Department of Microbiology and Immunology, University of Michigan Medical School, Arbor (D.C., Y.T., P.E.L., P.D.K.).
Center for Molecular Imaging, The Brown Foundation Institute of Molecular Medicine, The University of Texas Health Science Center at Houston, Houston (D.W., E.M.S.).
Circ Res. 2024 Nov 8;135(11):1048-1066. doi: 10.1161/CIRCRESAHA.124.325383. Epub 2024 Oct 18.
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.
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.
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.
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.
EPHB4（ephrin 受体 B4）和 RASA1（p120 Ras GTP 酶激活蛋白）对于淋巴管（LV）瓣膜的发育是必需的。然而，EPHB4 和 RASA1 调节 LV 瓣膜发育的确切机制尚不清楚。在这项研究中，我们研究了 EPHB4 和 RASA1 调节 LV 瓣膜发育的机制。
我们使用 LV 特异性诱导的 EphB4 缺陷型小鼠和表达不能与 RASA1 结合但保留蛋白酪氨酸激酶活性的 EphB4 形式的 EphB4 敲入小鼠（EPHB4 2YP），研究 EphB4 和 RASA1 在胚胎中 LV 瓣膜发育和成人中 LV 瓣膜维持中的作用。我们还使用体外培养的人真皮淋巴管内皮细胞，研究 EphB4 和 RASA1 作为振荡剪切力诱导的 LV 瓣膜特异性的调节剂的作用，这被认为是体内 LV 瓣膜特异性的触发因素。
LV 瓣膜特异性、持续的瓣膜发育和 LV 瓣膜维持在 LV 中诱导的 EphB4 缺失时被阻断。EPHB4 2YP 小鼠的 LV 瓣膜特异性和维持也受损。LV 瓣膜发育缺陷可通过抑制 Ras-MAPK（丝裂原激活蛋白激酶）信号通路逆转。在人真皮淋巴管内皮细胞中，EPHB4 或其 Ephrin b2 配体的表达缺失、RASA1 的表达缺失以及 EphB4 和 RASA1 之间物理相互作用的抑制导致失调的振荡剪切力诱导的 Ras-MAPK 激活和受损的 LV 特异性标志物的表达，这些可以通过 Ras-MAPK 途径抑制来挽救。当用 PIEZO1 振荡剪切力传感器的 Yoda1 激动剂刺激人真皮淋巴管内皮细胞时，也观察到相同的结果。虽然 Yoda1 在给予野生型胚胎时增加 LV 瓣膜的数量，但在给予 EphB4 2YP 胚胎时并未增加 LV 瓣膜的数量。
EPHB4 对于 LV 瓣膜的特异性、持续的瓣膜发育和瓣膜维持是必需的。LV 瓣膜的特异性需要 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。具体而言，EPHB4-RASA1 物理相互作用对于通过 PIEZO1 振荡剪切力传感器诱导的 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。
