Extracellular matrix and outflow resistance

 描述(申请人提供)：高眼压(IOP)是青光眼的主要危险因素，全世界有超过6600万人受到青光眼的影响。降低眼压仍然是阻止青光眼视力丧失进展的唯一有效的治疗策略。小梁网络(TM)是房水流出调节的主要部位，但我们仍然没有针对该TM的流出药物。如果我们要开发改变这种组织和降低眼压的新药，我们必须确定TM细胞调节流出阻力的分子机制。TM细胞的肌动蛋白细胞骨架与眼压调节密切相关。肌动蛋白微丝被组织成更高级的结构，包括应力纤维和丝足。肌动蛋白应力纤维在肌动蛋白中已被详细研究过，这些肌动球蛋白微丝的松弛增加了肌动蛋白的流出。然而，丝状足对流出阻力和眼压调节的相对贡献尚未被研究。我们使用培养的人TM细胞的活细胞成像的初步数据显示，TM细胞表面有非常丰富的丝状足细胞。其中一些形成了隧道纳米管(TNT)。TNTs是一种特殊的丝状支架，允许分子货物通过管状管道在细胞间直接转移。这是一种新的细胞通讯方法，以前还没有在TM细胞中进行过研究。我们的结果证实了荧光标记的囊泡和线粒体通过TNTs的单向转移。细胞有多种传递信号的机制。它们中的大多数利用细胞外扩散来允许分泌的因子以足够的浓度到达它们的目标细胞以引起效应。在TM组织中，AH是基于扩散的信号转导的主要障碍。任何分泌的因子都会在AH中稀释，然后被冲走。TNTs的识别避免了这个问题，因为信号直接通过管状管道在TM细胞之间传递，而不是分泌。这允许驻留在TM中的细胞与组织其他区域的细胞交流信号，包括那些没有沐浴在AH中的区域。在这 在应用方面，我们将表征TM细胞形成TNT的特征，并研究TNTs和丝状孔道是否有助于流出阻力调节。我们将使用先进的光学显微镜技术来确定哪些细胞器通过TNTs转移。接下来，我们将使用一种新的共培养方法来测量细胞器的转移。青光眼细胞和组织中TNT的形成和细胞器的转移将与正常的TM细胞进行比较。流式细胞术分离的小泡的蛋白质组学分析将确定哪些信号被传递。最后，我们将使用丝状伪足和TNT形成的特定抑制剂和诱导剂来测试这些肌动蛋白结构对正常TM细胞功能和眼灌流培养中流出阻力的影响。研究TM细胞TNT的形成将对肌动蛋白细胞骨架如何调节眼压提供重要的新的理解。这将导致开发新的、针对TM的治疗方法，用于降低青光眼患者的眼压和保护视力。

 DESCRIPTION (provided by applicant): Elevated intraocular pressure (IOP) is a primary risk factor for glaucoma, which affects over 66 million people worldwide. Lowering IOP remains the only effective therapeutic strategy to stop the progression of glaucomatous vision loss. The trabecular meshwork (TM) is the primary site of aqueous humor (AH) outflow regulation, but we still do not have an outflow drug that specifically targets the TM. If we are to develop new drugs that modify this tissue and lower IOP, we must determine the molecular mechanisms by which TM cells homeostatically adjust outflow resistance. The actin cytoskeleton of TM cells is highly involved in IOP regulation. Actin microfilaments are organized into higher ordered structures including stress fibers and filopodia. Actin stress fibers have been studied in detail in the TM and relaxation of these actomyosin filaments increases AH outflow. However, the relative contributions of filopodia to outflow resistance and IOP regulation have not been studied. Our preliminary data using live-cell imaging of cultured human TM cells show highly abundant filopodia at the TM cell surface. A few of these filopodia form tunneling nanotubes (TNTs). TNTs are specialized filopodia that allow direct intercellular transfer of molecular cargo through tubulr conduits. This is a novel method of cellular communication that has not been studied previously in TM cells. Our results demonstrate the unidirectional transfer of fluorescently-labeled vesicles and mitochondria via TNTs. Cells have multiple mechanisms to communicate signals. Most of these employ extracellular diffusion to allow secreted factors to reach their target cells at sufficient concentrations to elicit an effect. In the TM tissue, AH is a major barrier to diffusionl-based signaling. Any secreted factor is diluted in AH and washed away. Identification of TNTs circumvents this problem since signals are directly transferred between TM cells through tubular conduits without being secreted. This allows cells resident in the TM to communicate signals with cells in other regions of the tissue, including those areas that are not bathed in AH. In this application, we will characterize TNT formation by TM cells and investigate whether TNTs and filopodia contribute to outflow resistance regulation. We will determine which cellular organelles are transferred via TNTs using advanced light microscopy techniques. Next, we will measure organelle transfer using a novel co-culture assay. TNT formation and organelle transfer in glaucoma cells and tissue will be compared to normal TM cells. Proteomics analyses of flow cytometry-isolated vesicles will determine which signals are communicated. Finally, we will use specific inhibitors and inducers of filopodia and TNT formation to test the effects of these actin structures on normal TM cellular functions and on outflow resistance in ocular perfusion culture. Investigating TNT formation by TM cells will provide an important new understanding of how the actin cytoskeleton regulates IOP. This will lead to the development of novel, TM-specific therapeutic approaches for reducing IOP and preserving vision in patients with glaucoma.