Department of Molecular Pharmacology and Physiology, Morsani College of Medicine, University of South Florida, Tampa.
Arterioscler Thromb Vasc Biol. 2023 Nov;43(11):2197-2212. doi: 10.1161/ATVBAHA.123.319405. Epub 2023 Sep 28.
Lymphatic valves play a critical role in ensuring unidirectional lymph transport. Loss of lymphatic valves or dysfunctional valves are associated with several diseases including lymphedema, lymphatic malformations, obesity, and ileitis. Lymphatic valves first develop during embryogenesis in response to mechanotransduction signaling pathways triggered by oscillatory lymph flow. In blood vessels, eNOS (endothelial NO synthase; gene name: ) is a well-characterized shear stress signaling effector, but its role in lymphatic valve development remains unexplored.
We used global mice and cultured human dermal lymphatic endothelial cells to investigate the role of eNOS in lymphatic valve development, which requires oscillatory shear stress signaling.
Our data reveal a 45% reduction in lymphatic valve specification cell clusters and that loss of eNOS protein inhibited activation of β-catenin and its nuclear translocation. Genetic knockout or knockdown of eNOS led to downregulation of β-catenin target proteins in vivo and in vitro. However, pharmacological inhibition of NO production did not reproduce these effects. Co-immunoprecipitation and proximity ligation assays reveal that eNOS directly binds to β-catenin and their binding is enhanced by oscillatory shear stress. Finally, genetic ablation of the gene enhanced FOXC2 expression and partially rescued the loss of valve specification in the eNOS knockouts.
In conclusion, we demonstrate a novel, NO-independent role for eNOS in regulating lymphatic valve specification and propose a mechanism by which eNOS directly binds β-catenin to regulate its nuclear translocation and thereby transcriptional activity.
淋巴瓣膜在确保淋巴单向运输中起着至关重要的作用。淋巴瓣膜的缺失或功能障碍与多种疾病有关，包括淋巴水肿、淋巴管畸形、肥胖症和回肠炎。淋巴瓣膜在胚胎发育过程中首先形成，以响应由振荡性淋巴流触发的机械转导信号通路。在血管中，eNOS（内皮型一氧化氮合酶；基因名称：）是一种特征明确的切应力信号效应物，但它在淋巴瓣膜发育中的作用尚未被探索。
我们使用全局 敲除小鼠和培养的人真皮淋巴内皮细胞来研究 eNOS 在需要振荡切应力信号的淋巴瓣膜发育中的作用。
我们的数据显示，淋巴瓣膜特化细胞簇减少了 45%，并且 eNOS 蛋白的缺失抑制了 β-连环蛋白的激活及其核转位。eNOS 的基因敲除或敲低导致体内和体外 β-连环蛋白靶蛋白的下调。然而，NO 产生的药理学抑制并没有复制这些效应。共免疫沉淀和邻近连接分析显示，eNOS 直接与 β-连环蛋白结合，并且它们的结合在振荡切应力下增强。最后，FOXC2 基因的遗传消融增强了 FOXC2 的表达，并部分挽救了 eNOS 敲除体中瓣膜特化的缺失。
总之，我们证明了 eNOS 在调节淋巴瓣膜特化中的一种新型、NO 独立的作用，并提出了 eNOS 直接与 β-连环蛋白结合来调节其核转位从而调节其转录活性的机制。
