BACKGROUND EPHB4 (ephrin receptor B4) and the RASA1 (p120 Ras GTPase-activating protein) are necessary for the development of lymphatic vessel (LV) valves. However, precisely how EPHB4 and RASA1 regulate LV valve development is unknown. In this study, we examine the mechanisms by which EPHB4 and RASA1 regulate the development of LV valves. METHODS We used LV-specific inducible EPHB4-deficient mice and EPHB4 knockin mice that express a form of EPHB4 that is unable to bind RASA1 yet retains protein tyrosine kinase activity (EPHB4 2YP) to study the role of EPHB4 and RASA1 in LV valve development in the embryo and LV valve maintenance in adults. We also used human dermal lymphatic endothelial cells in vitro to study the role of EPHB4 and RASA1 as regulators of LV valve specification induced by oscillatory shear stress, considered the trigger for LV valve specification in vivo. RESULTS LV valve specification, continued valve development postspecification, and LV valve maintenance were blocked upon induced loss of EPHB4 in LV. LV valve specification and maintenance were also impaired in EPHB4 2YP mice. Defects in LV valve development were reversed by inhibition of the Ras-MAPK (mitogen-activated protein kinase) signaling pathway. In human dermal lymphatic endothelial cells, loss of expression of EPHB4 or its ephrin b2 ligand, loss of expression of RASA1, and inhibition of physical interaction between EPHB4 and RASA1 resulted in dysregulated oscillatory shear stress-induced Ras-MAPK activation and impaired expression of LV specification markers that could be rescued by Ras-MAPK pathway inhibition. The same results were observed when human dermal lymphatic endothelial cells were stimulated with the Yoda1 agonist of the PIEZO1 oscillatory shear stress sensor. Although Yoda1 increased the number of LV valves when administered to wild-type embryos, it did not increase LV valve number when administered to EPHB4 2YP embryos. CONCLUSIONS EPHB4 is necessary for LV valve specification, continued valve development postspecification, and valve maintenance. LV valve specification requires physical interaction between EPHB4 and RASA1 to limit activation of the Ras-MAPK pathway in lymphatic endothelial cells. Specifically, EPHB4-RASA1 physical interaction is necessary to dampen Ras-MAPK activation induced through the PIEZO1 oscillatory shear stress sensor. These findings reveal the mechanism by which EPHB4 and RASA1 regulate the development of LV valves.

中文翻译：

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EPHB4-RASA1 抑制 PIEZO1 Ras 激活驱动淋巴瓣生成。


背景 EPHB4 （肝配蛋白受体 B4） 和 RASA1 （p120 Ras GTP 酶激活蛋白） 是淋巴管 （LV） 瓣膜发育所必需的。然而，EPHB4 和 RASA1 究竟如何调节 LV 瓣膜的发育尚不清楚。在这项研究中，我们研究了 EPHB4 和 RASA1 调节 LV 瓣膜发育的机制。方法 我们使用 LV 特异性诱导型 EPHB4 缺陷小鼠和表达一种无法结合 RASA1 但保留蛋白酪氨酸激酶活性的 EPHB4 形式的 EPHB4 敲入小鼠 （EPHB4 2YP） 来研究 EPHB4 和 RASA1 在胚胎 LV 瓣膜发育和成人 LV 瓣膜维持中的作用。我们还在体外使用人真皮淋巴内皮细胞研究 EPHB4 和 RASA1 作为振荡剪切应力诱导的 LV 瓣膜规格调节剂的作用，被认为是体内 LV 瓣膜规格的触发因素。结果 LV 瓣膜规格、规格后持续瓣膜发育和 LV 瓣膜维护在 LV 中诱导 EPHB4 丢失后被阻断。EPHB4 2YP 小鼠的 LV 瓣膜规格和维护也受损。通过抑制 Ras-MAPK （丝裂原活化蛋白激酶） 信号通路，逆转了 LV 瓣膜发育的缺陷。在人真皮淋巴内皮细胞中，EPHB4 或其肝配蛋白 b2 配体表达缺失、RASA1 表达缺失以及 EPHB4 和 RASA1 之间物理相互作用的抑制导致振荡剪切应力诱导的 Ras-MAPK 激活失调和 LV 规格标志物表达受损，这些标志物可以通过 Ras-MAPK 通路抑制来挽救。当用 PIEZO1 振荡剪切应力传感器的 Yoda1 激动剂刺激人真皮淋巴内皮细胞时，观察到相同的结果。 尽管 Yoda1 在施用于野生型胚胎时增加了 LV 瓣膜的数量，但在施用于 EPHB4 2YP 胚胎时并没有增加 LV 瓣膜的数量。结论 EPHB4 对于 LV 瓣膜规范、规范后持续瓣膜开发和瓣膜维护是必需的。LV 瓣膜规格要求 EPHB4 和 RASA1 之间的物理相互作用，以限制淋巴内皮细胞中 Ras-MAPK 通路的激活。具体来说，EPHB4-RASA1 物理相互作用对于抑制通过 PIEZO1 振荡剪切应力传感器诱导的 Ras-MAPK 激活是必要的。这些发现揭示了 EPHB4 和 RASA1 调节 LV 瓣膜发育的机制。