Aims During embryonic development, arteriovenous (AV) differentiation ensures proper blood vessel formation and maturation. Defects in arterial or venous identity cause inappropriate fusion of vessels, resulting in atypical shunts, so-called arteriovenous malformations (AVM). Currently, the mechanism behind AVM formation remains unclear and treatment options are fairly limited. Mammalian AV differentiation is initiated before the onset of blood flow in the embryo; however, this pre-flow mechanism is poorly understood. Here, we aimed to unravel the role of Smad1/5 signalling in pre-flow arterial identity, and in the process uncovered an unexpected control mechanism of Smad1/5 signalling. Methods and Results We establish that despite Notch1 being expressed in the pre-flow mouse embryo, it is not activated, nor is it necessary for the expression of the earliest arterial genes in the dorsal aortae (i.e., Hey1 and Gja4). Furthermore, interrupting blood flow by using the Ncx1 KO model completely prevents the activation of Notch1 signalling, suggesting a strong role of shear stress in maintaining arterial identity. We demonstrate that early expression of Hey1 and Gja4 requires SMAD1/5 signalling. Using embryo cultures, we show that Smad1/5 signalling is activated through the Alk1/Alk5/TGFβR2 receptor complex, with TGFβ1 as a necessary ligand. Furthermore, our findings demonstrate that early arterial gene expression requires the acetylation of Smad1/5 proteins, rendering them more sensitive to TGFβ1 stimulation. Blocking acetyl-CoA production prevents pre-flow arterial expression of Hey1 and Gja4, while stabilizing acetylation rescues their expression. Conclusions Our findings highlight the importance of the acetyl-CoA production in the cell and provide a novel control mechanism of Smad1/5 signalling involving protein acetylation. As disturbed canonical Smad1/5 signalling is involved in several vascular conditions, our results offer new insights in treatment options for circumventing canonical Smad1/5 signalling.

中文翻译：

---

Smad1/5 在血流开始前在小鼠胚胎的背主动脉中被乙酰化，驱动早期动脉基因表达


目的 在胚胎发育过程中，动静脉 (AV) 分化可确保适当的血管形成和成熟。动脉或静脉特性缺陷会导致血管不适当融合，从而导致非典型分流，即所谓的动静脉畸形 (AVM)。目前，AVM 形成的机制仍不清楚，治疗选择也相当有限。哺乳动物 AV 分化在胚胎血流开始之前就开始了；然而，人们对这种预流机制知之甚少。在这里，我们的目的是阐明 Smad1/5 信号在流前动脉身份中的作用，并在此过程中发现 Smad1/5 信号的意想不到的控制机制。方法和结果我们确定，尽管Notch1在流前小鼠胚胎中表达，但它没有被激活，对于背主动脉中最早的动脉基因（即Hey1和Gja4）的表达也不是必需的。此外，使用 Ncx1 KO 模型中断血流完全阻止了 Notch1 信号的激活，这表明剪切应力在维持动脉特性方面发挥着重要作用。我们证明 Hey1 和 Gja4 的早期表达需要 SMAD1/5 信号传导。通过胚胎培养，我们发现 Smad1/5 信号通过 Alk1/Alk5/TGFβR2 受体复合物激活，其中 TGFβ1 作为必要的配体。此外，我们的研究结果表明，早期动脉基因表达需要 Smad1/5 蛋白的乙酰化，使它们对 TGFβ1 刺激更加敏感。阻断乙酰辅酶A的产生可以防止Hey1和Gja4在流动前动脉表达，而稳定乙酰化可以挽救它们的表达。 结论我们的研究结果强调了细胞中乙酰辅酶A产生的重要性，并提供了涉及蛋白质乙酰化的Smad1/5信号传导的新控制机制。由于受干扰的规范 Smad1/5 信号传导涉及多种血管疾病，因此我们的研究结果为规避规范 Smad1/5 信号传导的治疗方案提供了新的见解。