Glaucoma is an irreversible blinding disease that will affect over 79 million people worldwide by the year 2020. The factors or mechanisms that lead to this disease are not currently understood;however, the trabecular meshwork cells play a central role in the regulation of intraocular pressure in both the normal and disease state. The only proven method to slow the progression of the disease is to lower intraocular pressure, but there is evidence that drugs that lower the pressure without causing increased flow through the trabecular meshwork (TM) may ultimately cause progression of disease. Thus, therapeutics that would target the TM to increase flow and reduce pressure are actively being studied. Research in the field uses human cultured TM to test hypotheses because of the lack of a good animal model for human glaucoma. Unfortunately, human TM (HTM) cells are generally grown on flat plasticware. Cells in vivo do not experience the hard, flat surfaces that are present in vitro. Numerous reports have shown that cells respond to biophysical cues in the micro- to nano-scale range. Our studies have shown modified cell behaviors as well as gene and protein changes in HTM cells when they are grown on substrates that mimic the biomimetic features that are present in basement membrane. Additionally, a growing body of literature documents the profound impact of substratum compliance on cell behaviors. It has been reported that basement membrane is changed in glaucoma with a denser matrix observed with electron microscopy. The overall goal of this proposal is to 1. determine how biophysical properties of the substratum modulate cytoskeletal proteins in the HTM cells and 2. determine how these biophysical properties modulate HTM mediated extracellular matrix production and remodeling. Preliminary data indicate the compliance of the HTM is decreased with glaucoma and that HTM cells grown on less compliant substrates are less compliant as well. An increase in rigidity of the HTM cells might lead to increased resistance to aqueous humor outflow. The central idea of this proposal is that with progression of glaucoma HTM cells secrete matrix proteins that lessen the compliance of the HTM. This, in turn, decreases the compliance of the HTM cells and a feedback loop becomes initiated. This study would measure compliance of normal and glaucomatous HTM and then study HTM cell architecture, behavior and gene expression on surfaces that had similar compliances. We would focus on extracellular matrix proteins and remodeling of matrix by the HTM cells when they are supplied with biophysical cues (compliance and topography) in the biomimetic range as well as biochemical cues commonly used in glaucoma research. Our improved understanding of the consequences of the biophysical cues on the HTM cells will aid in the design of appropriate surfaces to use for glaucoma research. Additionally, these studies will help optimize therapeutic approaches to drugs targeting the HTM and lead to enhanced treatments for glaucoma.

Public Health Relevance

Glaucoma is an irreversible blinding disease that will affect almost 80 million people by the year 2020. Because of the unique features of the human eye, there are no good animal models to study this disease process. To date, research on the reduced outflow of aqueous humor seen in individuals with the most common form of this disease has centered on the perfused organ culture model or the use of trabecular meshwork cells. The organ culture model does not lend itself readily to exploratory research, but rather is used to confirm studies done with the cultured cells. Current work with cultured cells is done almost exclusively on flat tissue culture plasticware. In vivo, cells never see flat, hard surfaces that lack surface features in the micro to nanoscale range. Cells react to these biophysical cues and alter not only their behavior but also gene expression. Our investigation on the influence of biophysical cues on human trabecular meshwork cells will allow us to pattern substrates that better resemble the surfaces these cells sense in vivo. We believe this will allow researchers to improve the relevancy of their results with cultured cells;and additionally, we believe understanding of these cues will lead to novel therapeutic targets for glaucoma treatment.

Agency
National Institute of Health (NIH)
Institute
National Eye Institute (NEI)
Type
Research Project (R01)
Project #
5R01EY019475-04
Application #
8523884
Study Section
Anterior Eye Disease Study Section (AED)
Program Officer
Chin, Hemin R
Project Start
2010-08-03
Project End
2014-07-31
Budget Start
2013-08-01
Budget End
2014-07-31
Support Year
4
Fiscal Year
2013
Total Cost
$350,527
Indirect Cost
$117,303
Name
University of California Davis
Department
Veterinary Sciences
Type
Schools of Veterinary Medicine
DUNS #
047120084
City
Davis
State
CA
Country
United States
Zip Code
95618
Murphy, Kaitlin C; Morgan, Joshua T; Wood, Joshua A et al. (2014) The formation of cortical actin arrays in human trabecular meshwork cells in response to cytoskeletal disruption. Exp Cell Res 328:164-71
Morgan, Joshua T; Raghunathan, Vijay Krishna; Thomasy, Sara M et al. (2014) Robust and artifact-free mounting of tissue samples for atomic force microscopy. Biotechniques 56:40-2
Garland, Shaun P; McKee, Clayton T; Chang, Yow-Ren et al. (2014) A cell culture substrate with biologically relevant size-scale topography and compliance of the basement membrane. Langmuir 30:2101-8
Chang, Yow-Ren; Raghunathan, Vijay Krishna; Garland, Shaun P et al. (2014) Automated AFM force curve analysis for determining elastic modulus of biomaterials and biological samples. J Mech Behav Biomed Mater 37:209-18
Thomasy, Sara M; Raghunathan, Vijay Krishna; Winkler, Moritz et al. (2014) Elastic modulus and collagen organization of the rabbit cornea: epithelium to endothelium. Acta Biomater 10:785-91
Morgan, Joshua T; Wood, Joshua A; Walker, Naomi J et al. (2014) Human trabecular meshwork cells exhibit several characteristics of, but are distinct from, adipose-derived mesenchymal stem cells. J Ocul Pharmacol Ther 30:254-66
Garland, Shaun P; Wang, Royal Y; Raghunathan, Vijay Krishna et al. (2014) Photopatternable and photoactive hydrogel for on-demand generation of hydrogen peroxide in cell culture. Biomaterials 35:1762-70
Thomasy, Sara M; Morgan, Joshua T; Wood, Joshua A et al. (2013) Substratum stiffness and latrunculin B modulate the gene expression of the mechanotransducers YAP and TAZ in human trabecular meshwork cells. Exp Eye Res 113:66-73
Dreier, Britta; Gasiorowski, Joshua Z; Morgan, Joshua T et al. (2013) Early responses of vascular endothelial cells to topographic cues. Am J Physiol Cell Physiol 305:C290-8
Morgan, Joshua T; Murphy, Christopher J; Russell, Paul (2013) What do mechanotransduction, Hippo, Wnt, and TGF* have in common? YAP and TAZ as key orchestrating molecules in ocular health and disease. Exp Eye Res 115:1-12

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