Transcriptome Analysis of Retinal and Choroidal Pathologies in Aged BALB/c Mice Following Systemic Neonatal Murine Cytomegalovirus Infection.

Our previous studies have shown that systemic neonatal murine cytomegalovirus (MCMV) infection of BALB/c mice spread to the eye with subsequent establishment of latency in choroid/RPE. In this study, RNA sequencing (RNA-Seq) analysis was used to determine the molecular genetic changes and pathways affected by ocular MCMV latency. MCMV (50 pfu per mouse) or medium as control were injected intra-peritoneally (i.p.) into BALB/c mice at <3 days after birth. At 18 months post injection, the mice were euthanized, and the eyes were collected and prepared for RNA-Seq. Compared to three uninfected control eyes, we identified 321 differentially expressed genes (DEGs) in six infected eyes. Using the QIAGEN Ingenuity Pathway Analysis (QIAGEN IPA), we identified 17 affected canonical pathways, 10 of which function in neuroretinal signaling, with the majority of DEGs being downregulated, while 7 pathways function in upregulated immune/inflammatory responses. Retinal and epithelial cell death pathways involving both apoptosis and necroptosis were also activated. MCMV ocular latency is associated with upregulation of immune and inflammatory responses and downregulation of multiple neuroretinal signaling pathways. Cell death signaling pathways are also activated and contribute to the degeneration of photoreceptors, RPE, and choroidal capillaries.

Retinal and Choroidal Pathologies in Aged BALB/c Mice Following Systemic Neonatal Murine Cytomegalovirus Infection.

Although pathologies associated with acute virus infections have been extensively studied, the effects of long-term latent virus infections are less well understood. Human cytomegalovirus, which infects 50% to 80% of humans, is usually acquired during early life and persists in a latent state for the lifetime. The purpose of this study was to determine whether systemic murine cytomegalovirus (MCMV) infection acquired early in life disseminates to and becomes latent in the eye and if ocular MCMV can trigger in situ inflammation and occurrence of ocular pathology. This study found that neonatal infection of BALB/c mice with MCMV resulted in dissemination of virus to the eye, where it localized principally to choroidal endothelia and pericytes and less frequently to the retinal pigment epithelium (RPE) cells. MCMV underwent ocular latency, which was associated with expression of multiple virus genes and from which MCMV could be reactivated by immunosuppression. Latent ocular infection was associated with significant up-regulation of several inflammatory/angiogenic factors. Retinal and choroidal pathologies developed in a progressive manner, with deposits appearing at both basal and apical aspects of the RPE, RPE/choroidal atrophy, photoreceptor degeneration, and neovascularization. The pathologies induced by long-term ocular MCMV latency share features of previously described human ocular diseases, such as age-related macular degeneration.

Invasion of Human Retinal Pigment Epithelial Cells by Porphyromonas gingivalis leading to Vacuolar/Cytosolic localization and Autophagy dysfunction In-Vitro.

Recent epidemiological  studies link Periodontal disease(PD) to age-related macular degeneration (AMD). We documented earlier that Porphyromonas gingivalis(Pg), keystone oral-pathobiont, causative of PD, efficiently invades human gingival epithelial and blood-dendritic cells. Here, we investigated the ability of dysbiotic Pg-strains to invade human-retinal pigment epithelial cells(ARPE-19), their survival, intracellular localization, and the pathological effects, as dysfunction of RPEs leads to AMD. We show that live, but not heat-killed Pg-strains adhere to and invade ARPEs. This involves early adhesion to ARPE cell membrane, internalization and localization of Pg within single-membrane vacuoles or cytosol, with some nuclear localization apparent. No degradation of Pg or localization inside double-membrane autophagosomes was evident, with dividing Pg suggesting a metabolically active state during invasion. We found significant downregulation of autophagy-related genes particularly, autophagosome complex. Antibiotic protection-based recovery assay further confirmed distinct processes of adhesion, invasion and amplification of Pg within ARPE cells. This is the first study to demonstrate invasion of human-RPEs, begin to characterize intracellular localization and survival of Pg within these cells. Collectively, invasion of RPE by Pg and its prolonged survival by autophagy evasion within these cells suggest a strong rationale for studying the link between oral infection and AMD pathogenesis in individuals with periodontitis.

Ocular cytomegalovirus latency exacerbates the development of choroidal neovascularization.

Age-related macular degeneration (AMD) is a complex, multifactorial, progressive disease which represents a leading cause of irreversible visual impairment and blindness in older individuals. Human cytomegalovirus (HCMV), which infects 50-80% of humans, is usually acquired during early life and persists in a latent state for the life of the individual. In view of its previously described pro-angiogenic properties, we hypothesized that cytomegalovirus might be a novel risk factor for progression to an advanced form, neovascular AMD, which is characterized by choroidal neovascularization (CNV). The purpose of this study was to investigate if latent ocular murine cytomegalovirus (MCMV) infection exacerbated the development of CNV in vascular endothelial growth factor (VEGF)-overexpressing VEGF-Ahyper mice. Here we show that neonatal infection with MCMV resulted in dissemination of virus to various organs throughout the body including the eye, where it localized principally to the choroid in both VEGF-overexpressingVEGF-Ahyper and wild-type(WT) 129 mice. By 6 months post-infection, no replicating virus was detected in eyes and extraocular tissues, although virus DNA was still present in all eyes and extraocular tissues of both VEGF-Ahyper and WT mice. Expression of MCMV immediate early (IE) 1 mRNA was detected only in latently infected eyes of VEGF-Ahyper mice, but not in eyes of WT mice. Significantly increased CNV was observed in eyes of MCMV-infected VEGF-Ahyper mice compared to eyes of uninfected VEGF-Ahyper mice, while no CNV lesions were observed in eyes of either infected or uninfected WT mice. Protein levels of several inflammatory/angiogenic factors, particularly VEGF and IL-6, were significantly higher in eyes of MCMV-infected VEGF-Ahyper mice, compared to uninfected controls. Initial studies of ocular tissue from human cadavers revealed that HCMV DNA was present in four choroid/retinal pigment epithelium samples from 24 cadavers. Taken together, our data suggest that ocular HCMV latency could be a significant risk factor for the development of AMD. © 2020 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

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.

Macrophage activation associated with chronic murine cytomegalovirus infection results in more severe experimental choroidal neovascularization.

The neovascular (wet) form of age-related macular degeneration (AMD) leads to vision loss due to choroidal neovascularization (CNV). Since macrophages are important in CNV development, and cytomegalovirus (CMV)-specific IgG serum titers in patients with wet AMD are elevated, we hypothesized that chronic CMV infection contributes to wet AMD, possibly by pro-angiogenic macrophage activation. This hypothesis was tested using an established mouse model of experimental CNV. At 6 days, 6 weeks, or 12 weeks after infection with murine CMV (MCMV), laser-induced CNV was performed, and CNV severity was determined 4 weeks later by analysis of choroidal flatmounts. Although all MCMV-infected mice exhibited more severe CNV when compared with control mice, the most severe CNV developed in mice with chronic infection, a time when MCMV-specific gene sequences could not be detected within choroidal tissues. Splenic macrophages collected from mice with chronic MCMV infection, however, expressed significantly greater levels of TNF-α, COX-2, MMP-9, and, most significantly, VEGF transcripts by quantitative RT-PCR assay when compared to splenic macrophages from control mice. Direct MCMV infection of monolayers of IC-21 mouse macrophages confirmed significant stimulation of VEGF mRNA and VEGF protein as determined by quantitative RT-PCR assay, ELISA, and immunostaining. Stimulation of VEGF production in vivo and in vitro was sensitive to the antiviral ganciclovir. These studies suggest that chronic CMV infection may serve as a heretofore unrecognized risk factor in the pathogenesis of wet AMD. One mechanism by which chronic CMV infection might promote increased CNV severity is via stimulation of macrophages to make pro-angiogenic factors (VEGF), an outcome that requires active virus replication.

Disseminated Cryptococcus with ocular cryptococcoma in a human immunodeficiency virus-negative patient.

A human immunodeficiency virus-negative 63-year-old male with autoimmune hemolytic anemia presented with decreased vision, photophobia and hearing loss. After initial testing seemed consistent with sarcoidosis, he was found to have disseminated Cryptococcus with a cryptococcoma of the left eye. Treatment with systemic anti-fungal therapy improved the patient's condition.

Candida dubliniensis endophthalmitis: first case in North America.

To report an unusual case of endogenous fungal endophthalmitis due to Candida dubliniensis. Interventional case report of a 27-year-old immunocompetent male with loss of vision, dense vitritis, and chorioretinal infiltrates, who underwent a diagnostic pars plana vitrectomy. Microbiology cultures obtained by a diagnostic vitrectomy were positive for the growth of C. dubliniensis. This infectious process was then appropriately treated with intravitreal amphotericin B and systemic fluconazole with resolution of the endophthalmitis. Endogenous fungal endophthalmitis is a condition that can masquerade other more common causes of endophthalmitis. Atypical cases of endophthalmitis may benefit from diagnostic pars plana vitrectomy for prompt diagnosis and treatment.

Estrogen receptor beta 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 Bruch's 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.

Mouse retinal pigmented epithelial cell lines retain their phenotypic characteristics after transfection with human papilloma virus: a new tool to further the study of RPE biology.

Development of immortalized mouse retinal pigmented epithelial cell (RPE) lines that retain many of their in vivo phenotypic characteristics, would aid in studies of ocular diseases including age related macular degeneration (AMD). RPE cells were isolated from 18-month-old (estrogen receptor knockout) ERKOalpha and ERKObeta mice and their C57Bl/6 wildtype littermates. RPE65 and cellular retinaldehyde binding protein (CRALBP) expression, in vivo markers of RPE cells, were detected by real-time RT-PCR and western analysis. We confirmed the presence of epithelial cell markers, ZO1, cytokeratin 8 and 18 by immunofluorescence staining. In addition, we confirmed the distribution of actin filaments and the expression of ezrin. To develop cell lines, RPE cells were isolated, propagated and immortalized using human papilloma virus (HPV) 16 (E6/E7). RPE-specific markers and morphology were assessed before and after immortalization. In wildtype littermate controls, there was no evidence of any alterations in the parameters that we examined including MMP-2, TIMP-2, collagen type IV, and estrogen receptor (ER)alpha and ERbeta protein expression and ER copy number ratio. Therefore, immortalized mouse RPE cell lines that retain their in vivo phenotype can be isolated from either pharmacologically or genetically manipulated mice, and may be used to study RPE cell biology.

Dynamic indocyanine green angiography-guided focal thermal laser treatment of fibrotic choroidal neovascularization.

PURPOSE: Fibrotic choroidal neovascular membranes (CNV) are the end-stage outcomes of neovascular age-related macular degeneration (AMD). No treatment is currently available for fibrotic CNV. We investigated the role of focal thermal laser ablation of the perfusing afferent arteriole as determined by dynamic indocyanine green angiography (ICGA). METHODS: We conducted a retrospective study of 20 patients with fibrotic CNV associated with significant subretinal fluid or retinal edema, who also demonstrated well-defined perfusing arterioles by dynamic ICGA. Patients underwent focal thermal laser occlusion of the perfusing afferent arteriole. Six, 12 and 24 weeks post-treatment, eyes underwent repeat examination with optical coherence tomography (OCT) and visual acuity testing, and ICGA at 12 weeks. RESULTS: Therapeutic closure of the perfusing afferent arterioles was achieved in 17 of 20 eyes immediately post-treatment. All 17 of these eyes demonstrated significant resolution of retinal edema and subretinal fluid, as evidenced by OCT, which was dramatic in some cases. Seven eyes demonstrated an improvement in visual acuity of 1 line or more. While most eyes demonstrated reperfusion within 3 months, many lesions suggested reduced vascularity and flow. CONCLUSION: Eyes with fibrotic CNV and associated retinal edema often demonstrate well-defined vascularity of the fibrosis with discrete perfusing arterioles when imaged by dynamic ICGA. Thermal laser occlusion of these arterioles can result in resolution of subretinal fluid, and occasionally an improvement in vision. This represents a potential therapeutic intervention for an advanced stage of AMD currently regarded as stable.

Subtype specific estrogen receptor action protects against changes in MMP-2 activation in mouse retinal pigmented epithelial cells.

Eyes with age-related macular degeneration (AMD) demonstrate accumulation of specific deposits and extracellular matrix (ECM) molecules under the retinal pigment epithelium (RPE). AMD is about two times more prevalent in aging postmenopausal women. Therefore we studied whether 17beta-estradiol (E(2)) modulates the expression and activity of the trimolecular complex (MMP-2, TIMP-2 and MMP-14), molecules which are of major importance for ECM turnover in RPE. We used cell lines isolated from estrogen receptor knockout mice (ERKO) to determine which ER (estrogen receptor) subtype was important for ECM regulation in RPE cells. We found that mouse RPE sheets had higher baseline MMP-2 activity in the presence of ERbeta. This correlated with higher MMP-2 activity in RPE cell lines isolated from ERKOalpha mice. Exposure to E(2) increased MMP-2 activity in mouse RPE cell lines. In addition E(2) increased transcriptional activation of the MMP-2 promoter through a functional Sp1 site which required the presence of ERbeta, but not ERalpha. E(2) also maintained levels of pro MMP-2, and MMP-14 and TIMP-2 activity after oxidant injury. Since the direct effects of E(2) on MMP-2 transcriptional activation and the regulation of the trimolecular complex after oxidant-induced injury requires ERbeta, this receptor subtype may have a role as a potential therapeutic target to prevent changes in activation of MMP-2.

Smoking induces glomerulosclerosis in aging estrogen-deficient mice through cross-talk between TGF-beta1 and IGF-I signaling pathways.

Smoking is a known risk factor for the progression of chronic kidney diseases. However, its independent contribution to the development of ESRD and the underlying molecular mechanism have not been well elucidated. Although the risk for ESRD is higher in postmenopausal women according to the US Renal Data System, the number of women who smoke is on the rise worldwide. Therefore, the effects of smoking and estrogen status on glomerular function and structure were studied in female B6 mice that were ovariectomized at 3 (young) and 15 mo (aged) of age. The mice received either 17beta-estradiol (E(2)) replacement or placebo (Pla) and were divided further into groups that were exposed to cigarette smoke (S) and not exposed (NS). Six months of exposure to smoke had no effect on young mice, although aging S/Pla mice exhibited a phenotype of increased albumin excretion associated with a moderately increased glomerular collagen type IV deposition compared with NS/Pla mice. S/Pla mice also had a two-fold increase in glomerular TGF-beta, Smad3, and IGF-I receptor mRNA expression compared with the NS group. Mesangial cells that were isolated from S/Pla mice had an increase of IGF-I receptor protein, and IGF-I stimulated a TGF-beta reporter construct promoter three-fold. This was blocked by pretreatment with a neutralizing antibody to IGF-I, LY294002 (phosphatidylinositol-3 kinase inhibitor) or a dominant negative Smad construct. In addition, Smad3 activation was stimulated by IGF-I and blocked by LY294002, suggesting cross-talk between Smad and the phosphatidylinositol-3 kinase/AKT pathways. The smoking phenotype was reversed by E(2) replacement. In conclusion, smoking induces a phenotype in E(2)-deficient mice that is characterized by activation and cross-talk between the TGF-beta1 and IGF-I signaling pathways.

Repetitive nonlethal oxidant injury to retinal pigment epithelium decreased extracellular matrix turnover in vitro and induced sub-RPE deposits in vivo.

PURPOSE: To determine the impact of repetitive nonlethal oxidant injury with hydroquinone (HQ) on regulation of cell membrane blebbing and molecules, which are essential in extracellular matrix turnover (ECM) maintenance, especially matrix metalloproteinase (MMP)-2, tissue inhibitor of MMP (TIMP)-2, and type IV collagen in cultured RPE. In addition, to determine whether chronic oral HQ causes induction of sub-RPE deposit formation in a mouse model. METHODS: An ARPE-19 cell line stably expressing membrane-targeted green fluorescent protein (GFP) was challenged by exposure to HQ (100 microM). Repetitive acute (6 hours every 3 days for 4 weeks) or transient (6 hours followed by a recovery phase, every 5 days for 6 weeks) exposure to HQ were evaluated. An MTS assay, cell counts, and bromodeoxyuridine (BrdU) incorporation were used to detect cell viability and proliferation. Supernatants and cell homogenates were collected to assess MMP-2 and TIMP-2 activity by zymography and reverse zymography, proteins by Western blot, and type IV collagen accumulation by ELISA and immunostaining. Expression of MMP-2 and type IV collagen was examined by real-time RT-PCR on total RNA. Sixteen-month-old C57BL/6 female mice were fed a regular fat diet, with or without HQ (0.8%) in the drinking water, for 4 months. The eyes were removed for transmission electron microscopy of the retina and choroid after treatment. Semiquantitative grading of deposit severity was performed. RESULTS: In vitro, high doses of HQ (400-250 microM) killed a significant fraction of RPE cells ( approximately 60% of control). Low doses (50-100 microM) were nonlethal but induced significant blebbing. Both nonlethal repetitive acute and transient exposure to HQ were associated with diminished MMP-2 activity and increased collagen type IV accumulation. In vivo, mice exposed to oral HQ demonstrated moderately thick basal laminar deposits and a variable degree of deposits within Bruch's membrane (BrM). These homogeneous sub-RPE deposits accumulated in the eyes, consistent with early laminar deposits. CONCLUSIONS: In cultured RPE, nonlethal injury with HQ upregulated nonlethal blebbing and decreased ECM turnover. Similarly, in vivo exposure to oral HQ induced nonlethal bleb injury and sub-RPE deposits. These data support the hypothesis that HQ may regulate blebbing and molecules that influence ECM turnover. This study suggests that HQ may be another type of oxidant that causes injury to the RPE and may explain the association between environmental oxidants and early AMD.

Cigarette smoke-related oxidants and the development of sub-RPE deposits in an experimental animal model of dry AMD.

PURPOSE: Oxidative injury to the retinal pigment epithelium (RPE) has been proposed to be an important injury stimulus relevant to the accumulation of subretinal deposits in age-related macular degeneration (AMD). Cigarette smoking is a major risk factor for AMD, and cigarette smoke-related tar contains high concentrations of a potent oxidant, hydroquinone (HQ). This study was an investigation of the effects of cigarette smoke (CS) and HQ in the development of sub-RPE deposits in an experimental mouse model. METHODS: Sixteen-month-old C57BL/6 female mice were fed a high-fat diet (HFD) for 4.5 months. Mice were divided into two major experimental groups, one to examine the effects of cigarette smoke and one to study the effects of a defined cigarette smoke component such as HQ. In the first group, mice eyes were exposed to blue-green light (positive controls) or to whole cigarette smoke. A third group with no intervention served as the negative control. In the second experimental group, animals received a purified diet with HQ (0.8%) with low or high fat content for 4.5 months. Mice in both groups were euthanatized at 4.5 months and eyes processed for transmission electron microscopy. RESULTS: As previously demonstrated by our laboratory and others, most mice fed an HFD without other oxidant exposure demonstrated normal morphology or, in a few cases, small nodular basal laminar deposits. Eyes of mice exposed to whole cigarette smoke or to HQ in the food demonstrated a variable degree of basal laminar deposits and diffusely thickened Bruch's membrane. The choriocapillaris endothelium was variably hypertrophic. CONCLUSIONS: Exposure to cigarette smoke or the smoke-related redox molecule, HQ, results in the formation of sub-RPE deposits, thickening of Bruch's membrane, and accumulation of deposits within Bruch's membrane. Smoke-related oxidants may be another oxidative injury stimulus to the choriocapillaris and RPE, and may explain the association between cigarette smoking and early AMD.

Blood-derived macrophages infiltrate the retina and activate Muller glial cells under experimental choroidal neovascularization.

Inflammation is a major mechanism in the pathogenesis of age-related macular degeneration, the most important cause of blindness in the elderly. Previous studies have focused on the role of macrophages in regulating the growth of pathological new vessels over the retina, called choroidal neovascularization (CNV). However, no research has been done to evaluate the role of inflammation as a mechanism of vision loss and retinal degeneration in the retina underlying CNV. In other neuropathological conditions, hematogenous macrophages and/or resident microglia contribute to neurodegeneration. We have combined laser-induced CNV in mice and bone marrow transplantation with GFP-labeled bone marrow to determine the relative role of recruited blood-derived macrophages versus resident microglia in the retina associated with CNV. Using these chimeric mice, we have found that many GFP-labeled cells infiltrated the retina underlying CNV but not the retina unaffected by CNV. Immunostaining for the cell adhesion molecules VCAM 1, ICAM 1, and PECAM was strongly upregulated in retinal blood vessels under CNV. All GFP-labeled cells were immunoreactive for the macrophage marker F4/80. Most (70%) of the F4/80 immunoreactive cells were GFP-labeled under CNV. The density of resident microglia did not increase. Most GFP-labeled cells were found in close proximity to activated Muller cells. Depleting circulating macrophages with clodronic acid diminished the density of F4/80 immunoreactive cells as well as the density of pERK immunoreactive Muller cells in the retina under CNV. Thus, recruitment of blood-derived macrophages more than resident microglia seems to be associated with CNV.

Photoreceptor synapses degenerate early in experimental choroidal neovascularization.

Severe visual loss in patients with age-related macular degeneration is associated with the development of choroidal neovascularization (CNV). The pathogenic mechanisms for CNV formation have been extensively investigated, but remarkably little research has addressed the mechanisms for dysfunction of the retina in CNV. Using laser-induced CNV in mice, we evaluated the mechanisms of retinal dysfunction. At 3 days, 1 week, 2 weeks, and 4 weeks after laser application, retinas under experimental CNV were characterized physiologically (ERG recordings, synaptic uptake of the exocytotic marker FM1-43, and light-induced translocation of transducin), histologically, and immunohistochemically. ERG amplitudes were reduced by 20% at 1 week after CNV. Depolarization-induced FM1-43 uptake in photoreceptor synapses was selectively reduced by 45% at 1 week after CNV. Although photoreceptor outer segments were shortened by 36%, light adaptation as measured by transducin translocation was mostly preserved. Early in CNV (3 days to 1 week), Muller cells demonstrated induction of c-fos and pERK expression. Also, the density of macrophage-like, F4/80 immunoreactive cells increased approximately 3-fold. Minimal photoreceptor death occurred during the first week, and was variable thereafter. At later times in CNV formation (> or =2 weeks), expression of photoreceptor synaptic markers was reduced in the outer plexiform layer, indicating loss of photoreceptor synaptic terminals. ERG amplitudes, synaptic uptake of FM1-43, and the induction of c-fos and pERK in Muller cells were altered within 1 week of experimental CNV, suggesting that during CNV formation, deficits in retinal function, in particular photoreceptor synaptic function, precede degeneration of photoreceptor terminals and photoreceptor cell death.

Quantitative enumeration of vascular smooth muscle cells and endothelial cells derived from bone marrow precursors in experimental choroidal neovascularization.

Choroidal neovascularization (CNV) is characterized by the subretinal invasion of a pathologic new vessel complex from the choriocapillaris. Although CNV is traditionally considered to consist of endothelial cells, the cellular population of CNV is likely more complex in nature, comprising several different cell types. In addition, recent studies suggest that the CNV cell population has a dual origin (circulating versus resident populations). In this study we sought to determine the contribution and origin of different cell types in experimental CNV. Laser-induced CNV was performed on chimeric mice generated by reconstituting C57BL/6 mice with bone marrow from green fluorescent protein (GFP)-transgenic mice. In these mice, bone marrow-derived cells are GFP-labeled. Immunofluorescence staining was used to examine both flatmount preparations of the choroid and cross sections of the posterior pole for macrophages, endothelial cells, vascular smooth muscle cells, retinal pigment epithelial (RPE) cells, lymphocytes, or neutrophils at day 3, 7, 14 and 28 post-laser (n=5 per group). Cell types present in CNV included macrophages (20% of the cells in CNV), endothelial cells (25%), vascular smooth muscle cells (11%), RPE cells (12%) and non-labeled cells (32%). The macrophage population was mostly derived from circulating monocytes at all timepoints studied (70% were GFP labeled), while endothelial and vascular smooth muscle cells were partly bone marrow derived (50-60% were GFP labeled), and RPE cells appeared to be entirely derived from preexisting tissue resident cells. These results demonstrate that bone marrow-derived progenitor cells contribute significantly to the vascular and inflammatory components of CNV. Knowledge of the cellular composition and origin might help understand the pathogenic mechanisms controlling CNV severity as well as indicate potential targets for therapeutic intervention.

Gender and estrogen supplementation increases severity of experimental choroidal neovascularization.

Observational clinical studies suggest that post-menopausal women may be at risk for more severe age-related macular degeneration, and that estrogen loss due to menopause may contribute. We sought to determine the effect of gender and estrogen status on the severity of choroidal neovascularization (CNV) in a mouse model for experimental choroidal neovascularization. Laser-induced CNV was performed in mice with or without estrogen supplementation. At various times, eyes were removed for analysis of severity of CNV lesions or for extraction of choroidal mRNA to evaluate iNOS, TNF-alpha, MMP-9, and ER-alpha expression, which are molecules relevant to angiogenic processes. Also, splenic macrophages were analysed for iNOS to determine the effect of estrogen treatment in vitro. Finally, laser-induced CNV was performed in iNOS -/- mice. Our result showed that aged female mice had significantly larger CNV than age-matched males. Ovariectomy in adult mice did not increase severity, but paradoxically estrogen supplementation after ovariectomy did increase CNV severity. More severe CNV were associated with a significant decrease in choroidal iNOS mRNA. Splenic macrophages from estrogen supplemented mice showed a significant increased in TNF-alpha mRNA expression (eight fold difference compared to the control) but only a mild change in iNOS mRNA levels (2-3 fold difference). In vitro data further showed that nitric oxide production in splenic macrophages at different estrogen levels was not different from controls. Finally, CNV severity was significantly more severe in iNOS -/- mice, compared to iNOS +/+ mice after laser treatment. In conclusion, aged female mice developed more severe CNV than do males. Estrogen replacement seems to increase severity, possibly by suppressing the upregulation of choroidal iNOS and activating macrophages. The putative beneficial or detrimental role of estrogen biology in age-related macular degeneration must be more carefully evaluated and may vary with the stage of age-related macular degeneration (atrophic or neovascular) as well as with the specific target cell type (monocytes vs. endothelial cell or vascular smooth muscle cell).

The association of prior cytomegalovirus infection with neovascular age-related macular degeneration.

PURPOSE: To determine if prior exposure to pathogens associated with vascular disease, cytomegalovirus, Chlamydia pneumoniae, and Helicobacter pylori correlates with neovascular age-related macular degeneration (AMD). DESIGN: An experimental study. SETTING: Institutional. Bascom Palmer Eye Institute, October 2001 to December 2002. PATIENT POPULATION: 150 patients (47 neovascular amd, 36 dry amd, and 67 non-amd controls) were included in the study. exclusion criteria included hiv infection, malignancy, recent acute illness requiring hospitalization within 6 months, or immunosuppressive illness. PROCEDURE: Serum samples were obtained for analysis of cytomegalovirus, chlamydia pneumoniae, and helicobacter pylori igg antibody titers by elisa. MAIN OUTCOME MEASURE: Comparison of the distribution of igg titers between patients with wet amd, dry amd, and controls. RESULTS: The average cytomegalovirus IgG titer was higher in patients with wet AMD versus controls (p = 0.02, Student t-test, two-tailed) and patients with dry AMD (p = 0.06). Twenty-six (55%) of 47 subjects with wet AMD had high cytomegalovirus IgG titers compared with 14 (39%) of 36 patients with dry AMD (odds ratio [OR] = 2.23, 95% confidence interval [CI] = 0.77 to 6.44) and 23 (34%) of 67 control patients (OR = 2.49, 95% CI = 0.98 to 6.33). There was no major difference in the distribution of titers for Chlamydia pneumoniae IgG and Helicobacter pylori IgG in wet and dry AMD patients. Five of 47 patients with wet AMD (11%) had high antibody titers to all three pathogens, compared with only 1 of 36 patients with dry AMD (3%) (OR = 4.17, 95% CI = 0.46 to 37.36). CONCLUSIONS: There was a significant association of high cytomegalovirus IgG titer with neovascular AMD compared with dry AMD and control patients. Chronic infection with cytomegalovirus may be a novel risk factor for the progression from dry to neovascular AMD.