Retinal pigment epithelial cells respond to complement by an augmented production of vitronectin

Objectives: Genetic studies have demonstrated the role of activated complement on the alternative pathway during the development of age-related macular degeneration (AMD). The extracellular matrix component vitronectin can protect against activated complement. Drusen appear in the retina between the retinal pigment epithelial (RPE) cell layer and Bruch’s membrane. Drusen are hallmarks of early and late AMD and contain high amounts of vitronectin. Therefore this study addressed the influence of complement on the vitronectin production by RPE cells. Methods: ARPE-19 cells as model for RPE cells were cultivated with increasing amounts of human serum as complement source in its naïve and heat (and thereby complement) inactivated form. In another series of experiments zymosan as an activator of the alternative pathway of complement was tested alone and in combination with naïve human serum. Vitronectin was assayed in situ by immunohistochemistry, on protein level by western blot and by PCR after reverse transcription of total RNA. Results: A constitutive production of vitronectin by RPE cells was detected by all three tests. With naïve human serum increased vitronectin protein was found by immunohistochemistry and western blot while the number of mRNA transcripts was not significantly altered. The vitronectin production was further enhanced with the combination of zymosan and naïve human serum while heat inactivated serum showed lesser effect. Conclusion: Activated complement lead to an augmented vitronectin production by RPE cells on post-transcriptional level. Enhanced complement activation during AMD might also contribute in vivo to an enhanced production of vitronectin by RPE cells. On the one hand this can cause protection against activated complement but on the other hand the increased retinal vitronectin might contribute to thickening of Bruch’s membrane and may facilitate the development of drusen

Complement increases release of proinflammatory and proangiogenic mediators by retinal pigment epithelial cells

Objectives. A mutation in complement factor H (CFH) gene, leading to augmented complement activation, is correlated with development of age-related macular degeneration (AMD). Therefore, the influence of complement on retinal pigment epithelial (RPE) cells was examined concerning their production of proinflammatory and proangiogenic mediators relevant in AMD. Methods. ARPE-19 cells were cultured with human or fetal calf serum (FCS). Therefore, complement containing native serum as well as the heat-inactivated form with inoperable complement was used. Further, RPE cells were treated with zymosan, a complement activating yeast particle. Serum and zymosan in combination was also tested. Levels of interleukin (IL)-6, -8 and vascular endothelial growth factor (VEGF) in supernatants were examined by ELISA. Results. Untreated RPE cells produced IL-6, -8 and VEGF constitutively. FCS or human serum led to a concentration dependent release of all mediators. Thereby, FCS increased the cytokine production stronger than human serum, native serum stronger than heat-inactivated. Zymosan only intensified IL-6 and -8 secretion. Combined treatment with serum and zymosan resulted in an additive release of IL-8 and VEGF. In contrast, secretion of IL-6 was synergistic. Conclusion. The enhanced expression of IL-6, -8 and VEGF by RPE after exposure to complement might explain the correlation between augmented complement production and inflammatory processes accompanying AMD. IL-6 production was strongly increased due to activation of complement within the serum by zymosan. Thus, complement activation could stimulate inflammatory processes by activated RPE cells leading to AMD

The ratio of pro- and anti-angiogenic cytokines produced by retinal pigment epithelial cells is shifted to support angiogenesis by complement

Purpose The complement system of age-related macular degeneration (AMD) patients is marginally but chronically over-activated. Retinal pigment epithelial (RPE) cells and photoreceptor cells undergo cell death during the development of this potentially blinding eye disease. In this study the balance between the pro-angiogenic vascular endothelial growth factor (VEGF) and the anti-angiogenic pigment epithelium-derived factor (PEDF) by RPE cells in response to complement serum was analysed. Methods Increasing concentrations of complement competent human serum were incubated with human RPE cells. Controls with the addition of zymosan to activate the complement cascade, zymosan alone, and heat-treated serum with inoperative complement were included. The secretion of VEGF and PEDF was measured by sandwich ELISA. Immunocytochemistry was performed for the in situ detection of VEGF and PEDF. The experiments were supplemented by RT-PCR expression analysis and Western Blot detection of both antagonists. Results Human complement competent serum stimulated the RPE cells to produce enhanced amounts of VEGF while unspecific stimuli showed no influence on the secretion of VEGF. The combination of complement competent serum and zymosan was revealed as the most effective treatment for an increased VEGF production. The PEDF-specific staining of RPE cells decreased with augmented concentrations of complement competent serum. PCR data showed an enhanced amount of VEGF-encoding transcripts and an unaltered or lower amount of PEDF-specific transcripts. Western Blots confirmed the shift in favour of VEGF when compared to PEDF after complement treatment of RPE cells. Conclusions Activated complement may shift the balance between VEGF and PEDF produced by RPE cells towards the blood vessel chemoattractant VEGF. This finding may reveal a mechanism how enhanced complement activation might contribute to a pro-angiogenic retinal environment supporting neovascularisation during the late stage of exsudative AMD

A portable instrument for measuring macular pigment with central fixation

urpose: To evaluate the reliability and validity of a portable instrument for measuring macular pigment optical density. Methods: The instrument is small, uses light emitting diodes as light sources and the principles of heterochromatic flicker photometry of comparing foveal and extra-foveal minimum flicker matches. It uses central fixation for the extra-foveal matches, which subjects found easier than eccentric fixation. Subjects with healthy eyes used the instrument to measure their pigment density in a number of eye clinics. Results: The mean pigment density in 124 eyes in 124 individuals was 0.41 +/- 0.16 (mean +/- sd), there was no significant change with age but the density was less in females, those with light irides, smokers, subjects on diets low in precursor carotenoids and in those exposed to several hours of daylight every day or who used sun beds. Conclusions: The portable instrument gave valid and reliable data that confirmed published values for macular pigment. It was convenient to use in the clinic and has potential as a screening tool

Human Complement Sera stimulates Basolateral Secretion of VEGF by Retinal Pigment Epithelial Cells

Purpose. A mutation in the complement factor H (CFH) gene, leading to increased complement activation, is correlated with the development of age-related macular degeneration (AMD). Therefore, the influence of complement on human retinal pigment epithelial (RPE) cells was examined in respect to their polarized secretion of vascular endothelial growth factor (VEGF). Methods. RPE cells were cultured on transwell filters with DMEM and 1 % foetal calf serum. At six weeks post confluence, when the RPE have pigmented, the density of the cell monolayer was measured by a permeability assay using sodium fluorescein. The cells were treated with human complement sera for 24 hours. The amount of VEGF secreted into the media was quantified by enzyme-linked immunosorbent assay. Furthermore, the cellular distribution of VEGF in complement treated cells grown in chamber slides was detected by immunocytochemistry, and PCR analysis was used to determine the expression of the growth factor in RPE cells. Results. Untreated RPE cells produced VEGF constitutively. Basal stimulation of polarized cells with human complement sera led to a concentration dependent increased release of the growth factor towards the basal compartment. Immunocytochemical staining and PCR analysis for VEGF also demonstrated a concentration dependent enhancement in response to complement. Conclusions. VEGF production towards the basal side was strongly increased when RPE cells were exposed to human complement sera applied to the basal side. Therefore, complement might play a significant role in AMD, as VEGF is known to stimulate vessel growth in the choroid and support pro-angiogenic processes

Complement stimulates Retinal Pigment Epithelial Cells to undergo Pro-inflammatory Changes as in Early Age-Related Macular Degeneration

Purpose. A polymorphism in the complement factor H gene, leading to increased complement activation, is associated with the development of age-related macular degeneration (AMD). We therefore examined the effect of human complement sera (HCS) on retinal pigment epithelial (RPE) cells with respect to pro-inflammatory mediators relevant in early AMD. Methods. RPE cells were treated with HCS or heat-inactivated (HI)-HCS as a complement-deficient control. Cells were stained for C5b-9 using immunocytochemistry and immunofluorescence, and cell viability was determined. Interleukin (IL) -6, -8 and monocyte chemoattractant protein-1 (MCP-1) were quantified by ELISA and their expression was determined by RT-PCR. Intercellular adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule-1 (VCAM-1) and tumour necrosis factor-α (TNF-α) were analysed by western blotting. The intracellular distribution of nuclear factor (NF)-ƙB was investigated by immunofluorescence. Results. Concentration-dependent increased staining for C5b-9 was observed after HCS treatment, whereas cell viability decreased. ELISA and RT-PCR analysis revealed increased secretion and expression of IL-6, -8 and MCP-1. Western blot analysis showed a concentration-dependent enhancement in ICAM-1, VCAM-1 and TNF-α in response to HCS, and immunofluorescence staining revealed cytosolic to nuclear translocation of NF-ƙB. Conclusions. This study suggests that complement may stimulate RPE cells to create a pro-inflammatory environment via NF-ƙB activation which may support early AMD development

Macular Telangiectasia Type 2: A Classification System Using MultiModal Imaging MacTel Project Report Number 10

Purpose: To develop a severity classification for macular telangiectasia type 2 (MacTel) disease using multimodal imaging. Design: An algorithm was used on data from a prospective natural history study of MacTel for classification development. Subjects: A total of 1733 participants enrolled in an international natural history study of MacTel. Methods: The Classification and Regression Trees (CART), a predictive nonparametric algorithm used in machine learning, analyzed the features of the multimodal imaging important for the development of a classification, including reading center gradings of the following digital images: stereoscopic color and red-free fundus photographs, fluorescein angiographic images, fundus autofluorescence images, and spectral-domain (SD)-OCT images. Regression models that used least square method created a decision tree using features of the ocular images into different categories of disease severity. Main Outcome Measures: The primary target of interest for the algorithm development by CART was the change in best-corrected visual acuity (BCVA) at baseline for the right and left eyes. These analyses using the algorithm were repeated for the BCVA obtained at the last study visit of the natural history study for the right and left eyes. Results: The CART analyses demonstrated 3 important features from the multimodal imaging for the classification: OCT hyper-reflectivity, pigment, and ellipsoid zone loss. By combining these 3 features (as absent, present, noncentral involvement, and central involvement of the macula), a 7-step scale was created, ranging from excellent to poor visual acuity. At grade 0, 3 features are not present. At the most severe grade, pigment and exudative neovascularization are present. To further validate the classification, using the Generalized Estimating Equation regression models, analyses for the annual relative risk of progression over a period of 5 years for vision loss and for progression along the scale were performed. Conclusions: This analysis using the data from current imaging modalities in participants followed in the MacTel natural history study informed a classification for MacTel disease severity featuring variables from SD-OCT. This classification is designed to provide better communications to other clinicians, researchers, and patients. Financial Disclosure(s): Proprietary or commercial disclosure may be found after the references

Detection of heat shock protein 70 in choroidal neovascular membranes secondary to age related macular degeneration

<p>Abstract</p> <p>Background</p> <p>Heat shock proteins are acute phase proteins that are upregulated in inflammation or following thermal stress. We analyzed the presence of the heat shock protein 70 (Hsp 70) in choroidal neovascular (CNV) membranes secondary to AMD after treatment with verteporphin photodynamic therapy (PDT) or transpupillary thermo therapy (TTT) to determine whether treatment correlated with the presence of Hsp70.</p> <p>Results</p> <p>CNV membranes were removed by pars plana vitrectomy (ppV) and subretinal extraction. The membranes were analysed by light microscopy and the presence of Hsp 70 was examined using histochemistry. HeLa Cells served as controls.</p> <p>Of the 14 membranes analysed 11 were Hsp70 positive and 3 negative. In the no pre-treatment group of 8 membranes 6 were Hsp70 positive and 2 negative; in the PTD group all 4 membranes were positive and in the TTT group 1 membrane was positive and 1 membrane was negative for Hsp70.</p> <p>Conclusion</p> <p>Hsp70 is present in the most CNV membranes secondary to AMD. Pre-treatment of the membrane with PTD or TTT does not appear to influence the expression of Hsp70.</p