Search

The cellular events that dictate the initiation of the complement pathway in ocular degeneration, such as age-related macular degeneration (AMD), is poorly understood. Using gene expression analysis (single cell and bulk), mass spectrometry, and immunohistochemistry, we dissected the role of multiple retinal and choroidal cell types in determining the complement homeostasis. Our scRNA-seq data show that the cellular response to early AMD is more robust in the choroid, particularly in fibroblasts, pericytes and endothelial cells. In late AMD, complement changes were more prominent in the retina especially with the expression of the classical pathway initiators. Notably, we found a spatial preference for these differences. Overall, this study provides insights into the heterogeneity of cellular responses for complement expression and the cooperation of neighboring cells to complete the pathway in healthy and AMD eyes. Further, our findings provide new cellular targets for therapies directed at complement., Competing Interests: CC is a consultant for Apellis. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be constructed as a potential conflict of interest., (Copyright © 2022 Zauhar, Biber, Jabri, Kim, Hu, Kaplan, Pfaller, Schäfer, Enzmann, Schlötzer-Schrehardt, Straub, Hauck, Gamlin, McFerrin, Messinger, Strang, Curcio, Dana, Pauly, Grosche, Li and Stambolian.)

Aim: Treatment of exudative age-related macular degeneration by using vascular endothelial growth factor (VEGF) antagonists is the gold standard today. So far, several bioactive molecules have been approved for therapeutic use. In this study, we investigate the effects of ranibizumab (Lucentis®), bevacizumab (Avastin®), and aflibercept (Eylea®) on primary human retinal pigment epithelial (hRPE) cells in vitro., Methods: hRPE cells were prepared from donor eyes and cultured under standard culture conditions. Scleral fibroblasts also prepared from donor tissue served as physiological controls. The impact of the anti-VEGF molecules on cell viability was investigated with the trypan blue exclusion assay, whereas proliferation was measured using the MTT assay. Biological activity of the molecules was quantified in a VEGF-enzyme-linked immunosorbent assay (ELISA)., Results: All tested substances were biologically active in vitro. They displayed no cytotoxicity on RPE cells or scleral fibroblasts. However, proliferation of RPE cells was significantly decreased after treatment with ranibizumab or bevacizumab but not with aflibercept., Conclusions: The humanized antibodies (fragments) interfered specifically with the RPE cells. The thereby measured inhibition of cell proliferation may indicate possible side effects on the physiology of RPE cells., (© 2018 S. Karger AG, Basel.)

Age-related macular degeneration (AMD) is the leading cause of irreversible vision loss. The protein HtrA1 is enriched in retinal pigment epithelial (RPE) cells isolated from AMD patients and in drusen deposits. However, it is poorly understood how increased levels of HtrA1 affect the physiological function of the RPE at the intracellular level. Here, we developed hfRPE (human fetal retinal pigment epithelial) cell culture model where cells fully differentiated into a polarized functional monolayer. In this model, we fine-tuned the cellular levels of HtrA1 by targeted overexpression. Our data show that HtrA1 enzymatic activity leads to intracellular degradation of tubulin with a corresponding reduction in the number of microtubules, and consequently to an altered mechanical cell phenotype. HtrA1 overexpression further leads to impaired apical processes and decreased phagocytosis, an essential function for photoreceptor survival. These cellular alterations correlate with the AMD phenotype and thus highlight HtrA1 as an intracellular target for therapeutic interventions towards AMD treatment., (Copyright © 2017 The Authors. Published by Elsevier B.V. All rights reserved.)

Purpose: To investigate whether Chlamydia pneumoniae and complement factors were present in surgically removed choroidal neovascular membranes (CNV) of patients with age-related macular degeneration (AMD)., Methods: Paraffin sections of 26 CNV were stained for C. pneumoniae or the complement factors H (CFH) and C5, whereas macrophages were identified by positive CD68 staining. Clinical characteristics have been correlated to the immunohistochemical findings., Results: C. pneumoniae was found in 68% of the investigated membranes, and 88% of these membranes were also positive for CD68. Staining for CFH and C5 gave a positive reaction in 68 and 41% of the membranes, respectively. Patients with C5-positive membranes had significantly larger CNV mean area and were younger than patients with CFH-positive membranes at the operation time point., Conclusions: Correlations between clinical symptoms and complement factor C5 could be shown. The results strengthen the hypothesis of an involvement of the complement system in AMD.

Purpose: To evaluate whether complement Factor P (properdin) was present in surgically removed choroidal neovascular membranes of patients with age-related macular degeneration (AMD) and to investigate whether associated pre- and postoperative clinical characteristics can be correlated., Methods: The study population consisted of 26 AMD patients (26 eyes) with the exclusion criterion of prior treatment of the choroidal neovascular membranes. Factor P was detected immunocytochemically on paraffin sections (7 microm) by a polyclonal rabbit antihuman antibody., Results: Eleven of 22 assessable membranes showed a positive reaction for Factor P. The average percentage of Factor P-positive cells per membrane ranged from 0.65% to 4.09%. The duration of visual loss was significantly longer (8.6 +/- 2.7 vs. 3.9 +/- 0.8 months), and the size of postoperative measured area of atrophic retinal pigment epithelium was larger (27.6 +/- 7.6 vs. 15.0 +/- 6.9 mm2) in patients with Factor P-positive membranes compared with Factor P-negative ones., Conclusion: Factor P was expressed in 50% of choroidal neovascular membranes of patients with AMD. The group with Factor P-positive membranes differed significantly from the Factor P-negative group in key clinical outcomes. Additional studies need to investigate the role of Factor P in the development of AMD for potential therapeutic intervention.

Purpose: To investigate the appearance of geographic atrophy in high-resolution optical coherence tomography (OCT) images, the fundus autofluorescence (FAF) pattern, and infrared images simultaneously recorded with a novel combined OCT-scanning laser ophthalmology (SLO) system., Methods: Patients aged over 50 years with geographic atrophy secondary to dry age-related macular degeneration (ARMD) were assessed in a prospective cross-sectional study by means of simultaneous spectral OCT-SLO (Spectralis HRA+OCT; Heidelberg Engineering, Heidelberg, Germany). The integrity of the retinal layers was analyzed in the apparently normal areas, the junctional zone between the normal retina and the geographic atrophy, and the atrophic area. The presence and integrity of the external limiting membrane, the photoreceptor inner segments, the outer segments, and the retinal pigment epithelium were assessed., Results: Fifty-two eyes of 52 patients (28 women, 24 men) aged 51 to 92 years were examined. Retinal layer alterations were documented, not only in atrophic zones, but also in junctional zones surrounding the geographic atrophy. Disintegration of the retinal layers began in the RPE and adjacent retinal layers, such as the photoreceptor inner and outer segments and external limiting membrane., Conclusions: Novel imaging modalities will provide further valuable insight into ARMD pathogenesis. The key to understanding the morphologic change lies in in vivo depiction of retinal layers by spectral OCT technology in combination with other imaging modalities such as FAF.