Retinoic acid signaling in lymphangiogenesis

描述(申请人提供)：淋巴系统通过将间质液体排回循环，在组织液内稳中起主要作用。淋巴水肿是由淋巴畸形或阻塞引起的，通常与放射和手术有关；然而，迄今为止还没有针对潜在分子病理的有效治疗方法。我们最近报道，9-顺式维甲酸(RA)可以激活淋巴管内皮细胞(LECs)的增殖、迁移和管状形成，刺激体内淋巴管的生成，并通过促进淋巴管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功能是相当出人意料的，因为RAS已被认为对包括血管内皮细胞(BECs)在内的许多细胞类型具有抗增殖作用；其中RAS已被证明抑制BEC的增殖，而RA缺乏的小鼠胚胎表现出BECs的过度增殖。因此，在这个建议中，我们旨在解决两个主要问题(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可能调节Notch通路以促进淋巴管萌发，这表明这两个重要的形态发生信号之间存在新的串扰，而且淋巴管分化和发育的主要调节因子Prox1可以以RA控制的方式与RA结合的核受体RXR在物理和功能上相互作用。此外，LEC主要表达FABP4作为细胞质RA载体，PPARγ作为RXR的二聚化伙伴，已知促进RAS反应的细胞增殖，而BEC选择性表达CRABP-II和RARα，这两个分子配对诱导RAS反应细胞生长停滞。总之，我们提出了两个主要问题的工作假说：(1)RAS通过调节晶状体上皮细胞中Prox1和RxR的相互作用来调节Notch通路基因，从而刺激淋巴管萌发；(2)在LECs中，FABP4和PPARγ的优势表达将RA从抑制增殖的信号转变为促进生长的信号。在这里，我们旨在通过研究RAS通过RXRα和PPARγ(AIM1)促进淋巴管生成的作用，RAS对血管生成和淋巴管生成相反作用的机制(AIM 2)，以及RA控制的ProX1和RXRα之间的物理和功能相互作用(AIM 3)来验证这些工作假说。总之，我们的研究不仅将为Prox1作为核受体辅助调节因子作为淋巴管发育的主要调节因子提供重要信息，而且还将确定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将RAS作为潜在的淋巴循环不足的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.