Cells respond to chemical, mechanical and topographical cues both in vivo and in vitro. Much research has been carried out into the effects of chemical signals and to a lesser degree, mechanical. However, less is known about cell responses to topographical cues, particularly to topographies with nanoscale dimensions. Understanding how cells respond to topography is of particular interest to the field of tissue engineering, where it is crucial to characterise the effects that biomaterial surfaces have on the cells that they come into contact with. Observations of the impact that topographic signalling has on cells, within two tissue engineering systems, are discussed in this thesis. These systems are: polymer conduits for peripheral nerve regeneration and thin films for the replacement of the retinal pigment epithelium. Understanding the effects that micro and nano scaled topographies have on nerve and retinal cell regeneration is important for successful development and implementation of appropriate tissue engineered devices. In order to fabricate topographical patterns on biomaterial surfaces, a number of fabrication techniques were investigated. The fundamental requirement of these techniques was for reliable production of uniform nano and micro scale topographical patterns over large lateral areas (millimeter scale). Initially, the suitability of electrohydrodynamic lithography (EHDL) was assessed. EHDL is a relatively new technique, first published in 2000, which employs electrostatic forces to pattern thin polymer films. Subsequently, techniques traditionally associated with the computing industry, such as e-beam lithography and reactive ion etching, were evaluated. Following successful pattern fabrication, NG108-15 and ARPE-19 cells were cultured on grooved topographies. Against a baseline parameter of elapsed time, the cell morphologies and their propensity for alignment with the grooves was rigorously assessed and compared. ARPE-19 and NG108-15 cell responses differed from one another, and were sensitive to varying groove dimensions. Ultimately, the developing morphologies (for both cell types) proved to be clearly dependent on groove dimensions and elapsed time.

The thesis describes the effects of various plant flavonoids (curcumin, epigallocatechin-3-gallate [EGCG], luteolin, apigenin, myricetin, quercetin, and cyanidin) on the physiological properties and viability of cultured human retinal pigment epithelial (RPE) cells. It is described that, with the exception of EGCG, all flavonoids tested decrease dose-dependently the RPE cell proliferation, migration, and secretion of VEGF. Luteolin, apigenin, myricetin, and quercetin decreased the viability of RPE cells at higher concentrations, by triggering cellular necrosis. Curcumin decreased the viability of RPE cells via induction of early necrosis and delayed apoptosis. The cytotoxic effect of curcumin involved activation of caspase-3 and calpain, intracellular calcium signaling, mitochondrial permeability, oxidative stress, and increased phosphorylation of p38 MAPK and decreased phosphorylation of Akt protein. Myricetin caused caspase-3 independent RPE cell necrosis mediated by free radical generation and activation of calpain and phospholipase A2. The myricetin- and quercetin-induced RPE cell necrosis was partially inhibited by necrostatin-1, a blocker of programmed necrosis. The author concludes that the intake of curcumin, luteolin, apigenin, myricetin, and quercetin as supplemental cancer therapy or in the treatment of retinal diseases should be accompanied by careful monitoring of the retinal function. Possible beneficial effects of EGCG and cyanidin in the treatment of retinal diseases should be examined in further investigations.

Age related macular degeneration (AMD) is the most common cause of irreversible vision loss in the elderly. At present, there are an estimated one million people in Canada with some form of AMD and this number is expected to double to two million by 2031. These estimates are sobering, and it is predicted that costs for treatment and care of individuals who suffer vision loss from AMD will have significant impact on the social and public health systems in Canada in the next two decades. There are treatments to slow the progression of vision loss, but unfortunately, there are currently no cures available for AMD. In order to develop effective second generation therapies and cures, further insights into how and why AMD develops are greatly needed. Recent studies have provided novel insights into the role of inflammation in the pathogenesis of AMD. Inflammation, or swelling of the retinal tissues, causes harmful processes that promote macular degeneration. The proposed studies will focus on the triggers of inflammation in the retina. It is hypothesized that macular degeneration may be slowed or stopped by eliminating the molecules that cause inflammation in the retina. This study will focus on amyloid beta (Aβ), a toxic molecule that has been implicated in retinal inflammation, and the role that it may play in gene expression of the retinal pigment epithelial cell. Amyloid beta is a well studied peptide in another age related disorder, Alzheimer’s disease. It is the major extracellular deposit in Alzheimer’s disease plaques, and has recently been discovered as a component of drusen, the hallmark extracellular deposits in the retina of patients with the ‘dry’ form of AMD. These studies will allow the development of new treatment regimens that target retinal inflammation and thus minimize the processes that ‘trigger’ the onset of macular degeneration.

Age related macular degeneration (AMD) is the most common cause of irreversible vision loss in the elderly. At present, there are an estimated one million people in Canada with some form of AMD and this number is expected to double to two million by 2031. These estimates are sobering, and it is predicted that costs for treatment and care of individuals who suffer vision loss from AMD will have significant impact on the social and public health systems in Canada in the next two decades. There are treatments to slow the progression of vision loss, but unfortunately, there are currently no cures available for AMD. In order to develop effective second generation therapies and cures, further insights into how and why AMD develops are greatly needed. Recent studies have provided novel insights into the role of inflammation in the pathogenesis of AMD. Inflammation, or swelling of the retinal tissues, causes harmful processes that promote macular degeneration. The proposed studies will focus on the triggers of inflammation in the retina. It is hypothesized that macular degeneration may be slowed or stopped by eliminating the molecules that cause inflammation in the retina. This study will focus on amyloid beta (Aβ), a toxic molecule that has been implicated in retinal inflammation, and the role that it may play in gene expression of the retinal pigment epithelial cell. Amyloid beta is a well studied peptide in another age related disorder, Alzheimer’s disease. It is the major extracellular deposit in Alzheimer’s disease plaques, and has recently been discovered as a component of drusen, the hallmark extracellular deposits in the retina of patients with the ‘dry’ form of AMD. These studies will allow the development of new treatment regimens that target retinal inflammation and thus minimize the processes that ‘trigger’ the onset of macular degeneration.

Age related macular degeneration (AMD) is the most common cause of irreversible vision loss in the elderly. At present, there are an estimated one million people in Canada with some form of AMD and this number is expected to double to two million by 2031. These estimates are sobering, and it is predicted that costs for treatment and care of individuals who suffer vision loss from AMD will have significant impact on the social and public health systems in Canada in the next two decades. There are treatments to slow the progression of vision loss, but unfortunately, there are currently no cures available for AMD. In order to develop effective second generation therapies and cures, further insights into how and why AMD develops are greatly needed. Recent studies have provided novel insights into the role of inflammation in the pathogenesis of AMD. Inflammation, or swelling of the retinal tissues, causes harmful processes that promote macular degeneration. The proposed studies will focus on the triggers of inflammation in the retina. It is hypothesized that macular degeneration may be slowed or stopped by eliminating the molecules that cause inflammation in the retina. This study will focus on amyloid beta (Aβ), a toxic molecule that has been implicated in retinal inflammation, and the role that it may play in gene expression of the retinal pigment epithelial cell. Amyloid beta is a well studied peptide in another age related disorder, Alzheimer’s disease. It is the major extracellular deposit in Alzheimer’s disease plaques, and has recently been discovered as a component of drusen, the hallmark extracellular deposits in the retina of patients with the ‘dry’ form of AMD. These studies will allow the development of new treatment regimens that target retinal inflammation and thus minimize the processes that ‘trigger’ the onset of macular degeneration. / Medicine, Faculty of / Graduate

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第22004号 / 医博第4518号 / 新制||医||1038(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 辻川 明孝, 教授 中川 一路, 教授 高橋 淳 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

Melatonin is an indolamine hormone presumably synthesized by retinal photoreceptors, and may act as a paracrine signal of darkness within the retina. Previous studies have suggested that melatonin, acting through specific receptors, may be involved in cyclic retinal functions such as photoreceptor outer segment disc shedding and phagocytosis, and modulation of neurotransmitter release in the inner retina. The goal of this study was to determine if melatonin receptor mRNA is expressed in the neural retina and retinal pigment epithelium (RPE) of Xenopus laevis. Sheets of RPE, devoid of contaminating cells, were obtained from Xenopus eyes, and epithelial cultures were subsequently established on microporous membrane filters in a defined medium. Total RNA was isolated from whole brain, neural retina, fresh RPE sheets, and cultured RPE cells. RNA expression of the three known Xenopus melatonin receptor subtypes (MEL1A, 1B, and 1C) was determined by reverse- transcription/polymerase chain reaction (RT/PCR) amplification, followed by Southern hybridization with RNA probes. PCR-amplified cDNA encoding melatonin receptor subtypes 1B and 1C, but not 1A, were detected in reverse-transcribed RNA obtained from brain, neural retina and RPE. RPE cells grown in culture for two weeks also demonstrated 1B and 1C receptor RNA expression. This study suggests that RNA encoding the 1B and 1C melatonin receptor subtypes is expressed in the neural retina and RPE of Xenopus retina, and the expression persists in RPE cells when grown in culture. The expression of melatonin receptor RNA in the RPE may reflect a regulatory role for melatonin in some diurnal events that occur in this tissue, such as phagocytosis of photoreceptor outer segment membranes, and intracellular migration of pigment granules.

Yes-associated protein (Yap) transcriptional co-activator, a major downstream effector of Hippo signaling pathway, controls organ size by modulating cell proliferation and apoptosis. The Hippo signaling cascade phosphorylates Yap, and this phosphorylation inhibits the nuclear retention of Yap, which is essential for cell proliferation. Thus, the loss of Hippo pathway components leads to enlarged organs through increased Yap activity in the nucleus. Our initial study showed that Yap was expressed in the developing retina and retinal pigment epithelium (RPE), suggesting Yap's tissue-specific roles during the eye development. Intriguingly, Yap proteins were localized at the apical junctions in addition to the nucleus and cytosol of the retinal progenitor cells, adding another level of regulation. To uncover the tissue- and localization-specific functions of Yap, we generated a Yap conditional knockout mouse with Rx-Cre for the ablation of the Yap gene in the developing retina and RPE. Upon deletion of Yap, the retina showed severe lamination defects with numerous folding, which is reminiscent of the polarity and adhesion loss. The RPE, a single pigmented cell layer overlying the retina, lost pigmentation and changed into a multi-layered epithelium. The marker analysis revealed that 1) in the retina, the localization of the polarity complex proteins such as Pals1, Crb1 and atypical PKC were disrupted, suggesting Yap's indispensable role in junctional stability, and 2) the level of Otx2 in RPE decreased while those of Chx10 and beta-tubulin increased, suggesting transdifferentiation of RPE into the retina. In addition, the deletion of Yap induced a decrease in proliferation and an increase in apoptosis, ultimately resulting in microphthalmia. In conclusion, our results are consistent with the model that Yap functions in the stabilization of apical proteins for maintenance of the laminar organization, determination of RPE territory, and regulation of proliferation and apoptosis during the eye development. / Cell Biology

INTRODUCTION: Retinal Pigment Epithelia (RPE) degeneration is a key event in the development of age-related macular degeneration (AMD). RPE dysfunction in AMD is thought to occur through the accumulation of reactive oxygen species (ROS) and oxidative damage. The transcriptional co-activators, PGC-1α and PGC-1β, are important regulators of mitochondrial biogenesis and anti-oxidant capacity. Our group has previously shown that the PGC-1α protein promotes RPE oxidative metabolism and that overexpression of the PGC-1α gene protects cells from AMD-associated pro-oxidants. On the other hand, PGC-1β gene expression has been found to be upregulated in patients with neovascular AMD, and in-vitro overexpression of PGC-1β damages cells and induces pro-oxidant conditions. OBJECTIVE: Given the divergence of PGC-1α and PGC-1β functions in RPE and their clinical relevance in AMD pathogenesis, this project will seek to investigate the impact of the upregulation of PGC-1α and PGC-1β in RPE metabolism. PGC-1α will be upregulated through treatment with compound ZLN005. A new methodology for PGC-1β expression will be developed to closely modulate in-vitro PGC-1β induction. METHODS: In-vitro experiments were performed on the ARPE-19 cell line. Cells were treated with 10µM of ZLN005 for 24 hours. Oxidative stress was induced by exposure to H2O2 and NaIO3 under serum-free conditions. Lactate dehydrogenase (LDH) levels were used to quantify cell death. Quantitative PCR (qPCR) and Western Blot were performed to measure changes in gene and protein expression respectively. Superoxide production by the mitochondria was measured to evaluate ROS levels within the cell. Intravitreal injections of 20µM ZLN005 were performed on eight-week old male C57BL/6J mice. After 24 and 72 hours of treatment, the mice were euthanized and the enucleated eyes were dissected to obtain the RPE and neural retina layers. Total RNA was extracted from these layers and qPCR was performed to measure gene expression. A tetracycline-inducible PGC-1β plasmid was designed and transfected into ARPE-19 cells. The cells were exposed to 0.01-100µg/ml doxycycline for 48-hours and qPCR was used to measure gene expression. Transfected cells were treated with ZLN005 and cell death upon exposure to oxidative stress was quantified. RESULTS: Gene expression analysis on ARPE-19 cells treated with ZLN005 showed robust upregulation of PGC-1α, PGC-1β and their associated transcription factors and enzymes. Induction of PGC-1α at the protein level was also confirmed. ZLN005 efficiently protected ARPE-19 cells from H2O2 and NaIO3 cytotoxicity and its protection was negated in PGC-1α-silenced cells. Treatment with ZLN005 also decreased mitochondrial superoxide production. ZLN005 intravitreal injections were safely administered to the animals and did not cause cataracts or other damage to the ocular tissues. While statistical significance in gene expression changes was limited due to the small sample size, anti-oxidants GPX1 and TXN2, and electron transport chain gene, ATP50, were found to be potentially induced in the neuro-retina, while FOXO3 was found to be downregulated. Evaluation of our novel tetracycline-inducible PGC-1β adenoviral vector showed that upregulation of PGC-1β was efficiently controlled by the addition of doxycycline to transfected cells. Upon exposure to H2O2, transfected cells treated with doxycycline experienced greater cell death than transfected cells not exposed to doxycycline. ZLN005 treatment was able to decrease cell death in both conditions. CONCLUSION: The present study shows that ZLN005 efficiently protects RPE cells from oxidative damage through selective induction of PGC-1α. While still preliminary, the in-vivo study indicates that ZLN005 is safe to be injected into the eye and may be able to increase the expression of mito-protective and anti-oxidant genes in the neuronal retina. In addition, our design of the tetracycline inducible PGC-1β plasmid allows for tight control over PGC-1β expression through doxycycline addition. Upregulation of PGC-1β at levels similar to those observed in clinical conditions caused increased pro-oxidant induced cell death and treatment with ZLN005 was able to protect against cell death. / 2021-06-30T00:00:00Z

Le récepteur MerTK est impliqué dans la phagocytose des segments externes des photorécepteurs (SEP) par l'épithélium pigmentaire rétinien (EPR), fonction cruciale pour la survie des photorécepteurs et la vision. Dans la rétine, ces deux tissus sont en contact permanent et la phagocytose ne survient qu'une fois par jour, cette fonction nécessite donc d'être contrôlée précisément. Le pic de phagocytose est lié à l'activation intracellulaire de MerTK via l'intégrine αvβ5. Ce projet a eu pour but d'étudier les mécanismes extracellulaires régulant la fonction de MerTK au cours de cette phagocytose.Nous avons montré que MerTK est clivé à la surface des cellules d'EPR in vivo avant et après le pic de phagocytose. Ceci permettrait d'éviter une phagocytose trop prononcée des SEP.Nous avons démontré le rôle opposé des ligands de MerTK, spécifique à l'EPR. Gas6 semble inhibiteur, il stimule le clivage de MerTK et inhibe la phagocytose in vitro, et son expression in vivo est faible au moment du pic de phagocytose. Au contraire, Protéine S, dont l'expression augmente in vivo au moment du pic, inhibe le clivage de MerTK et stimule la phagocytose in vitro, et pourrait ainsi potentialiser cette fonction.Parmi les protéases étudiées, l'inhibition d'ADAM17 in vitro engendre une diminution du clivage de MerTK corrélée à une augmentation de sa biodisponibilité à la surface cellulaire et de son activité. Cependant, cet effet n'étant pas total, l'implication d'une autre protéase n'est pas exclue.Ainsi, mes travaux de Doctorat permettent de mieux comprendre la régulation complexe de l'activité de MerTK dans la phagocytose rétinienne, essentielle pour le rythme circadien de cette fonction. / The MerTK receptor is involved in the daily phagocytosis of photoreceptor outer segments (POS) by the retinal pigment epithelium (RPE), an indispensable process for photoreceptors survival and vision. In the retina, the contact between POS and RPE is permanent, and POS phagocytosis occurs once a day, requiring a precise control of this function. The phagocytic peak is initiated by activation of MerTK via the αvβ5 integrin receptor. This project aimed at studying extracellular mechanisms that control MerTK function during POS phagocytosis. We have shown that MerTK can be cleaved from the RPE cell surface in vivo before and just after the phagocytic peak. This process might avoid an excess of POS phagocytosis. We have also shown the opposite role of MerTK ligands, specific to RPE cells. Gas6 appears to act as an inhibitor as it stimulates MerTK cleavage and inhibits POS phagocytosis in vitro. Moreover, in vivo, Gas6 expression is weak at peak phagocytosis time. In contrast, Protein S, which in vivo expression increases at the time of the phagocytic peak, inhibits MerTK cleavage and stimulates POS phagocytosis in vitro, and thus might potentiate phagocytosis. Among the protease candidates we studied, in vitro inhibition of ADAM17 results in decreased MerTK cleavage associated with the increase of full-length receptors available at cell surface and of MerTK activation. However, as cleavage still occurs in these conditions, we cannot exclude the implication of another protease. Taken together, my PhD data allows us to better understand the complex regulation of MerTK activity during retinal phagocytosis, which is essential for the circadian rhythm of this function.