Michele C. Madigan

Immunological and aetiological aspects of macular degeneration.

Aetiological and immunological aspects of AMD, a leading cause of blindness in Western countries, have been reviewed. Developmental studies suggest that anatomical features unique to the fovea result in a critical relationship between metabolic demand and blood supply at the macula, which is maintained throughout life. Recent studies show a sufficient degree of consistency in the link between smoking and both dry and wet AMD to regard it as causative. Dry AMD is considered to be the natural endstage of the disease; epidemiological and morphological studies point to choroidal vascular atrophy as the causative event and it is suggested that signals associated with acute vascular compromise lead to the development of subretinal neovascularisation. The relationship between sub-pigment epithelial deposits, including basal laminar deposit, and the pathogenesis of AMD is examined. Much of the literature is consistent with a choroidal origin for the constituents of drusen. The blood-retinal barrier preserves the physiological environment of the neural retina and limits inflammatory responses. The factors, including cytokines, adhesion molecules and the presence of resident immunocompetent cells (microglia), which determine the immune status of the retina are considered. Historical descriptions of the involvement of inflammatory cells are provided, evidence implicating inflammation in the pathogenesis of AMD involving macrophages, giant cells and microglia has been derived from observations of human and animal subretinal neovascular lesions. The role of humoral factors such as anti-retinal autoantibodies and acute phase proteins together with clinical observations has been surveyed. Taken together these data demonstrate the involvement of both cellular and humoral immunity in the pathogenesis of AMD. It remains to be determined to what degree the influence of immunity is causative or contributory in both wet and dry AMD, however, the use of anti-inflammatory agents to ameliorate the condition further indicates the existence of an inflammatory component.

DICER1 deficit induces Alu RNA toxicity in age-related macular degeneration

Geographic atrophy (GA), an untreatable advanced form of age-related macular degeneration, results from retinal pigmented epithelium (RPE) cell degeneration. Here we show that the microRNA (miRNA)-processing enzyme DICER1 is reduced in the RPE of humans with GA, and that conditional ablation of Dicer1, but not seven other miRNA-processing enzymes, induces RPE degeneration in mice. DICER1 knockdown induces accumulation of Alu RNA in human RPE cells and Alu-like B1 and B2 RNAs in mouse RPE. Alu RNA is increased in the RPE of humans with GA, and this pathogenic RNA induces human RPE cytotoxicity and RPE degeneration in mice. Antisense oligonucleotides targeting Alu/B1/B2 RNAs prevent DICER1 depletion-induced RPE degeneration despite global miRNA downregulation. DICER1 degrades Alu RNA, and this digested Alu RNA cannot induce RPE degeneration in mice. These findings reveal a miRNA-independent cell survival function for DICER1 involving retrotransposon transcript degradation, show that Alu RNA can directly cause human pathology, and identify new targets for a major cause of blindness.

Anatomy and development of the macula: specialisation and the vulnerability to macular degeneration

The central retina in primates is adapted for high acuity vision. The most significant adaptations to neural retina in this respect are: 1. The very high density of cone photoreceptors on the visual axis; 2. The dominance of Midget pathways arising from these cones and 3. The diminishment of retinal blood supply in the macula, and its absence on the visual axis. Restricted blood supply to the part of the retina that has the highest density of neural elements is paradoxical. Inhibition of vascular growth and proliferation is evident during foetal life and results in metabolic stress in ganglion cells and Müller cells, which is resolved during formation of the foveal depression. In this review we argue that at the macula stressed retinal neurons adapt during development to a limited blood supply from the choriocapillaris, which supplies little in excess of metabolic demand of the neural retina under normal conditions.

Differential expression of GFAP in early v late AMD: a quantitative analysis

Background/aims: Glial fibrillary acidic protein (GFAP) is an established indicator of retinal stress; its expression in retinal astrocytes and Müller cells has been demonstrated to be modulated by cytokines and retinal pathology, including age related macular degeneration (AMD). This study aims to quantify the modulation of GFAP expression in retinas with drusen and atrophic AMD versus normal age matched controls. Methods: Following a histopathological survey, 17 donor retinas were classified into four groups: drusen (n=5), geographic atrophy (GA) (n=6), aged normal (n=3), and young normal (n=3). Paramacular cryosections were immunolabelled with GFAP antibody, examined by confocal microscopy, and quantified by NIH digital image analysis. Groups were matched for potential confounding factors including age, sex, and postmortem delay. Results: A significant increase in GFAP immunolabelling of macroglia was noted in aged normal compared with young normal retinas (p<0.04). Upregulation of GFAP immunoreactivity involving astrocytes was observed in drusen retinas compared with control retinas (p<0.03). GFAP was also upregulated in retinas with GA compared with controls (p<0.05) and in retinas with GA compared with drusen (p<0.04), both involving Müller cells. Discrete regions of GFAP upregulation in Müller cells were associated with drusen formation. In GA specimens atrophied retinal pigment epithelium (RPE) was substituted by GFAP immunoreactive Müller cell processes (gliosis). Conclusion: This study provides a quantitative assessment of GFAP modulation in ageing and AMD affected retinas. Morphological observations were consistent with quantitative analyses indicating differential modulation of GFAP immunoreactivity in inner and outer retina. Upmodulation of GFAP in inner retina and astroglial processes was predominantly associated with drusen, while in outer retina Müller glia upmodulation of GFAP was associated with disruption of the RPE and blood-retinal barrier.

Co-expression of CD147 (EMMPRIN), CD44v3-10, MDR1 and monocarboxylate transporters is associated with prostate cancer drug resistance and progression

Background:The aim of this study is to seek an association between markers of metastatic potential, drug resistance-related protein and monocarboxylate transporters in prostate cancer (CaP).Methods:We evaluated the expression of invasive markers (CD147, CD44v3-10), drug-resistance protein (MDR1) and monocarboxylate transporters (MCT1 and MCT4) in CaP metastatic cell lines and CaP tissue microarrays (n=140) by immunostaining. The co-expression of CD147 and CD44v3-10 with that of MDR1, MCT1 and MCT4 in CaP cell lines was evaluated using confocal microscopy. The relationship between the expression of CD147 and CD44v3-10 and the sensitivity (IC50) to docetaxel in CaP cell lines was assessed using MTT assay. The relationship between expression of CD44v3-10, MDR1 and MCT4 and various clinicopathological CaP progression parameters was examined.Results:CD147 and CD44v3-10 were co-expressed with MDR1, MCT1 and MCT4 in primary and metastatic CaP cells. Both CD147 and CD44v3-10 expression levels were inversely related to docetaxel sensitivity (IC50) in metastatic CaP cell lines. Overexpression of CD44v3-10, MDR1 and MCT4 was found in most primary CaP tissues, and was significantly associated with CaP progression.Conclusions:Our results suggest that the overexpression of CD147, CD44v3-10, MDR1 and MCT4 is associated with CaP progression. Expression of both CD147 and CD44v3-10 is correlated with drug resistance during CaP metastasis and could be a useful potential therapeutic target in advanced disease.

Angiogenesis in normal human retinal development the involvement of astrocytes and macrophages

Recent studies have suggested a role for mononuclear phagocytes series (MPS) cells in neovascularisation associated with retinal pathology and experimentally induced subretinal neovascularisation. The present study is concerned with the normal development of the human retinal vasculature. Morphological details are provided of developing vascular structures including the formation of tight junctions and canalisation of angioblast cords. The relationships of astrocytes and pericytes to developing structures and the presence of a perivascular collagenous matrix are described. Ultrastructural and histochemcal analyses reveal an association between MPS cells and developing vascular structures. It is suggested that MPS cells may influence angiogenesis in normal retinal development, as well as in retinal pathology.

Intermediate filament expression in human retinal macroglia. Histopathologic changes associated with age-related macular degeneration.

Purpose Intermediate filament expression by retinal macroglia was studied in normal eyes, normal eyes of older subjects, and eyes of subjects with age-related macular degeneration (AMD), classified on the basis of histopathologic assessment of the retinal pigment epithelium (RPE), choroid, and donor age. Methods. Adult human retinae (N = 43) were divided into three groups: normal (< 50 years of age, with normal RPE and choroid); normal eyes of older subjects (> 50 years with normal RPE and choroid); and eyes of subjects with AMD (>50 years with histopathologic findings indicative of AMD), on the basis of histopathologic assessment of the RPE/choroid and donor age. Intermediate filament expression by retinal macroglia was studied in cryostat sections and in retinal flatmounts using avidin-biotinperoxidase immunolabeling of antibodies to glial fibrillary acidic protein (GFAP) and vimentin. Results. Analyses of immunohistochemically labeled retinal sections revealed similar distributions of vimentin reactivity in retinae of each group. Distributions of GFAP in normal and normal aged retinae were similar, but sections of AMD-affected retinae showed evidence of GFAP expression by Müller cells. In flatmounts, vimentin distribution was similar in the three groups, but GFAP labeling revealed hypertrophic astrocytes, which were absent from normal retinae, in 17% of aged retinae and 55% of AMD-affected retinae. Deeply penetrating, GFAP-positive processes were observed in 17%, 27%, and 55% of normal, normal aged, and AMD-affected retinae, respectively. Conclusions Variation in GFAP and vimentin expression in retinal macroglia is affected by increasing age, and a distinctive variation of intermediate filament expression in retinal macroglia is associated with the pathogenesis of AMD.

TUMOR NECROSIS FACTOR-ALPHA (TNF-ALPHA )-INDUCED OPTIC NEUROPATHY IN RABBITS

Both in vitro and in vivo studies have implicated a role for tumor necrosis factor-alpha (TNF alpha) in various demyelinating diseases, including HIV-related encephalopathy. To investigate whether intravitreal TNF alpha can induce optic nerve axonal damage in a rabbit eye model, fifteen rabbit eyes were initially injected with TNF alpha (final concentrations: 2U, 20U, and 200U respectively) and studied at varying time intervals for up to 24 weeks post-injection, using light and electron microscopy. Control optic nerves (no injection or diluent injection only) had normal myelinated axons and glia; the myelinated regions, neural retina, retinal glia and vasculature of control retinas were normal. In TNF alpha-exposed optic nerves, intact, degenerating and demyelinated axons were interspersed. Astrogliosis was present, particularly from 8 weeks p.i. and was noted up to 24 weeks. Oligodendrocytes were not severely affected in TNF alpha-exposed optic nerves, and activated macrophages or microglia were not obvious. Axonal degeneration was visible among the more superficial myelinated fibers in TNF alpha-exposed retinas however the neural retina glia were unaffected. These observations suggest that the axonal degeneration induced in TNF alpha-exposed rabbit optic nerves over a 24 week period was most likely related to direct effects of TNF alpha on optic nerve axons, and not primarily due to anterograde degeneration from retinal lesions. In-so-far as neurological pathology in general, and optic nerve degeneration in particular, has been described in AIDS, and TNF alpha levels may be elevated in this disease, it is of great clinical significance that TNF alpha has the capacity to mediate neuronal or axonal injury. If so, strategies to block or inhibit TNF alpha can be pursued for treatment for the neurological symptoms of AIDS.

Antibodies to human leucocyte antigens indicate subpopulations of microglia in human retina

Monoclonal antibodies to human leucocyte antigens, including anti-CD45 and anti-CD68, have been used to describe microglia in flatmounts of normal adult human retina for the first time. Anti-CD45 (the leucocyte common antigen) intensely labeled large numbers of cells in a regular distribution across the retina; anti-CD68 and anti-macrophage antibodies labeled fewer cells with distinctive morphologies, suggesting the presence of subpopulations of microglia in the human retina expressing leucocyte antigens.

Effect of Müller cell co-culture on in vitro permeability of bovine retinal vascular endothelium in normoxic and hypoxic conditions.

Müller cell dysfunction may contribute to the early pathological changes associated with conditions such as diabetes, that cause breakdown of the blood-retinal barrier. In this study we used an in vitro model of the blood-retinal barrier to investigate Müller cell effects on retinal vascular endothelial cell monolayer permeability under normoxic (20% oxygen) and hypoxic (1% oxygen) conditions. Second passage bovine retinal capillary endothelial cells were co-cultured with retinal Müller cells on opposite sides of a 0.4 microm pore size polycarbonate Transwell filter or in medium that was continually conditioned by Müller cells. Permeability changes were observed for up to 24h of hypoxia by measurement of [(3)H]-inulin and [(14)C]-albumin flux across the endothelial cell monolayer. Endothelial cell barrier function was enhanced by co-culturing with Müller cells under normoxic conditions. Under hypoxic conditions however, the barrier was significantly impaired after 12h of co-culture with Müller cells. These results shed more light on the trophic effect of Müller cells on the blood-retinal barrier, suggesting a critical role in the maintenance and regulation of the barrier in health and during disease.