Targeted Disruption of Components Involved in Aqueous Humor Outflow Resistance Using an Organ Culture System

Janice Vranka, PhD
Oregon Health and Science University (Portland, OR)
Year Awarded:
2014
Grant Duration:
July 1, 2014 to June 30, 2016
Disease:
Glaucoma
Award Amount:
$100,000
Grant Reference ID:
G2014058
Award Type:
Standard
Award Region:
US Northwestern
Janice Vranka, PhD

Altered Versican Fibrillar Interactions in the Outflow Pathway

Summary

Our research focuses on the trabecular meshwork (TM), a spongy tissue located in the eye around the base of the cornea, near the ciliary body. The TM is responsible for draining a fluid called aqueous humor from the eye via the anterior chamber, and is believed to be the tissue that regulates intraocular pressure, a critical factor in the development of glaucoma. Our laboratory is investigating the hypothesis that a specific subset of complex molecules that reside in the TM, and that interact with each other, are important in the regulation of aqueous humor outflow resistance. When these processes are disrupted, there will be changes in the tissue that lead to alterations in outflow facility. Thus our primary objective is to improve the overall understanding of how aqueous humor outflow resistance is regulated and to determine the triggers of glaucoma in order to hasten the development of new therapies that help glaucoma patients.

Details

Our research focuses on the trabecular meshwork (TM), a discrete tissue located circumferentially around the base of the cornea and near the ciliary body, which is responsible for draining the aqueous humor from the anterior chamber of the eye. The TM is believed to be the tissue that regulates intraocular pressure, a critical factor in the development of glaucoma. The goal of this project is to define how a specific subset of interacting and complex molecules in the TM contribute to the regulation of aqueous humor outflow resistance.

Because the availability of human eye tissue is limited and costly, the experiments in the first and second aims are being performed in primary cell cultures. TM cells can be grown in primary culture and used to evaluate the effects of targeted gene silencing. The objective in the first specific aim is to measure gene-specific changes in primary cultured TM cells in response to gene silencing of versican and fibrillin-1 using quantitative polymerase chain reaction (PCR) arrays. This approach allows us to study the consequences of silencing specific genes involved in outflow resistance on a genetic level. The objective of the second specific aim is identifying proteins altered in response to targeted gene silencing of versican and fibrillin-1 in primary cultured human TM cells. Proteins secreted from untreated and gene-silenced TM cells in culture will be compared and quantitated. This approach allows us to study the consequences of silencing specific genes involved in outflow resistance on a protein level. Finally, the objective in the third specific aim is determining structural changes in TM tissue after versican or fibrillin-1 gene silencing. In this aim we will use an in vitro organ culture model system in which outflow rates are measured before and after gene silencing. Untreated and gene-silenced tissues will then be imaged by confocal microscopy and super-resolution microscopy to assess differences in ultrastructure as a result of targeted gene silencing.

This proposal is innovative in that it uses targeted gene silencing in cultured cells and quantitative PCR arrays, combined with protein identification, to provide new insight into the events that may determine outflow resistance mechanisms. Additionally, anterior segment organ culture is unique in that it is the only ex vivo model system available to test for outflow facility in response to targeted gene silencing. Our primary objective is to improve the overall understanding of how aqueous humor outflow resistance is regulated and what are the triggers in the development of glaucoma. Once completed, the knowledge gained from this study will facilitate the development of novel therapies aimed at helping glaucoma patients.

About the Researcher

As a graduate student in the Biochemistry and Molecular Biology Department at Oregon Health & Science University (OHSU), Janice Vranka gained experience in the Acott lab and was involved in projects focusing on characterizing the enzymatic activity of matrix metalloproteinases and tissue inhibitors of metalloproteinases in the retina and trabecular meshwork. She then joined the Bächinger lab at the Shriners Hospital in Portland as a postdoctoral fellow and worked for many years on the biophysical characterization of extracellular matrix molecules and their involvement in human diseases affecting the skeleton. While there, Dr. Vranka participated in a number projects that led to publications and gained considerable experience in a wide range of lab techniques. In 2009, she was recruited to OHSU as a research assistant professor of ophthalmology on a two-year National Institutes of Health American Recovery & Reinvestment Act Supplement and since then has had multiple years of experience investigating the cell biology of the trabecular meshwork (TM) and using advanced imaging techniques to investigate nanoparticle segmental flow patterns and molecular differences of high and low outflow regions in the (TM). This work led to her current interest in uncovering the linkage between the putative primary outflow resistance molecule, versican, and the fibrillar structural components of the outflow pathway, and their potential role in the development of glaucoma. Dr. Vranka’s long-term goal is to become a completely independent investigator pursuing her primary interests in basic and translational vision research, ultimately to improve the outcomes for glaucoma patients.
Don't miss out.
Receive research updates, inspiring stories, and expert advice
Please enter your first name.
Please enter your last name.
Keep me informed about: *
Please select at least one.
You must select at least one disease category.