Extracellular matrix and outflow resistance

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 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.