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

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

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

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

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

Continuous evaluation of large-scale information access systems : a case for living labs

A/B testing is currently being increasingly adopted for the evaluation of commercial information access systems with a large user base since it provides the advantage of observing the efficiency and effectiveness of information access systems under real conditions. Unfortunately, unless university-based researchers closely collaborate with industry or develop their own infrastructure or user base, they cannot validate their ideas in live settings with real users. Without online testing opportunities open to the research communities, academic researchers are unable to employ online evaluation on a larger scale. This means that they do not get feedback for their ideas and cannot advance their research further. Businesses, on the other hand, miss the opportunity to have higher customer satisfaction due to improved systems. In addition, users miss the chance to benefit from an improved information access system. In this chapter, we introduce two evaluation initiatives at CLEF, NewsREEL and Living Labs for IR (LL4IR), that aim to address this growing “evaluation gap” between academia and industry. We explain the challenges and discuss the experiences organizing these living labs

Retinal Vascular Occlusion after COVID-19 Vaccination : More Coincidence than Causal Relationship? Data from a Retrospective Multicentre Study

Background: To investigate whether vaccination against SARS-CoV-2 is associated with the onset of retinal vascular occlusive disease (RVOD). Methods: In this multicentre study, data from patients with central and branch retinal vein occlusion (CRVO and BRVO), central and branch retinal artery occlusion (CRAO and BRAO), and anterior ischaemic optic neuropathy (AION) were retrospectively collected during a 2-month index period (1 June–31 July 2021) according to a defined protocol. The relation to any previous vaccination was documented for the consecutive case series. Numbers of RVOD and COVID-19 vaccination were investigated in a case-by-case analysis. A case– control study using age- and sex-matched controls from the general population (study participants from the Gutenberg Health Study) and an adjusted conditional logistic regression analysis was conducted. Results: Four hundred and twenty-one subjects presenting during the index period (61 days) were enrolled: one hundred and twenty-one patients with CRVO, seventy-five with BRVO, fifty-six with CRAO, sixty-five with BRAO, and one hundred and four with AION. Three hundred and thirty-two (78.9%) patients had been vaccinated before the onset of RVOD. The vaccines given were BNT162b2/BioNTech/Pfizer (n = 221), followed by ChadOx1/AstraZeneca (n = 57), mRNA1273/Moderna (n = 21), and Ad26.COV2.S/Johnson & Johnson (n = 11; unknown n = 22). Our case–control analysis integrating population-based data from the GHS yielded no evidence of an increased risk after COVID-19 vaccination (OR = 0.93; 95% CI: 0.60–1.45, p = 0.75) in connection with a vaccination within a 4-week window. Conclusions: To date, there has been no evidence of any association between SARS-CoV-2 vaccination and a higher RVOD risk

Cigarette Smoke-Related Hydroquinone Dysregulates MCP-1, VEGF and PEDF Expression in Retinal Pigment Epithelium in Vitro and in Vivo

Age-related macular degeneration (AMD) is the leading cause of legal blindness in the elderly population. Debris (termed drusen) below the retinal pigment epithelium (RPE) have been recognized as a risk factor for dry AMD and its progression to wet AMD, which is characterized by choroidal neovascularization (CNV). The underlying mechanism of how drusen might elicit CNV remains undefined. Cigarette smoking, oxidative damage to the RPE and inflammation are postulated to be involved in the pathophysiology of the disease. To better understand the cellular mechanism(s) linking oxidative stress and inflammation to AMD, we examined the expression of pro-inflammatory monocyte chemoattractant protein-1 (MCP-1), pro-angiogenic vascular endothelial growth factor (VEGF) and anti-angiogenic pigment epithelial derived factor (PEDF) in RPE from smoker patients with AMD. We also evaluated the effects of hydroquinone (HQ), a major pro-oxidant in cigarette smoke on MCP-1, VEGF and PEDF expression in cultured ARPE-19 cells and RPE/choroids from C57BL/6 mice.MCP-1, VEGF and PEDF expression was examined by real-time PCR, Western blot, and ELISA. Low levels of MCP-1 protein were detected in RPE from AMD smoker patients relative to controls. Both MCP-1 mRNA and protein were downregulated in ARPE-19 cells and RPE/choroids from C57BL/6 mice after 5 days and 3 weeks of exposure to HQ-induced oxidative injury. VEGF protein expression was increased and PEDF protein expression was decreased in RPE from smoker patients with AMD versus controls resulting in increased VEGF/PEDF ratio. Treatment with HQ for 5 days and 3 weeks increased the VEGF/PEDF ratio in vitro and in vivo.We propose that impaired RPE-derived MCP-1-mediated scavenging macrophages recruitment and phagocytosis might lead to incomplete clearance of proinflammatory debris and infiltration of proangiogenic macrophages which along with increased VEGF/PEDF ratio favoring angiogenesis might promote drusen accumulation and progression to CNV in smoker patients with dry AMD