Flow Dependent Development and Maintenance of Schlemm's Canal

项目总结 目的：探讨房水流出(Aho)的分子机制。 通过KLF4形成Schlemm管(SC)。SC是一种特殊的血管结构，它排出 房水从前房进入循环，对眼内的调节起着关键作用 压力(眼压)。由于衰老或疾病导致的SC功能障碍会严重升高眼压，通常会导致 眼神经受损，可能导致青光眼。更好地理解人与人之间的相互作用 SC的发展和AHO因此将对预防引起的青光眼产生变革性的影响 由高眼压引起。 原理：在结构上，SC直接连接到房水静脉以排出房水。因为 在这种直接的血管连接中，SC一直被认为是一种特殊的静脉延伸，其内部 壁上排列着血管内皮细胞。然而，有趣的是，几项研究表明 区分SC与典型血管的证据，并将SC重新归类为一种新的淋巴样 血管结构。这些研究促使我们仔细地重新检查分子和细胞特征 来自SC.作为结果，我们和其他人最近发现SC是出生后从角膜缘衍生而来的 上调淋巴管发育的主要调节因子Prox1，从而形成血管丛。重要的是，这 血管内皮细胞(BECs)到SC内皮细胞(SCECs)的淋巴重编程似乎 由最优的AHO触发和维护。因此，我们在这里要解决的主要问题是如何 来自AHO的机械信号调节指定SCEC身份的遗传程序。 策略与目标：除了我们已发表的淋巴管特异性荧光小鼠模型 最近，我们建立了一个转基因大鼠模型，其淋巴管和SC被基因标记为 绿色荧光蛋白。从这两个新的小鼠模型中，我们将在体外纯化和培养SCECs，以获得各种分子和 啮齿动物SCECs的细胞学特征。我们最近报道了一种剪切应力反应的KLF4 转录调节因子，在干细胞前体细胞中高度表达，并且与KLF4物理上相互作用 PROX1.因此，我们假设流体流动诱导的机械信号可能通过KLF4结合 进入Prox1介导的细胞命运指定程序，该程序共同控制角膜缘血液的分化 容器BEC到SCEC。在Aim1中，我们将阐明流介导的SCEC-Fate规范的机制 通过使用纯化的SCECs的KLF4。作为初步研究，我们成功地分离了小鼠SCECs和 证实了SCECs存在两种独特的超微结构，即巨大的空泡和跨细胞的毛孔。 我们将研究KLF4在分离的SCEC的各种分子和细胞特性中的作用。在AIM2中，我们 将使用两组组织特异性的、可诱导的KLF4基因敲除小鼠模型来研究 KLF4至SC器官发生的初始阶段。总之，我们提议的研究将提供一个独特的 能够解决关于供应链发展的重要悬而未决的问题，并产生有价值的信息 为了更好地了解SC开发和IOP控制之间的功能交互 青光眼的治疗意义。

PROJECT SUMMARY OBJECTIVE: We will determine the molecular mechanism by which the aqueous humor outflow (AHO) directs the formation of Schlemm’s canal (SC) through Klf4. SC is a specialized vascular structure that drains the aqueous humor from the anterior chamber into the circulation, and plays a key role in regulating the intraocular pressure (IOP). Dysfunctional SC due to aging or diseases could critically elevate the IOP and often causes ocular nerve damage, possibly leading to glaucoma. Better understanding of the reciprocal interaction between SC development and the AHO would thus have a transformative impact on the prevention of glaucoma caused by elevated IOP. RATIONALE: Structurally, SC is directly connected to the aqueous vein to drain the aqueous humor. Because of this direct vascular joining, SC has long been thought to be a specialized venous extension, whose inner wall is lined by blood vascular endothelial cells. Interestingly, however, several studies have demonstrated evidences, which distinguish SC from typical blood vessels and re-categorize SC as a new lymphatic-like vascular structure. These studies have prompted us to carefully re-examine the molecular and cellular features of SC. As the results, we and others have recently uncovered that SC is postnatally derived from the limbal vascular plexus by upregulating Prox1, the master regulator of lymphatic development. Importantly, this lymphatic reprograming of blood vessel endothelial cells (BECs)-to-SC endothelial cells (SCECs) appeared to be triggered and maintained by the optimal AHO. Accordingly, our main question to address here is how the mechanical signal from the AHO regulates the genetic program that specifies the SCEC identity. STRATEGY & GOAL: In addition to our lymphatic-specific fluorescent mouse model that was published recently, we have created a transgenic rat model whose lymphatic vessels and SC are genetically labeled with GFP. From these two novel murine models, we will purify and culture SCECs in vitro for various molecular and cellular characterizations of rodent SCECs. We have recently reported that Klf4, a shear stress responsive transcription regulator, is highly expressed in the SC precursor cells, and that Klf4 physically interacts with Prox1. Accordingly, we hypothesize that the fluid flow-induced mechanical signal may be incorporated via Klf4 into Prox1-mediated cell fate specification program, which together controls differentiation of limbal blood vessel BECs to SCECs. In Aim1, we will elucidate the mechanism of flow-mediated SCEC-fate specification through Klf4 using purified SCECs. As a preliminary study, we successfully isolated mouse SCECs and confirmed the presence of two unique ultrastructures of SCECs, namely giant vacuole and trans-cellular pores. We will study the roles of Klf4 in various molecular and cellular characteristics of isolated SCECs. In Aim2, we will employ two cohorts of tissue-specific, inducible Klf4 knockout mouse models to study the contribution of Klf4 to the initial stage of the SC organogenesis. Together, our proposed studies will provide a unique capability to address important unanswered questions on SC development, and generate valuable information to better understand the functional interaction between SC development and the IOP control with a possible therapeutic implication toward glaucoma.