The retinal pigment epithelial (RPE) cells of several species, including: chicken, rat, mouse and newt, have been observed to undergo a phenomenon known as transdifferentiation. This involves the re-specification of the RPE phenotype towards a neuroretinal, or lens phenotype, in response to various cues, including basic fibroblast growth factor (bFGF). Transdifferentiation has yet to be described in human RPE cells, however, this investigation has demonstrated that human cells, including primary fetal, and human embryonic stem cell-derived (HESC), RPE, may retain the capacity to undergo some level of bFGF-mediated transdifferentiation. It appears that the process is likely comparable to that observed in the embryonic chick model of the phenomenon, given that human transdifferentiation appears to be restricted to the earliest stages of RPE development (approximately 6 weeks). Additionally, the features of transdifferentiated RPE observed in the earliest available human tissue are shown to resemble that of a similar stage in chick development (HH27), which is shown to display limited transdifferentiation for the first time, in contrast with previous studies which report the loss of potential for transdifferentiation at this stage. It remains unclear as to why RPE cells lose the capacity for transdifferentiation with development, however, it appears to be linked to, but not exclusively a result of, a loss in the expression of Pax6 across the RPE monolayer, given both capacity for transdifferentiation, and Pax6 expression, are both variable in different regions of the same monolayer, in the chick model of transdifferentiation. Known RPE augmentation signaling pathways, including bone morphogenic protein (BMP) and Sonic hedgehog (Shh) were analysed for their potential involvement in the restriction of transdifferentiation, however, neither appeared to be directly involved. Further studies in the embryonic chick model of the phenomenon will be necessary to unlock this potential in human RPE cells.

A genetic predisposition to Rhegmatogenous Retinal Detachment (RD) has been suggested for over 40 years. Ectopia Lentis (EL) is known to have a genetic aetiology as part of Marfan Syndrome, other ocular syndromes and when occurring in isolation. This work investigates the genetic aetiology to these conditions in Mendelian and non Mendelian inheritance. The work in this thesis establishes a clear genotype-phenotype correlation between isolated EL and its most important causative gene: ADAMTSL4. This suggests that mutations in this gene result in a more severe phenotype than other genes causing EL. In doing so, a novel clinical grading system for this condition has also been developed. The expression of ADAMTSL4 and distribution of its protein within ocular tissue has also been investigated and suggests further roles for this protein in ocular development. Modelling of the protein was undertaken and provides insights for future investigations. Traditional and novel next generation investigative tools have also been employed to examine families with Mendelian inherited phenotypes including RD and EL. The role of a deleted exon in ADAMTS17 has been identified as playing a role in Weill-Marchesani Like syndrome. A novel ocular phenotype has also been defined in three families demonstrated to be caused by mutations in ADAMTS1 8. This gene has previously been described in few probands with ophthalmic phenotypes, and this work has further delineated the role of this gene. It is becoming clear that members of the ADAMTS family of proteins play a significant role in ocular development. Finally, over 1300 probands with non-Mendelian RD were recruited and closely phenotyped as part of this work. It has demonstrated novel racial differences in the phenotypes of those affected. This cohort contributed significantly towards the first genome wide association study (GWAS) into RD; and established for the first time the genetic contribution to this condition. Further funding has now been acquired to investigate this cohort further using a novel exome array. Preliminary quality control analysis has been performed; allowing a platform for further detailed analysis to identify putative functional variants associated with RD.

There are a number of surgical methods for treating glaucoma, including trabeculectomy and the insertion of drainage devices. At the current time, these procedures are still associated with post-operative problems for a significant number of patients, particularly with the control of aqueous humour flow and pressure e.g. hypotony. The main aim of this work was to look at ways of improving the outcomes of the procedures. The usual approach in assessing surgical techniques is to test them on live human or animal eyes. This is inherently complex, with a significant challenge to keep some of the major parameters e.g. aqueous humour inflow and wound healing consistent throughout a series of tests, and with this the problem of reproducibility. The approach used in this work includes a review and analysis of the different surgical methods and devices from an engineering perspective, the use of scaling analysis and a large-scale physical model coupled with image processing to study trabeculectomy scleral flap characteristics and its effect on flow and pressure, and also the use of model drainage devices in ex vivo settings to look at flow and pressure. For each experiment, implications for clinical practice are discussed. Applying engineering principles to glaucoma procedures and devices provided novel insight into their functions. It was found that the trabeculectomy scleral flap acts as a valve to guard from excessive aqueous humour outflow and low pressures, and parameters such as the number and position of sutures and scleral flap geometry can be tailored to alter aqueous humour outflow. Additionally, 50 μm internal diameter tubes show promise for controlled aqueous humour flow into the subconjunctival space with avoidance of low pressures. Engineering methodology can be used in the development of new treatments and devices. However, some results may not translate exactly into the more complex living eye.

Background: The spectrum of ocular allergy includes reversible conditions (seasonal, perennial and giant papillary conjunctivitis) and irreversible chronic severe forms of disease involving the cornea (vernal and atopic keratoconjunctivitis). Conjunctival remodelling may play a role in the pathogenesis of chronic allergic eye disease (CAED). Based on findings on asthmatic airway epithelia, it is suggested in chronic asthma that activation of the epithelial-mesenchymal trophic unit (EMTU hypothesis) and EGFR may participate in disease development and account for chronicity, severity and poor response to steroid treatment. This thesis investigates the potential application of the EMTU hypothesis to CAED. Aims and Methods: Conjunctival biopsies of healthy subjects, SAC, GPC (controls group) were compared to VKC and AKC (CAED group) and conjunctival tissues of active and treated OCP. Expression of EGFR, TGFa and CD44 was assessed by means of immunohistochemistry. Cells of two human epithelial conjunctival cell lines (IOBA-NHC & CHwK) received single and multiple cytokine treatments (PMA, IL-17A, TNFQ/IL-1β) and secretion of EGFR, VEGF, CD44, TGFa and p21waf of harvested supernatants were assessed by means of ELISA. Statistical analysis was performed using Mann-Whitney test for the immunohistochemistry and t-test for the ELISA results. Results: Immunohistochemistry studies revealed that there was increased expression of all EMTU remodelling markers in the CAED group compared to Controls (p<0.01). OCP conjunctival biopsies showed no expression for EGFR and TGFa. There was increased secretion of the remodelling molecules by both cell lines after single/multiple cytokine treatments. Conclusions: Epithelial conjunctival remodelling may be responsible for disease severity in CAED as shown in chronic asthma. Epithelial cell EGFR-mediated remodelling parallels the observations made in chronic asthmatic epithelia and the results are in agreement with previous studies in biopsies and tears of VKC patients. Finally suggestions for new treatment strategies are proposed to prevent or inhibit disease perpetuation.

Processes of wound healing or scarring cause many blinding eye conditions and can limit the success of surgical eye treatments. The mechanisms underlying matrix remodelling and contraction by connective tissue cells (fibroblasts) are not fully understood. Two main theories dominate the field: firstly, that fibroblasts in a wound begin to express alpha smooth muscle actin and transdifferentiate into myofibroblasts with enhanced contractile properties, and secondly that fibroblast migration might underly matrix condensation. The purpose of this work was to develop novel imaging techniques to investigate the mechanisms of tissue contraction and force generation using live cell and matrix imaging during fibroblast-mediated collagen gel contraction, and to determine the role played by growth factors and mechanical tension in that process. We have shown that fibroblasts from different parts of the eye display marked differences in macroscopic matrix contraction profiles, and that four factors determine the early matrix contraction profile: 1) cell size. 2) intrinsic cellular force. 3) growth factor-mediated actomyosin-based dynamic cell protrusive activity and 4) net pericellular matrix displacement. Intrinsic cellular force and dynamic activity appear to be independent unique characteristics of each cell type and might serve as predictors of matrix contraction. In addition, in corneal fibroblasts, we have shown that the recently identified PIXil rab5 RN I re-pathway mediates a specific type of cell protrusive activity leading to matrix contraction. Specific inhibitors of actomyosin- and rab5'RNTre-pathways are likely candidates to prevent corneal haze formation after surgery, trauma or inflammation, finally, we explored novel models of contraction involving real tissue by developing techniques to maintain and microscopically study ocular surface tissue ex vivo. We achieved successful concomitant live imaging of resident cells and extra-cellular matrix behaviour at high resolution in both conjunctival and corneal stroma by combining optimized strategies for tissue maintenance, fluorescent cell labeling, and confocal laser and/or two-photon microscopy. In summary, this thesis presents novel pathways of matrix contraction and imaging techniques to study these events in vitro and ex vivo.

Several of the multitude of functions attributed to Vascular Endothelial Growth Factor-A (VEGF-A) are coordinated by its various isoforms, which are generated as a result of alternative splicing from a single gene. Despite the fact that the VEGF isoforms exhibit distinct biochemical properties, little has been done to clarify their functions and their contributions to physiological and pathological processes. In this thesis I describe the biochemical and biological characterization of the heparin-binding domain of mouse VEGF 164 through structure-function analysis. To investigate the functional significance of heparin binding, mutations were introduced into exon 7 of VEGF 164 to identify essential residues for heparin binding. Three key amino acids involved in this interaction were identified. Mutants with alterations in these amino acids were unable to bind heparin and were compromised in their ability to bind to cell-surface heparan sulfate. These mutants, however, retained wild-type like potency in inducing tissue factor expression in vitro and microvessel growth ex vivo, and maintained the capacity to bind to the receptors neuropilin-1, VEGFR-1, and VEGFR-2. A second goal of this work was to better define the role of VEGF 164 in mediating inflammatory processes during pathological vascularization of the retina. Analysis of VEGF 164-deficient (VEGF1ZU/ iao) mice subjected to neovascularization-inducing conditions and rats injected intravitreally with recombinant VEGF variants demonstrated that endogenous and exogenous VEGF 164 increases leukocyte adhesiveness to retinal vessels compared to the non-heparin-binding isoform, VEGF 120. Interestingly, the three basic residues that confer heparin binding of VEGF 164, appear to be critical for its pro inflammatory activity, but not for its angiogenic activity. In addition, both mutants (and VEGF 120) exhibited a reduced affinity for VEGFR-1, a leukocyte-expressed receptor that mediates VEGF-induced migration. Results of in vivo experiments using P1GF, VEGF-E, as well as a VEGFR-1 neutralizing antibody, further demonstrate a role for VEGFR-1 in retinal leukostasis.

Background: The vascular endothelial growth factor (VEGF) inhibitor ranibizumab is emerging as an efficacious treatment for diabetic macular oedema. Large clinical trials have shown improvements in visual acuity and reduced central retinal thickness. Details of its effect on other retinal functional parameters are lacking. There is a concern that repeated ranibizumab treatment could exacerbate macular ischaemia or lead to global retinal dysfunction by inhibiting physiological isoforms of VEGF. Outcomes of surgery for advanced proliferative retinopathy remain variable and post-operative complications including recurrent haemorrhage can limit visual recovery. VEGF is strongly implicated in the pathogenesis of advanced retinopathy, so VEGF inhibition prior to surgery may improve outcomes. Trials have failed to demonstrate a clear benefit for bevacizumab, so investigation of the licensed intraocular agent ranibizumab represents a logical next step. Aims: To investigate the effects of ranibizumab and laser treatment in diabetic macular oedema on the following parameters: visual acuity, protan and tritan colour contrast sensitivity, 4° and 12° macular sensitivity by microperimetry, electrophysiological indices from pattern and full field electroretinograms. To report structural retinal changes following ranibizumab and laser treatment in terms of qualitative and quantitative optical coherence tomography outcomes, and to quantify macular ischaemia by fluorescein angiography. To investigate the effect on visual acuity at three months post-surgery of ranibizumab pre-treatment in patients undergoing vitrectomy for advanced proliferative diabetic retinopathy. Methods: Randomised clinical trial of intravitreal ranibizumab vs. laser in 36 subjects with centre-involving diabetic macular oedema (The LUCIDATE study). Randomised clinical trial of pre-operative intravitreal ranibizumab vs. subconjunctival saline injection in 30 subjects undergoing vitrectomy-delamination for advanced proliferative diabetic retinopathy (The RaDiVit study). Results: Thirty six subjects with diabetic macular oedema were recruited and 33 completed the trial. Ranibizumab treated subjects gained a mean of 6 letters compared with 0.9 letter loss for laser at 48 weeks. Retinal sensitivity improved in the central macular 4° and 12° in both groups but to a greater extent with ranibizumab. There was no evidence of worsening global retinal dysfunction by electroretinograms in either group. Retinal thickness decreased in both groups: there was a 132 µm reduction in central macular thickness with ranibizumab compared with 103 µm for laser. Fluorescein angiography showed no evidence of significantly increased macular ischaemia in either group. Thirty subjects with advanced proliferative diabetic retinopathy were recruited, underwent surgery, and completed the study. At three months post-surgery, visual acuity in the ranibizumab group was 53 letters compared with 47 letters in the control group. Conclusion: In diabetic macular oedema, there is evidence that ranibizumab leads to greater improvements in visual acuity and retinal sensitivity than laser, with a corresponding greater reduction in retinal thickness. There is no evidence that it worsens macular ischaemia or indices of global retinal electrophysiological function, but larger trials designed to address each of the outcomes investigated here would be required to confirm these findings. In proliferative diabetic retinopathy, there is evidence from this small pilot study that ranibizumab treatment leads to better visual acuity at 3 months post-surgery. An appropriately powered trial would be required to confirm this.

Limbal epithelial stem cells (LESCs) produce progeny (human limbal epithelial cells, hLEC) responsible for corneal repair and maintenance. The loss, or dysfunction, of LESCs results in LESC deficiency (LESCD) characterised by corneal opacification, neovascularisation and vision loss. Current treatments include LESC transplantation through cultivated limbal epithelial transplantation (CLET). There remains a number of manufacturing and regulatory challenges with CLET, therefore, this research was performed to address these challenges and improve existing protocols. This thesis aims were; to improve the starting number of LESCs by investigating to what extent corneal storage media preserve LESCs. Secondly, to decrease risk of zoonotic agent transmission through animal-derived materials, the potential of human-derived feeders (MRC5 fibroblasts) and serum for hLEC expansion was assessed. The final aim was to examine the feasibility of collagen-based tissue equivalents (TE) containing surrogate niche cells (human dermal fibroblasts, hDFs) as alternative delivery method to current protocols utilising human amniotic membrane, a substrate predisposed to inter-and intra-donor variability. Experiments showed different corneal storage formulations can preserve poorly differentiated cells; however, investigations into other factors (donor age/ hLEC isolation) may further improve the starting number of LESCs. Human derived serum was effective in maintaining hLECs and is an adequate replacement for animal-derived serum in CLET protocols. However, human-derived MRC5 feeders should not replace animal-derived feeders in CLET manufacture due to unfavourable hLEC characteristics observed. Furthermore, future investigations into cytokines expressed by human MRC5 feeders may elucidate factors influencing hLEC behaviour. Finally, changes in hLEC characteristics and increased inflammation-associated mediator expression demonstrated hDFs were not a suitable TE surrogate niche cell; however, the potential role of such mediators in epithelial-stromal interactions should be explored. To conclude; these experiments have improved existing CLET protocols, highlighted lower-risk materials may not always be effective, and that the surrounding culture environment is integral for CLET graft success.

Adenosine 5’ triphosphate (ATP)-gated P2X7 receptors (P2X7Rs) are known to act as conduits for photoreceptor and retinal ganglion cell (RGC) damage, consequences of various neurodegenerative conditions within the visual pathway. Growing evidence supports the notion that P2X7Rs and associated inflammatory mediators may coordinate microglia, the resident immune cells of the central nervous system (CNS), to play a genuine role in modulating neurotransmission. This study aimed to characterise the role of P2X7Rs in modulating outer and inner retinal function within the rod-mediated pathway, and in the putative microglial-mediated modulation of signal transmission in the retina. Excitatory components of outer and inner retinal function were assessed by recording light-evoked, extracellular transretinal electroretinogram (ERG), and ON and OFF retinal ganglion cell (RGC) field excitatory postsynaptic potential (fEPSP) responses from the acutely isolated, dark-adapted, mouse retina. Alterations to microglial morphology, under similar conditions, were also explored. Initial experiments confirmed the excitatory responses as predominantly mediated via the ‘classic’ rod photoreceptor – rod-ON bipolar cell – AII amacrine cell pathway. With the use of selective P2X7R antagonists, it was shown that P2X7R activation directly modulated photoreceptor, ON bipolar cell and ON RGC function, but not OFF RGC function, through partially independent mechanisms. A novel finding of this study demonstrated that acute application of the microglia-activating bacterial component, lipopolysaccharide (LPS) modulated inner retinal function, possibly through a P2X7R- and Pannexin-1-associated mechanism of microglial ATP release. These results were supported by observations of early morphometric changes to microglia caused by P2X7R activation and LPS, as revealed by immunofluorescence labelling and confocal laser scanning microscopy. Since changes in neurotransmission and microglial function are early indicators of neuropathology, these results contribute to the understanding of early neural-immune interactions in retinal disease, and in the central nervous system as a whole.

Fibroproliferative diseases including pulmonary fibrosis and scleroderma are leading causes of mortality. Mucous membrane pemphigoid (MMP) is a systemic autoimmune disease characterised by inflammation and variable blistering and scarring of one or more of the mucous membranes and the skin. MMP affecting the conjunctiva was formally termed Ocular Cicatricial Pemphigoid (OCP). Ocular MMP (OcMMP) will be used in this thesis to describe studies carried out on MMP patients having ocular involvement as a major component of their disease. Therefore OcMMP is synonymous with the term OCP used in previous studies. OcMMP affects the readily accessible conjunctiva; providing a model disease for investigating the role and control of inflammation and fibrosis. Current immunosuppressive therapy fails to halt the aggressive and visually devastating scarring. This study aimed to identify novel pro-fibrotic genes and to use functional surrogate scarring assays to assess the gene products for their therapeutic potential. OcMMP fibroblasts maintained their phenotype in-vitro and exhibited increased collagen production and secretion, decreased matrix reorganisation and reduced rates of proliferation and myofibroblast differentiation. Gene expression profiling revealed that Aldehyde dehydrogenase 1 family, member A3 (ALDH1A3) is up regulated in OcMMP. Elevated expression of ALDH1A3 was confirmed by qPCR and protein levels assessed by western blot. Addition of disulfiram, a selective aldehyde dehydrogenase inhibitor restored phenotype and function of OcMMP fibroblasts to control cell levels. Conversely, aldehyde dehydrogenase activation using retinoic acid added to control conjunctival fibroblasts cultures promoted OcMMP-like phenotype in the scarring assays. Protein phosphorylation profiling also uncovered potential ALDH1A3 signalling pathways and that ALDH1A3 may be mediating the fibrotic response in OcMMP by orchestrating impaired fibroblast/dendritic cell interactions and metabolic stress. These significant findings not only show that ALDH1A3 is a novel anti-scarring target in OcMMP but also that ALDH inhibition may be a novel therapeutic approach to fibrosis with application in OcMMP and one that should be evaluated in other scarring disorders.