Flow Dependent Development and Maintenance of Schlemm's Canal

项目摘要 目的：我们将确定房水流出（AHO）的分子机制， 通过Klf4形成Schlemm管（SC）。SC是一种专门的血管结构， 眼内液从前房进入循环，并起着调节眼内压的关键作用。 压力（IOP）。由于衰老或疾病引起的SC功能障碍可能严重升高IOP， 眼神经损伤可能导致青光眼更好地理解相互作用， 因此，SC的发展和AHO将对青光眼的预防产生变革性的影响。 眼压升高 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长期以来被认为是一种专门的静脉延伸，其内部 血管壁由血管内皮细胞构成。然而，有趣的是，一些研究表明， 这些证据将SC与典型的血管区分开来，并将SC重新归类为一种新的类血管， 血管结构这些研究促使我们仔细重新审视分子和细胞特征 作为结果，我们和其他人最近发现SC是出生后来源于角膜缘 通过上调Prox 1（淋巴发育的主要调节因子）来调节血管丛。重要的是这 血管内皮细胞（BEC）向SC内皮细胞（SCEC）的淋巴重编程似乎 由最佳AHO触发和维持。因此，我们在这里要解决的主要问题是， 来自AHO的机械信号调节指定SCEC身份的遗传程序。 战略和目标：除了我们发表的荧光小鼠模型， 最近，我们建立了一种转基因大鼠模型，其淋巴管和SC被基因标记为 GFP。从这两种新的小鼠模型中，我们将在体外纯化和培养SCEC，用于各种分子和 啮齿类SCEC的细胞特征。我们最近报道了Klf 4，一种剪切应力响应的 转录调节因子，在SC前体细胞中高度表达，并且Klf4与之物理相互作用 近端1。因此，我们假设流体流动引起的机械信号可以通过Klf4并入 Prox1介导的细胞命运特化程序，其共同控制角膜缘血的分化 从BEC到SCEC在Aim 1中，我们将阐明血流介导的SCEC命运特化的机制。 通过Klf4纯化SCEC。作为初步研究，我们成功分离了小鼠SCEC， 证实了SCEC存在两种独特的超微结构，即巨空泡和跨细胞孔。 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。在目标2中，我们 将采用两组组织特异性、诱导型Klf4敲除小鼠模型来研究 Klf4在SC器官发生初期的作用。总之，我们提出的研究将提供一个独特的 解决供应链发展中尚未解决的重要问题并生成有价值信息的能力 为了更好地理解SC发展和IOP控制之间的功能相互作用， 对青光眼的治疗意义。

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.