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.

Public Health Relevance

Glaucoma is a leading source of vision loss worldwide and is frequently associated with elevated intraocular pressure. This study aims to investigate a novel form of cellular communication in the trabecular meshwork, the tissue responsible for regulating pressure in the eye. Results may provide new information about how normal eyes regulate intraocular pressure, while providing new avenues to explore to develop novel therapies to reduce elevated pressure in glaucoma patients.

Agency
National Institute of Health (NIH)
Institute
National Eye Institute (NEI)
Type
Research Project (R01)
Project #
5R01EY019643-07
Application #
9258447
Study Section
Biology of the Visual System Study Section (BVS)
Program Officer
Liberman, Ellen S
Project Start
2010-08-01
Project End
2021-04-30
Budget Start
2017-05-01
Budget End
2018-04-30
Support Year
7
Fiscal Year
2017
Total Cost
Indirect Cost
Name
Oregon Health and Science University
Department
Ophthalmology
Type
Schools of Medicine
DUNS #
096997515
City
Portland
State
OR
Country
United States
Zip Code
97239
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Keller, Kate E; Bhattacharya, Sanjoy K; BorrĂ¡s, Theresa et al. (2018) Consensus recommendations for trabecular meshwork cell isolation, characterization and culture. Exp Eye Res 171:164-173
Keller, Kate E; Bradley, John M; Sun, Ying Ying et al. (2017) Tunneling Nanotubes are Novel Cellular Structures That Communicate Signals Between Trabecular Meshwork Cells. Invest Ophthalmol Vis Sci 58:5298-5307
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Jayaram, Hari; Phillips, Jay I; Lozano, Diana C et al. (2017) Comparison of MicroRNA Expression in Aqueous Humor of Normal and Primary Open-Angle Glaucoma Patients Using PCR Arrays: A Pilot Study. Invest Ophthalmol Vis Sci 58:2884-2890
Keller, Kate E; Wirtz, Mary K (2017) Working your SOCS off: The role of ASB10 and protein degradation pathways in glaucoma. Exp Eye Res 158:154-160
Yang, Yong-Feng; Sun, Ying Ying; Acott, Ted S et al. (2016) Effects of induction and inhibition of matrix cross-linking on remodeling of the aqueous outflow resistance by ocular trabecular meshwork cells. Sci Rep 6:30505
Vranka, Janice A; Bradley, John M; Yang, Yong-Feng et al. (2015) Mapping molecular differences and extracellular matrix gene expression in segmental outflow pathways of the human ocular trabecular meshwork. PLoS One 10:e0122483
Vranka, Janice A; Kelley, Mary J; Acott, Ted S et al. (2015) Extracellular matrix in the trabecular meshwork: intraocular pressure regulation and dysregulation in glaucoma. Exp Eye Res 133:112-25
Sun, Ying Ying; Keller, Kate E (2015) Hyaluronan cable formation by ocular trabecular meshwork cells. Exp Eye Res 139:97-107

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