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by Xu Xun. / Thesis (M.Phil.)--Chinese University of Hong Kong, 1994. / Includes bibliographical references (leaves 78-87). / Chapter CHAPTER 1 --- INTRODUCTION --- p.1 / Chapter CHAPTER 2 --- LITERATURE REVIEW --- p.6 / Chapter 2.1 --- Anatomy of retina and vitreous --- p.6 / Chapter 2.2 --- Rhegmatogenous retinal detachment --- p.8 / Chapter 2.2.1 --- Retinal breaks --- p.8 / Chapter 2.2.2 --- Retinal detachment and subretinal fluid --- p.8 / Chapter 2.3 --- Proliferative vitreoretinopathy --- p.9 / Chapter 2.4 --- Total protein in subretinal fluid --- p.11 / Chapter 2.5 --- Fibroblast growth factor --- p.12 / Chapter 2.5.1 --- Structure of b and a FGF and their gene --- p.13 / Chapter 2.5.2 --- Expression of bFGF and aFGF in neuroretinal and pigment epithelial cells --- p.14 / Chapter 2.5.3 --- The FGF receptors --- p.15 / Chapter 2.5.4 --- In vitro biological effect of FGF --- p.15 / Chapter 2.5.5 --- FGF in retinal diseases --- p.16 / Chapter 2.6 --- Cellular study of proliferative vitreoretinopathy --- p.18 / Chapter 2.6.1 --- Experimental study --- p.18 / Chapter 2.6.2 --- Pathogenesis of intravitreal proliferation --- p.20 / Chapter 2.6.3 --- Cellular components of proliferative tissue --- p.20 / Chapter 2.6.4 --- Cellular components of subretinal fluid --- p.22 / Chapter CHAPTER 3 --- MATERIALS AND METHODS --- p.24 / Chapter 3.1 --- Specimens --- p.24 / Chapter 3.2 --- Determination of total protein --- p.24 / Chapter 3.2.1 --- Study population --- p.24 / Chapter 3.2.2 --- Quantitation of total protein --- p.25 / Chapter 3.3 --- Determination of bFGF --- p.27 / Chapter 3.3.1 --- Study population --- p.27 / Chapter 3.3.2 --- Quantitation of bFGF --- p.28 / Chapter 3.4 --- Celular study --- p.30 / Chapter 3.4.1 --- Study population --- p.30 / Chapter 3.4.2 --- Fixation of samples --- p.30 / Chapter 3.4.3 --- Immunocytology --- p.32 / Chapter 3.4.4 --- Examination of autofluorescence --- p.35 / Chapter 3.4.5 --- Hematoxylin and eosin staining --- p.36 / Chapter CHAPTER 4 --- RESULTS --- p.37 / Chapter 4.1 --- Total protein --- p.37 / Chapter 4.1.2 --- Total protein in subretinal fluids --- p.38 / Chapter 4.1.3 --- Total protein in normal vitreous of autopsy and sera of patients --- p.40 / Chapter 4.1.4 --- Relationship of protein level and size of retinal break --- p.40 / Chapter 4.1.5 --- Relationship of protein level and duration of retinal detachment --- p.43 / Chapter 4.1.6 --- Relationship of protein level and degree of PVR --- p.45 / Chapter 4.2 --- Basic FGF in subretinal fluids --- p.47 / Chapter 4.2.1 --- Standard curve for determination of bFGF in SRF --- p.47 / Chapter 4.2.2 --- The levels of bFGF in both SRF and controls --- p.48 / Chapter 4.2.3 --- Levels of bFGF in different degrees of PVR --- p.50 / Chapter 4.2.4 --- Levels of bFGF in SRF of RD with and without previous cryotherapy --- p.54 / Chapter 4.2.5 --- The relationship of level of protein and bFGF --- p.55 / Chapter 4.3 --- Results of cytological examination --- p.57 / Chapter 4.3.1 --- Pigment examingnation by autofluorescence --- p.57 / Chapter 4.3.2 --- Cellular study of subretinal fluids --- p.59 / Chapter 4.3.3 --- Cellular study of subretinal fluid in eyes with prior cryotherapy --- p.62 / Chapter CHAPTER 5 --- DISCUSSION --- p.68 / Chapter 5.1 --- Evaluaton of method for obtaining specimens --- p.68 / Chapter 5.2 --- Total protein of SRF in retinal detachment --- p.69 / Chapter 5.3 --- Basic FGF in subretinal fluids --- p.71 / Chapter 5.4 --- Elevated level of bFGF in eye after cryotherapy --- p.74 / Chapter 5.5 --- Cell components in SRF of PVR --- p.75 / REFERENCES --- p.78

Bei der proliferativen Retinopathie (PVR) kommt es aufgrund einer Netzhautablösung zu einer Störung der Blut-Retina-Schranke. Durch die daraus resultierende Milieuänderung wandern retinale Zellen und Bestandteile des Blutes in den Glaskörper ein. Wachstumsfaktoren regen die mitotisch inaktiven RPE-Zellen zur Migration und Proliferation an. Im weiteren Verlauf der Erkrankung führt dies zur Ausbildung von Traktionsmembranen und zur Ablösung der Netzhaut. Die dadurch entstehenden Zugkräfte verhindern eine Wiederanlegung der Netzhaut. Ziel dieser Arbeit war es, zu untersuchen, welchen Einfluss Wachstumsfaktoren und deren Kombinationen auf die Proliferation und Migration humaner retinaler Pigmentepithelzellen, die VEGF-Sekretion durch RPE-Zellen und die mRNA-Expression von Kollagenen haben. Die beteiligten Signalwege sollten ebenfalls ermittelt werden. Im Ergebnis sollten Faktoren identifiziert werden, die die Entstehung einer PVR begünstigen. Migration, Proliferation, VEGF-Sekretion und die TGF-ß-Sekretion konnten durch den Wachstumsfaktor PDGF (Platelet-derived growth factor) gesteigert werden. Die Wirkung des PDGF-Signals wurde durch die Aktivierung verschiedener intrazellulärer Signalwege (MAPK, p38MAPK, PI3K/AKT) vermittelt. PDGF wirkte hemmend auf die Kollagen mRNA-Expression. Für TGF-ß (Transforming growth factor-ß) wurde eine hemmende Wirkung auf die Proliferation und eine induzierende Wirkung auf die Kollagensynthese nachgewiesen. Beide Faktoren – PDGF und TGF-ß – steigerten die VEGF-Sekretion. Eine additive Erhöhung der VEGF-Sekretion wurde durch die Kombination beider Faktoren nachgewiesen. Die untersuchten Wachstumsfaktoren interagieren mit den humanen retinalen Pigmentepithelzellen und tragen somit maßgeblich zur Ausbildung und auch zur Beschleunigung des Verlaufs der proliferativen Retinopathie sowie der pathologischen Gefäßneubildung bei.

Bei der proliferativen Retinopathie (PVR) kommt es aufgrund einer Netzhautablösung zu einer Störung der Blut-Retina-Schranke. Durch die daraus resultierende Milieuänderung wandern retinale Zellen und Bestandteile des Blutes in den Glaskörper ein. Wachstumsfaktoren regen die mitotisch inaktiven RPE-Zellen zur Migration und Proliferation an. Im weiteren Verlauf der Erkrankung führt dies zur Ausbildung von Traktionsmembranen und zur Ablösung der Netzhaut. Die dadurch entstehenden Zugkräfte verhindern eine Wiederanlegung der Netzhaut. Ziel dieser Arbeit war es, zu untersuchen, welchen Einfluss Wachstumsfaktoren und deren Kombinationen auf die Proliferation und Migration humaner retinaler Pigmentepithelzellen, die VEGF-Sekretion durch RPE-Zellen und die mRNA-Expression von Kollagenen haben. Die beteiligten Signalwege sollten ebenfalls ermittelt werden. Im Ergebnis sollten Faktoren identifiziert werden, die die Entstehung einer PVR begünstigen. Migration, Proliferation, VEGF-Sekretion und die TGF-ß-Sekretion konnten durch den Wachstumsfaktor PDGF (Platelet-derived growth factor) gesteigert werden. Die Wirkung des PDGF-Signals wurde durch die Aktivierung verschiedener intrazellulärer Signalwege (MAPK, p38MAPK, PI3K/AKT) vermittelt. PDGF wirkte hemmend auf die Kollagen mRNA-Expression. Für TGF-ß (Transforming growth factor-ß) wurde eine hemmende Wirkung auf die Proliferation und eine induzierende Wirkung auf die Kollagensynthese nachgewiesen. Beide Faktoren – PDGF und TGF-ß – steigerten die VEGF-Sekretion. Eine additive Erhöhung der VEGF-Sekretion wurde durch die Kombination beider Faktoren nachgewiesen. Die untersuchten Wachstumsfaktoren interagieren mit den humanen retinalen Pigmentepithelzellen und tragen somit maßgeblich zur Ausbildung und auch zur Beschleunigung des Verlaufs der proliferativen Retinopathie sowie der pathologischen Gefäßneubildung bei.

Central to ocular health is the vitreous body, a complex, gelatinous tissue filling the space between the lens and retina. It is a natural polymeric hydrogel whose delicate architecture of collagen and hyaluronic acid loses its mechanical structure under the influence of degeneration or destruction, leaving the retina vulnerable to injury and disease. Since World War II, combat ocular trauma has increased six-fold while the population of aging veterans continues to grow in tandem. Compared to injuries in the civilian sector, injuries in theaters of combat operations are sustained in dirty, dusty, high-stress environments under hostile fire. These penetrating and perforating ocular injuries have predicable consequences, cascading into scarring on or under the retina (known as proliferative vitreoretinopathy or PVR). The growing population of aging veterans also faces a multitude of vitreous-related and vision-threatening pathologies. Current standards of care call for the removal and replacement of the vitreous, but contemporary substitutes are ill suited for long-term use. As such, there is critical need for development of a successful, long-term vitreous substitute. A biomimetic in situ forming hydrogel has been developed that utilizes a reversible disulfide cross-linker, enabling easy injection into the vitreous cavity. Recently, a copolymer has been introduced with this new formulation that possesses a unique comb-like structure whose characteristic bristles inhibit protein adsorption and cellular adherence, perfectly suited for the inhibition of PVR. The objective of this dissertation was to evaluate select formulations of this unique in situ forming hydrogel as potential vitreous substitutes. This was accomplished through rigorous in vivo rabbit modeled testing of long-term biocompatibility and bioperformance utilizing electroretinography, clinical examination, and histopathological assessment. We hypothesized that the in situ forming hydrogels would serve as a substantial improvement over the current gold standard, silicone oil, in terms of biocompatibility and the ability to inhibit PVR. / Bioengineering

Norrin is a secreted cystine-knot growth factor that plays critical roles in vascular development in the brain, retina, and cochlea, as well as the uterus. Although Norrin is unrelated to the lipid-modified morphogens Wnts, Norrin activates the canonical Wnt/β-catenin pathway by binding to receptor Frizzled4 cysteine-rich domain (Fz4-CRD) and co-receptors of low density lipoprotein receptor related protein 5/6 ectodomain (Lrp5/6-ECD) in conjunction with Tetraspanin-12 (Tspan-12). Like Wnts, Norrin has limited extracellular diffusion properties as a result of associating with heparan sulfate proteoglycans (HSPGs). Mutations lead to inherited disordered retinal vascularization diseases such as Norrie disease, familial exudative vitreoretinopathy and coats' disease. However, the molecular mechanism of how Norrin initiates signalling by engagement with Fz4, Lrp5/6, and HSPGs has remained unresolved. Here, novel strategies for protein production of recombinant human Norrin and Fz4-CRD as well as the complex are developed. The crystal structures of Norrin and its complex with Fz4-CRD, plus complex bound with the heparin mimic sucrose octasulphate, and unliganded structures of Fz4-CRD are presented. These structural data together with biophysical and cellular assays not only reveal the Fz4 and Lrp5/6 binding sites on distinct patches of the Norrin surface, but also indicate the HSPGs binding site on Norrin and Fz4-CRD as well as providing a framework to explain numerous disease-related mutations. Structural comparison with Xenopus Wnt8 in complex with mouse Fz8-CRD provides molecular insights for our understanding of ligand-receptor binding specificity and promiscuity, which has important implications for developing therapeutic strategies against Norrin dependent retinal disorders, and cancers caused by abnormal Wnt signaling.