Sulforaphane inhibits blue light-induced inflammation and apoptosis by upregulating the SIRT1/PGC-1α/Nrf2 pathway and autophagy in retinal pigment epithelial cells.

The present study elucidated mechanisms through which sulforaphane (SFN) protects retinal pigment epithelial (RPE) cells from blue light-induced impairment. SFN could activate the nuclear translocation of nuclear factor erythroid 2-related factor 2 (Nrf2) and increase the expression of the heme oxygenease-1 (HO-1) gene and production of glutathione. SFN reduced blue light-induced oxidative stress, and effectively activated cytoprotective components including Nrf-2, HO-1, thioredoxin-1, and glutathione. The protective effect of SFN on blue light-induced injury was blocked by the Nrf2 inhibitor ML385, suggesting that the SFN-induced Nrf2 pathway is involved in the cytoprotective effect of SFN. SFN inhibited intercellular adhesion molecule-1 expression induced by TNF-α or blue light, suggesting the anti-inflammatory activity of SFN. The inhibitory effect of SFN was associated with the blocking of NF-κB p65 nuclear translocation in blue light-exposed RPE cells. SFN protected RPE cells from blue light-induced interruption of the mitochondrial membrane potential and reduction of the Bcl-2/Bax ratio and cleaved caspase-3 and PARP-1 expression, suggesting the antiapoptotic activity of SFN. SFN alone or together with blue light exposure increased the expression of the autophagy-related proteins LC3BII and p62. An autophagy inhibitor, 3-MA, inhibited the protective effect of SFN on blue light-induced cell damage. SFN increased sirtuin-1 (SIRT1) expression; however, treatment with blue light induced peroxisome proliferator-activated receptor gamma coactivator-1α (PGC-1α) expression. Our study results demonstrated that SFN exerts its protective effect under blue light exposure by maintaining the Nrf2-related redox state and upregulating SIRT1 and PGC-1α expression and autophagy.

Carbon monoxide­releasing molecules protect against blue light exposure and inflammation in retinal pigment epithelial cells.

The most common cause of vision loss among the elderly is age­related macular degeneration (AMD). The aim of the present study was to investigate the potential cytoprotective and anti­inflammatory effects of carbon monoxide­releasing molecules (CORMs), and their ability to activate the expression of nuclear factor erythroid 2­related factor 2 (Nrf2)­related genes in human retinal pigment epithelium (RPE) cells, as well as the inhibition of endothelial cell migration. It was first determined that CORM2 and CORM3 suppressed blue light­induced cell damage. In addition, a decrease in the level of cleaved poly(ADP­ribose) polymerase 1 protein and dissipation of mitochondrial membrane potential were considered to reflect the anti­apoptotic activity of CORMs. Furthermore, CORM2 induced Nrf­2 activation and the expression of the Nrf2­related genes heme oxygenase­1 and glutamate­cysteine ligase. Pretreatment with CORM2 abolished the blue light­induced increase in oxidative stress, suggesting that CORM2­induced antioxidant activity was involved in the cytoprotection against blue light. It was also demonstrated that CORMs markedly suppressed tumor necrosis factor (TNF)α­induced intercellular adhesion molecule­1 expression. Moreover, it was further observed that CORMs exert their inhibitory effects through blocking nuclear factor­κB/p65 nuclear translocation and IκBα degradation in TNFα­treated RPE cells. It was observed that CORM2, but not CORM3, protected against oxidative stress­induced cell damage. CORMs abolished vascular endothelial growth factor­induced migration of endothelial cells. The findings of the present study demonstrated the cytoprotective, antioxidant and anti­inflammatory effects of CORMs on RPE cells and anti­angiogenic effects on endothelial cells, suggesting the potential clinical application of CORMs as anti­AMD agents.

Lycopene inhibits NF-κB activation and adhesion molecule expression through Nrf2-mediated heme oxygenase-1 in endothelial cells.

The endothelial expression of cell adhesion molecules plays a leading role in atherosclerosis. Lycopene, a carotenoid with 11 conjugated double bonds, has been shown to have anti-inflammatory properties. In the present study, we demonstrate a putative mechanism for the anti-inflammatory effects of lycopene. We demonstrate that lycopene inhibits the adhesion of tumor necrosis factor α (TNFα)-stimulated monocytes to endothelial cells and suppresses the expression of intercellular cell adhesion molecule-1 (ICAM-1) at the transcriptional level. Moreover, lycopene was found to exert its inhibitory effects by blocking the degradation of the inhibitory protein, IκBα, following 6 h of pre-treatment. In TNFα-stimulated endothelial cells, nuclear factor-κB (NF-κB) nuclear translocation and transcriptional activity were abolished by up to 12 h of lycopene pre-treatment. We also found that lycopene increased the intracellular glutathione (GSH) level and glutamate-cysteine ligase expression. Subsequently, lycopene induced nuclear factor-erythroid 2 related factor 2 (Nrf2) activation, leading to the increased expression of downstream of heme oxygenase-1 (HO-1). The use of siRNA targeting HO-1 blocked the inhibitory effects of lycopene on IκB degradation and ICAM-1 expression. The inhibitory effects of lycopene thus appear to be mediated through its induction of Nrf2-mediated HO-1 expression. Therefore, the findings of the present study indicate that lycopene suppresses the activation of TNFα-induced signaling pathways through the upregulation of Nrf2-mediated HO-1 expression.

Carbon monoxide releasing molecule induces endothelial nitric oxide synthase activation through a calcium and phosphatidylinositol 3-kinase/Akt mechanism.

The production of nitric oxide (NO) by endothelial NO synthase (eNOS) plays a major role in maintaining vascular homeostasis. This study elucidated the potential role of carbon monoxide (CO)-releasing molecules (CORMs) in NO production and explored the underlying mechanisms in endothelial cells. We observed that 25µM CORM-2 could increase NO production and stimulate an increase in the intracellular Ca2+ level. Furthermore, ethylene glycol-bis(ß-aminoethyl ether)-N,N,N',N'-tetra acetic acid caused CORM-2-induced NO production, which was abolished by 1,2-bis(2-aminophenoxy) ethane-N,N,N',N'-tetraacetic acid tetraacetoxy-methyl ester (BAPTA-AM), indicating that intracellular Ca2+ release plays a major role in eNOS activation. The inhibition of the IP3 receptor diminished the CORM-2-induced intracellular Ca2+ increase and NO production. Furthermore, CORM-2 induced eNOS Ser1179 phosphorylation and eNOS dimerization, but it did not alter eNOS expression. CORM-2 (25µM) also prolonged Akt phosphorylation, lasting for at least 12h. Pretreatment with phosphatidylinositol 3-kinase inhibitors (wortmannin or LY294002) inhibited the increases in NO production and phosphorylation but did not affect eNOS dimerization. CORM-2-induced eNOS Ser1179 phosphorylation was intracellularly calcium-dependent, because pretreatment with an intracellular Ca2+ chelator (BAPTA-AM) inhibited this process. Although CORM-2 increases intracellular reactive oxygen species (ROS), pretreatment with antioxidant enzyme catalase and N-acetyl-cysteine did not abolish the CORM-2-induced eNOS activity or phosphorylation, signifying that ROS is not involved in this activity. Hence, CORM-2 enhances eNOS activation through intracellular calcium release, Akt phosphorylation, and eNOS dimerization.

Lycopene inhibits ICAM-1 expression and NF-κB activation by Nrf2-regulated cell redox state in human retinal pigment epithelial cells.

AIMS: Age-related macular degeneration (AMD) is one of the most common diseases leading to blindness in elderly people. The progression of AMD may be prevented through anti-inflammation and antioxidation in retinal pigment epithelium (RPE) cells. Lycopene, a carotenoid, has been shown to possess both antioxidative and anti-inflammatory properties. This research was conducted to detail the mechanisms of these effects of lycopene-treated RPE cells. MAIN METHODS: We exposed ARPE-19 cells to TNFα after pretreatment with lycopene, and measured monocyte adhesion, ICAM-1 expression, NF-κB nuclear translocation, and transcriptional activity. Cell viability was assayed with Alamar Blue. The cell redox state was tested by glutathione (GSH) and reactive oxygen species (ROS) levels. The importance of the Nrf2 pathway was tested in nuclear translocation, promoter reporter assay, and siRNA. KEY FINDINGS: Lycopene could reduce TNF-α-induced monocyte adhesion and H2O2- induced cell damage in RPE cells. Furthermore, lycopene inhibits ICAM-1 expression and abolishes NF-κB activation for up to 12h in TNFα-treated RPE cells. Lycopene upregulates Nrf2 levels in nuclear extracts and increases the transactivity of antioxidant response elements. The use of Nrf2 siRNA blocks the inhibitory effect of lycopene in TNF-α-induced ICAM-1 expression and NF-κB activation. Glutamate-cysteine ligase (GCL) is the rate-limiting enzyme in the de novo synthesis of GSH. We found that lycopene increases intracellular GSH levels and GCL expression. Following lycopene treatment, TNF-α-induced ROS production was abolished. SIGNIFICANCE: The Nrf2-regulated antioxidant property plays a pivotal role in the anti-inflammatory mechanism underlying the inhibition of NF-κB activation in lycopene-treated ARPE-19 cells.

Carbon monoxide induces heme oxygenase-1 to modulate STAT3 activation in endothelial cells via S-glutathionylation.

IL-6/STAT3 pathway is involved in a variety of biological responses, including cell proliferation, differentiation, apoptosis, and inflammation. In our present study, we found that CO releasing molecules (CORMs) suppress IL-6-induced STAT3 phosphorylation, nuclear translocation and transactivity in endothelial cells (ECs). CO is a byproduct of heme degradation mediated by heme oxygenase (HO-1). However, CORMs can induce HO-1 expression and then inhibit STAT3 phosphorylation. CO has been found to increase a low level ROS and which may induce protein glutathionylation. We hypothesized that CORMs increases protein glutathionylation and inhibits STAT3 activation. We found that CORMs increase the intracellular GSSG level and induce the glutathionylation of multiple proteins including STAT3. GSSG can inhibit STAT3 phosphorylation and increase STAT3 glutathionylation whereas the antioxidant enzyme catalase can suppress the glutathionylation. Furthermore, catalase blocks the inhibition of STAT3 phosphorylation by CORMs treatment. The inhibition of glutathione synthesis by BSO was also found to attenuate STAT3 glutathionylation and its inhibition of STAT3 phosphorylation. We further found that HO-1 increases STAT3 glutathionylation and that HO-1 siRNA attenuates CORM-induced STAT3 glutathionylation. Hence, the inhibition of STAT3 activation is likely to occur via a CO-mediated increase in the GSSG level, which augments protein glutathionylation, and CO-induced HO-1 expression, which may enhance and maintain its effects in IL-6-treated ECs.

CO-releasing molecules and increased heme oxygenase-1 induce protein S-glutathionylation to modulate NF-κB activity in endothelial cells.

Protein glutathionylation is a protective mechanism that functions in response to mild oxidative stress. Carbon monoxide (CO) can increase the reactive oxygen species concentration from a low level via the inhibition of cytochrome c oxidase. We therefore hypothesized that CO would induce NF-κB-p65 glutathionylation and then show anti-inflammatory effects. In this study, we found that CO-releasing molecules suppress TNFα-induced monocyte adhesion to endothelial cells (ECs) and reduce ICAM-1 expression. Moreover, CO donors were further found to exert their inhibitory effects by blocking NF-κB-p65 nuclear translocation, but do so independent of IκBα degradation, in TNFα-treated ECs. In addition, p65 protein glutathionylation represents the response signal to CO donors and is reversed by the reducing agent dithiothreitol. Thiol modification of the cysteine residue in the p65 RHD region was required for the CO-modulated NF-κB activation. The suppression of p65 glutathionylation by a GSH synthesis inhibitor, BSO, and by catalase could also attenuate TNFα-induced p65 nuclear translocation and ICAM-1 expression. CO donors induce Nrf2 activation and Nrf2 siRNA suppresses CO-induced p65 glutathionylation and inhibition. Furthermore, we found that the CO donors induce heme oxygenase-1 (HO-1) expression, which increases p65 glutathionylation. In contrast, HO-1 siRNA attenuates CO donor- and hemin-induced p65 glutathionylation. Our results thus indicate that the glutathionylation of p65 is likely to be responsible for CO-mediated NF-κB inactivation and that the HO-1-dependent pathway may prolong the inhibitory effects of CO donors upon TNFα treatment of ECs.

Myopexy of the vertical rectus muscles using expanded polytetrafluoroethylene for management of a lost medial rectus muscle.

Strabismus due to a lost rectus muscle is uncommon. Surgical treatment of the condition by means of scleral augmented myopexy of the vertical rectus muscles can reduce the potential for anterior segment ischemia by leaving the vascular supply intact. We report a successful use of expanded polytetrafluoroethylene in this procedure to correct long-term exotropia caused by a lost medial rectus muscle.

Effects of topoisomerase II inhibitors on retinal pigment epithelium and experimental proliferative vitreoretinopathy.

AIMS: The aim of this study was to compare the effect of several commercially available topoisomerase II inhibitors on the proliferation of retinal pigment epithelium (RPE) cells in vitro and to test the toxicity and efficacy of the inhibitor against experimental proliferative vitreoretinopathy (PVR). METHODS: Three different topoisomerase II inhibitors (etoposide, doxorubicin, and daunorubicin) were tested in vitro. Rabbit RPE cells were cultured with or without the drugs at various concentrations. An MTT assay was used to determine the cell viability at 48 h and 96 h. Etoposide, a drug which showed a broad therapeutic range in vitro, was injected to the rabbit eye for the evaluation of the toxicity in vivo. Therapeutic effects of an intravitreal injection of etoposide were evaluated in an experimental PVR model induced by the intravitreal implantation of RPE cells in rabbits. RESULTS: All tested topoisomerase II inhibitors showed a significant reduction of cell viability in vitro. The slope of the dose-response curve was slowly declined for etoposide, and declined sharply for doxorubicin and daunorubicin. Therefore, etoposide was selected for further toxicity and efficacy studies in vivo. There was no significant change in b-wave amplitudes in the etoposide-injected eyes (0.02 mg, 10 microg/mL) after 2 weeks, but a significant reduction occurred in the etoposide-injected eyes (0.2 mg, 100 microg/mL). In the study of the experimental model of PVR, the rabbit eyes injected with RPE cells and etoposide (0.02 mg, 10 microg/mL) showed a significantly lower grading of PVR than that of the control eyes (injected RPE cells and PBS). CONCLUSIONS: These results indicate that etoposide would be an adjunctive for the prevention of PVR. Further pharmacokinetic study of the intravitreal injection of etoposide is required.

Pneumatic displacement of a dense submacular hemorrhage with or without tissue plasminogen activator.

BACKGROUND: To evaluate the efficacy of treating a dense submacular hemorrhage with pneumatic displacement with or without tissue plasminogen activator (tPA). METHODS: Twenty-four patients with a dense submacular hemorrhage were treated with intravitreal expansile gas, with or without an intravitreal injection of tPA, in order to displace the submacular blood. The main outcome measurements include preoperative and postoperative visual acuity, postoperative fluorescein angiography (FAG) results and additional postoperative treatments. RESULTS: Total or subtotal subfoveal blood displacement was achieved in all 24 eyes. After a mean follow-up of 15.5 months (range 6-50 months), final visual acuity had improved two or more lines in 11 (45.8%) of the 24 eyes, and measured 20/100 or better in 10 (41.7%) of the 11 eyes. Based on the FAG results for 14 cases, nine eyes (64.3%) received additional postoperative laser treatment. Final visual acuity of 20/100 or better was achieved in four (40%) of the 10 eyes, with a choroidal neovascular membrane (CNVM) detected on FAG, and dye leakage not detected in three (75%) of the four eyes. CONCLUSIONS: Pneumatic displacement, with or without intravitreal injection of tPA, seems useful in displacing dense submacular hemorrhage and facilitating visual improvement, although the visual result is often limited by the progression of the underlying macular disease. In patients with age-related macular degeneration, more treatable CNVM may be detected on postoperative FAG.