Extracellular matrix and outflow resistance

 描述（由申请人提供）：眼内压（IOP）升高是青光眼的主要风险因素，影响全球超过6600万人。降低IOP仍然是阻止青光眼性视力丧失进展的唯一有效治疗策略。小梁网（TM）是房水（AH）流出调节的主要部位，但我们仍然没有专门针对TM的流出药物。如果我们要开发新的药物来改变这种组织并降低眼压，我们必须确定TM细胞稳态调节流出阻力的分子机制。TM细胞的肌动蛋白细胞骨架高度参与IOP调节。肌动蛋白微丝被组织成更高级的结构，包括应力纤维和丝状伪足。肌动蛋白应力纤维已被详细研究，在TM和这些肌动球蛋白丝的松弛增加AH流出。然而，尚未研究丝状伪足对流出阻力和IOP调节的相对贡献。我们的初步数据使用活细胞成像培养的人TM细胞显示高度丰富的丝状伪足在TM细胞表面。这些丝状伪足中的一些形成隧道纳米管（TNT）。TNT是一种特殊的丝状伪足，允许分子货物通过微管导管直接在细胞间转移。这是一种新的细胞通信方法，以前没有在TM细胞中研究过。我们的研究结果表明，通过TNT的荧光标记的囊泡和线粒体的单向转移。细胞有多种传递信号的机制。这些中的大多数采用细胞外扩散以允许分泌的因子以足够的浓度到达其靶细胞以引起效应。在TM组织中，AH是基于扩散的信号传导的主要屏障。任何分泌的因子在AH中稀释并洗掉。TNT的鉴定避免了这个问题，因为信号通过管状导管在TM细胞之间直接转移而不被分泌。这使得TM中的细胞能够与组织其他区域的细胞进行信号交流，包括那些没有浸泡在AH中的区域。在这 应用，我们将表征由TM细胞形成的TNT，并研究TNT和丝状伪足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确定哪些细胞器通过TNT转移。接下来，我们将使用一种新的共培养测定来测量细胞器转移。将青光眼细胞和组织中的TNT形成和细胞器转移与正常TM细胞进行比较。流式细胞术分离的囊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形成的特异性抑制剂和诱导剂来测试这些肌动蛋白结构对正常TM细胞功能和眼灌注培养中流出阻力的影响。研究TM细胞的TNT形成将为肌动蛋白细胞骨架如何调节IOP提供重要的新认识。这将导致开发新的TM特异性治疗方法，用于降低青光眼患者的IOP并保护视力。

 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.