift88 小鼠心脏和肺中分离的内皮细胞在内皮细胞中表现出 Ift88 表达的缺失。Ift88 小鼠按预期孟德尔比例出生，在基线时没有任何心脏表型异常。从 Ift88 小鼠中分离的心脏和肺内皮细胞表现出 EndMT 的迹象，博来霉素处理加剧了 Ift88 小鼠的肺纤维化。主动脉缩窄形式的压力超负荷应激并未显示 Ift88 小鼠与对照小鼠之间在心脏纤维化方面有显著差异。我们的研究结果表明内皮纤毛与 EndMT 和细胞增殖之间存在新的关联，并表明内皮纤毛相关的 EndMT 增加特异性地导致肺纤维化。
