bromochloroacetic-acid and Macular-Degeneration

Age related macular degeneration (AMD) is one of the leading causes of blindness in Western society. A hallmark of early stage AMD are drusen, extracellular deposits that accumulate in the outer retina. Advanced glycation endproducts (AGE) accumulate with aging and are linked to several age-related diseases such as Alzheimer's disease, osteoarthritis, atherosclerosis and AMD. AGE deposits are found in drusen and in Bruch's membrane of the eye and several studies have suggested its role in promoting oxidative stress, apoptosis and lipofuscin accumulation. Recently, complement activation and chronic inflammation have been implicated in the pathogenesis of AMD. While AGEs have been shown to promote inflammation in other diseases, whether it plays a similar role in AMD is not known. This study investigates the effects of AGE stimulation on pro- and anti-inflammatory pathways in primary culture of human retinal pigment epithelial cells (RPE). Differential gene expression studies revealed a total of 41 up- and 18 down-regulated RPE genes in response to AGE stimulation. These genes fell into three categories as assessed by gene set enrichment analysis (GSEA). The main categories were inflammation (interferon-induced, immune response) and proteasome degradation, followed by caspase signaling. Using suspension array technology, protein levels of secreted cytokines and growth factors were also examined. Anti-inflammatory cytokines including IL10, IL1ra and IL9 were all overexpressed. Pro-inflammatory cytokines including IL4, IL15 and IFN-γ were overexpressed, while other pro-inflammatory cytokines including IL8, MCP1, IP10 were underexpressed after AGE stimulation, suggesting a para-inflammation state of the RPE under these conditions. Levels of mRNA of chemokine, CXCL11, and viperin, RSAD2, were up-regulated and may play a role in driving the inflammatory response via the NF-kB and JAK-STAT pathways. CXCL11 was strongly immunoreactive and associated with drusen in the AMD eye. The pathways and novel genes identified here highlight inflammation as a key response to AGE stimulation in primary culture of human RPE, and identify chemokine CXCL11 as putative novel agent associated with the pathogenesis of AMD.

Topics: Cell Survival; Cells, Cultured; Chemokine CXCL11; Down-Regulation; Gene Expression Profiling; Gene Expression Regulation; Glycation End Products, Advanced; Humans; Inflammation; Keratins; Lysine; Macular Degeneration; Pigment Epithelium of Eye; Postmortem Changes; Reproducibility of Results; Retinal Drusen; Serum Albumin, Bovine; Tissue Donors; Up-Regulation

To compare RPE derived from human embryonic stem cells (hES-RPE) and fetal RPE (fRPE) behavior on human Bruch's membrane (BM) from aged and AMD donors.. hES-RPE of 3 degrees of pigmentation and fRPE were cultured on BM explants. Explants were assessed by light, confocal, and scanning electron microscopy. Integrin mRNA levels were determined by real-time polymerase chain reaction studies. Secreted proteins in media were analyzed by multiplex protein analysis after 48-hour exposure at culture day 21.. hES-RPE showed impaired initial attachment compared to fRPE; pigmented hES-RPE showed nuclear densities similar to fRPE at day 21. At days 3 and 7, hES-RPE resurfaced BM to a limited degree, showed little proliferation (Ki-67), and partial retention of RPE markers (MITF, cytokeratin, and CRALBP). TUNEL-positive nuclei were abundant at day 3. fRPE exhibited substantial BM resurfacing at day 3 with decreased resurfacing at later times. Most fRPE retained RPE markers. Ki-67-positive nuclei decreased with time in culture. TUNEL staining was variable. Increased integrin mRNA expression did not appear to affect cell survival at day 21. hES-RPE and fRPE protein secretion was similar on equatorial BM except for higher levels of nerve growth factor and thrombospondin-2 (TSP2) by hES-RPE. On submacular BM, fRPE secreted more vascular endothelial growth factor (VEGF), brain-derived neurotrophic factor, and platelet-derived growth factor; hES-RPE secreted more TSP2.. Although pigmented hES-RPE and fRPE resurfaced aged and AMD BM to a similar, limited degree at day 21, cell behavior at earlier times was markedly dissimilar. Differences in protein secretion may indicate that hES-RPE may not function identically to native RPE after seeding on aged or AMD BM.

Topics: Aged; Aged, 80 and over; Aging; Bruch Membrane; Carrier Proteins; Cells, Cultured; Embryonic Stem Cells; Female; Fetal Stem Cells; Fluorescent Antibody Technique, Indirect; Humans; In Situ Nick-End Labeling; Integrins; Keratins; Ki-67 Antigen; Macular Degeneration; Male; Microphthalmia-Associated Transcription Factor; Microscopy, Confocal; Microscopy, Electron, Scanning; Middle Aged; Organ Culture Techniques; Retinal Pigment Epithelium; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger

Up-regulation of pro-angiogenic cytokine expression occurring secondary to hypoxia in physiologic and pathophysiologic conditions is mediated by the family of transcription regulators know as hypoxia inducible factors (HIF). The present study was undertaken to investigate the expression of HIF occurring in human choroidal neovascularization (CNV) and the posterior segment of young and old eyes.. Surgically excised CNV from patients with either age-related macular degeneration (AMD; n = 9), punctuate inner choroidopathy (PIC; n = 3) and young normal eyes were immunohistochemically probed with monoclonal antibodies against HIF-1alpha and -2alpha and compared to that for cell markers specific for vascular endothelial cells (CD34), macrophages (CD68), retinal pigment epithelial cells (RPE; panel cytokeratins/CK18) and VEGF. Following secondary antibody amplification, reactions were visualized with fast red chromogen.. Cellular immunoreactivity of membranes for HIF-2alpha was strong in eight out of nine AMD specimens but it was only weakly positive for HIF-1alpha in five specimens. In contrast, two out of three PIC specimens were weakly positive for HIF-1alpha but demonstrated no staining for HIF-2alpha. Immunohistochemical analysis revealed areas within the CNV membranes that were predominantly immunopositive for CD68 and cytokeratin indicating the presence of RPE and/or macrophages and that these cells strongly co-localized with the presence of HIF and VEGF. No immunochemical co-localization was observed with HIF and the endothelial cell marker CD34 in any membranes studied. Normal globes also demonstrated HIF-2 positivity to be predominantly localized to the central RPE rather than peripheral RPE irrespective of age of donor.. The localization of HIF expression supports the concept that hypoxia is a major stimulus for the development of submacular wound healing and within this context CNV is but one component of this process.

Topics: Adult; Aged; Aged, 80 and over; Antigens, CD; Antigens, CD34; Antigens, Differentiation, Myelomonocytic; Aryl Hydrocarbon Receptor Nuclear Translocator; Choroid; Choroidal Neovascularization; Eye Banks; Female; Humans; Hypoxia-Inducible Factor 1, alpha Subunit; Immunohistochemistry; In Vitro Techniques; Keratins; Macular Degeneration; Male; Middle Aged; Retinal Pigment Epithelium; Staining and Labeling; Tissue Distribution

Endothelial progenitor cells (EPCs) have been shown to contribute to experimentally induced choroidal neovascularisation (CNV) in animal models. The recruitment pathway for EPCs is dependent on the chemokine stromal cell derived factor 1-alpha (SDF) and its receptor CXCR4 on the progenitor cell. We examined 23 specimens of CNV occurring secondary to a variety of aetiologies (10 secondary to age-related macular degeneration (AMD), 4 inflammatory, 4 idiopathic and 5 melanoma-associated) for the presence and distribution of SDF and CXCR4 in order to determine if this pathway may play a role in neovascularisation. Specimens were examined by immunohistochemistry using a panel of antibodies against SDF, CXCR4, vascular endothelial growth factor receptor 2 (VEGFR-2), CD34 (endothelial cells), CD68 (macrophages) and cytokeratins (retinal pigment epithelium; RPE). SDF was detected in 2 cases of CNV in AMD, 1 inflammatory CNV, 3 idiopathic CNVs and in 3 cases of CNV associated with melanoma. A significant association was found between SDF and VEGFR-2 immunostaining in individual membranes (p<0.001). Localisation of SDF immunostaining to the presumed RPE was also significant (p<0.05). CXCR4 immunostaining was widespread in all membranes in keeping with the published work of other investigators. Our study suggests that SDF, which may be produced by the RPE, could play a role in CNV.

Topics: Adult; Aged; Aged, 80 and over; Antigens, CD; Biomarkers; Chemokine CXCL12; Child; Choroid Neoplasms; Choroidal Neovascularization; Choroiditis; Endothelium, Vascular; Female; Humans; Keratins; Macular Degeneration; Male; Melanoma; Mesenchymal Stem Cells; Middle Aged; Receptors, CXCR4; Vascular Endothelial Growth Factor Receptor-2

The accumulation of macrophages is known to be involved in the pathogenesis of age-related macular degeneration (AMD), but the reasons why macrophages accumulate in AMD lesions have not been determined. Because the histopathology of AMD has some factors common with those of atherosclerosis, the authors hypothesized that macrophages accumulate to take up oxidized lipoproteins in the eyes of patients with AMD, as has been demonstrated in atherosclerosis.. Immunohistochemistry was performed on 10 surgically excised choroidal neovascular (CNV) membranes from eyes with AMD. An antibody against oxidized lipoprotein and antibodies against the scavenger receptors SR-PSOX and LOX-1 were used. Antibodies against cytokeratin, CD68, and von Willebrand factor were used to identify retinal pigment epithelium (RPE), macrophages, and vascular endothelial cells, respectively. RT-PCR was performed to detect the mRNAs of the scavenger receptors in the CNV membranes.. Oxidized lipoproteins were immunohistochemically detected in the CNV membranes. Intense immunostaining was observed at the surface of the CNV membranes with the SR-PSOX antibody, whereas LOX-1 immunostaining was weak. Cells expressing scavenger receptors were found to be predominantly macrophages with a minority of RPE. Both SR-PSOX and LOX-1 mRNAs were detected in CNV membranes.. Oxidized lipoproteins are present in AMD lesions. Macrophages and RPE in the CNV membranes express cell surface scavenger receptors for oxidized lipoproteins. These findings suggest that macrophages may accumulate to take up oxidized lipoproteins in AMD and that the control of oxidative stress and macrophage responses may therefore be potential treatments for AMD.

Topics: Aged; Aged, 80 and over; Antigens, CD; Antigens, Differentiation, Myelomonocytic; Chemokine CXCL16; Chemokines, CXC; Choroidal Neovascularization; Female; Humans; Immunoenzyme Techniques; Keratins; Lipoproteins, LDL; Macrophages; Macular Degeneration; Male; Middle Aged; Pigment Epithelium of Eye; Receptors, Scavenger; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Scavenger Receptors, Class E; von Willebrand Factor

Endostatin is an endogenous angiogenesis inhibitor which requires E-selectin for its antiangiogenic activity. The aim of this study was to investigate the expression of endostatin in human choroidal neovascular membranes (CNV) secondary to age-related macular degeneration (AMD) with regard to vascularization and proliferative activity. An interventional case series of 36 patients who underwent removal of CNV were retrospectively investigated. Thirty-six CNV were analyzed by light microscopic immunohistochemistry for the expression of CD34 (endothelial cells, EC), CD105 (activated EC), Ki-67 (cell proliferation), Cytokeratin 18 (epithelial cells), VEGF (vascular endothelial growth factor), E-selectin and endostatin. Donor eyes (n=7) including one with AMD were used as controls. Endostatin immunoreactivity was present in choroidal vessels of five as well as in the retinal pigment epithelium (RPE)-Bruch's membrane complex of two donor eyes without AMD. In one eye with AMD, endostatin was detected in RPE, Bruch's membrane and choroidal vessels. Ninety-two percent (33/36) of CNV disclosed endostatin staining. RPE-Bruch's membrane complex, choroidal vessels and stroma were positive in 50% (18/36), 72% (26/36), and 78% (28/36) of the membranes, respectively. Both control eyes and CNV expressed all the investigated markers except E-selectin being positive only in membranes. Endostatin, an endogenous angiogenesis inhibitor, is expressed in CNV and its therapeutic up-regulation may be a new strategy in the treatment of neovascular AMD.

Topics: Adult; Antigens, CD; Antigens, CD34; Bruch Membrane; Cell Division; Cell Membrane; Choroid; Choroidal Neovascularization; E-Selectin; Endoglin; Endostatins; Endothelial Cells; Eye Proteins; Female; Humans; Immunohistochemistry; Keratins; Ki-67 Antigen; Macular Degeneration; Male; Middle Aged; Pigment Epithelium of Eye; Receptors, Cell Surface; Retrospective Studies; Vascular Endothelial Growth Factor A

To examine the impact of photodynamic therapy (PDT) on pigment epithelium derived factor (PEDF) expression in human choroidal neovascularization (CNV) membranes with regard to vascular endothelial growth factor (VEGF) expression.. Interventional case series.. Retrospective review of interventional case series of 42 patients (42 eyes) who underwent removal of CNV. CNV was secondary to age-related macular degeneration (AMD) in all cases. Fifteen patients were treated with PDT, 3 to 246 days before surgery. CNV were stained for CD34, CD105, cytokeratin 18, VEGF, and PEDF. Twenty-seven CNV without previous treatment were used as control.. Specimens without pretreatment disclosed varying degrees of vascularization, VEGF, and PEDF expression by different cells. Specimens treated by PDT, three days previously showed mostly occluded vessels lined with damaged endothelial cells (EC). In contrast, specimens excised at later time points after PDT were highly vascularized with healthy EC. This chronology was associated with an impressive VEGF immunoreactivity increased considerably in retinal pigment epithelial cells as well as significantly reduced PEDF expression in EC and stroma.. PDT induces a selective vascular damage in CNV. The effectiveness of PDT, however, seems to be jeopardized by a rebound effect initiated by an enhanced VEGF and reduced PEDF expression in CNV.

Topics: Aged; Aged, 80 and over; Antigens, CD; Antigens, CD34; Choroidal Neovascularization; Endoglin; Endothelium, Vascular; Eye Proteins; Female; Humans; Immunoenzyme Techniques; Keratins; Macular Degeneration; Male; Middle Aged; Nerve Growth Factors; Photochemotherapy; Photosensitizing Agents; Pigment Epithelium of Eye; Porphyrins; Receptors, Cell Surface; Retrospective Studies; Serpins; Vascular Endothelial Growth Factor A; Verteporfin

To determine the gene expression profiles of primary retinal pigment epithelium (RPE) and iris pigment epithelium (IPE) using microarrays.. Primary RPE and IPE from 6 human donor eyes were collected, and total RNA was isolated. Differences in gene expression were determined using a human genechip (human U95Av2 [12 600 probes]; Affymetrix Inc, Santa Clara, Calif).. Hierarchical cluster analysis differentiated the gene expression profiles of RPE and IPE clusters into 2 distinct groups. A mean +/- SD of 5308 +/- 416 gene probes were expressed in RPE vs 6130 +/- 205 in IPE. Sixty-eight genes were expressed only in RPE; 154 genes were expressed only in IPE. Twenty-two additional genes had greater than 3-fold increased expression in RPE vs IPE, and 147 genes had greater than 3-fold decreased expression in RPE vs IPE.. There are major differences in the gene expression profiles of primary RPE vs IPE. Clinical Relevance The different gene expression profiles of primary RPE vs IPE harvested from the same donor eyes infer that it may be difficult for IPE to replace all aspects of damaged RPE function in transplantation studies.

Topics: Aged; Aged, 80 and over; Cells, Cultured; Eye Proteins; Gene Expression; Gene Expression Profiling; Humans; Iris; Keratins; Macular Degeneration; Middle Aged; Oligonucleotide Array Sequence Analysis; Pigment Epithelium of Eye; Quality Control; Retina; Reverse Transcriptase Polymerase Chain Reaction; RNA

The objective of the study was to gather further information about the pathogenesis of choroidal neovascularisations (CNV), which is still not clearly understood, and to establish criteria for making decisions on a appropriate therapy. Immunohistochemical characteristation should allow a more comprehensive evaluation of cellular components of the membranes and their functional role.. In 29 patients (16 women, 13 men) with age-related macular degeneration ranging in age from 46 to 91 years (mean age, 76.4 years), CNV were excised by pars-plana vitrectomy. Sections were stained with hematoxylin-eosin (HE) and periodic acid-Schiff (PAS) and examined by light microscopy. For the immunohistochemical characterisation of the surgical specimens the following anti-genetic determinants were used: glial fibrillary acid protein (GFAP) for glial cells, synaptophysin for neuronal cells, neuron-specific enolase (NSE) for neuronal and neuroectodermal cells, CD 31 for endothelial cells and pancytokeratin (KL1) for cells of the retinal pigment epithelium (RPE). Cells undergoing apoptosis were labeled with the TUNEL technique.. 22 (76%) surgical specimens showed TUNEL positive cells in the connective tissue, vascular endothelium and retinal pigment epithelium. Positive immunostaining of neuronal antigenetic determinants was found for glial fibrillary acid protein in 22 patients (76%), for synaptophysin in 28 patients (97%) and for neuron-specific enolase in 21 patients (72%) CNV. The epithelial marker KL1 was positive in 28 patients (97%) and the endothelial marker CD 31 in 20 patients (69%).. The immunohistochemical analyses of CNV showed that in the majority of cases during the excision of choroidal neovascularizations in addition to scar tissue and connective tissue also parts of the native retinal pigment epithelium and of the neurosensory retina are removed which is only partly visible with standard staining techniques. These findings suggest that the mostly not satisfying postoperative results are partly due to the damage of neuronal cells and a partial loss of the retinal pigment epithelium. Apoptosis as a regulating mechanism in choroidal neovascularization. The variable appearence of apoptosis suggests that it is possibly related to the degree of activity of CNV.

Topics: Aged; Aged, 80 and over; Choroidal Neovascularization; DNA Fragmentation; Ectoderm; Endothelium, Vascular; Female; Glial Fibrillary Acidic Protein; Humans; Immunohistochemistry; In Situ Nick-End Labeling; Keratins; Macular Degeneration; Male; Middle Aged; Neuroglia; Neurons; Phosphopyruvate Hydratase; Pigment Epithelium of Eye; Platelet Endothelial Cell Adhesion Molecule-1; Synaptophysin; Vitrectomy

To investigate the localization of N epsilon-(carboxymethyl)lysine (CML), a component and major immunologic epitope of advanced glycation end products, in aged eyes and choroidal neovascular membranes (CNVMs) surgically excised from eyes with age-related macular degeneration.. Immunohistochemistry for CML was performed using 8 snap-frozen, surgically excised CNVMs. Twelve eyes from patients aged 69 to 82 years and 2 donor eyes, 1 each from a 23-week-old fetus and 21-year-old patient, without age-related macular degeneration or diabetic retinopathy were also examined. To determine if retinal pigment epithelial cells in CNVMs accumulate advanced glycation end products, cytokeratin and CML were stained in paired serial sections.. Soft, macular drusen and/or basal laminar and basal linear deposits were observed in 8 of 12 aged eyes. Each case showed CML accumulation, while overlying retinal pigment epithelial cells showed no accumulation in all 12 eyes. In CNVMs, however, retinal pigment epithelial cells showed CML accumulation in their cytoplasm.. The additional accumulation of advanced glycation end products in soft, macular drusen and/or retinal pigment epithelial cells may play a role in the pathogenesis of CNVM formation in age-related macular degeneration.. Recently, advanced glycation end products have been found to play a role both in aging changes and neovascularization. Localization of advanced glycation end products in the above-mentioned tissue may lead to a better understanding of the pathogenesis of age-related macular degeneration.

Topics: Adult; Aged; Aged, 80 and over; Choroidal Neovascularization; Glycation End Products, Advanced; Humans; Immunoenzyme Techniques; Keratins; Lysine; Macular Degeneration; Pigment Epithelium of Eye

To determine the cellular origin and the vascular endothelial growth factor (VEGF) immunoreactivity of the nonvascular stromal cells in surgically excised age-related macular degeneration (ARMD)-associated choroidal neovascular membranes (CNVMs).. Immunohistochemical analysis was performed on frozen sections of eight surgically excised ARMD-related CNVMs.. Cytokeratin-positive, smooth muscle actin-positive polygonal or fibroblastic (transdifferentiated RPE) cells were the principal nonvascular stromal cells detected. The polygonal cells were more commonly found in active (highly vascularized) regions and were strongly immunoreactive for VEGF. The fibroblastic cells were predominantly found in fibrotic (hypovascular) regions and were minimally immunoreactive for VEGF.. Transdifferentiated RPE cells are the principal nonvascular stromal cells of both vascular and fibrotic ARMD-related CNVMs. Preferential localization of VEGF immunoreactivity with the cytoplasm of the polygonal transdifferentiated RPE cells in the highly vascularized regions of the surgically excised CNVMs suggests an important angiogenic role of these cells and this growth factor in the progression of ARMD-related choroidal neovascularization.

Topics: Actins; Aged; Aged, 80 and over; Antibodies, Monoclonal; Cell Differentiation; Choroid; Endothelial Growth Factors; Female; Fluorescent Antibody Technique; Glial Fibrillary Acidic Protein; Humans; Immunoenzyme Techniques; Keratins; Lymphokines; Macular Degeneration; Male; Neovascularization, Pathologic; Pigment Epithelium of Eye; Vascular Endothelial Growth Factor A; Vascular Endothelial Growth Factors