Sandra R. Montezuma

Review of Genetics in Age Related Macular Degeneration

Age-related macular degeneration (AMD) is a degenerative disease of the retina and the leading cause of blindness in industrialized countries. AMD is a complex disease caused by the combination of genetic predisposition and environmental factors. The prevalence of AMD increases with age. The adverse effect of smoking is well established. Genetic predisposition has been demonstrated by familial aggregation studies and twin studies. Using genome linkage scan and association studies, multiple potentially causative genes have been identified. The chromosomes most commonly implicated are 1q25-31 and 10q26. In particular, variants in the gene for the complement factor H (CFH) and the genes PLEKHA1/LOC387715/HTRA1, Factor B (BF) and complement component 2 (C2) have been implicated as major risk or protective factors for the development of AMD. There have been some advances in the treatment of this condition; however, a complete cure remains remote but hopeful. Understanding the causative environmental and genetic interactions will facilitate the development of future preventive methods and treatments.

Investigating mitochondria as a target for treating age-related macular degeneration

Age-related macular degeneration (AMD) is the leading cause of blindness among older adults in the developed world. Although the pathological mechanisms have not been definitively elucidated, evidence suggests a key role for mitochondrial (mt) dysfunction. The current study used our unique collection of human retinal samples graded for the donors stage of AMD to address fundamental questions about mtDNA damage in the retina. To evaluate the distribution of mtDNA damage in the diseased retina, damage in the retinal pigment epithelium (RPE) and neural retina from individual donors were compared. To directly test a long-held belief that the macula is selectively damaged with AMD, RPE mtDNA damage was measured in the macula and peripheral sections from individual donors. Small segments of the entire mt genome were examined to determine whether specific regions are preferentially damaged. Our results show that mtDNA damage is limited to the RPE, equivalent mtDNA damage is found in the macular and peripheral RPE, and sites of damage are localized to regions of the mt genome that may impact mt function. These results provide a scientific basis for targeting the RPE mitochondria with therapies that protect and enhance mt function as a strategy for combating AMD.

Review of the Ocular Angiogenesis Animal Models

Increasing interest in developing reliable and reproducible models to study angiogenesis has emerged due to recent advances in the treatment of eye disease with pathologic angiogenesis. This review provides a summary of the principal ocular animal models for angiogenesis. Models of anterior segment neovascularization include the corneal micropocket assay, used to study the influence of specific molecules/proteins in angiogenesis, and corneal chemical and suture induced injury, which mimic more closely the complex nature of the human disease. Angiogenesis models of the posterior segment include the well-known laser-induced injury of the choroid/Bruchs membrane, as well as the oxygen induced retinopathy and models of injections of pro-angiogenic/inflammatory molecules. In addition, knockout or knock-in transgenic mice provide powerful tools in studying the role of specific proteins in angiogenesis.

Evaluation of Subretinal Implants Coated with Amorphous Aluminum Oxide and Diamond-like Carbon

Retinal prostheses may be used to support patients suffering from age-related macular degeneration (AMD) or retinitis pigmentosa (RP). A hermetic encapsulation of the poly(imide) (PI)-based prosthesis is important in order to prevent the leakage of water and ions into the electric circuitry embedded in the poly(imide) matrix. The deposition of amorphous aluminum oxide (by sputtering) and diamond like carbon (by pulsed laser ablation) were made for applications in retinal prostheses. The thin films obtained were characterized for composition, thickness, adhesion and smoothness by scanning electron microscopy-energy dispersive spectroscopy, atomic force microscopy, profilometry and light microscopy. Biocompatibility was tested in vivo by implanting coated specimen subretinally in the eye of Yucatan pigs. While amorphous aluminum oxide is more readily deposited with sufficient adhesion quality, superior biocompatibility behavior was shown by diamond-like carbon. Amorphous aluminum oxide had more adverse effects and caused more severe damage to the retinal tissue.

A Novel Nonradioactive Method to Evaluate Vascular Barrier Breakdown and Leakage

PURPOSE To identify a novel, sensitive, nonradioactive leakage assay that can be used in the assessment of retinal vascular permeability in rats and mice. METHODS Breakdown of the vascular barrier was induced by vascular endothelial growth factor (VEGF), lipopolysaccharide (LPS), or diabetes. Biotinylated bovine serum albumin (bBSA) was administered as a tracer. After perfusion with lactated Ringers solution, extravasated bBSA was detected with immunoprecipitation and Western blot analysis or sandwich ELISA. The results were then normalized against the final bBSA plasma concentration, the circulation time, and the protein concentration of the tissue. RESULTS Six hours after VEGF injection, BRB breakdown was quantified in the injected eye and was 2.5-fold higher than in the contralateral phosphate-buffered saline (PBS)-injected eye (n = 6 rats, P < 0.01). Intravitreal LPS injection induced severe inflammation in the directly injected eye and moderate inflammation in the contralateral untreated eye. Leakage was six- and threefold higher, respectively, compared with that in the untreated control animals (n = 5 rats, P < 0.01). Nine-month diabetic rats had a threefold increase in vascular leakage compared with age-matched control animals (n = 6 retinas, P < 0.05). Twenty-four hours after intraperitoneal administration of LPS in mice, the animals showed increased vascular leakage in all tissue organs examined (retina, 1.7-fold; brain, 1.5-fold; and kidney, 1.3-fold). CONCLUSIONS bBSA can serve as an effective alternative to the current methods used for quantitating vascular leakage and especially the blood-retinal barrier breakdown. It is reasonably easy to perform, low in cost, and adaptable to experiments in mice.

Increased retinal mtDNA damage in the CFH variant associated with age-related macular degeneration

Age-related macular degeneration (AMD) is a major cause of blindness among the elderly in the developed world. Genetic analysis of AMD has identified 34 high-risk loci associated with AMD. The genes at these high risk loci belong to diverse biological pathways, suggesting different mechanisms leading to AMD pathogenesis. Thus, therapies targeting a single pathway for all AMD patients will likely not be universally effective. Recent evidence suggests defects in mitochondria (mt) of the retinal pigment epithelium (RPE) may constitute a key pathogenic event in some AMD patients. The purpose of this study is to determine if individuals with a specific genetic background have a greater propensity for mtDNA damage. We used human eyebank tissues from 76 donors with AMD and 42 age-matched controls to determine the extent of mtDNA damage in the RPE that was harvested from the macula using a long extension polymerase chain reaction assay. Genotype analyses were performed for ten common AMD-associated nuclear risk alleles (ARMS2, TNFRSF10A, CFH, C2, C3, APOE, CETP, LIPC, VEGF and COL10A1) and mtDNA haplogroups. Sufficient samples were available for genotype association with mtDNA damage for TNFRSF10A, CFH, CETP, VEGFA, and COL10A1. Our results show that AMD donors carrying the high risk allele for CFH (C) had significantly more mtDNA damage compared with donors having the wild-type genetic profile. The data from an additional 39 donors (12 controls and 27 AMD) genotyped for CFH alleles further supported these findings. Taken together, these studies provide the rationale for a more personalized approach for treating AMD by uncovering a significant correlation between the CFH high risk allele and accelerated mtDNA damage. Patients harboring this genetic risk factor may benefit from therapies that stabilize and protect the mt in the RPE.

Silent sinus syndrome presenting as enophthalmos long after orbital trauma

Late enophthalmos is a well-known consequence of large orbital floor fractures. In rare cases, late enophthalmos can occur after direct trauma to the maxillary ostiomeatal complex and present as silent sinus syndrome (SSS). We report two cases of SSS manifesting as enophthalmos years after facial trauma. The first patient developed SSS 4 years after a minimally displaced orbital floor fracture. The second patient had progressive enophthalmos as a result of atelectasis of the maxillary sinus years after facial trauma and surgical repair of nasal fractures. There have been two prior reports of SSS presenting after orbital trauma. Our patients differ from these prior reports in that the enophthalmos was discovered years after the initial facial trauma. In the first patient, surgery addressing the blockage of the ostiomeatal complex arrested the enophthalmos; in the second patient, it reversed the enophthalmos.

Pseudo-hypopyon as the presenting feature of recurrent B-cell lymphoma

Purpose: To report a case of a pseudo-hypopyon as the presentation for recurrent B-cell lymphoma. Design: Case report. Methods: Case report with anterior segment photograph. Results: A 74-year-old man with past medical history of peripheral B-cell lymphoma developed a hypopyon 3 months after R-CHOP chemotherapy and prophylactic intrathecal chemotherapy. Anterior chamber aspirate was consistent with B-cell lymphoma. Conclusions: Pseudo-hypopyon as a manifestation of recurrent B-cell lymphoma is a rare clinical presentation of an intraocular malignancy. A high index of suspicion along with intraocular fluid analysis are essential to establish the diagnosis.

Differential recovery of the electroretinogram, visually evoked cortical potential, and electrically evoked cortical potential following vitrectomy: Implications for acute testing of an implanted retinal prosthesis

To determine the extent to which electrophysiologic tests of the afferent visual pathway are affected by vitrectomy, the procedure was performed in 15 eyes of 11 adult Dutch-belted rabbits. An electroretinogram (ERG), visually evoked cortical potential (VECP), and electrically evoked cortical potential (EECP) were obtained preoperatively and sequentially after surgery. For electrical stimulations, biphasic impulses were delivered to the retina. Post-vitrectomy declines of 49, 25, and 41% from the median baseline amplitudes and increases of 13, 18, and 17% from the median baseline latency values were found for ERG, VECP, and EECP, respectively. At 90 min, 13 to 30% of eyes still had an amplitude more than 10% below baseline on at least one of the three tests, whereas 10 to 47% of eyes had an abnormal latency more than 10% above baseline on at least one of the three tests. Amplitudes were more likely than latencies to return to near baseline, but for eyes that remained subnormal, the decline was greater for amplitudes than latencies. Significant alterations in retinal function, manifested by declines in amplitudes and increases in latencies of the ERG, VECP, and EECP, persist in a large proportion of eyes up to 90 min post-vitrectomy.

Altered bioenergetics and enhanced resistance to oxidative stress in human retinal pigment epithelial cells from donors with age-related macular degeneration

Age-related macular degeneration (AMD) is the leading cause of blindness among older adults. It has been suggested that mitochondrial defects in the retinal pigment epithelium (RPE) underlies AMD pathology. To test this idea, we developed primary cultures of RPE to ask whether RPE from donors with AMD differ in their metabolic profile compared with healthy age-matched donors. Analysis of gene expression, protein content, and RPE function showed that these cultured cells replicated many of the cardinal features of RPE in vivo. Using the Seahorse Extracellular Flux Analyzer to measure bioenergetics, we observed RPE from donors with AMD exhibited reduced mitochondrial and glycolytic function compared with healthy donors. RPE from AMD donors were also more resistant to oxidative inactivation of these two energy-producing pathways and were less susceptible to oxidation-induced cell death compared with cells from healthy donors. Investigation of the potential mechanism responsible for differences in bioenergetics and resistance to oxidative stress showed RPE from AMD donors had increased PGC1α protein as well as differential expression of multiple genes in response to an oxidative challenge. Based on our data, we propose that cultured RPE from donors phenotyped for the presence or absence of AMD provides an excellent model system for studying “AMD in a dish”. Our results are consistent with the ideas that (i) a bioenergetics crisis in the RPE contributes to AMD pathology, and (ii) the diseased environment in vivo causes changes in the cellular profile that are retained in vitro.