Keratoconus is the degeneration of the corneal stroma, which is the layer that provides the structure for the cornea. This degeneration causes the cornea to slowly change from its normal curved shape to a more conical shape, leading to vision distortion. Unfortunately, to-date, the cause of the disease is not known, and in most cases, patients require some kind of surgical intervention. Currently, there are no in vitro or animal models available, limiting the number of feasible studies. Therefore, we propose to develop a novel in vitro 3-dimensional (3D) culture system consisting of human keratoconus cells (HKCs), which will allow us to identify vital proteins and metabolites that are driving the disease in vitro and compare these results to in vivo tear samples. Our preliminary data consists of some exciting observations that potentially link the defects in vivo to in vitro findings. Firsty, we have identified significant inherent cellular defects in HKCs when compared to normal primary human corneal fibroblasts (HCFs). HKCs were unable to secrete and self-assemble a functional extracellular matrix (ECM) when stimulated with vitamin C (VitC), while HCFs assembled high amounts of collagenous ECM. Secondly, we identified TGF-?3 (T3) as a potential growth factor for stimulating HKCs to self-assemble an ECM. Thirdly, we have identified metabolites and proteins that are similarly regulated in our in vitro model when compared to in vivo, some of which may be new markers for identifying keratoconus disease. The hypotheses we propose to test are as follows: (1) T3 stimulates HKCs to adopt a keratocyte-like phenotype and secrete a functional ECM, (2) Regulation of metabolic activity is a key component in the understanding of Keratoconus defects and (3) Identification of new markers for keratoconus disease is vital for early detection and treatment. We propose three specific aims in order to address the following questions: First, what are the inherent differences of HKCs and how may these be regulated by T3? Second, can metabolic activity regulate HKCs to assemble ECM? Third, what are the key proteins regulating keratoconus in vivo and in vitro? The novelty of this proposal is that for the first time, we can establish a 3D in vitro model that can be used to represent the events during keratoconus disease in vivo. Indirect- immunofluorescence microscopy, real-time PCR and transmission electron microscopy will be the major analysis tools for the in vitro model. Metabolomics and Proteomics will be the tools for analyzing both the in vitro and in vivo samples. The combination of the above will link the in vitr to in vivo studies and provide new, novel markers for the diagnosis and prevention of keratoconus at early stages. Relevance to Public Health - Improvement in the ability to detect and diagnose suspicious to advanced keratoconus will enable the exclusion of patients at risk for corneal ectasia after corneal refractive surgery. Also, earlier detection of forme fruste keratoconus may lead to earlier intervention.

Public Health Relevance

Keratoconus disease is the degeneration of the stroma, which causes the cornea to slowly change from its normal curved shape to a more conical shape, leading to vision distortion. Unfortunately, to-date, the cause of the disease is not known, and in most cases, patients require some kind of surgical intervention. Currently, there are no in vitro or animal models available;therefore, we propose to develop a novel in vitro 3-dimensional culture system consisting of human keratoconus cells, that will allow us to identify vital proteins and metabolites that are driving the disease in vitro and compare these results to in vivo tear samples.

Agency
National Institute of Health (NIH)
Type
Research Project (R01)
Project #
5R01EY023568-02
Application #
8737270
Study Section
(BVS)
Program Officer
Mckie, George Ann
Project Start
Project End
Budget Start
Budget End
Support Year
2
Fiscal Year
2014
Total Cost
Indirect Cost
Name
University of Oklahoma Health Sciences Center
Department
Ophthalmology
Type
Schools of Medicine
DUNS #
City
Oklahoma City
State
OK
Country
United States
Zip Code
73117
McKay, Tina B; Hjortdal, Jesper; Sejersen, Henrik et al. (2016) Endocrine and Metabolic Pathways Linked to Keratoconus: Implications for the Role of Hormones in the Stromal Microenvironment. Sci Rep 6:25534
Sarker-Nag, Akhee; Hutcheon, Audrey E K; Karamichos, Dimitrios (2016) Mitochondrial Profile and Responses to TGF-β Ligands in Keratoconus. Curr Eye Res 41:900-7
Priyadarsini, Shrestha; McKay, Tina B; Sarker-Nag, Akhee et al. (2016) Complete metabolome and lipidome analysis reveals novel biomarkers in the human diabetic corneal stroma. Exp Eye Res 153:90-100
Karamichos, D (2015) Ocular tissue engineering: current and future directions. J Funct Biomater 6:77-80
Priyadarsini, Shrestha; McKay, Tina B; Sarker-Nag, Akhee et al. (2015) Keratoconus in vitro and the key players of the TGF-β pathway. Mol Vis 21:577-88
Karamichos, D; Zieske, J D; Sejersen, H et al. (2015) Tear metabolite changes in keratoconus. Exp Eye Res 132:1-8
Priyadarsini, Shrestha; Sarker-Nag, Akhee; Allegood, Jeremy et al. (2015) Description of the sphingolipid content and subspecies in the diabetic cornea. Curr Eye Res 40:1204-10
Drevets, Peter; Chucair-Elliott, Ana; Shrestha, Priyadarsini et al. (2015) The use of human cornea organotypic cultures to study herpes simplex virus type 1 (HSV-1)-induced inflammation. Graefes Arch Clin Exp Ophthalmol 253:1721-8
McKay, T B; Lyon, D; Sarker-Nag, A et al. (2015) Quercetin attenuates lactate production and extracellular matrix secretion in keratoconus. Sci Rep 5:9003
McKay, Tina B; Sarker-Nag, Akhee; Lyon, Desiree' et al. (2015) Quercetin modulates keratoconus metabolism in vitro. Cell Biochem Funct 33:341-50

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