Denis Jean-François, Diagbouga Mannekomba R, Molica Filippo, Hautefort Aurélie, Linnerz Tanja, Watanabe Masakatsu, Lemeille Sylvain, Bertrand Julien Y, Kwak Brenda R
Department of Pathology and Immunology, University of Geneva, Geneva, Switzerland.
Graduate School of Frontier Biosciences, Osaka University, Osaka, Japan.
Front Physiol. 2019 Feb 12;10:80. doi: 10.3389/fphys.2019.00080. eCollection 2019.
Shear stress, a blood flow-induced frictional force, is essential in the control of endothelial cell (EC) homeostasis. High laminar shear stress (HLSS), as observed in straight parts of arteries, assures a quiescent non-activated endothelium through the induction of Krüppel-like transcription factors (KLFs). Connexin40 (Cx40)-mediated gap junctional communication is known to contribute to a healthy endothelium by propagating anti-inflammatory signals between ECs, however, the molecular basis of the transcriptional regulation of Cx40 as well as its downstream effectors remain poorly understood. Here, we show that flow-induced KLF4 regulated Cx40 expression in a mouse EC line. Chromatin immunoprecipitation in ECs revealed that KLF4 bound to three predicted KLF consensus binding sites in the Cx40 promoter. HLSS-dependent induction of Cx40 expression was confirmed in primary human ECs. The downstream effects of Cx40 modulation in ECs exposed to HLSS were elucidated by an unbiased transcriptomics approach. Cell cycle progression was identified as an important downstream target of Cx40 under HLSS. In agreement, an increase in the proportion of proliferating cell nuclear antigen (PCNA)-positive ECs and a decrease in the proportion of ECs in the G0/G1 phase were observed under HLSS after Cx40 silencing. Transfection of communication-incompetent HeLa cells with Cx40 demonstrated that the regulation of proliferation by Cx40 was not limited to ECs. Using a zebrafish model, we finally showed faster intersegmental vessel growth and branching into the dorsal longitudinal anastomotic vessel in embryos knock-out for the Cx40 orthologs and . Most significant effects were observed in embryos with a mutant encoding for a channel with reduced gap junctional function. Faster intersegmental vessel growth in mutant embryos was associated with increased EC proliferation as assessed by PH3 immunostaining. Our data shows a novel evolutionary-conserved role of flow-driven KLF4-dependent Cx40 expression in endothelial quiescence that may be relevant for the control of atherosclerosis and diseases involving sprouting angiogenesis.
剪切应力是一种血流诱导的摩擦力，对维持内皮细胞（EC）的内环境稳定至关重要。在动脉的直管部分观察到的高切应力（HLSS），通过诱导Krüppel样转录因子（KLFs）确保内皮细胞处于静止、未激活状态。已知连接蛋白40（Cx40）介导的缝隙连接通讯通过在EC之间传递抗炎信号，有助于维持健康的内皮细胞，但Cx40转录调控的分子基础及其下游效应器仍知之甚少。在此，我们表明血流诱导的KLF4调节小鼠EC系中的Cx40表达。EC中的染色质免疫沉淀显示，KLF4与Cx40启动子中的三个预测的KLF共有结合位点结合。在原代人EC中证实了HLSS依赖的Cx40表达诱导。通过无偏转录组学方法阐明了暴露于HLSS的EC中Cx40调节的下游效应。细胞周期进程被确定为HLSS下Cx40的一个重要下游靶点。同样，在Cx40沉默后的HLSS条件下，观察到增殖细胞核抗原（PCNA）阳性EC的比例增加，而处于G0/G1期的EC比例减少。用Cx40转染无通讯能力的HeLa细胞表明，Cx40对增殖的调节不限于EC。使用斑马鱼模型，我们最终表明，在敲除Cx40直系同源基因的胚胎中，节间血管生长更快，并分支进入背侧纵向吻合血管。在编码缝隙连接功能降低的通道的突变体胚胎中观察到最显著的影响。通过PH3免疫染色评估，突变体胚胎中更快的节间血管生长与EC增殖增加有关。我们的数据显示了血流驱动的KLF4依赖的Cx40表达在内皮细胞静止中的一种新的进化保守作用，这可能与动脉粥样硬化和涉及芽生血管生成的疾病的控制有关。
