Retinoic acid signaling in lymphangiogenesis

描述（由申请人提供）：淋巴系统通过将组织液引流回循环系统，在组织液稳态中起主要作用。淋巴水肿，由淋巴畸形或阻塞引起，通常与放疗和手术有关；然而，有效的治疗方法，解决潜在的分子病理是不可用的。我们最近报道了9-顺式维甲酸（RA）可以激活淋巴内皮细胞（LECs）的细胞增殖、迁移和管状形成，刺激体内淋巴管生成，并通过促进小鼠淋巴再生来改善继发性淋巴水肿。然而，9-cisRA的这些促淋巴管生成的特征是相当出乎意料的，因为众所周知，RAs对许多细胞类型具有抗增殖作用，包括血管内皮细胞（BECs）；RAs抑制BEC增殖，而RAs缺陷小鼠胚胎显示BEC过度增殖。因此，在本提案中，我们的目标是解决两个主要问题(1)ra诱导淋巴管生成的分子机制是什么，以及(2)RAs如何选择性地诱导淋巴管生成，同时抑制血管生成。我们的初步研究表明，RAs可能通过调控Notch通路促进淋巴发芽，这表明这两个重要的形态发生信号之间存在新的串扰，并且淋巴分化和发育的主要调控因子Prox1可以通过ra控制的方式与ra结合的核受体RXR进行物理和功能上的相互作用。此外，LECs主要表达作为细胞质ra载体的FABP4和作为RXR二聚化伙伴的PPARγ，已知其在RAs响应下促进细胞增殖，而BECs选择性表达CRABP-II和RARα，这是一种分子对，在RAs响应下诱导细胞生长停滞。总之，我们提出了解决我们的两个主要问题的工作假设：(1)RAs通过调节LECs中Prox1和RXR的相互作用来调节Notch通路基因，从而刺激淋巴发芽；(2)LECs中FABP4和PPARγ的主要表达将RA从LECs中的抗增殖信号转化为促生长信号。本研究旨在通过研究RAs通过RXRα和PPARγ促进淋巴管生成的作用（ai1）， RAs对血管生成和淋巴管生成的相反作用机制（aim 2），以及RAs控制的Prox1和RXRα之间的物理和功能相互作用（aim 3）来验证这些工作假设。总之，我们的研究不仅将提供Prox1如何作为核受体共调节剂作为淋巴发育的主要调节剂的重要信息，而且还将定义ra介导的选择性促进淋巴管生成的分子机制。从长远来看，我们的研究将有助于为RAs作为淋巴循环功能不全的潜在治疗剂奠定必要的实验基础。

DESCRIPTION (provided by applicant): The lymphatic system plays the major role in tissue fluid homeostasis by draining the interstitial fluid back to the circulation. Lymphedema, caused by lymphatic malformation or obstruction, is often associated with radiation and surgery; however effective treatments that address the underlying molecular pathology are not available to date. We have recently reported that 9-cis retinoic acid (RA) can activate cell proliferation, migration and tube formation of lymphatic endothelial cells (LECs), stimulate lymphangiogenesis in vivo, and ameliorate secondary lymphedema by promoting lymphatic regeneration in a mouse model. These pro-lymphangiogenic features of 9-cisRA, however, are quite unexpected, because RAs have been known for their anti-proliferative effects on many cell types, including blood vascular endothelial cells (BECs); where RAs have been shown to suppress BEC proliferation, and RA-deficient mouse embryos display hyper-proliferation of BECs. In this proposal, therefore, we aim to address two main questions (1) what is the molecular mechanism underlying RA-induced lymphangiogenesis, and (2) how can RAs selectively induce lymphangiogenesis, while concurrently suppressing angiogenesis. Our preliminary studies revealed that RAs may regulate Notch pathway to promote lymphatic sprouting, suggesting novel crosstalk between the two important morphogenic signals, and also that Prox1, the master regulator of lymphatic differentiation and development, can physically and functionally interact with a RA-binding nuclear receptor RXR in a RA-controlled manner. Furthermore, LECs predominantly express FABP4 as a cytoplasmic RA-carrier, and PPARγ as a dimerization partner of RXR, which is known to promote cell proliferation in response to RAs, whereas BECs selectively express CRABP-II and RARα, a molecular pairing that induces cell growth arrest in response to RAs. Together, we propose working hypotheses addressing our two main questions that (1) RAs stimulate lymphatic sprouting by modulating Notch pathway genes through regulation of the interactions of Prox1 and RXR in LECs and (2) the predominant expression of FABP4 and PPARγ in LECs converts RA from an anti-proliferative signal to a pro-growth cue in LECs. Here, we aim to validate these working hypotheses by studying the role of RAs in promoting lymphangiogenesis through RXRα and PPARγ (Aim1), mechanism underlying the opposing effects of RAs on angiogenesis vs. lymphangiogenesis (Aim 2), and RA-controlled physical and functional interactions between Prox1 and RXRα (Aim 3). Together, our studies will not only provide important information on how Prox1 functions as the master regulator of lymphatic development by functioning as a nuclear receptor coregulator, but also define the molecular mechanism underlying RA-mediated selective promotion of lymphangiogenesis. In the long run, our study will help lay an essential experimental foundation to repurpose RAs as potential therapeutic agents for lymphatic circulation insufficiency.