Peter Saloupis

Daunorubicin treatment in a refined experimental model of proliferative vitreoretinopathy.

A condition similar to proliferative vitreoretinopathy (PVR) in man can be produced by injecting 25000 homologous dermal fibroblasts into rabbit eyes following gas compression of the vitreous. Daunorubicin (15 nmol) was effective in preventing retinal detachment in this model when injected simultaneously with the fibroblasts or in two doses (10 nmol followed by 5 nmol 4 h later) on the 3rd day after fibroblast injection. A single dose of 15 nmol on the 3rd day was not effective in preventing retinal detachment. These results suggest that daunorubicin may be clinically useful in preventing PVR when given by injection both at the time of vitrectomy as well as later, when protein exudation and pigment clumps in the vitreous cavity herald the onset of PVR.

Aryl hydrocarbon receptor deficiency causes dysregulated cellular matrix metabolism and age-related macular degeneration-like pathology

Significance Age-related Macular Degeneration (AMD) is the leading cause of vision loss. In its early stage, extracellular deposits accumulate below the retinal pigment epithelial layer (RPE), nurse cells to the retina. Identification of therapeutic treatments targeting deposit removal, which when left untreated exacerbate RPE and retinal damage, necessitates the discovery of pathways regulating deposit formation. We show that the activity of a nuclear receptor, essential to xenobiotic/toxin metabolism and cellular debris clearance, is critical to maintaining RPE cell health and that its deficiency in mice causes AMD pathology. This model provides a better understanding of AMD pathogenic mechanisms and a platform for testing novel therapeutics. The aryl hydrocarbon receptor (AhR) is a nuclear receptor that regulates xenobiotic metabolism and detoxification. Herein, we report a previously undescribed role for the AhR signaling pathway as an essential defense mechanism in the pathogenesis of early dry age-related macular degeneration (AMD), the leading cause of vision loss in the elderly. We found that AhR activity and protein levels in human retinal pigment epithelial (RPE) cells, cells vulnerable in AMD, decrease with age. This finding is significant given that age is the most established risk factor for development of AMD. Moreover, AhR−/− mice exhibit decreased visual function and develop dry AMD-like pathology, including disrupted RPE cell tight junctions, accumulation of RPE cell lipofuscin, basal laminar and linear-like deposit material, Bruch’s membrane thickening, and progressive RPE and choroidal atrophy. High-serum low-density lipoprotein levels were also observed in AhR−/− mice. In its oxidized form, this lipoprotein can stimulate increased secretion of extracellular matrix molecules commonly found in deposits from RPE cells, in an AhR-dependent manner. This study demonstrates the importance of cellular clearance via the AhR signaling pathway in dry AMD pathogenesis, implicating AhR as a potential target, and the mouse model as a useful platform for validating future therapies.

Estrogen receptor β protects against in vivo injury in RPE cells

Epidemiological data suggest that estrogen deficiency in postmenopausal women may contribute to the severity of AMD. We discovered that 17beta-estradiol (E2) was a crucial regulator of the severity of extracellular matrix turnover (ECM) dysregulation both in vivo and in vitro. We also found in vitro that the presence of estrogen receptor (ER)beta regulates MMP-2 activity. Therefore in an attempt to delineate the role of the ER subtypes, female estrogen receptor knockout (ERKO) mice were fed a high-fat diet, and the eyes were exposed to seven 5-second doses of nonphototoxic levels of blue-green light over 2 weeks. Three months after cessation of blue light treatment, transmission electron microscopy was performed to assess severity of deposits, Bruchs membrane changes, and choriocapillaris endothelial morphology. We found that changes in the trimolecular complex of pro-MMP-2, MMP-14 and TIMP-2 correlated with increased Bruchs membrane thickening or sub-retinal deposit formation (basal laminar deposits) in ERKObeta mice. In addition RPE isolated from ERKObeta mice had an increase in expression of total collagen and a decrease in MMP-2 activity. Finally we found that ERK an intermediate signaling molecule in the MMP pathway was activated in RPE isolated from ERKObeta mice. These data suggest that mice which lack ERbeta are more susceptible to in vivo injury associated with environmental light and high fat diet.

Bone Marrow Transplantation Transfers Age-Related Susceptibility to Neovascular Remodeling in Murine Laser- Induced Choroidal Neovascularization

PURPOSE Neovascular remodeling (NVR), the progression of small capillaries into large-caliber arterioles with perivascular fibrosis, represents a major therapeutic challenge in neovascular age-related macular degeneration (AMD). Neovascular remodeling occurs after laser-induced choroidal neovascularization (CNV) in aged but not young mice. Additionally, bone marrow-derived cells, including macrophages, endothelial precursor cells, and mesenchymal precursor cells, contribute to CNV severity. In this study, we investigated the impact of aged bone marrow transplantation (BMT) on the degree of fibrosis, size, and vascular morphology of CNV lesions in a mouse model of laser-induced CNV. METHODS Young (2 months) and old (16 months) mice were transplanted with green fluorescent protein (GFP)-labeled bone marrow isolated from either young or old donors. Laser CNV was induced 1 month following transplant, and eyes were analyzed via choroidal flat mounts and immunohistochemistry 1 month postlaser. The identity of cells infiltrating CNV lesions was determined using specific markers for the labeled transplanted cells (GFP+), macrophages (F4/80+), perivascular mesenchymal-derived cells (smooth muscle actin, SMA+), and endothelial cells (CD31+). RESULTS Bone marrow transplantation from aged mice transferred susceptibility to NVR into young recipients. Inversely, transplantation of young marrow into old mice prevented NVR, preserving small size and minimal fibrosis. Mice with NVR demonstrated a greater relative contribution of marrow-derived SMA+ perivascular mesenchymal cells as compared to other cells. CONCLUSIONS Our findings indicate that the status of bone marrow is an important determining factor of neovascular severity. Furthermore, we find that perivascular mesenchymal cells, rather than endothelial cells, derived from aged bone marrow may contribute to increased CNV severity in this murine model of experimental neovascularization.

Initial Observations of Key Features of Age-Related Macular Degeneration in APOE Targeted Replacement Mice

Age-related macular degeneration (AMD) is a late-onset, neurodegenerative disease of the retina and is the leading cause of catastrophic vision loss in the elderly. AMD usually occurs in people over the age of 65 years (Javitt et al., 2003) and accounts for approximately 50% of registered blindness in Western Europe and North America (Mitchell et al., 1998; Vingerling et al., 1995a). It develops as either dry AMD, geographic atrophy, or wet AMD (exudative) (Bird et al., 1995; Green, 1999). Dry AMD is characterized by the presence of sub-retinal pigment epithelium (RPE) deposits including drusen, basal linear deposits and basal laminar deposits (Curcio and Millican, 1999; Sarks, 1976). Geographic atrophy is characterized by RPE atrophy and wet or exudative AMD is characterized by choroidal neovascularization (CNV) (1991) and more recently, retinal angiomatous proliferation (Yannuzzi et al., 2001). In AMD with CNV, tufts of newly formed, functionally incompetent, blood vessels proliferate from the choroid, break through a thickened and fragile Bruch’s membrane (Grossniklaus and Green, 2004), and extend laterally into the sub-RPE (Sarks et al., 1980; Tobe et al., 1998a). These vessels in turn, may erode through the RPE, infiltrate the neural sensory retina, and communicate with the retinal circulation in what has been referred to as a retinal-choroidal anastomosis. This is common in the end stage of disciform disease (Yannuzzi et al., 2001).

Low dose aldosterone exposure causes increased retinal edema following laser-induced retinal vein occlusion in mice.

Purpose Cataract blindness accounts for a substantial proportion of blindness worldwide. Understanding the correlations between national levels of socioeconomic development with the quantity and quality of cataract surgery may provide insight for the prioritization and resource allocation for blindness prevention programs. Methods The relationships between human development index (HDI), gross domestic product (GDP) per capita, and cataract surgical coverage (CSC) and visual outcome of cataract surgery were examined in a multinational study utilizing secondary data from the repository for Rapid Assessment of Avoidable Blindness (RAAB), World Health Organization, Global Burden of Disease, United Nations, and the World Bank. Results A total of 266 RAAB studies across 73 countries/territories were retrieved. Linear regression model results revealed strong associations of HDI with prevalence of cataract blindness (β = -7.056, P < 0.001), CSC (β = 60.808, P = 0.004), proportion of intraocular lens (IOL) implantation (β = 87.040, P = 0.001), and proportion of cases with good vision outcomes among operated eyes (β = 73.351, P < 0.001) in studies performed between 1995 and 2009. Similar associations were observed for studies performed between 2010 and 2015. In addition, countries with lower GDP per capita showed a higher rate of cataract blindness (β = -0.527, P = 0.001), lower CSC (β = 9.800, P < 0.001), lower percentage of IOL implantation (β = 6.871, P = 0.001), and fewer patients with good vision outcomes after surgery (β = 7.959, P < 0.001). After controlling survey year, country, and other factors, GDP per capita and HDI were also found to be significantly associated with CSC and visual outcomes after cataract surgery (all P < 0.05). Conclusions We documented the strong associations of socioeconomic indices with quantity and quality of cataract surgery. These socioeconomic indicators should be considered as important factors for developing strategies aimed to improve worldwide cataract surgery service delivery.

Aldosterone Exposure Causes Increased Retinal Edema and Severe Retinopathy Following Laser-Induced Retinal Vein Occlusion in Mice

Purpose To determine the effects of aldosterone exposure on retinal edema and retinopathy in a mouse model of retinal vein occlusion (RVO). Methods RVO was induced immediately following intravenous injection of Rose bengal (66 mg/kg) using a 532-nm wavelength laser to place three to seven applications at 80 mW and 50-μm spot size directed at the superior retinal vein one disc diameter away from the nerve. Negative control consisted of placing an equal number of laser spots without targeting the vein. Male and female C57BL/6J mice aged 7 to 9 months with confirmed absence of Crb1rd8 were used. Aldosterone pellets releasing a daily dose of 0.83 μg/day were implanted subcutaneously 4 weeks prior to RVO. Retinal imaging by optical coherence tomography (OCT) was performed using a Micron IV rodent imaging system. Retinas were analyzed by immunohistochemistry using standard techniques. Retinal imaging and tissue analysis were performed 2, 4, and 7 days following RVO. Comparisons were made using Students t-test, ANOVA, and Pearsons χ2. Results RVO caused retinal edema in the form of cystic spaces and retinal thickening detectable by both OCT and histology. RVO also caused Müller glia (MG) dysfunction manifest as upregulated glial fibrillary acidic protein (GFAP) and altered localization of aquaporin 4 (AQP4) and Kir4.1. Treatment with aldosterone caused a significant increase in retinal edema and more severe retinopathy manifest as retinal whitening and extensive intraretinal hemorrhage. MG dysfunction was more severe and persistent in aldosterone-treated mice. Finally, aldosterone greatly increased the number of infiltrating mononuclear phagocytes following RVO. Conclusions Systemic aldosterone exposure causes a more severe RVO phenotype manifest as increased severity and duration of retinal edema and more severe retinopathy. The effects of aldosterone may be mediated by MG dysfunction and increased infiltration of mononuclear phagocytes. This suggests that small increases in aldosterone levels may be a risk factor for severe RVO.

Apolipoprotein E allele-dependent pathogenesis: a model for age-related retinal degeneration

Age-related macular degeneration (AMD) is a late-onset, multifactorial, neurodegenerative disease of the retina and the leading cause of irreversible vision loss in the elderly in the Western world. We describe here a murine model that combines three known AMD risk factors: advanced age, high fat cholesterol-rich (HF-C) diet, and apolipoprotein E (apoE) genotype. Eyes of aged, targeted replacement mice expressing human apoE2, apoE3, or apoE4 and maintained on a HF-C diet show apoE isoform-dependent pathologies of differential severity. ApoE4 mice are the most severely affected. They develop a constellation of changes that mimic the pathology associated with human AMD. These alterations include diffuse sub-retinal pigment epithelial deposits, drusenoid deposits, thickened Bruchs membrane, and atrophy, hypopigmentation, and hyperpigmentation of the retinal pigment epithelium. In extreme cases, apoE4 mice also develop marked choroidal neovascularization, a hallmark of exudative AMD. Neither age nor HF-C diet alone is sufficient to elicit these changes. We document choroidal neovascularization and other AMD-like ocular pathologies in an animal model that exploits known AMD risk factors. The model is additionally attractive because it is not complicated by invasive experimental intervention. Our findings in this model implicate the human apoE E4 allele as a susceptibility gene for AMD and support the hypothesis that common pathogenic mechanisms may underlie AMD and Alzheimers disease.