understanding the mechanisms of pore formation and how pore formation may be induced with drugs may yield new insights into the pathogenesis and treat

Lead Research Organisation: Imperial College London
Department Name: Dept of Bioengineering

Abstract

Glaucoma is a leading cause of blindness that affects roughly 60 million people worldwide (Quigley & Broman, 2006), and 6.59 million people in the Caribbean and Latin America alone(Tham et al., 2014; Allingham et al., 1992). Glaucoma is typically associated with elevated eye pressure, which all current glaucoma treatments aim to reduce. However, many treatments eventually fail, allowing blindness to progress or forcing patients to undergo risky glaucoma drainage surgery. Current glaucoma medications likely fail because they do not effectively target the root cause of elevated eye pressure.
Although the detailed mechanisms of eye pressure regulation remain largely unknown, the hydraulic resistance to aqueous humour drainage from the eye, known as outflow resistance, is the primary determinant of eye pressure. Outflow resistance is increased in glaucoma, leading to the elevated eye pressure that is characteristic of the disease. The source of outflow resistance generation lies near the cells lining Schlemm's Canal (SC), a vessel that runs along the outer border of the iris that collects aqueous humour as it drains from the eye (Figure 1).
To enter SC, aqueous humour must cross the continuous cell layer that comprises SC wall. Because aqueous humour flows upwards from below the cell layer, the cells experience an upward-directed force that pushes the cells away from their underlying supporting tissue, causing the cells to form dome-shaped outpouchings known as giant vacuoles (GVs) that are typically associated with micrometer-sized pores (Figure 1, right panel).
These pores provide the pathway for aqueous humour flow across the cellular wall of SC (Braakman 2015). In glaucoma, the number of pores is reduced (Johnson et al., 2002; Allingham et al., 1992), which may explain the source of increased outflow resistance. Thus, understanding the mechanisms of pore formation and how pore formation may be induced with drugs may yield new insights into the pathogenesis and treatment of glaucoma.

The Overby lab at Imperial College London has discovered that pore formation is driven by mechanical force. As SC cells are stretched during GV formation, this stretch triggers pores to form (Braakman, 2014). In glaucoma, the biomechanical stiffness of SC cells increases, and the increased stiffness correlates with reduced pore-forming ability (Overby et al., 2014). This suggests that drugs that target the biomechanical properties of SC cells, particularly cell stiffness, may be useful to promote pore formation and treat glaucoma. However, it remains unclear which particular biomechanical structures within the SC cells would have the greatest effect on pore formation.
The goal of this project is to determine which sub-cellular structures are responsible for controlling pore formation. These structures include the cell membrane surrounding the cell, the contractile filamentous network within the cell known as the cytoskeleton, and adhesions between the cell and its underlying tissue. Each of these structures may influence the biomechanical stiffness and hence the stretch experienced by SC cells to affect pore formation. We hypothesise that by selectively targeting specific biomechanical components of SC cells, we may pharmacologically promote pore formation. Knowing which cellular biomechanical structures to target will identify new drugs that could be used to treat glaucoma, for example using FDA-approved drugs that have already been shown to modulate cell membrane tension or contractility.
Currently, an apparatus for inducing giant vacuole formation in SC cells in vitro has been established (Pedrigi et al., 2011), but there is currently no method to measure the stretch experienced by SC cells. The proposed work will thus develop a microscopy and image analysis system to quantitatively measure the stretch experienced by SC cells during GV formation. We will measure stretch as the SC cells are treated with compounds that selectively alter t

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Studentship Projects

Project Reference Relationship Related To Start End Student Name
EP/N509486/1 01/10/2016 30/09/2021
1975830 Studentship EP/N509486/1 01/10/2017 20/10/2021 Justino Rodrigues
 
Description Elevated intraocular pressure is the main risk factor associated with glaucoma. Intraocular pressure is regulated by the conventional aqueous humor outflow pathway. It is thought that the inner wall of Schlemm's canal plays a pivotal role in the modulation of intraocular pressure through its ability to form openings for aqueous humor to flow across, or "pores". It is known that pore formation is impaired in cultured Schlemm's canal cells from glaucoma patients, suggesting a link between Schlemm's canal pore formation and glaucoma. Schlemm's canal pore formation is currently a poorly understood phenomenon.

Previously, it has been shown that Schlemm's Canal pore formation is dependent on the strain applied to cells. Our research shows that for a given strain, Schlemm's canal cells open and close pores continuously in a competing dynamic system. Through novel fluorescent assaying methods we developed, we have found that pore lifetime, pore formation and pore closure time scales are on the order of minutes or less.

Studying pores using fluorescent assays produces large amounts of data that are difficult and time consuming to analyze objectively. We have found that It is possible to streamline the analysis of pore formation data using machine learning techniques. We have created a system leveraging random forest classification that achieves similar performance to expert human analysis in hours instead of weeks.
Exploitation Route Confirmation that Schlemm's canal pore formation is a dynamic process deepens our understanding of pore formation. It focuses the investigation of the mechanisms behind pore formation away from simple mechanotransduction and towards the interaction of strain-modulated competing mechanisms of pore formation and closure. It also allows us to investigate how glaucoma theraputics affect pore formation, which will aid in the search for glaucoma drugs that act at the root cause of elevated intraocular pressure.

Our data analysis system allows for future studies using our assays to produce useful data faster than previously possible, accelerating the rate that research can progress. It also allows for the extraction of more quantiative information from existing data, allowing us to investigate trends in pore size, location and number, which may provide further insight into how the pore formation ability of glaucomatous cells is damaged.
Sectors Healthcare,Pharmaceuticals and Medical Biotechnology