James T. Handa

Complement factor H binds malondialdehyde epitopes and protects from oxidative stress.

Oxidative stress and enhanced lipid peroxidation are linked to many chronic inflammatory diseases, including age-related macular degeneration (AMD). AMD is the leading cause of blindness in Western societies, but its aetiology remains largely unknown. Malondialdehyde (MDA) is a common lipid peroxidation product that accumulates in many pathophysiological processes, including AMD. Here we identify complement factor H (CFH) as a major MDA-binding protein that can block both the uptake of MDA-modified proteins by macrophages and MDA-induced proinflammatory effects in vivo in mice. The CFH polymorphism H402, which is strongly associated with AMD, markedly reduces the ability of CFH to bind MDA, indicating a causal link to disease aetiology. Our findings provide important mechanistic insights into innate immune responses to oxidative stress, which may be exploited in the prevention of and therapy for AMD and other chronic inflammatory diseases.

New steady-hand Eye Robot with micro-force sensing for vitreoretinal surgery

In retinal microsurgery, surgeons are required to perform micron scale maneuvers while safely applying forces to the retinal tissue that are below sensory perception. Real-time characterization and precise manipulation of this delicate tissue has thus far been hindered by human limits on tool control and the lack of a surgically compatible endpoint sensing instrument. Here we present the design of a new generation, cooperatively controlled microsurgery robot with a remote center-of-motion (RCM) mechanism and an integrated custom micro-force sensing surgical hook. Utilizing the forces measured by the end effector, we correct for tool deflections and implement a micro-force guided cooperative control algorithm to actively guide the operator. Preliminary experiments have been carried out to test our new control methods on raw chicken egg inner shell membranes and to capture useful dynamic characteristics associated with delicate tissue manipulations.

Development and Application of a New Steady-Hand Manipulator for Retinal Surgery

This paper describes the development and initial testing of a new and optimized version of a steady-hand manipulator for retinal microsurgery. In the steady-hand paradigm, the surgeon and the robot share control of a tool attached to the robot through a force sensor. The robot controller senses forces exerted by the operator on the tool and uses this information in various control modes to provide smooth, tremor-free, precise positional control and force scaling. The steady-hand manipulator reported here has been specifically designed with the unique constraints of retinal microsurgery in mind. In particular, the system makes use of a compact wrist design that places the bulk of the robot away from the operating field. The resulting system has high efficacy, flexibility and ergonomics while meeting the accuracy and safety requirements of microsurgery. We have now tested this robot on a biological model system and we report a protocol for reliably cannulating ~80 mum OD veins (the size of veins in the human retina) using the system

Glycation-altered proteolysis as a pathobiologic mechanism that links dietary glycemic index, aging, and age-related disease (in nondiabetics)

Epidemiologic studies indicate that the risks for major age‐related debilities including coronary heart disease, diabetes, and age‐related macular degeneration (AMD) are diminished in people who consume lower glycemic index (GI) diets, but lack of a unifying physiobiochemical mechanism that explains the salutary effect is a barrier to implementing dietary practices that capture the benefits of consuming lower GI diets. We established a simple murine model of age‐related retinal lesions that precede AMD (hereafter called AMD‐like lesions). We found that consuming a higher GI diet promotes these AMD‐like lesions. However, mice that consumed the lower vs. higher GI diet had significantly reduced frequency (P < 0.02) and severity (P < 0.05) of hallmark age‐related retinal lesions such as basal deposits. Consuming higher GI diets was associated with > 3 fold higher accumulation of advanced glycation end products (AGEs) in retina, lens, liver, and brain in the age‐matched mice, suggesting that higher GI diets induce systemic glycative stress that is etiologic for lesions. Data from live cell and cell‐free systems show that the ubiquitin–proteasome system (UPS) and lysosome/autophagy pathway [lysosomal proteolytic system (LPS)] are involved in the degradation of AGEs. Glycatively modified substrates were degraded significantly slower than unmodified substrates by the UPS. Compounding the detriments of glycative stress, AGE modification of ubiquitin and ubiquitin‐conjugating enzymes impaired UPS activities. Furthermore, ubiquitin conjugates and AGEs accumulate and are found in lysosomes when cells are glycatively stressed or the UPS or LPS/autophagy are inhibited, indicating that the UPS and LPS interact with one another to degrade AGEs. Together, these data explain why AGEs accumulate as glycative stress increases.

Cigarette smoking, oxidative stress, the anti-oxidant response through Nrf2 signaling, and Age-related Macular Degeneration.

Age-related Macular Degeneration (AMD) is the leading cause of blindness among the elderly. While excellent treatment has emerged for neovascular disease, treatment for early AMD is lacking due to an incomplete understanding of the early molecular events. Cigarette smoking is the strongest epidemiologic risk factor, yet we do not understand how smoking contributes to AMD. Smoking related oxidative damage during the early phases of AMD may play an important role. This review explores how cigarette smoking and oxidative stress to the retinal pigmented epithelium (RPE) might contribute to AMD, and how the transcription factor Nrf2 can activate a cytoprotective response.

Long-Term Results from an Epiretinal Prosthesis to Restore Sight to the Blind

PURPOSE Retinitis pigmentosa (RP) is a group of inherited retinal degenerations leading to blindness due to photoreceptor loss. Retinitis pigmentosa is a rare disease, affecting only approximately 100 000 people in the United States. There is no cure and no approved medical therapy to slow or reverse RP. The purpose of this clinical trial was to evaluate the safety, reliability, and benefit of the Argus II Retinal Prosthesis System (Second Sight Medical Products, Inc, Sylmar, CA) in restoring some visual function to subjects completely blind from RP. We report clinical trial results at 1 and 3 years after implantation. DESIGN The study is a multicenter, single-arm, prospective clinical trial. PARTICIPANTS There were 30 subjects in 10 centers in the United States and Europe. Subjects served as their own controls, that is, implanted eye versus fellow eye, and system on versus system off (native residual vision). METHODS The Argus II System was implanted on and in a single eye (typically the worse-seeing eye) of blind subjects. Subjects wore glasses mounted with a small camera and a video processor that converted images into stimulation patterns sent to the electrode array on the retina. MAIN OUTCOME MEASURES The primary outcome measures were safety (the number, seriousness, and relatedness of adverse events) and visual function, as measured by 3 computer-based, objective tests. RESULTS A total of 29 of 30 subjects had functioning Argus II Systems implants 3 years after implantation. Eleven subjects experienced a total of 23 serious device- or surgery-related adverse events. All were treated with standard ophthalmic care. As a group, subjects performed significantly better with the system on than off on all visual function tests and functional vision assessments. CONCLUSIONS The 3-year results of the Argus II trial support the long-term safety profile and benefit of the Argus II System for patients blind from RP. Earlier results from this trial were used to gain approval of the Argus II by the Food and Drug Administration and a CE mark in Europe. The Argus II System is the first and only retinal implant to have both approvals.

Chronic cigarette smoke causes oxidative damage and apoptosis to retinal pigmented epithelial cells in mice

The purpose of this study was to determine whether mice exposed to chronic cigarette smoke develop features of early age-related macular degeneration (AMD). Two month old C57Bl6 mice were exposed to either filtered air or cigarette smoke in a smoking chamber for 5 h/day, 5 days/week for 6 months. Eyes were fixed in 2.5% glutaraldehyde/2% paraformaldehyde and examined for ultrastructural changes by transmission electron microscopy. The contralateral eye was fixed in 2% paraformaldehyde and examined for oxidative injury to the retinal pigmented epithelium (RPE) by 8-oxo-7,8-dihydro-2′-deoxyguanosine (8-OHdG) immunolabeling and apoptosis by TUNEL labeling. Mice exposed to cigarette smoke had immunolabeling for 8-OHdG in 85±3.7% of RPE cells counted compared to 9.5±3.9% in controls (p<0.00001). Bruch membrane was thicker in mice exposed to smoke (1086±332 nm) than those raised in air (543±132 nm; p = 0.0069). The two most pronounced ultrastructural changes (severity grading scale from 0–3) seen were a loss of basal infoldings (mean difference in grade = 1.98; p<0.0001), and an increase in intracellular vacuoles (mean difference in grade = 1.7; p<0.0001). Ultrastructural changes to Bruch membrane in cigarette-smoke exposed mice were smaller in magnitude but consistently demonstrated significantly higher grade injury in cigarette-exposed mice, including basal laminar deposits (mean difference in grade = 0.54; p<0.0001), increased outer collagenous layer deposits (mean difference in grade = 0.59; p = 0.002), and increased basal laminar deposit continuity (mean difference in grade = 0.4; p<0.0001). TUNEL assay showed a higher percentage of apoptotic RPE from mice exposed to cigarette smoke (average 8.0±1.1%) than room air (average 0±0%; p = 0.043). Mice exposed to chronic cigarette smoke develop evidence of oxidative damage with ultrastructural degeneration to the RPE and Bruch membrane, and RPE cell apoptosis. This model could be useful for studying the mechanism of smoke induced changes during early AMD.

Different effects of angiopoietin-2 in different vascular beds: new vessels are most sensitive

In this study, we used double transgenic mice with inducible expression of angiopoietin‐2 (Ang2) to investigate the role of Ang2 in the retinal and choroidal circulations and in three models of ocular neovascularization (NV). Mice with induced expression of Ang2 ubiquitously, or specifically in the retina, survived and appeared grossly normal. They also had normal‐appearing retinal and choroidal circulations, demonstrating that high levels of Ang2 did not induce regression of mature retinal or choroidal vessels. When Ang2 expression was induced soon after birth, there was increased density of the deep capillary bed on postnatal day (P) 11 that returned to normal by P18, the time that retinal vascular development is usually completed. In mice with ischemic retinopathy, induction of Ang2 during the ischemic period resulted in a significant increase in retinal NV, but induction of Ang2 at a later time point when ischemia (and vascular endothelial growth factor [VEGF]) was less, hastened regression of NV. In triple transgenic mice that coexpressed VEGF and Ang2, the increased expression of Ang2 inhibited VEGF‐induced NV in the retina. Increased expression of Ang2 also resulted in regression of choroidal neovascularization. These data suggest that ocular neovascularization, but not mature retinal or choroidal vessels, is sensitive to Ang2; a high Ang2/VEGF ratio promotes regression, while high Ang2 in the setting of hypoxia and/or concomitantly high Ang2 and VEGF stimulate neovascularization.

Histologic localization of indocyanine green dye in aging primate and human ocular tissues with clinical angiographic correlation

OBJECTIVE This study aimed to histologically localize indocyanine green (ICG) dye in the geriatric primate and human eye and to correlate these findings with clinical ICG angiography. DESIGN The study design was a clinicopathologic correlation. PARTICIPANTS Six eyes of three geriatric monkeys (Maccaca mulatta) with macular drusen, 19 to 29 years of age, housed at the California Primate Research Center and an enucleated human eye from a 66-year-old patient with choroidal melanoma were examined. INTERVENTION All six monkey eyes and the human eye underwent clinical ICG angiography. Five monkey eyes were enucleated at varying intervals after intravenous ICG dye injection for histologic examination. One monkey eye was removed without prior ICG injection as an age-matched control. The human eye was enucleated after intravenous injection of ICG dye. MAIN OUTCOME MEASURES Infrared fluorescence microscopy of freeze-dried tissue sections was performed to detect ICG fluorescence. Histologic sections were stimulated with an 810-nm diode laser, and the fluorescence emitted was detected with a Hamamatsu infrared camera. The images were digitally recorded. The distribution of fluorescence on histologic examination was correlated with the fluorescence of the clinical ICG angiogram. RESULTS Infrared fluorescence microscopy of monkey sections localized fluorescence within retinal and choroidal vessels early after injection of ICG dye. The ICG fluorescence was seen in the extravascular choroidal stroma within 10 minutes after injection. The stromal fluorescence persisted in sections obtained 50 minutes after injection of ICG. The retinal pigment epithelium (RPE)-Bruchs membrane complex was brightly fluorescent in the middle- and late-stage histologic sections. Drusen deposits were brightly fluorescent at all timepoints examined. Similar findings were observed in freeze-dried tissue sections of the human eye. The fluorescence detected on histologic sections correlated closely with the fluorescence of the clinical ICG angiograms for the same interval. CONCLUSIONS The ICG dye does not remain solely within the choroidal intravascular space but extravasates into the choroidal stroma and accumulates within the RPE. Extravascular ICG binds to drusen material. These findings will enhance the interpretation of clinical ICG angiography.

Microarray analysis of H2O2-, HNE-, or tBH-treated ARPE-19 cells

Oxidative stress plays a key role in aging diseases of the posterior pole of the eye such as age-related macular degeneration. The oxidative stress response of in vitro RPE cells has been studied for a small number of genes. However, a comprehensive transcriptional response has yet to be elucidated. The purpose of this study was to determine if the transcription of a common set of genes is altered by exposure of ARPE-19 cells to three major generators of oxidative stress, hydrogen peroxide (H2O2), 4-hydroxynonenal (HNE), and tert-butylhydroperoxide (tBH). As expected, a common response was observed that included 35 genes differentially regulated by all three treatments. Of these, only one gene was upregulated, and only by one oxidant, while all other responses were downregulation. The majority of these genes fell into five functional categories: apoptosis, cell cycle regulation, cell-cell communication, signal transduction, and transcriptional regulation. Additionally, a large number of genes were differentially regulated by one oxidant only, including the majority of the conventional oxidative stress response genes present on the Clontech Human 1.2 microarray. This study raises questions regarding the generality of results that involve the use of a single oxidant and a single cell culture condition.