A mechanistic understanding of corneal pathobiology and the development of therapeutic strategies for the treatment of connective tissue disorders

The cornea is the transparent window at the front of the eye and is its main focussing element. To fulfil its role it has to be transparent, strong and precisely shaped. Transparency and strength are controlled by the collagen fibrils that make up the cornea, and by the small molecules between them. Shape is also controlled by the collagen arrangement, but we have now discovered a complex system of small elastic fibres that we believe helps to restore shape when the cornea is distorted, for example as blood is pumped round the body, during blinking or after eye rubbing. These properties of the cornea are controlled at different structural levels: collagen molecules form fibrils, which in turn form larger structures called lamellae, which are then stacked up to form the tissue itself. Elastic fibres that contain the protein elastin are concentrated around the edge of the cornea in the form of sheets, which we have shown are connected across the human cornea by fine filaments rich in proteins called fibrillins. We want to test our hypothesis that this arrangement allows distortion and recovery mainly at the edge of the cornea, maintaining the shape of the central cornea that controls the focussing of the incoming light. From previous work by us and others, we know a lot about why the cornea is transparent and are beginning to understand the arrangement of collagen lamellae and elastic fibres that gives rise to the cornea's shape and thus its focusing abilities. However, the contribution of different elements of the structure to the overall function is still not known and, until we elucidate this, it will not be possible to understand why, in numerous diseases of the cornea, or after different types of surgery on the cornea, transparency, strength and/or shape are abnormal and vision is lost or very blurred. We have pioneered the use of several sophisticated techniques to study the cornea at every structural level from the molecules upwards: x-ray scattering, serial block face scanning electron microscopy and two photon fluorescence light microscopy. We propose now to build equipment that will allow us to measure which constituents of the structure change when the cornea is distorted by known forces, either during its normal functioning or due to disease and/or surgery. We will also explain how lamellae are arranged to provide form and strength, how the elastic fibres are structured in different parts of the cornea, and what role they play in health and disease. We showed that abnormalities of the elastic fibres occur in corneal diseases such as keratoconus, and we will test our idea that they play a role in other diseases of the eye, such as glaucoma. In addition, we will investigate treatments for corneal disorders, for example by developing new chemical crosslinking methods. To address the world-wide shortage of donor corneas, biological artificial corneas are being developed. However, for corneal replacements to function normally, we must fully understand how nature utilises the constituents of a tissue to achieve its vital properties. This means elucidating the exact relationships between its various components and its function, including how cells communicate with other cells during development, wound healing and tissue regeneration. In the case of the cornea, the knowledge that we will obtain by discovering the exact relationship between its various structural components and its function is crucial for our understanding of corneal transparency and biomechanical stability as related to corneal development, surgical manipulation and implantation, and tissue engineering. Finally, we will demonstrate how cornea is an excellent model system for connective tissues more generally, by collaborating with other groups around the world, using our new techniques to aid our understanding of function/dysfunction in other parts of the body.

As the primary refractive component of the eye, the well-functioning cornea must be precisely curved, strong and transparent. Our previous studies have shown that these intrinsic qualities are governed by the hierarchical ultrastructure and complex microanatomy of its cells and extracellular matrix. Building on our existing knowledge of healthy corneal function, our main objectives are to (1) understand the structural basis of normal corneal function and dysfunction, and elucidate the role of a previously identified but as yet, uncharacterised corneal elastic fibre system that we believe may help to restore shape when the cornea is distorted; (2) increase our understanding of the role of extracellular vesicle signalling in the development, regeneration and repair of the cornea; (3) develop photochemical therapies for corneal ectasia through laboratory studies and the collection of clinical data into our UK cross-linking consortium audit tool; (4) develop synchrotron x-ray scattering and microscopy techniques to carry out time-resolved biomechanics/structure correlations to study in three-dimensions, at high-resolution, and in an orchestrated, hierarchical manner, the response of the normal and pathological corneas to different loads; (5) collaboratively demonstrate the applicability of these new techniques to other areas of connective tissue research. Most of these will be achieved by combining state-of-the-art imaging techniques, including microfocus synchrotron x-ray scattering, x-ray spectroscopy, optical coherence tomography, multiphoton laser microscopy, high-pressure cryopreservation electron microscopy and volume serial block face scanning electron microscopy, immunofluorescence microscopy, with established biomechanical and biochemical testing procedures (extensometry, interferometry, inflation testing and immunohistochemisty) to study karyotypically normal and abnormal human foetal and adult corneas, pathological corneas and corneal tissue alternatives.

Our research will have a significant number of non-academic beneficiaries, the principal ones being members of the general public suffering or caring for those with corneal disorders, the practitioners treating them and the charities providing them with advice and support. Corneal problems blind more people worldwide than any other condition apart from cataract. Research into the fundamental structural basis of corneal pathologies and disease mechanisms will have ongoing impact, as it will inform and drive the development of better treatments from which practitioners, and ultimately patients, will benefit. Through public engagement our research will have added societal impact by inspiring interest in STEM subjects and highlighting the importance of organ donation.
Corneal cross-linking has significantly cut costs to the health services by reducing the number of grafts required for keratoconus patients. We predict, that in the short-to-medium term, our cross-linking work will lead to safer treatments, more predictable outcomes, better patient satisfaction and a significant increase in the number of keratoconus patients who can undergo treatment. Our research into new therapies will also offer hope to keratoconus patients with more severe forms of the disease that cannot be treated with conventional cross-linking and if left unchecked, can progress to a devastating corneal perforation. Understanding why the corneas of children and young adults with Down's syndrome are particularly susceptible to developing keratoconus may lead to changes in clinical practice and health care policy, such as advice on how and when to treat. Our research will also be useful to charity and support groups such as the UK Keratoconus Self Help and Support Group, National Keratoconus Foundation and the Down's Syndrome Association, that seek to empower and support sufferers by providing them with the most up-to-date information about their conditions. Our further development of the UK Cross-Linking Consortium and the national keratoconus e-registry will benefit the NHS through the collection of auditable data on cross-linking outcomes, in line with NICE guidelines. It is anticipated that the data set will be used as a resource by governmental bodies to help inform decision-making processes relating to the cost-effectiveness of corneal cross-linking and the provision of cross-linking services.
Corneal surgery has been carried out on millions of people worldwide, not always without post-operative complications. As in the past, where we have helped to improve surgical outcomes in the fields of ex vivo expanded corneal epithelial transplantation and treatments for corneal endothelial dysfunction, our proposed research will benefit public health by driving the development of new and improved surgical approaches. In the same way that our published studies of corneal ultrastructure have been used to develop commercial products (eg. Lumaxis, a device to properly orientate donor corneas prior to surgery), it is anticipated that our newly generated structural and biomechanical data will also be used by commercial entities as a basis for product development. As alluded to above, our research and development of cross-linking therapies continues to generate commercial interest as new products are released into a rapidly evolving market, and as such, it has the potential to stimulate commercial benefit for British medical device companies and economic benefit for the UK.
Finally, due to a world-wide shortage of donor tissue there is a pressing need for the development of artificial corneal replacements. Our research will examine ways of encouraging endogenous regeneration and thus lead to improved corneal-tissue alternatives. This research has high translational potential and will have a lasting impact on quality of life, particularly in developing countries where the shortage of donor tissue is already critical.