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Thesis (Ph.D.)--The University of Texas Southwestern Medical Center at Dallas, 2006. / Embargoed. Vita. Bibliography.

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The purpose of this study is to examine the association between serum bilirubin and the development of age-related macular degeneration (AMD). The study design includes the utilization of a USA-nationally representative population based cross-sectional study in the National Health and Nutrition Examination Survey: specifically, the NHANES III and continuous NHANES from years 2005-2008. 15,501 survey participants from the NHANES studies chosen for this analysis were interviewed for demographic, behavioral, and medical information, put through a comprehensive medical examination segment, and a laboratory analysis portion. The 15,501 participants were chosen based on their age (40 and above) and the presence of fundus photographs. Fundus photographs were graded using standardized protocol to diagnose early and later AMD, which were combined to form the outcome "AMD" in a binary variable. Serum bilirubin levels were measured using spectrophotometry. Of the 15,501 participants in the study, 1305 (8.9%) were diagnosed with AMD. In a multivariate logistic regression adjusted for age, sex, smoking status, race, and serum C-reactive protein (CRP) levels, bilirubin was significantly associated with AMD (odds ratio, 0.728; confidence interval, 0.547-0.969; P value, 0.0296). The findings of this study indicate that the antioxidative effects of bilirubin may play protective role in the pathology of AMD.

Age-related macular degeneration (AMD) is the leading cause of blindness worldwide, affecting an estimated 50 million individuals aged over 65 years. Environmental and genetic risk-factors contribute to the development of AMD. An AMD-risk locus on chromosome 10q26 spans two genes, ARMS2 and HTRA1, and controversy exists as to which variants are responsible for increased risk of disease. Recent work suggests that HTRA1 expression levels are significantly increased in carriers of the risk haplotype associated with AMD. However, relatively little is known about the interactions, substrate specificity and roles in disease played by this secreted serine protease. This thesis aims to elucidate the potential role played by HTRA1 in AMD pathogenesis. A combination of tandem affinity purification (TAP) and yeast two-hybrid techniques was used to identify interacting partners of HTRA1. A number of proteins, with diverse roles in the alternative complement pathway, cell signaling, cell-matrix interactions, inflammation, angiogenesis and fibrosis, were identified. These are attractive candidates for further study as such processes are disturbed in AMD, implicating HTRA1 and its binding partners in disease development. One interacting partner, Complement Factor D (CFD), is a key activator in the alternative complement pathway. CFD, a 24 kDa serine protease, is expressed as an inactive zymogen, from which a signal peptide and activation peptide are cleaved before release of the mature, active protein into the circulation. In vitro studies show that CFD interacts with, and can be a substrate for, HTRA1. The interacting domain between the two proteins is localised to a region of 30 amino acids at the N-terminal end of proCFD. The 5 amino acid pro-peptide of CFD appears to be both necessary and sufficient for proteolysis of CFD by HTRA1. Investigation of the functional relevance of the interaction between HTRA1 and CFD shows that proCFD is cleaved by HTRA1, whilst mature CFD is not subjected to proteolysis. HTRA1-mediated cleavage of CFD forms an active protease, leading to activation of factor B in the alternative complement pathway in in vitro assays. Furthermore, a normal complement response is restored to CFD-depleted serum by addition of proCFD activated by HTRA1. Thus, an HTRA1- mediated increase in alternative complement pathway activity may explain a proportion of the AMD-risk attributed to the chr10q26 locus. Genetic and protein-based approaches were used to study the potential role of CFD in AMD pathogenesis, independent of an interaction with HTRA1. An intronic SNP, rs3826945, was significantly associated with increased risk of AMD in two British case-control cohorts, and in a combined meta-analysis with 4 additional cohorts from North America and Europe (p-value = 0.032, Odds Ratio = 1.112 in 4765 cases and 2693 controls). Assessment of copy number variation and sequencing of CFD did not identify any functional variants which may explain the association with disease. However, plasma levels of CFD were measured by ELISA in 751 AMD cases and 474 controls, and were found to be significantly elevated in AMD cases compared to controls (p-value = 0.00025). This further implicates complement activation in AMD pathogenesis, and makes CFD an attractive candidate for therapeutic intervention. An alteration in the level of activated CFD, possibly mediated via an interaction with HTRA1, either at the systemic or local tissue level, may play a role in disease development and progression.

Both proliferative diabetic retinopathy and exudative age-related macular degeneration are major causes of blindness which are caused by growth of defective, leaky and tortuous blood vessels in the retina. Hypoxia is implicated in triggering both of these diseases and results in induction of HIF-1alpha transcription factor in addition to the angiogenic factor VEGF. Müller cells are the major glial cell in the retina and they contribute to neovascularization in hypoxic regions of the retina through eliciting secretion of growth factors, cytokines and angiogenic factors. As Müller cells span the breadth of the retina they can secrete angiostatic factors as well as neuroprotective trophic factors, the Müller cell is a valuable cell type for targeting by potential new gene therapies. The current investigation tests the hypoxia responsiveness of an AAV vector containing a hybrid hypoxia response element together with a GFAP promoter, and this vector encodes the angiostatic protein decorin, a well characterized multi-receptor tyrosine kinase inhibitor. Decorin may have advantages over other key angiostatic factors such as endostatin or angiostatin by virtue of its multiple anti-angiogenic signaling modalities. We employed Q-RT-PCR to evaluate the cell specificity and hypoxia responsiveness of an AAV-Vector termed AAV-REG-Decorin containing a hybrid HRE and GFAP promoter driving expression of the decorin transgene. The vector also contains a silencer element between the HRE and the GFAP domains to enable low basal expression in normoxia as well as high level inducibility in hypoxia. AAV-REGDecorin was found to elicit high level expression of decorin mRNA in hypoxia with greater than 9 – fold induction of the transgene in hypoxic conditions in astrocytes by comparison to normoxic astrocytes. AAV-REG-Decorin showed low levels of transgene expression by comparison to the positive control vector AAV-CMV -decorin containing the ubiquitously active CMV-promoter. The expression levels of decorin mRNA from AAV-REG-Decorin and from AAV-GFAP-Decorin were low in the PC12 neuronal cell model and in the ARPE19 line of retinal pigment epithelial cells with respect to those of AAV-CMV-decorin and no induction of Decorin mRNA was found with AAV-REGDecorin in these two control cell lines. Our novel gene therapy vector will serve as a platform for testing efficacy in rodent disease models (OIR and laser induced choroidal neovascularization) for assessment of the benefits of tightly regulated antiangiogenic gene therapy eliciting decorin transgene expression, both in terms of timing and the cellular source of production, during the progression of the retinal pathophysiology. / Includes bibliography. / Dissertation (PhD)--Florida Atlantic University, 2021. / FAU Electronic Theses and Dissertations Collection

C1QTNF5 is a 25kDa short chain collagen of unknown function which is mutated in late-onset retinal macular degeneration (L-ORMD). L-ORMD is an autosomal dominant disease characterised by sub-retinal pigment epithelial deposits leading to photoreceptor death and visual loss and shows several similarities to age-related macular degeneration (AMD). A Tyr402His polymorphism in complement factor H (CFH), a regulatory protein in the innate immune system, has been associated with increased risk of AMD. C1QTNF5 and CFH are both expressed and secreted by the retinal pigment epithelium (RPE) which supports photoreceptors and is responsible for phagocytosis of shed rod photoreceptor outer segments (ROS). The properties of the normal C1QTNF5 and disease-associated Ser163Arg mutation were examined in detail, including protein characterisation, cellular processing and function. Recombinant wild type and mutant C1QTNF5 were produced and their multimerisation and solubility functions compared. Both proteins were found to be soluble and to form similar multimeric species which were resistant to reducing conditions, as seen in other short chain collagens. Due to the similarities between LORMD and AMD, a proposed interaction between C1QTNF5 and CFH was investigated. CFH is composed of 20 short consensus repeats (SCR) and interactions were confirmed between C1QTNF5 and both CFH and SCR modules 7-8 and 19-20. CFH showed a greater affinity for mutant C1QTNF5 compared with wild type on the basis of surface plasmon resonance assays. Stably transfected RPE-derived cell lines were created which expressed either wild type or mutant C1QTNF5. Both proteins were found to be secreted and showed similar cellular processing with no evidence of aggregation or retention of the mutant protein within the endoplasmic reticulum. In order to investigate C1QTNF5 function, phagocytosis of ROS by the stably transfected cell lines was carried out. Cells expressing wild type C1QTNF5 showed greater ROS phagocytosis compared with mutant C1QTNF5-expressing or untransfected cells. Addition of anti-C1QTNF5 antibody increased ROS phagocytosis further. In summary, it is proposed that wild type and mutant C1QTNF5 are secreted by the RPE where they interact with CFH. C1QTNF5 is also shown to have a role in ROS phagocytosis, with mutation in C1QTNF5 affecting phagocytosis efficiency, which may contribute to sub-RPE deposit formation. The results suggest that CFH may also be involved in this process, suggesting a common pathogenic pathway between L-ORMD and AMD.

Submitted by Natalia Vieira (natalia.vieira@famerp.br) on 2016-05-19T19:07:09Z No. of bitstreams: 1 sabrinamayaracezario_dissert.pdf: 2283086 bytes, checksum: 130b0d9b52a17f23980c982605a0dad9 (MD5) / Made available in DSpace on 2016-05-19T19:07:10Z (GMT). No. of bitstreams: 1 sabrinamayaracezario_dissert.pdf: 2283086 bytes, checksum: 130b0d9b52a17f23980c982605a0dad9 (MD5) Previous issue date: 2015-05-27 / Fundação de Amparo à Pesquisa do Estado de São Paulo - FAPESP / Background - Age-related macular degeneration (AMD) is a complex disease. The identification of risk factors may contribute to the prognosis and treatment. Objectives – Evaluate the influence of genetic variants related to lipid metabolism, angiogenesis and inflammation and its relation with clinical and lipid profile and lifestyle beyond their gene expression in patients with AMD. Casuistic and Methods – We studied 333 individuals aged ≥50 years, 108 with AMD in exudative form (G1); 45 with AMD in dry form (G2), and 180 individuals without clinical and angiographic signs of the disease (G3). The polymorphisms of apolipoprotein E (APOE-rs429358/rs7412), triphosphate binding cassette sub -family A transport -member 4 (ABCA4-rs472908), complement factor H (CFH-rs1061170) and vascular endothelial growth factor (VEGF-rs3025039/rs1570360) were analyzed by PCR/RFLP (polymerase chain reaction/restriction fragments length polymorphism), while the respective gene expression in blood by PCR/RT (reverse transcription-PCR) and serum levels of apo E, ABCR, CFH, VEGF by ELISA (enzyme linked immuno sorbent assay). Clinical and lipid profile data in addition to lifestyle were obtained from medical records and questionnaire. Level of significance was accepted for P<0.05. Results – Systemic arterial hypertension (SAH) and smoking prevailed in patients with AMD exudative form (P<0.05). Genetic polymorphisms: APOE- rs429358/rs7412 - APOE*3/3 was noted in all groups, followed by APOE*3/4, as well APOE*4 (P>0.05). ABCA4-rs472908 - Genotype A/G was more frequent in G3 (68%) versus G2 (44%; P<0.0001), while A/A in G2 (36%) versus G1 (19%; P=0.04) and G3 (14%; P=0.003). The mutant genotype (G/G) prevailed in combination (G1+G2) versus G3 (P< 0.0001), and allele G in all groups (P>0.05). CFH-rs1061170 – The wild homozygous (TT) was in evidence in G3 (58%) versus G1 (39%, P=0.003); as well the homozygous mutant (CC) in G1 (27 %) versus G3 (14%; P=0.002) and allele T in G3 (0.72) versus G1 (0.56; P=0.0002). VEGF-rs3025039 – Genotypic and allelic distribution were similar between groups (P>0.05), highlighting the CC genotype and allele C. VEGF-rs1570360 – Mutant homozygote (A/A) prevailed in G2 (21%) versus G1 (5%; P=0.002) and G3 (8%; P=0.015) as well as the wild type allele (G) G1 (0.75) and G3 (0.71) versus G2 (0.57, P=0.004, P=0.020, respectively). Gene expression – Similar values between groups for all analyzed genes (P>0.05). Serum levels (median values in ng/mL) – ApoE– Increased level in G1 (270.6) versus G2 (196.5; P<0.0001) and G3 (242.8; P=0.035), and G3 versus G2 (P=0.0002). ABCR – High levels in G1 (0.30) versus G2 (0.25; P=0.003) and G3 (0.25, P<0.0001). CFH – Increase in G1 (1198.9) versus G2 (859.8; P=0.0069), both higher than in G3 (618.3; P<0.001, P=0.001, respectively). VEGF – Similar values between groups (P>0.05). Lipid profile – G3 showed the highest level of HDLc (median=71mg /dL), compared to G2 (60mg/dL), and G1 (45mg/dL; P=0.003, P=0.029, respectively). Conclusion – Genetic variants of ABCA4, VEGF and CFH, besides SAH, smoking and increased serum levels of apo E, ABCR and CFH are associated with AMD, whereas the expression of the respective genes not differentiate AMD exsudative and dry forms, in contrast CFH (homozygous wild-type) has a protective character, as well as serum levels of HDLc. / Introdução – Degeneração macular relacionada à idade (DMRI) é uma doença complexa. A identificação de fatores de risco poderá contribuir no seu prognóstico e tratamento. Objetivos – Avaliar a influência de variantes genéticas, relacionadas com o metabolismo de lipídios, angiogênese e inflamação e sua relação com perfil clínico e lipídico e hábitos de vida, além das respectivas expressões gênicas em pacientes com DMRI. Casuística e Métodos – Foram estudados 333 indivíduos com idade ≥50 anos, sendo 108 com DMRI na forma exsudativa (G1); 45 com DMRI na forma seca (G2) e 180 indivíduos sem sinais clínicos e angiográficos da doença (G3). Os polimorfismos de apolipoproteína E (APOE-rs429358/rs7412), triphosphate binding cassette transporte sub-family A-member 4 (ABCA4-rs472908), complemento do fator H (CFH-rs1061170) e fator de crescimento endotelial vascular (VEGF-rs3025039/rs1570360) foram analisados por PCR/RFLP (polymerase chain reaction/restriction fragments lengh polymorphism), enquanto as respectivas expressões gênicas no sangue por PCR/RT (reverse transcription-PCR) e níveis séricos de apo E, ABCR, CFH, VEGF por ELISA (enzyme linked immuno sorbent assay). Dados de perfil clínico e lipídico, além de hábitos de vida, foram obtidos em prontuário médico e questionário. Admitiu-se nível de significância para P<0,05. Resultados – Hipertensão arterial sistêmica (HAS) e tabagismo prevaleceram em pacientes com DMRI exsudativa (P<0,05). Polimorfismos genéticos: APOE-rs429358/rs7412–APOE*3/3 destacou-se em todos os grupos, seguido de APOE*3/4, assim como o alelo APOE*4 (P>0,05). ABCA4-rs472908–O genótipo A/G foi mais frequente em G3 (68%) versus G2 (44%; P<0,0001), enquanto A/A em G2 (36%) versus G1 (19%; P=0,043) e G3 (14%; P=0,003). O genótipo mutante (G/G) prevaleceu na combinação (G1+G2) versus G3 (P<0,0001), e o alelo G em todos os grupos (P>0,05). CFH-rs1061170–O homozigoto selvagem (TT) destacou-se em G3 (58%) versus G1 (39%, P=0,003); já o homozigoto mutante (CC) em G1 (27%) versus G3 (14%; P=0,002), e o alelo T em G3 (0,72) versus G1 (0,56; P=0,0002). VEGF-rs3025039–Distribuição genotípica e alélica semelhante entre os grupos (P>0,05), destacando-se o genótipo CC e alelo C. VEGF-rs1570360–Homozigoto mutante (A/A) prevaleceu em G2 (21%) versus G1 (5%; P=0,002) e G3 (8%; P=0,015), assim como o alelo selvagem (G) em G1 (0,75) e G3 (0,71) versus G2 (0,57; P=0,004; P=0,020, respectivamente). Expressão gênica–Valores semelhantes entre os grupos para todos os genes analisados (P>0,05). Níveis séricos (valores de mediana em ng/mL)–ApoE–Aumento em G1 (270,6) versus G2 (196,5; P<0,0001) e G3 (242,8; P=0,035), e G3 versus G2 (P=0,0002). ABCR–Níveis elevados em G1 (0,30) versus G2 (0,25; P=0,003) e G3 (0,25; P<0,0001). CFH–Aumento em G1 (1.198,9) versus G2 (859,8; P=0,0069), ambos com acréscimo em relação a G3 (618,3; P<0,001; P=0,001, respectivamente). VEGF–Valores semelhantes entre os grupos (P>0,05). Perfil lipídico–G3 mostrou valor mais elevado de HDLc (mediana=71mg/dL), comparado a G2 (60mg/dL) e G1 (45mg/dL) (P=0,003; P=0,029, respectivamente). Conclusão – Variantes genéticas de ABCA4, VEGF e CFH, além de HAS, tabagismo e nível sérico elevado de apoE, ABCR e CFH associam-se a DMRI, enquanto a expressão dos referidos genes não diferencia DMRI exsudativa e seca, em contrapartida CFH (homozigoto selvagem) tem caráter protetor, assim como nível sérico de HDLc.

Age-related macular degeneration (AMD) is the leading cause of vision loss in individuals aged 50 years and older and is estimated to affect as many as 11 million individuals in the United States. The purpose of this study was to examine the association between vitamin D and AMD disease progression. The life course epidemiology framework model was used to explore how vitamin D level as a risk factor may have an association to AMD disease through time. Data in the 2005-2008 National Health and Nutrition Examination Survey (NHANES) database were collected on vitamin D levels and identified stages of AMD level based on graded fundus eye exams from an available sample size of 5,604 participants. A quantitative cross-sectional study approach was used to address this gap in knowledge. A bivariate analysis was used to examine each independent variable (age, race/ethnicity, smoking status, and diabetes) to the dependent variable AMD from the 2005-2008 NHANES dataset. A multivariate logistic regression analysis was performed with AMD including each independent variable found to be significant. The findings from this study failed to suggest an association between vitamin D levels to AMD, with or without the covariates included in the model. There was not an association found between vitamin D level and presence of AMD. An association was found between age, smoking, and race to presence of AMD in each of the bivariate models. The findings from this study could be used for positive social change by encouraging medical and public health agencies to target screening programs at high-risk age, smoking, and race groups. There remains to be conflicting data in the literature. This study adds to the body of literature suggesting that higher levels of vitamin D are not necessarily beneficial as they pertains to AMD.