Selective retina therapy and thermal stimulation of the retina: different regenerative properties - implications for AMD therapy.

BACKGROUND: Selective Retina Therapy (SRT), a photodisruptive micropulsed laser modality that selectively destroys RPE cells followed by regeneration, and Thermal Stimulation of the Retina (TSR), a stimulative photothermal continuous wave laser modality that leads to an instant sublethal temperature increase in RPE cells, have shown therapeutic effects on Age-related Macular Degeneration (AMD) in mice. We investigate the differences between both laser modalities concerning RPE regeneration. METHODS: For PCR array, 6 eyes of murine AMD models, apolipoprotein E and nuclear factor erythroid-derived 2- like 2 knock out mice respectively, were treated by neuroretina-sparing TSR or SRT. Untreated litter mates were controls. Eyes were enucleated either 1 or 7 days after laser treatment. For morphological analysis, porcine RPE/choroid organ cultures underwent the same laser treatment and were examined by calcein vitality staining 1 h and 1, 3 or 5 days after irradiation. RESULTS: TSR did not induce the expression of cell-mediators connected to cell death. SRT induced necrosis associated cytokines as well as inflammation 1 but not 7 days after treatment. Morphologically, 1 h after TSR, there was no cell damage. One and 3 days after TSR, dense chromatin and cell destruction of single cells was seen. Five days after TSR, there were signs of migration and proliferation. In contrast, 1 h after SRT a defined necrotic area within the laser spot was seen. This lesion was closed over days by migration and proliferation of adjacent cells. CONCLUSIONS: SRT induces RPE cell death, followed by regeneration within a few days. It is accompanied by necrosis induced inflammation, RPE proliferation and migration. TSR does not induce immediate RPE cell death; however, migration and mitosis can be seen a few days after laser irradiation, not accompanied by necrosis-associated inflammation. Both might be a therapeutic option for the treatment of AMD.

Response of Retinal Pigment Epithelium (RPE)-Choroid Explants to Thermal Stimulation Therapy of the RPE (TSR).

BACKGROUND AND OBJECTIVES: The thermal stimulation therapy of the retinal pigment epithelium (TSR) is a sublethal laser technique for thermal stimulation of the retinal pigment epithelium (RPE)-Bruch's membrane (BrM)-complex. The aim of this study was to investigate the influence of TSR on the release of age-related macular degeneration (AMD)-relevant cell mediators. STUDY DESIGN/MATERIALS AND METHODS: Porcine RPE-BrM-choroid explants were irradiated with a 532 nm continuous wave laser using different spot sizes (100-300 µm, duration 100 milliseconds, 15-100 mW). Cell death was investigated by calcein staining. Explants were treated with grids of sublethal spots and cultivated in modified Ussing chambers. The effect on matrix metalloproteinase-2 (MMP-2) and -9 was investigated by zymography and quantitative reverse transcription polymerase chain reaction. Secretion of vascular endothelial growth factor (VEGF), pigment epithelium derived factor (PEDF), and transforming growth factor-ß (TGF-ß) was analyzed by enzyme-linked immunosorbent assay and expression of HSP70 was examined by western blot. Integrity of the RPE/BrM-complex was analyzed by scanning electron microscopy. RESULTS: Laser powers of 15 mW (100 µm) and 45 mW (300 µm) did not induce RPE cell death. The integrity of the RPE/BrM-complex was not impaired after TSR. After TSR with 300 µm spot size, we observed a significant increase of active MMP-2 in the basal compartments. The content of PEDF significantly increased in treated explants in both compartments with 100 and 300 µm spot sizes. VEGF and TGF-ß secretion was not triggered by TSR. CONCLUSIONS: TSR represents a possible RPE stimulating treatment for dry AMD. TSR increases the basal release of active MMP-2, which might reverse age-related thickening of BrM. VEGF secretion was not triggered by TSR while anti-angiogenic PEDF was increased, indicating an induction of an anti-angiogenic and neuroprotective environment. Lasers Surg. Med. © 2020 Wiley Periodicals LLC.

[Effect of Selective Retina Therapy (SRT) on the Inflammatory Microenvironment of the Subretinal Space]. / Einfluss der Selektiven Retinatherapie (SRT) auf inflammatorische Zellmediatoren des subretinalen Raums.

PURPOSE: To investigate the effect of Selective Retina Therapy (SRT) on inflammatory key factors such as complement factor-C3 (CC3), tumor growth factor-beta2 (TGF-ß2), tumor necrosis factor-alpha (TNF-α) and interferon-gamma (IFN-γ). MATERIALS AND METHODS: Porcine RPE-Bruch's membrane-choroid explants were irradiated with two SRT laser systems, SRTYLF and SRTYAG (Nd : YLF laser, wave length 527 nm, pulse duration 1.7 µs and Nd : YAG laser, wave length 532 nm, pulse duration 2.4 - 3 µs). Laser irradiation was performed on a spot size of 200 × 200 µm, 30 pulses, with a repetition rate of 100 Hz, and a radiant exposure of 140 (threshold RPE death) and 180 mJ/cm2 per pulse (above threshold RPE death). Explants were cultivated in modified Ussing chambers and culture viability was assessed by calcein-AM cell staining. Secretion of inflammatory factors was analyzed by ELISA. Protein expression of tissue explants was assessed by Western blot. RESULTS: Regeneration of RPE was observed after 4 days. One day after SRT with 140 mJ/cm2 per pulse the secretion of basal CC3 decreased in ELISA. Following 180 mJ/cm2 radiant exposure, the level of IFN-γ decreased at day 4. CONCLUSION: SRT does not induce the release of the pro-inflammatory factors analyzed in this in-vitro study.

Compromised Barrier Function in Human Induced Pluripotent Stem-Cell-Derived Retinal Pigment Epithelial Cells from Type 2 Diabetic Patients.

In diabetic patients, high blood glucose induces alterations in retinal function and can lead to visual impairment due to diabetic retinopathy. In immortalized retinal pigment epithelial (RPE) cultures, high glucose concentrations are shown to lead to impairment in epithelial barrier properties. For the first time, the induced pluripotent stem-cell-derived retinal pigment epithelium (hiPSC-RPE) cell lines derived from type 2 diabetics and healthy control patients were utilized to assess the effects of glucose concentration on the cellular functionality. We show that both type 2 diabetic and healthy control hiPSC-RPE lines differentiate and mature well, both in high and normal glucose concentrations, express RPE specific genes, secrete pigment epithelium derived factor, and form a polarized cell layer. Here, type 2 diabetic hiPSC-RPE cells had a decreased barrier function compared to controls. Added insulin increased the epithelial cell layer tightness in normal glucose concentrations, and the effect was more evident in type 2 diabetics than in healthy control hiPSC-RPE cells. In addition, the preliminary functionality assessments showed that type 2 diabetic hiPSC-RPE cells had attenuated autophagy detected via ubiquitin-binding protein p62/Sequestosome-1 (p62/SQSTM1) accumulation, and lowered pro- matrix metalloproteinase 2 (proMMP2) as well as increased pro-MMP9 secretion. These results suggest that the cellular ability to tolerate stress is possibly decreased in type 2 diabetic RPE cells.

Intravitreal injection of anti-Interleukin (IL)-6 antibody attenuates experimental autoimmune uveitis in mice.

PURPOSE: To evaluate the effect of an intravitreally applied anti-IL-6 antibody for the treatment of experimental autoimmune uveitis (EAU). METHODS: EAU was induced in female B10.RIII mice by Inter-Photoreceptor-Binding-Protein (IRBP) in complete Freund's adjuvant, boosted by Pertussis toxin. Single blinded intravitreal injections of anti-IL-6 antibody were applied 5-7days as well as 8-10days (3day interval) after EAU induction into the randomized treatment eye and phosphate buffered saline (PBS) into the fellow control eye. Clinical and fluorescein angiography scoring (6 EAU grades) was done at each injection day and at enucleation day 14. Enucleated eyes were either scored histologically (6 EAU grades) or examined by ELISA for levels of IL-6, IL-17 and IL-6 soluble Receptor (sIL-6R). RESULTS: Uveitis developed in all 12 mice. Clinical uveitis score was significantly reduced (p=0.035) in treated eyes (median 2.0, range 0-4.0, n=12) compared to the fellow control eyes (median 3.0, range 1.0-4.0, n=12). Angiography scores were reduced in 9/12 treated eyes and histological scores in 3/4 treated eyes compared to the fellow control eyes. Cytokine levels were determined in 8 mice, of which 4 responded to anti-IL-6 treatment and 4 did not respond. All mice responding to treatment had a significant reduction of IL-6 (p<0.01) and IL-17 (p=0.01) levels in treated eyes compared to the fellow control eyes. This difference was not seen in non-responding mice. CONCLUSIONS: Intravitreal anti-IL-6 treatment significantly attenuates experimental autoimmune uveitis in mice. EAU activity correlates with ocular IL-6 and IL-17 levels.

Reduction of GAPDH in lenses of Parkinson's disease patients: A possible new biomarker.

BACKGROUND: We previously showed that the protein pattern of lenses removed in cataract surgery differs between patients with Parkinson's disease and age-matched controls. In this study, we identified the protein reduced in abundance in the 34- to 37-kDa gel band. METHODS: During cataract surgery (phacoemulsification), we collected the rinsing fluid and lens particles from the eyes of PD patients and controls. Residual lens fragments in the supernatant of 3 PD patients and aged-matched controls were studied for protein profiles using liquid chromatography-mass spectrometry and Western blots. RESULTS: We identified glyceraldehyde-3-phosphate dehydrogenase by mass spectrometry as the protein reduced in abundance and verified this finding in Western blots. CONCLUSIONS: Glyceraldehyde-3-phosphate dehydrogenase has been implicated in PD development. The reduction of glyceraldehyde-3-phosphate dehydrogenase in the lenses of PD patients may be a new biomarker for PD and might also indicate an important role for this protein in PD development. © 2016 International Parkinson and Movement Disorder Society.

Fucoidan Does Not Exert Anti-Tumorigenic Effects on Uveal Melanoma Cell Lines.

BACKGROUND: The polysaccharide fucoidan is widely investigated as an anti-cancer agent. Here, we tested the effect of fucoidan on uveal melanoma cell lines. METHODS: The effect of 100 µM fucoidan was investigated on five cell lines (92.1, Mel270 OMM1, OMM2.3, OMM2.5) and of 1 µg/mL-1 mg/mL fucoidan in two cell lines (OMM1, OMM2.3). Cell proliferation and viability were investigated with a WST-1 assay, migration in a wound healing (scratch) assay. Vascular Endothelial Growth Factor (VEGF) was measured in ELISA. Angiogenesis was evaluated in co-cultures with endothelial cells. Cell toxicity was induced by hydrogen-peroxide. Protein expression (Akt, ERK1/2, Bcl-2, Bax) was investigated in Western blot. RESULTS: Fucoidan increased proliferation in two and reduced it in one cell line. Migration was reduced in three cell lines. The effect of fucoidan on VEGF was cell type and concentration dependent. In endothelial co-culture with 92.1, fucoidan significantly increased tubular structures. Moreover, fucoidan significantly protected all tested uveal melanoma cell lines from hydrogen-peroxide induced cell death. Under oxidative stress, fucoidan did not alter the expression of Bcl-2, Bax or ERK1/2, while inducing Akt expression in 92.1 cells but not in any other cell line. CONCLUSION: Fucoidan did not show anti-tumorigenic effects but displayed protective and pro-angiogenic properties, rendering fucoidan unsuitable as a potential new drug for the treatment of uveal melanoma.

CRB1rd8 mutation influences the age-related macular degeneration phenotype of NRF2 knockout mice and favors choroidal neovascularization.

PURPOSE: We examined the influence of retinal degeneration 8 (rd8) mutation of crumbs homolog 1 (CRB1) gene on age-related macular degeneration (AMD) phenotype in nuclear factor E2-related factor 2 knock out (NRF2-/-) mouse model. METHODS: CRB1rd8 mutation genotype was determined by polymerase chain reaction from tail clips in 73 NRF2-/- mice originating from C57BL/6J background on mixed C57BL/6J and C57BL/6N ancestry. The clinical grade of AMD-like fundus alterations was determined by funduscopy, optical coherence tomography (OCT) and fluorescein angiography (FLA) at the age of 9 or 12 months. RESULTS: Twelve NRF2-/- mice were wildtype CRB1+/+, 61 NRF2-/- were homozygous CRB1rd8/rd8. NRF2-/-CRB1rd8/rd8 mice had a significantly higher probability to show an advanced grade (grade 4 and 5) of AMD-like fundus alterations known to appear in NRF2-/- mice. Choroidal neovascularization (CNV) was only detected in NRF2-/-CRB1rd8/rd8 homozygous mice. CONCLUSIONS: Homozygous CRB1rd8/rd8 mutation is common in commercial vendor mice strains of C57BL/6J origin if partly on C57BL/6N ancestry. The mutation has an influence on the extent of AMD-like retinal alterations in NRF2-/- mice and favors CNV formation.

Modulation of inflammatory processes by thermal stimulating and RPE regenerative laser therapies in age related macular degeneration mouse models.

PURPOSE: Inflammatory processes play a major role within the multifactorial pathogenesis of age-related macular degeneration (AMD). Neuroretina sparing laser therapies, thermal stimulation of the retina (TSR) and selective retina therapy (SRT), are known to reduce AMD-like pathology in vitro and in vivo. We investigated the effect of TSR and SRT on inflammatory processes in AMD mouse models. METHODS: One randomized eye of 8 months old apolipoprotein (Apo)E and 9 months old nuclear factor (erythroid-derived 2) -like 2 (NRF2) knock out mice were treated by TSR (10 ms, 532 nm, 50 µm2 spot size, mean 4.5 W, ~200 spots) or SRT (~1.4 µs pulses, 532 nm, 50 µm spot size, 100 Hz over 300 ms, mean 2.5 µJ per pulse, ~200 spots). Fellow eyes, untreated knock out mice and wild-type BL/6J mice acted as controls. All mice were examined funduscopically and by optical coherence tomography (OCT) at the day of laser treatment. Mice were euthanized and enucleated either 1 day or 7 days after laser treatment and examined by gene expression analysis of 84 inflammatory genes. RESULTS: The inflammatory gene expression profile of both knock out models compared to healthy BL/6J mice suggests a regulation of pro- and anti-inflammatory processes especially concerning T-cell activity and immune cell recruitment. TSR resulted in downregulation of several pro-inflammatory cell-mediators both in ApoE -/- and NRF2-/- mice compared to treatment naïve litter mates one day after treatment. In contrast, SRT induced pro-inflammatory cell-mediators connected with necrosis one day after treatment as expected following laser-induced selective RPE cell death. Seven days after laser treatment, both findings were reversed. CONCLUSIONS: Both TSR and SRT influence inflammatory processes in AMD mouse models. However, they act conversely. TSR leads to anti-inflammatory processes shortly after laser therapy and induces immune-cell recruitment one week after treatment. SRT leads to a quick inflammatory response to laser induced RPE necrotic processes. One week after SRT inflammation is inhibited. It remains unclear, if and to what extent this might play a role in a therapeutic or preventive approach of both laser modalities on AMD pathology.

Selective Retina Therapy Reduces Bruch's Membrane Thickness and Retinal Pigment Epithelium Pathology in Age-Related Macular Degeneration Mouse Models.

PURPOSE: To investigate the effect of selective retina therapy (SRT) on age-related macular degeneration (AMD)-like alterations of retinal pigment epithelium (RPE) and Bruch's membrane (BrM) in AMD mouse models as therapeutic approach for the treatment of dry AMD. METHODS: In B6.129P2-Apoetm1Unc /J (ApoE-/-) and B6.129X1-Nfe2I2 tm1Ywk /J (NRF2-/-), one randomized eye of each mouse in groups of 15 mice was treated by SRT (532 nm, 300 ms, ∼1.4-µs pulse, 100 Hz, 50-µm spot), the fellow eye and healthy C57BL/6J mice served as controls. Clinical examinations were obtained at treatment day and 1 month later, followed by enucleation to analyze BrM thickness and ultrastructural RPE morphology. RESULTS: Nearly all ApoE-/- and NRF2-/- mice showed AMD-like retinal alterations. BrM thickness was increased in both mouse models, RPE had vacuoles within the cell body and shortened apical microvilli. SRT neither affected neuroretinal anatomy nor function. BrM thickness as well as AMD-like ultrastructural alterations of the RPE were significantly reduced in laser-treated eyes compared with fellow control and untreated control eyes. CONCLUSIONS: SRT reduces BrM thickness and AMD-like RPE alterations in AMD mouse models without damage to structural or functional properties of neuroretina. It may be a prophylactic or therapeutic option for dry AMD. TRANSLATIONAL RELEVANCE: SRT shows therapeutic effectivity in murine AMD models and might therefore become an option for the treatment of dry AMD.

Thermal Stimulation of the Retina Reduces Bruch's Membrane Thickness in Age Related Macular Degeneration Mouse Models.

PURPOSE: To investigate the effect of thermal stimulation of the retina (TS-R) on Bruch's membrane (BrM) thickness in age-related macular degeneration (AMD) mouse models as a novel concept for the prophylaxis and treatment of dry AMD. METHODS: Two knockout AMD mouse models, B6.129P2-Apoetm1Unc/J (ApoE-/-) and B6.129X1-Nfe2I2tm1Ywk/J (NRF2-/-), were chosen. One randomized eye of each mouse in four different groups (two of different age, two of different genotype) of five mice was treated by TS-R (532 nm, 10-ms duration, 50-µm spot size), the fellow eye served as control. Laser power was titrated to barely visible laser burns, then reduced by 70% to guarantee for thermal elevation without damage to the neuroretina, then applied uniformly to the murine retina. Fundus, optical coherence tomography (OCT), and fluorescein angiography (FLA) images were obtained at the day of treatment and 1 month after treatment. Eyes were enucleated thereafter to analyze BrM thickness by transmission electron microscopy (TEM) in a standardized blinded manner. RESULTS: Fundus images revealed that all ApoE-/- and NRF2-/- mice had AMD associated retinal alterations. BrM thickness was increased in untreated controls of both mouse models. Subvisible TS-R laser spots were not detectable by fundus imaging, OCT, or FLA 2 hours or 1 month after laser treatment. TEM revealed a significant reduction of BrM thickness in laser-treated eyes of all four groups compared to their fellow control eyes. CONCLUSIONS: TS-R reduces BrM thickness in AMD mouse models ApoE-/- and NRF2-/- without damage to the neuroretina. It may become a prophylactic or even therapeutic treatment option for dry AMD. TRANSLATIONAL RELEVANCE: TS-R may become a prophylactic or even therapeutic treatment option for dry AMD.

Release of Different Cell Mediators During Retinal Pigment Epithelium Regeneration Following Selective Retina Therapy.

Purpose: To investigate the effect of selective retina therapy (SRT) on the release of AMD-relevant cell mediators, such as matrix metalloproteinases (MMPs), VEGF, and pigment epithelium derived factor (PEDF) using different laser spot sizes and densities. Methods: Porcine RPE-choroid explants were treated with a pulsed 532 nm Nd:YAG laser using (1) large spot sizes, (2) small spot sizes with a high-density (hd) treatment, and (3) small spot sizes with a low-density (ld) treatment. Explants were cultivated in modified Ussing chambers. RPE regeneration and RPE cell death were investigated by calcein-AM staining and immunofluorescence. The MMP release was examined via zymography and immunofluorescence. VEGF and PEDF secretion was analyzed by ELISA. Results: During pigment epithelium regeneration (PER), mitosis and RPE cell migration were observed. Four days after SRT (large spot size) the content of active MMP2 increased significantly (P < 0.01). Hd treatment with small spot sizes resulted also in an increase of active MMP2 (P < 0.05). In immunofluorescence explants showed a localized expression of MMP2 within the healing lesions after irradiation. The PEDF level increased significantly (P = 0.01) after SRT with large spot sizes. VEGF secretion decreased significantly (P < 0.05) following SRT with large spot sizes and with hd treatment of small spot sizes. Conclusions: SRT induces a cytokine profile, which may improve the flux across Bruch's membrane, slows down progression of early AMD by RPE regeneration, and inhibits the formation of choroidal neovascularization. The cytokine release depends on the size and density of applied laser spots.

Effects of aflibercept on primary RPE cells: toxicity, wound healing, uptake and phagocytosis.

BACKGROUND/AIM: Anti-VEGF treatment is the therapy of choice in age-related macular degeneration, and is also applied in diabetic macular oedema or retinal vein occlusion. Recently, the fusion protein, aflibercept, has been approved for therapeutic use. In this study, we investigate the effects of aflibercept on primary RPE cells. METHODS: Primary RPE cells were prepared from freshly slaughtered pigs' eyes. The impact of aflibercept on cell viability was investigated with MTT and trypan blue exclusion assay. The influence of aflibercept on wound healing was assessed with a scratch assay. Intracellular uptake of aflibercept was investigated in immunohistochemistry and its influence on phagocytosis with a phagocytosis assay using opsonised latex beads. RESULTS: Aflibercept displays no cytotoxicity on RPE cells but impairs its wound healing ability. It is taken up into RPE cells and can be intracellularly detected for at least 7 days. Intracellular aflibercept impairs the phagocytic capacity of RPE cells. CONCLUSIONS: Aflibercept interferes with the physiology of RPE cells, as it is taken up into RPE cells, which is accompanied by a reduction of the phagocytic ability. Additionally, it impairs the wound healing capacity of RPE cells. These effects on the physiology of RPE cells may indicate possible side effects.

Fucoidan reduces secretion and expression of vascular endothelial growth factor in the retinal pigment epithelium and reduces angiogenesis in vitro.

Fucoidan is a polysaccharide isolated from brown algae which is of current interest for anti-tumor therapy. In this study, we investigated the effect of fucoidan on the retinal pigment epithelium (RPE), looking at physiology, vascular endothelial growth factor (VEGF) secretion, and angiogenesis, thus investigating a potential use of fucoidan for the treatment of exudative age-related macular degeneration. For this study, human RPE cell line ARPE-19 and primary porcine RPE cells were used, as well as RPE/choroid perfusion organ cultures. The effect of fucoidan on RPE cells was investigated with methyl thiazolyl tetrazolium--assay, trypan blue exclusion assay, phagocytosis assay and a wound healing assay. VEGF expression was evaluated in immunocytochemistry and Western blot, VEGF secretion was evaluated in ELISA. The effect of fucoidan on angiogenesis was tested in a Matrigel assay using calcein-AM vital staining, evaluated by confocal laser scanning microcopy and quantitative image analysis. Fucoidan displays no toxicity and does not diminish proliferation or phagocytosis, but reduces wound healing in RPE cells. Fucoidan decreases VEGF secretion in RPE/choroid explants and RPE cells. Furthermore, it diminishes VEGF expression in RPE cells even when co-applied with bevacizumab. Furthermore, fucoidan reduces RPE-supernatant- and VEGF-induced angiogenesis of peripheral endothelial cells. In conclusion, fucoidan is a non-toxic agent that reduces VEGF expression and angiogenesis in vitro and may be of interest for further studies as a potential therapy against exudative age-related macular degeneration.