Protective roles of autophagy in retinal pigment epithelium under high glucose condition via regulating PINK1/Parkin pathway and BNIP3L

BACKGROUND: Our study aimed to investigate the roles of autophagy against high glucose induced response in retinal pigment epithelium (ARPE-19 cells). METHODS: The morphological changes and reactive oxygen species (ROS) generation in ARPE-19 cells under high glucose treatment were respectively detected using the transmission electron microscopy and flow cytometry. The expression levels of Parkin, PINK1, BNIP3L, LC3-I and LC3-II in ARPE-19 cells received high glucose treatment were measured by western blot after pretreatment of carbonyl cyanide m-chlorophenylhydrazone (CCCP), 3-methyladenine (3-MA), N-acetyl cysteine (NAC) or cyclosporin A (CsA) followed by high glucose treatment. RESULTS: ARPE-19 cells subjected to high glucose stress showed an obvious reduction in the LC3-I expression and significant increase in the number of autophagosomes, in the intracellular ROS level, and in the expression levels of Parkin, PINK1, BNIP3L and LC3-II (p < 0.05). Pretreatment with CCCP significantly reduced the LC3-I expression and increased the expression levels of Parkin, PINK1, BNIP3L and LC3-II (p < 0.05). ARPE-19 cells pretreated with CsA under high glucose stress showed markedly down-regulated expressions of Parkin, PINK1 and BNIP3L compared with the cells treated with high glucose (p < 0.05). Pretreatment of ARPE-19 cells with NAC or 3-MA under high glucose stress resulted in a marked reduction in the expression levels of PINK1, BNIP3L and LC3-II (p < 0.05). Meanwhile, the expression level of Parkin in the ARPE-19 cells pretreated with NAC under high glucose stress was comparable with that in the control cells. CONCLUSION: Autophagy might have protective roles against high glucose induced injury in ARPE19 cells via regulating PINK1/Parkin pathway and BNIP3L.

Efficacy of aflibercept on exudative age-related macular degeneration in patients exhibiting complete ranibizumab resistance and tachyphylaxis / Eficácia do aflibercept em pacientes portadores de degeneração macular relacionada à idade exsudativa com resistência completa e taquifilaxia ao ranibizumab

ABSTRACT Purpose: The present study compared the efficacy of aflibercept for neovascular age-related macular degeneration (NV-AMD) in patients with complete ranibizumab resistance and tachyphylaxis. Methods: Forty-four eyes of 38 neovascular age-related macular degeneration patients were evaluated. Eyes were divided into a complete resistance group (n=23 eyes) and tachyphylaxis group (n=21 eyes). Results: After three injections, eight (38.1%) patients in the tachyphylaxis group and nine (39.1%) in the complete resistance group presented with macular dryness. After the first injection of aflibercept, the mean visual acuity improved significantly in the tachyphylaxis group (p=0.018) but remained unchanged in the complete resistance group (p=0.37). There was a non-significant trend towards improved mean visual acuity in both groups after the second and third injections relative to the acuity at the final visit for ranibizumab treatment. In the tachyphylaxis group, the presence of subfoveal pigmented epithelium detachment (PED) decreased significantly after intravitreal aflibercept treatment. Conclusions: Although treatment with aflibercept yielded generally positive anatomical results in both groups, no significant increase in visual acuity was achieved.

RESUMO Objetivo: O presente estudo comparou a eficácia do aflibercept na degeneração macular neovascular relacionada à idade (NV-AMD) com de resistência completa ao ranibizumab e taquifilaxia ao ranibizumab. Método: Quarenta e quatro olhos de 38 pacientes com degeneração macular neovascular relacionada à idade foram inscritos. Eles foram divididos em dois grupos: grupo de resistência completa (n=23 olhos) e grupo taquifilaxia (n=21 olhos). Resultados: Depois de três injeções, 8 (38,1%) olhos no grupo de taquifilaxia e 9 (39,1%) olhos no grupo de resistência completa, apresentaram mácula seca. Após a primeira injeção de aflibercept, a acuidade visual média melhorou significativamente no grupo taquifilaxia (p=0,018) e manteve-se inalterada no grupo de resistência completa (p=0,37). Houve uma tendência de melhora da acuidade visual média em ambos os grupos após a segunda e terceira injeções em comparação com a última visita do tratamento com ranibizumab, mas isso não foi estatisticamente significativo. A presença de descolamento do epitélio pimentado subfoveal (PED) em olhos com taquifilaxia ao ranibizumab diminuiu significativamente após o tratamento aflibercept intravítreo. Conclusões: Embora o tratamento com aflibercept tenha mostrado resultados anatômicos positivas em ambos os grupos, não foi obtida melhora significativa da acuidade visual.

Anatomical and visual outcomes of ranibizumab injections in retinal pigment epithelium tears / Resultados anatômicos e visuais de injeções de ranibizumab em roturas do epitélio pigmentado da retina

ABSTRACT Purpose: To report the anatomical and visual results in patients diagnosed as having retinal pigment epithelium (RPE) tears after receiving ranibizumab injections. Methods: Eyes diagnosed as having RPE tears with a minimum 6-month follow-up were retrospectively evaluated. Each eye was treated with at least three doses of ranibizumab at monthly intervals. Best-corrected visual acuity (BCVA), anterior segment findings, intraocular pressure, and fundus examination results were evaluated during control visits. Color fundus photography, fundus fluorescein angiographies, fundus autofluorescence, and spectral domain optical coherence tomography (SD-OCT) images were obtained. The height of pigment epithelial detachment (PED) was measured by SD-OCT. Results: Twelve eyes with RPE tears were studied. Nine eyes (75%) developed RPE tears during ranibizumab injections for choroidal neovascularization (eight eyes with vascularized PED and one eye with choroidal osteoma), and tears occurred in three eyes before any injections. The median number of ranibizumab injections after diagnosis of RPE tears was 3 (min 2, max 5). In the most recent follow-up visit, there was no statistically significant correlation between the grade of RPE and logMAR of BCVA (p>0.05, r=0.112). Eight of twelve eyes had PED, and seven of these had irregular PED contours before injection therapy. The mean PED height was 447 ± 122 µm. Conclusions: In this series, RPE tears developed mostly after intravitreal anti-VEGF injections for vascularized PED. Increased vertical height and irregular contours of the PEDs can be risk factors for the formation of RPE tears. The continuation of anti-VEGF therapy after tear formation is beneficial for vision improvement in eyes with RPE tears. .

RESUMO Objetivo: Apresentar os resultados anatômicos e visuais de injeções de ranibizumab em pacientes que foram diagnosticados com roturas do epitélio pigmentado da retina (RPE). Métodos: Olhos com um mínimo de seis meses de acompanhamento após diagnóstico de roturas do RPE foram avaliados retrospectivamente. Cada olho foi tratado com, pelo menos, três doses de ranibizumab em intervalos mensais. Acuidade visual com a melhor correção (BCVA), achados do segmento anterior, pressão intraocular e exames de fundo de olho foram avaliados nas visitas de controle. Retinografia colorida, angiografias fluoresceínicas, autofluorescência de polo posterior e tomografia de coerência óptica imagens de domínio espectral (SD-OCT) foram obtidos. A altura do descolamento do epitélio pigmentado (PED) foi medida com SD-OCT. Resultados: Doze olhos com roturas do epitélio pigmentado da retina foram incluídos no estudo. Nove olhos (75%) desenvolveram roturas do epitélio pigmentado da retina durante as injeções ranibizumab para neovascularização de coroide (oito olhos com descolamento do epitélio pigmentado vascularizado e um olho com osteoma de coroide), a rotura ocorreu em três olhos antes de quaisquer injeções. A mediana do número de injeções de ranibizumab após o diagnóstico da rotura do RPE foi de 3 (mínimo 2, máximo 5). Na visita de acompanhamento mais recente, não houve correlação estatisticamente significante entre o grau de RPE e logMAR de BCVA (p>0,05, r=0,112). Oito dos doze olhos tinham descolamento do epitélio pigmentado, desses, 7 olhos tinham PEDs com contornos irregulares antes da injeção. A altura média do PED foi 447 ± 122 µm. Conclusões: Nesta série, as roturas de epitélio pigmentado da retina aconteceram principalmente após a injeção intravítrea anti-VEGF para descolamento do epitélio pigmentado vascularizado. O aumento da altura vertical e contornos irregulares dos PEDs podem ser considerados fatores de risco para a formação da rotura ...

Effects of Bevacizumab on Bcl-2 Expression and Apoptosis in Retinal Pigment Epithelial Cells under Oxidative Stress

PURPOSE: To evaluate the effects of bevacizumab on expression of B-cell leukemia/lymphoma (Bcl)-2 and apoptosis in retinal pigment epithelial (RPE) cells under oxidative stress conditions. METHODS: RPE cells were treated with H2O2 (0, 100, 200, 300, and 400 microM) and bevacizumab at or above the doses normally used in clinical practice (0, 0.33, 0.67, 1.33, and 2.67 mg/mL). Cell apoptosis was measured using flow cytometry with annexin V-fluorescein isothiocyanate. The expression of Bcl-2 mRNA was determined using reverse transcription polymerase chain reaction. RESULTS: Under low oxidative stress conditions (H2O2 100 microM), cell apoptosis was not significantly different at any concentration of bevacizumab, but Bcl-2 mRNA expression decreased with increasing concentration of bevacizumab (0.33, 0.67, 1.33, and 2.67 mg/mL). Under moderate oxidative stress conditions (H2O2 200 microM), Bcl-2 mRNA expression decreased with increasing concentration of bevacizumab (0.33, 0.67, 1.33, and 2.67 mg/mL), but cell apoptosis increased only at 2.67 mg/mL of bevacizumab. Under high oxidative stress (300 microM) conditions, cell apoptosis increased at high concentrations of bevacizumab (1.33 and 2.67 mg/mL), but it did not correlate with Bcl-2 expression. CONCLUSIONS: Withdrawal of vascular endothelial growth factor can lead to RPE cell apoptosis and influences the expression of anti-apoptotic genes such as Bcl-2 under oxidative stress conditions. Since oxidative stress levels of each patient are unknown, repeated injections of intravitreal bevacizumab, as in eyes with age-related macular degeneration, might influence RPE cell survival.

Resveratrol Inhibits Hypoxia-Induced Vascular Endothelial Growth Factor Expression and Pathological Neovascularization

PURPOSE: To investigate the effects of resveratrol on the expression of hypoxia-inducible factor 1alpha (HIF-1alpha) and vascular endothelial growth factor (VEGF) in human adult retinal pigment epithelial (ARPE-19) cells, and on experimental choroidal neovascularization (CNV) in mice. MATERIALS AND METHODS: ARPE-19 cells were treated with different concentrations of resveratrol and then incubated under hypoxic conditions with subsequent evaluation of cell viability, expression of HIF-1alpha, and expression of VEGF. The effects of resveratrol on the synthesis and degradation of hypoxia-induced HIF-1alpha were evaluated using inhibitors of the PI3K/Akt/mTOR and the ubiquitin proteasome pathways. In animal studies, CNV lesions were induced in C57BL/6 mice by laser photocoagulation. After 7 days of oral administration of resveratrol or vehicle, which began one day after CNV induction, image analysis was used to measure CNV areas on choroidal flat mounts stained with isolectin IB4. RESULTS: In ARPE-19 cells, resveratrol significantly inhibited HIF-1alpha and VEGF in a dose-dependent manner, by blocking the PI3K/Akt/mTOR signaling pathway and by promoting proteasomal HIF-1alpha degradation. In mice experiments, orally administered resveratrol significantly inhibited CNV growth in a dose-dependent manner. CONCLUSION: Resveratrol may have therapeutic value in the management of diseases involving pathological neovascularization.

SIRT1 negatively regulates amyloid-beta-induced inflammation via the NF-B pathway

Chronic inflammation induced by amyloid-beta (Aβ) plays a key role in the development of age-related macular degeneration (AMD), and matrix metalloproteinase-9 (MMP-9), interleukin (IL)-6, and IL-8 may be associated with chronic inflammation in AMD. Sirtuin 1 (SIRT1) regulates inflammation via inhibition of nuclear factor-kappa B (NF-κB) signaling, and resveratrol has been reported to prevent Aβ-induced retinal degeneration; therefore, we investigated whether this action was mediated via activation of SIRT1 signaling. Human adult retinal pigment epithelial (RPE) cells were exposed to Aβ, and overactivation and knockdown of SIRT1 were performed to investigate whether SIRT1 is required for abrogating Aβ-induced inflammation. We found that Aβ-induced RPE barrier disruption and expression of IL-6, IL-8, and MMP-9 were abrogated by the SIRT1 activator SRT1720, whereas alterations induced by Aβ in SIRT1-silenced RPE cells were not attenuated by SRT1720. In addition, SRT1720 inhibited Aβ-mediated NF-κB activation and decrease of the NF-κB inhibitor, IκBα. Our findings suggest a protective role for SIRT1 signaling in Aβ-dependent retinal degeneration and inflammation in AMD.

Effectiveness and safety of iodopovidone in an experimental pleurodesis model

OBJECTIVES: Chemical pleurodesis is an important therapeutic tool to control recurrent malignant pleural effusion. Among the various sclerosing agents, iodopovidone is considered effective and safe. However, in a recent study, ocular changes were described after iodopovidone was used in recurrent pneumothorax. The aim of the study was to evaluate the efficacy and morbidity of iodopovidone pleurodesis in an experimental model. METHODS: New Zealand rabbits were submitted to intrapleural injection of iodopovidone at concentrations of 2%, 4% and 10%. Biochemical (lactic dehydrogenase, proteins, triiodothyronine, free thyroxine, urea and creatinine) and immunological (Interleukin-8 [IL-8], VEGF and TGFβ) parameters were measured in the pleural fluid and blood. After 1, 3, 7, 14 and 28 days, groups of animals were euthanized, and macro- (pleura) and microscopic (pleura and retina) analyses were performed. RESULTS: An early pleural inflammatory response with low systemic repercussion was observed without corresponding changes in thyroid or renal function. The higher concentrations (4% and 10%) correlated with greater initial exudation, and maximum pleural thickening was observed after 28 days. No changes were observed in the retinal pigment epithelium of the rabbits. CONCLUSION: Iodopovidone is considered to be an effective and safe sclerosing agent in this animal model. However, its efficacy, tolerance and safety in humans should be further evaluated. .

Effects of hydroquinone on retinal and vascular cells in vitro.

Aim: To explore the molecular pathophysiology that might explain the epidemiologic association between cigarette smoke and age-related macular degeneration (AMD) by examining the effects of hydroquinone (HQ), a toxic compound present in high concentration in cigarette smoke-related tar, on human retinal pigment epithelial cells (ARPE-19), rat retinal neurosensory cells (R-28), and human microvascular endothelial cells (HMVEC). Materials and Methods: ARPE-19, R-28, and HMVEC were treated for 24 h with four different concentrations of HQ (500 μM, 200 μM, 100 μM, 50 μM). Cell viability, caspase-3/7 activation, DNA laddering patterns, and lactate dehydrogenase (LDH) levels were analyzed. Results: At 50 μM HQ, R-28 cells showed a significant decrease in cell viability compared with the dimethyl sulfoxide (DMSO)-treated controls. At the 100–500 μM concentrations, all three cell lines showed significant cell death (P < 0.001). In the ARPE-19, R-28, and HMVEC cultures, the caspase-3/7 activities were not increased at any of the HQ concentration. Conclusion: Our findings suggest that the mechanism of cell death in all three cell lines was through non-apoptotic pathway. In addition, neuroretinal R-28 cells were more sensitive to HQ than the ARPE-19 and HMVEC cultures.

The Effect of Brimonidine on Transepithelial Resistance in a Human Retinal Pigment Epithelial Cell Line

PURPOSE: To investigate the effects of brimonidine, an alpha-2-adrenergic agonist, on barrier function in ARPE-19 cells by measuring transepithelial resistance (TER). METHODS: ARPE-19 cells were cultured into a confluent monolayer on a microporous filter. Brimonidine was added to the apical medium, and the barrier function of the cells was evaluated by measuring TER. A subset of cells was treated under hypoxic conditions, and the TER changes observed upon administration of brimonidine were compared to those observed in cells in normoxic conditions. RESULTS: The ARPE cell membrane reached a peak resistance of 29.1+/-7.97 Omega cm2 after four weeks of culture. The TER of the cells treated under normoxic conditions increased with brimonidine treatment; however, the TER of the cells treated under hypoxic conditions did not change following the administration of brimonidine. CONCLUSIONS: Barrier function in ARPE-19 cells increased with brimonidine treatment. Understanding the exact mechanism of this barrier function change requires further investigation.

Intravitreal bevacizumab in pigmented rabbit eyes: histological analysis 90 days after injection / Bevacizumabe intravítreo em olhos de coelhos não albinos: análise histológica 90 dias após a injeção

PURPOSE: To evaluate bevacizumab toxicity in neurosensorial retina and retinal pigment epithelium in pigmented rabbit eyes by means of histological studies. METHODS: Thirty eyes of fifteen rabbits were distributed into three groups: sham group (S), that received a 0.1 ml balanced saline solution (BSS) intravitreal injection (10 eyes); group 1, that received a 1.25 mg (0.1 ml) bevacizumab intravitreal injection (10 eyes); and group 2, that received a 2.5 mg (0.1 ml) bevacizumab intravitreal injection (10 eyes). Rabbits were sacrificed 90 days after the procedure and both eyes of each rabbit were enucleated. A histological examination of neurosensorial retina and retinal pigmented epithelium (RPE) was performed. Its morphological features and layer thickness were also analyzed. RESULTS: No histological differences in neurosensorial retina or in retinal pigmented epithelium were found and layer thickness did not differ significantly between balanced saline solution-injected eyes and bevacizumab-injected eyes. CONCLUSION: After a 90-day follow-up period, a single 1.25 or 2.5 mg bevacizumab intravitreal injection did not lead to toxic damage in the neurosensorial retina and retinal pigment epithelium of pigmented rabbit eyes, and it appears to be a safe procedure for retinal neovascular diseases.

OBJETIVOS: Avaliar a toxicidade do bevacizumabe na retina neurossensorial e epitélio pigmentado da retina (EPR) em olhos de coelhos não albinos pelos estudos histológicos. MÉTODOS: Trinta olhos de 15 coelhos foram distribuídos em três grupos: 10 olhos no grupo placebo (P), que recebeu uma injeção intravítrea de 0,1 ml de solução salina balanceada (SSB); 10 olhos no grupo 1, que recebeu uma injeção intravítrea de 1,25 mg (0,1 ml) de bevacizumabe; e 10 olhos no grupo 2, que recebeu uma injeção intravítrea de 2,5 mg (0,1 ml) de bevacizumabe. Os coelhos tiveram seus dois olhos enucleados sob anestesia geral e submetidos à eutanásia 90 dias após a injeção. Foi realizada avaliação histológica na retina neurossensorial e no epitélio pigmentado da retina e seus aspectos morfológicos e espessuras das camadas retinianas foram analisadas. RESULTADOS: Não foi observada diferença significante entre a morfologia histológica e espessura das camadas retinianas entre o grupo P (SSB) e os grupos 1 e 2 (bevacizumabe 1,25 mg e 2,5 mg, respectivamente). CONCLUSÃO: Após um seguimento de 90 dias, uma única injeção intravítrea de bevacizumabe com 1,25 e 2,5 mg não levou a danos histológicos na retina neurossensorial e epitélio pigmentado da retina em olhos de coelhos não albinos e parece ser um procedimento seguro para o tratamento das doenças neovasculares da retina.

Maculopatia tóxica por antimaláricos: detecção pela tomografia óptica espectral de alta resolução: relato de casos / Toxic maculopathy caused by antimalarial drugs: detection using spectral domain OCT: case reports

As drogas antimaláricas, como a cloroquina, são úteis no controle de doenças reumáticas, mas podem causar lesões oculares potencialmente graves como a retinopatia cloroquínica (RC). Neste relato, são descritos os achados à tomografia de coerência óptica convencional e na espectral de alta resolução (HD-OCT) em aparelho CirrusTM HD-OCT de duas pacientes com RC. Em um dos casos, chama a atenção à similaridade entre a imagem de atrofia do epitélio pigmentado retiniano (EPR) na angiofluoresceinografia e a obtida na HD-OCT por segmentação "en -face" (reconstruções coronais). Em outra paciente, com sinais clínicos de maculopatia, mas sem anormalidades na angiofluoresceinografia, os sinais de atrofia do EPR foram também observados nos "scans" da HD-OCT, levantando a possibilidade de que esta técnica possa permitir a detecção precoce desta doença.

Antimalarial drugs, such as chloroquine, are useful in the management of rheumatic diseases, but may cause a potentially blinding condition known as toxic maculopathy. This report describes the findings on standard and on high-resolution spectral domain ocular coherence tomography performed in a CirrusTM HD-OCTdevice in two patients with chloroquine maculopathy. In one case, a very similar aspect of retinal pigment epithelium (RPE) plaque atrophy occured in both angiographic and HD-OCT images obtained by "en-face" (coronal) segmentation. In another patient with clinical signs of maculopathy and no angiographic abnormalities, signs of RPE atrophy could also be observed in HD-OCT scans, raising the possibility that this technique may allow the early detection of the disease.

Ocular drug delivery; impact of invitro cell culture models

Normal vision depends on the optimal function of ocular barriers and intact membranes that selectively regulate the environment of ocular tissues. Novel pharmaco-therapeutic modalities have aimed to overcome such biological barriers which impede efficient ocular drug delivery. To determine the impact of ocular barriers on research related to ophthalmic drug delivery and targeting, herein we provide a review of the literature on isolated primary or immortalized cell culture models which can be used for evaluation of ocular barriers. In vitro cell cultures are valuable tools which serve investigations on ocular barriers such as corneal and conjunctival epithelium, retinal pigment epithelium and retinal capillary endothelium, and can provide platforms for further investigations. Ocular barrier-based cell culture systems can be simply set up and used for drug delivery and targeting purposes as well as for pathological and toxicological research