Corneal biomechanics and diagnostics: a review.

PURPOSE: Corneal biomechanics is an emerging field and the interest into physical and biological interrelations in the anterior part of the eye has significantly increased during the past years. There are many factors that determine corneal biomechanics such as hormonal fluctuations, hydration and environmental factors. Other factors that can affect the corneas are the age, the intraocular pressure and the central corneal thickness. The purpose of this review is to evaluate the factors affecting corneal biomechanics and the recent advancements in non-destructive, in vivo measurement techniques for early detection and improved management of corneal diseases. METHODS: Until recently, corneal biomechanics could not be directly assessed in humans and were instead inferred from geometrical cornea analysis and ex vivo biomechanical testing. The current research has made strides in studying and creating non-destructive and contactless techniques to measure the biomechanical properties of the cornea in vivo. RESULTS: Research has indicated that altered corneal biomechanics contribute to diseases such as keratoconus and glaucoma. The identification of pathological corneas through the new measurement techniques is imperative for preventing postoperative complications. CONCLUSIONS: Identification of pathological corneas is crucial for the prevention of postoperative complications. Therefore, a better understanding of corneal biomechanics will lead to earlier diagnosis of ectatic disorders, improve current refractive surgeries and allow for a better postoperative treatment.

Macrophages coordinate immune response to laser-induced injury via extracellular traps.

BACKGROUND: Retinal degeneration results from disruptions in retinal homeostasis due to injury, disease, or aging and triggers peripheral leukocyte infiltration. Effective immune responses rely on coordinated actions of resident microglia and recruited macrophages, critical for tissue remodeling and repair. However, these phagocytes also contribute to chronic inflammation in degenerated retinas, yet the precise coordination of immune response to retinal damage remains elusive. Recent investigations have demonstrated that phagocytic cells can produce extracellular traps (ETs), which are a source of self-antigens that alter the immune response, which can potentially lead to tissue injury. METHODS: Innovations in experimental systems facilitate real-time exploration of immune cell interactions and dynamic responses. We integrated in vivo imaging with ultrastructural analysis, transcriptomics, pharmacological treatments, and knockout mice to elucidate the role of phagocytes and their modulation of the local inflammatory response through extracellular traps (ETs). Deciphering these mechanisms is essential for developing novel and enhanced immunotherapeutic approaches that can redirect a specific maladaptive immune response towards favorable wound healing in the retina. RESULTS: Our findings underscore the pivotal role of innate immune cells, especially macrophages/monocytes, in regulating retinal repair and inflammation. The absence of neutrophil and macrophage infiltration aids parenchymal integrity restoration, while their depletion, particularly macrophages/monocytes, impedes vascular recovery. We demonstrate that macrophages/monocytes, when recruited in the retina, release chromatin and granular proteins, forming ETs. Furthermore, the pharmacological inhibition of ETosis support retinal and vascular repair, surpassing the effects of blocking innate immune cell recruitment. Simultaneously, the absence of ETosis reshapes the inflammatory response, causing neutrophils, helper, and cytotoxic T-cells to be restricted primarily in the superficial capillary plexus instead of reaching the damaged photoreceptor layer. CONCLUSIONS: Our data offer novel insights into innate immunity's role in responding to retinal damage and potentially help developing innovative immunotherapeutic approaches that can shift the immune response from maladaptive to beneficial for retinal regeneration.

Modulation of Extracellular Matrix Composition and Chronic Inflammation with Pirfenidone Promotes Scar Reduction in Retinal Wound Repair.

Wound repair in the retina is a complex mechanism, and a deeper understanding of it is necessary for the development of effective treatments to slow down or even prevent degenerative processes leading to photoreceptor loss. In this study, we harnessed a laser-induced retinal degeneration model (532-nm laser photocoagulation with 300 µm spot size, 60 ms duration and 60 mV pulse), enabling a profound molecular elucidation and a comprehensive, prolonged observation of the wound healing sequence in a murine laser-induced degeneration model (C57BL/6J mice, 6-12 weeks) until day 49 post-laser. Our observations included the expression of specific extracellular matrix proteins and myofibroblast activity, along with an analysis of gene expression related to extracellular matrix and adhesion molecules through RNA measurements. Furthermore, the administration of pirfenidone (10 mg/kg via drinking water), an anti-inflammatory and anti-fibrotic compound, was used to modulate scar formation after laser treatment. Our data revealed upregulated collagen expression in late regenerative phases and sustained inflammation in the damaged tissue. Notably, treatment with pirfenidone was found to mitigate scar tissue formation, effectively downregulating collagen production and diminishing the presence of inflammatory markers. However, it did not lead to the regeneration of the photoreceptor layer.

Assessing the role of T cells in response to retinal injury to uncover new therapeutic targets for the treatment of retinal degeneration.

BACKGROUND: Retinal degeneration is a disease affecting the eye, which is an immune-privileged site because of its anatomical and physiological properties. Alterations in retinal homeostasis-because of injury, disease, or aging-initiate inflammatory cascades, where peripheral leukocytes (PL) infiltrate the parenchyma, leading to retinal degeneration. So far, research on PL's role in retinal degeneration was limited to observing a few cell types at specific times or sectioning the tissue. This restricted our understanding of immune cell interactions and response duration. METHODS: In vivo microscopy in preclinical mouse models can overcome these limitations enabling the spatio-temporal characterization of PL dynamics. Through in vivo imaging, we assessed structural and fluorescence changes in response to a focal injury at a defined location over time. We also utilized minimally invasive techniques, pharmacological interventions, and knockout (KO) mice to determine the role of PL in local inflammation. Furthermore, we investigated PL abundance and localization during retinal degeneration in human eyes by histological analysis to assess to which extent our preclinical study translates to human retinal degeneration. RESULTS: We demonstrate that PL, especially T cells, play a detrimental role during retinal injury response. In mice, we observed the recruitment of helper and cytotoxic T cells in the parenchyma post-injury, and T cells also resided in the macula and peripheral retina in pathological conditions in humans. Additionally, we found that the pharmacological PL reduction and genetic depletion of T-cells reduced injured areas in murine retinas and rescued the blood-retina barrier (BRB) integrity. Both conditions promoted morphological changes of Cx3cr1+ cells, including microglial cells, toward an amoeboid phenotype during injury response. Interestingly, selective depletion of CD8+ T cells accelerated recovery of the BRB compared to broader depletions. After anti-CD8 treatment, the retinal function improved, concomitant to a beneficial immune response. CONCLUSIONS: Our data provide novel insights into the adaptive immune response to retinal injury in mice and human retinal degeneration. Such information is fundamental to understanding retinal disorders and developing therapeutics to modulate immune responses to retinal degeneration safely.

Modelling MOG antibody-associated disorder and neuromyelitis optica spectrum disorder in animal models: Spinal cord manifestations.

Antibodies to myelin oligodendrocyte glycoprotein (MOG-IgG) or aquaporin 4 (AQP4-IgG) are associated with CNS inflammatory disorders. We directly compared MOG35-55-induced experimental autoimmune encephalomyelitis exacerbated by MOG- and AQP4-IgG (versus isotype IgG, Iso-IgG). Disease severity was highest after MOG-IgG application. MOG- and AQP4-IgG administration increased disease incidence compared to Iso-IgG. Inflammatory lesions appeared earlier and with distinct localizations after AQP4-IgG administration. AQP4 intensity was more reduced after AQP4- than MOG-IgG administration at acute disease phase. The described models are suitable for comparative analyses of pathological features associated with MOG- and AQP4-IgG and the investigation of therapeutic interventions.

Modeling MOG Antibody-Associated Disorder and Neuromyelitis Optica Spectrum Disorder in Animal Models: Visual System Manifestations.

BACKGROUND AND OBJECTIVES: Mechanisms of visual impairment in aquaporin 4 antibody (AQP4-IgG) seropositive neuromyelitis optica spectrum disorder (NMOSD) and myelin oligodendrocyte glycoprotein antibody (MOG-IgG)-associated disorder (MOGAD) are incompletely understood. The respective impact of optic nerve demyelination and primary and secondary retinal neurodegeneration are yet to be investigated in animal models. METHODS: Active MOG35-55 experimental autoimmune encephalomyelitis (EAE) was induced in C57BL/6Jrj mice, and monoclonal MOG-IgG (8-18C5, murine), recombinant AQP4-IgG (rAb-53, human), or isotype-matched control IgG (Iso-IgG, human) was administered 10 days postimmunization. Mobility impairment was scored daily. Visual acuity by optomotor reflex and ganglion cell complex thickness (GCC, 3 innermost retinal layers) by optical coherence tomography (OCT) were longitudinally assessed. Histopathology of optic nerve and retina was investigated during presymptomatic, acute, and chronic disease phases for immune cells, demyelination, complement deposition, natural killer (NK) cell, AQP4, and astrocyte involvement, retinal ganglion cells (RGCs), and Müller cell activation. Groups were compared by nonparametric tests with a p value <0.05 indicating statistical significance. RESULTS: Visual acuity decreased from baseline to chronic phase in MOG-IgG (mean ± standard error of the mean: 0.54 ± 0.01 to 0.46 ± 0.02 cycles/degree, p < 0.05) and AQP4-IgG EAE (0.54 ± 0.01 to 0.43 ± 0.02, cycles/degree, p < 0.05). Immune cell infiltration of optic nerves started in presymptomatic AQP4-IgG, but not in MOG-IgG EAE (5.85 ± 2.26 vs 0.13 ± 0.10 macrophages/region of interest [ROI] and 1.88 ± 0.63 vs 0.15 ± 0.06 T cells/ROI, both p < 0.05). Few NK cells, no complement deposition, and stable glial fibrillary acid protein and AQP4 fluorescence intensity characterized all EAE optic nerves. Lower GCC thickness (Spearman correlation coefficient r = -0.44, p < 0.05) and RGC counts (r = -0.47, p < 0.05) correlated with higher mobility impairment. RGCs decreased from presymptomatic to chronic disease phase in MOG-IgG (1,705 ± 51 vs 1,412 ± 45, p < 0.05) and AQP4-IgG EAE (1,758 ± 14 vs 1,526 ± 48, p < 0.01). Müller cell activation was not observed in either model. DISCUSSION: In a multimodal longitudinal characterization of visual outcome in animal models of MOGAD and NMOSD, differential retinal injury and optic nerve involvement were not conclusively clarified. Yet optic nerve inflammation was earlier in AQP4-IgG-associated pathophysiology. Retinal atrophy determined by GCC thickness (OCT) and RGC counts correlating with mobility impairment in the chronic phase of MOG-IgG and AQP4-IgG EAE may serve as a generalizable marker of neurodegeneration.

Characterization of Macroglia Response during Tissue Repair in a Laser-Induced Model of Retinal Degeneration.

Reactive gliosis is a hallmark of chronic degenerative diseases of the retina. As gliosis involves macroglia, we investigated their gliotic response to determine the role of S100ß and intermediate filaments (IFs) GFAP, vimentin, and nestin during tissue repair in a laser-induced model of retinal degeneration. We validated the results with human retinal donor samples. Experiments were performed in zebrafish and mice using an argon laser (532 nm) to induce focal lesions in the outer retina. At different time points following injury induction, the kinetics of retinal degeneration and regeneration were assessed using hematoxylin and eosin staining (H&E). Immunofluorescence was performed to evaluate Müller cell (GS) and astrocyte (GFAP) injury response and to distinguish between both cell types. Additionally, staining was performed in human retinal sections containing drusen. Focal laser treatment elevated the expression of gliotic markers in the area of the damage, which was associated with increased expression of S100ß, GFAP, vimentin, and nestin in mice and humans. In zebrafish, we detected S100ß at the first time point, but not GFAP or nestin. Double-positive cells with the selected glia markers were detected in all models. However, in zebrafish, no double-positive GFAP/GS cells were found on days 10 and 17, nor were S100ß/GS double-positive cells found on day 12. Macroglia cells showed a different pattern in the expression of IFs in degenerative and regenerative models. In particular, S100ß may prove to be a target for suppressing chronic gliosis in retinal degeneration.

Animal model of subretinal fibrosis without active choroidal neovascularization.

Subretinal fibrosis can occur during neovascular age-related macular degeneration (nAMD) and consequently provokes progressing deterioration of AMD patient's vision. Intravitreal anti-vascular endothelial growth factor (VEGF) injections decrease choroidal neovascularization (CNV), however, subretinal fibrosis remains principally unaffected. So far, no successful treatment nor established animal model for subretinal fibrosis exists. In order to investigate the impact of anti-fibrotic compounds on solely fibrosis, we refined a time-dependent animal model of subretinal fibrosis without active choroidal neovascularization (CNV). To induce CNV-related fibrosis, wild-type (WT) mice underwent laser photocoagulation of the retina with rupture of Bruch's membrane. The lesions volume was assessed with optical coherence tomography (OCT). CNV (Isolectin B4) and fibrosis (type 1 collagen) were separately quantified with confocal microscopy of choroidal whole-mounts at every time point post laser induction (day 7-49). In addition, OCT, autofluorescence and fluorescence angiography were carried out at designated timepoints (day 7, 14, 21, 28, 35, 42, 49) to monitor CNV and fibrosis transformation over time. From 21 to 49 days post laser lesion leakage in the fluorescence angiography decreased. Correspondingly, Isolectin B4 decreased in lesions of choroidal flat mounts and type 1 collagen increased. Fibrosis markers, namely vimentin, fibronectin, alpha-smooth muscle actin (α-SMA) and type 1 collagen were detected at different timepoints of tissue repair in choroids and retinas post laser. These results prove that the late phase of the CNV-related fibrosis model enables screening of anti-fibrotic compounds to accelerate the therapeutic advancement for the prevention, reduction, or inhibition of subretinal fibrosis.

Retinal regions shape human and murine Müller cell proteome profile and functionality.

The human macula is a highly specialized retinal region with pit-like morphology and rich in cones. How Müller cells, the principal glial cell type in the retina, are adapted to this environment is still poorly understood. We compared proteomic data from cone- and rod-rich retinae from human and mice and identified different expression profiles of cone- and rod-associated Müller cells that converged on pathways representing extracellular matrix and cell adhesion. In particular, epiplakin (EPPK1), which is thought to play a role in intermediate filament organization, was highly expressed in macular Müller cells. Furthermore, EPPK1 knockout in a human Müller cell-derived cell line led to a decrease in traction forces as well as to changes in cell size, shape, and filopodia characteristics. We here identified EPPK1 as a central molecular player in the region-specific architecture of the human retina, which likely enables specific functions under the immense mechanical loads in vivo.

As in Real Estate, Location Matters: Cellular Expression of Complement Varies Between Macular and Peripheral Regions of the Retina and Supporting Tissues.

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.

Antineonatal Fc Receptor Antibody Treatment Ameliorates MOG-IgG-Associated Experimental Autoimmune Encephalomyelitis.

BACKGROUND AND OBJECTIVES: Myelin oligodendrocyte glycoprotein antibody-associated disorder (MOGAD) is a rare, autoimmune demyelinating CNS disorder, distinct from multiple sclerosis and neuromyelitis optica spectrum disorder. Characterized by pathogenic immunoglobulin G (IgG) antibodies against MOG, a potential treatment strategy for MOGAD is to reduce circulating IgG levels, e.g., by interference with the IgG recycling pathway mediated by the neonatal Fc receptor (FcRn). Although the optic nerve is often detrimentally involved in MOGAD, the effect of FcRn blockade on the visual pathway has not been assessed. Our objective was to investigate effects of a monoclonal anti-FcRn antibody in murine MOG-IgG-associated experimental autoimmune encephalomyelitis (EAE). METHODS: We induced active MOG35-55 EAE in C57Bl/6 mice followed by the application of a monoclonal MOG-IgG (8-18C5) 10 days postimmunization (dpi). Animals were treated with either a specific monoclonal antibody against FcRn (α-FcRn, 4470) or an isotype-matched control IgG on 7, 10, and 13 dpi. Neurologic disability was scored daily on a 10-point scale. Visual acuity was assessed by optomotor reflex. Histopathologic hallmarks of disease were assessed in the spinal cord, optic nerve, and retina. Immune cell infiltration was visualized by immunohistochemistry, demyelination by Luxol fast blue staining and complement deposition and number of retinal ganglion cells by immunofluorescence. RESULTS: In MOG-IgG-augmented MOG35-55 EAE, anti-FcRn treatment significantly attenuated neurologic disability over the course of disease (mean area under the curve and 95% confidence intervals (CIs): α-FcRn [n = 27], 46.02 [37.89-54.15]; isotype IgG [n = 24], 66.75 [59.54-73.96], 3 independent experiments), correlating with reduced amounts of demyelination and macrophage infiltration into the spinal cord. T- and B-cell infiltration and complement deposition remained unchanged. Compared with isotype, anti-FcRn treatment prevented reduction of visual acuity over the course of disease (median cycles/degree and interquartile range: α-FcRn [n = 16], 0.50 [0.48-0.55] to 0.50 [0.48-0.58]; isotype IgG [n = 17], 0.50 [0.49-0.54] to 0.45 [0.39-0.51]). DISCUSSION: We show preserved optomotor response and ameliorated course of disease after anti-FcRn treatment in an experimental model using a monoclonal MOG-IgG to mimic MOGAD. Selectively targeting FcRn might represent a promising therapeutic approach in MOGAD.

Complement Factor H-Related 3 Enhanced Inflammation and Complement Activation in Human RPE Cells.

Complement Factor H-Related 3 (FHR-3) is a major regulator of the complement system, which is associated with different diseases, such as age-related macular degeneration (AMD). However, the non-canonical local, cellular functions of FHR-3 remained poorly understood. Here, we report that FHR-3 bound to oxidative stress epitopes and competed with FH for interaction. Furthermore, FHR-3 was internalized by viable RPE cells and modulated time-dependently complement component (C3, FB) and receptor (C3aR, CR3) expression of human RPE cells. Independently of any external blood-derived proteins, complement activation products were detected. Anaphylatoxin C3a was visualized in treated cells and showed a translocation from the cytoplasm to the cell membrane after FHR-3 exposure. Subsequently, FHR-3 induced a RPE cell dependent pro-inflammatory microenvironment. Inflammasome NLRP3 activation and pro-inflammatory cytokine secretion of IL-1ß, IL-18, IL-6 and TNF-α were induced after FHR-3-RPE interaction. Our previously published monoclonal anti-FHR-3 antibody, which was chimerized to reduce immunogenicity, RETC-2-ximab, ameliorated the effect of FHR-3 on ARPE-19 cells. Our studies suggest FHR-3 as an exogenous trigger molecule for the RPE cell "complosome" and as a putative target for a therapeutic approach for associated degenerative diseases.

The TGFß/Notch axis facilitates Müller cell-to-epithelial transition to ultimately form a chronic glial scar.

BACKGROUND: Contrasting with zebrafish, retinal regeneration from Müller cells (MCs) is largely limited in mammals, where they undergo reactive gliosis that consist of a hypertrophic response and ultimately results in vision loss. Transforming growth factor ß (TGFß) is essential for wound healing, including both scar formation and regeneration. However, targeting TGFß may affect other physiological mechanisms, owing its pleiotropic nature. The regulation of various cellular activities by TGFß relies on its interaction with other pathways including Notch. Here, we explore the interplay of TGFß with Notch and how this regulates MC response to injury in zebrafish and mice. Furthermore, we aimed to characterize potential similarities between murine and human MCs during chronic reactive gliosis. METHODS: Focal damage to photoreceptors was induced with a 532 nm diode laser in TgBAC (gfap:gfap-GFP) zebrafish (ZF) and B6-Tg (Rlbp1-GFP) mice. Transcriptomics, immunofluorescence, and flow cytometry were employed for a comparative analysis of MC response to laser-induced injury between ZF and mouse. The laser-induced injury was paired with pharmacological treatments to inhibit either Notch (DAPT) or TGFß (Pirfenidone) or TGFß/Notch interplay (SIS3). To determine if the murine laser-induced injury model translates to the human system, we compared the ensuing MC response to human donors with early retinal degeneration. RESULTS: Investigations into injury-induced changes in murine MCs revealed TGFß/Notch interplay during reactive gliosis. We found that TGFß1/2 and Notch1/2 interact via Smad3 to reprogram murine MCs towards an epithelial lineage and ultimately to form a glial scar. Similar to what we observed in mice, we confirmed the epithelial phenotype of human Müller cells during gliotic response. CONCLUSION: The study indicates a pivotal role for TGFß/Notch interplay in tuning MC stemness during injury response and provides novel insights into the remodeling mechanism during retinal degenerative diseases.

Sodium Iodate-Induced Degeneration Results in Local Complement Changes and Inflammatory Processes in Murine Retina.

Age-related macular degeneration (AMD), one of the leading causes of blindness worldwide, causes personal suffering and high socioeconomic costs. While there has been progress in the treatments for the neovascular form of AMD, no therapy is yet available for the more common dry form, also known as geographic atrophy. We analysed the retinal tissue in a mouse model of retinal degeneration caused by sodium iodate (NaIO3)-induced retinal pigment epithelium (RPE) atrophy to understand the underlying pathology. RNA sequencing (RNA-seq), qRT-PCR, Western blot, immunohistochemistry of the retinas and multiplex ELISA of the mouse serum were applied to find the pathways involved in the degeneration. NaIO3 caused patchy RPE loss and thinning of the photoreceptor layer. This was accompanied by the increased retinal expression of complement components c1s, c3, c4, cfb and cfh. C1s, C3, CFH and CFB were complement proteins, with enhanced deposition at day 3. C4 was upregulated in retinal degeneration at day 10. Consistently, the transcript levels of proinflammatory ccl-2, -3, -5, il-1ß, il-33 and tgf-ß were increased in the retinas of NaIO3 mice, but vegf-a mRNA was reduced. Macrophages, microglia and gliotic Müller cells could be a cellular source for local retinal inflammatory changes in the NaIO3 retina. Systemic complement and cytokines/chemokines remained unaltered in this model of NaIO3-dependent retinal degeneration. In conclusion, systemically administered NaIO3 promotes degenerative and inflammatory processes in the retina, which can mimic the hallmarks of geographic atrophy.

Diverse Signaling by TGFß Isoforms in Response to Focal Injury is Associated with Either Retinal Regeneration or Reactive Gliosis.

Müller cells may have stem cell-like capability as they regenerate photoreceptor loss upon injury in some vertebrates, but not in mammals. Indeed, mammalian Müller cells undergo major cellular and molecular changes summarized as reactive gliosis. Transforming growth factor beta (TGFß) isoforms are multifunctional cytokines that play a central role, both in wound healing and in tissue repair. Here, we studied the role of TGFß isoforms and their signaling pathways in response to injury induction during tissue regeneration in zebrafish and scar formation in mouse. Our transcriptome analysis showed a different activation of canonical and non-canonical signaling pathways and how they shaped the injury response. In particular, TGFß3 promotes retinal regeneration via Smad-dependent canonical pathway upon regulation of junb gene family and mycb in zebrafish Müller cells. However, in mice, TGFß1 and TGFß2 evoke the p38MAPK signaling pathway. The activation of this non-canonical pathway leads to retinal gliosis. Thus, the regenerative versus reparative effect of the TGFß pathway observed may rely on the activation of different signaling cascades. This provides one explanation of the different injury response in zebrafish and mouse retina.

Properdin Modulates Complement Component Production in Stressed Human Primary Retinal Pigment Epithelium Cells.

The retinal pigment epithelium (RPE) maintains visual function and preserves structural integrity of the retina. Chronic dysfunction of the RPE is associated with retinal degeneration, including age-related macular degeneration (AMD). The AMD pathogenesis includes both increased oxidative stress and complement dysregulation. Physiological sources of oxidative stress in the retina are well known, while complement sources and regulation are still under debate. Using human primary RPE (hpRPE) cells, we have established a model to investigate complement component expression on transcript and protein level in AMD-risk and non-risk hpRPE cells. We evaluated the effect of properdin, a complement stabilizer, on the hpRPE cell-dependent complement profile exposed to oxidative stress. hpRPE cells expressed complement components, receptors and regulators. Complement proteins were also stored and secreted by hpRPE cells. We associated AMD-risk single nucleotide polymorphisms with an increased secretion of complement factors D (CFD) and I (CFI). Furthermore, we detected hpRPE cell-associated complement activation products (C3a, C5a) independent of any extracellularly added complement system. Exogenous properdin increased the mRNA expression of CFI and CFD, but decreased levels of complement components (C1Q, C3), receptors (C3AR, C5AR1, CD11B) and inflammation-associated transcripts (NLRP3, IL1B) in hpRPE cells exposed to oxidative stress. This properdin effect was time-dependently counter regulated. In conclusion, our data unveiled a local, genotype-associated complement component production in hpRPE cells, regulated by exogenous properdin. The local complement production and activation via blood-independent mechanisms can be a new therapeutic target for AMD.

Oxidative Stress Increases Endogenous Complement-Dependent Inflammatory and Angiogenic Responses in Retinal Pigment Epithelial Cells Independently of Exogenous Complement Sources.

Oxidative stress-induced damage of the retinal pigment epithelium (RPE) and chronic inflammation have been suggested as major contributors to a range of retinal diseases. Here, we examined the effects of oxidative stress on endogenous complement components and proinflammatory and angiogenic responses in RPE cells. ARPE-19 cells exposed for 1-48 h to H2O2 had reduced cell-cell contact and increased markers for epithelial-mesenchymal transition but showed insignificant cell death. Stressed ARPE-19 cells increased the expression of complement receptors CR3 (subunit CD11b) and C5aR1. CD11b was colocalized with cell-derived complement protein C3, which was present in its activated form in ARPE-19 cells. C3, as well as its regulators complement factor H (CFH) and properdin, accumulated in the ARPE-19 cells after oxidative stress independently of external complement sources. This cell-associated complement accumulation was accompanied by increased nlrp3 and foxp3 expression and the subsequently enhanced secretion of proinflammatory and proangiogenic factors. The complement-associated ARPE-19 reaction to oxidative stress, which was independent of exogenous complement sources, was further augmented by the poly(ADP-ribose) polymerase (PARP) inhibitor olaparib. Our results indicate that ARPE-19 cell-derived complement proteins and receptors are involved in ARPE-19 cell homeostasis following oxidative stress and should be considered as targets for treatment development for retinal degeneration.

Transcriptome Analysis Did Not Show Endogenous Stem Cell Characteristics in Murine Lgr5+ Retinal Cells.

Lgr5, an intestinal adult stem cell marker, was recently also found in neuronal tissues. We investigated whether retinal Lgr5+ cells express properties of neural stem cells (NSC) and/or of differentiated interneurons during retinal development. RNA was isolated from Lgr5+ and Lgr5- populations from postnatal day 5 (PN5) and adult retinas of Lgr5EGFP-Ires-CreERT2 knock-in mice sorted by fluorescence-activated cell sorting (FACS). Transcriptome analyses were performed on two RNA samples of each developmental stage (PN5 and adult). The online platform PANTHER (Protein ANalysis THrough Evolutionary Relationships) was used to determine overrepresented gene ontology (GO) terms of biological processes within the set of differentially expressed genes. The detailed evaluation included gene expression in regard to stem cell maintenance/proliferation, cell cycle, and Wnt signaling but also markers of differentiated retinal neurons. None of the enriched GO terms of upregulated genes of Lgr5+ cells showed a positive association to NSC. On the contrary, NSC maintenance and proliferation rather prevail in the Lgr5- cell population. Furthermore, results suggesting that Wnt signaling is not active in the Lgr5+ population. Therefore, our transcriptome analysis of Lgr5+ retinal cells suggest that these cells are differentiated neurons, specifically glycinergic amacrine cells.

Extraocular muscle function is impaired in ryr3 -/- mice.

Calcium is an ubiquitous second messenger mediating numerous physiological processes, including muscle contraction and neuronal excitability. Ca2+ is stored in the ER/SR and is released into the cytoplasm via the opening of intracellular inositol trisphosphate receptor and ryanodine receptor calcium channels. Whereas in skeletal muscle, isoform 1 of the RYR is the main channel mediating calcium release from the SR leading to muscle contraction, the function of ubiquitously expressed ryanodine receptor 3 (RYR3) is far from clear; it is not known whether RYR3 plays a role in excitation-contraction coupling. We recently reported that human extraocular muscles express high levels of RYR3, suggesting that such muscles may be useful to study the function of this isoform of the Ca2+ channel. In the present investigation, we characterize the visual function of ryr3-/- mice. We observe that ablation of RYR3 affects both mechanical properties and calcium homeostasis in extraocular muscles. These changes significantly impact vision. Our results reveal for the first time an important role for RYR3 in extraocular muscle function.