Ana L. Gramajo

Effects of Benzo(e)Pyrene, a Toxic Component of Cigarette Smoke, on Human Retinal Pigment Epithelial Cells In Vitro

PURPOSE To better understand the cellular and molecular basis for the epidemiologic association between cigarette smoke and age-related macular degeneration (AMD), the authors examined the effects of Benzo(e)Pyrene (B(e)P), a toxic element in cigarette smoke, on human retinal pigment epithelial cells (ARPE-19). METHODS ARPE-19 cells were cultured in Dulbecco modified Eagle medium containing 10% fetal bovine serum. Cells were treated for 24 hours with 1000 microM, 400 microM, 200 microM, and 100 microM B(e)P. Cell viability was determined by a trypan blue dye-exclusion assay. Activities of caspase-3/7, caspase-8, caspase-9, and caspase-12 were measured by a fluorescence image scanner, and DNA laddering was evaluated by electrophoresis on 3% agarose gel. RESULTS The mean percentage of cell viabilities of ARPE-19 cells was decreased in a dose-dependent manner after exposure to B(e)P at the higher concentrations of 1000 microM (20.0 +/- 0.4; P < 0.001), 400 microM (35.6 +/- 6.4; P < 0.001), and 200 microM (58.7 +/- 2.3; P < 0.001) but not at 100 microM (95.9 +/- 0.7; P > 0.05) compared with the equivalent dimethyl sulfoxide (DMSO)-treated control cultures. There were significant increases in caspase-3/7, -8, -9, and -12 activities compared with the DMSO-treated controls (P < 0.001). DNA laddering revealed bands at 200-bp intervals. CONCLUSIONS These results show that B(e)P is a toxicant to human retinal pigment epithelial cells in vitro. It causes cell death and induces apoptosis by the involvement of multiple caspase pathways.

Differential effects of nicotine on retinal and vascular cells in vitro.

The purpose of the current study is to understand the effects of nicotine in human retinal pigment epithelial (ARPE-19), human microvascular endothelial cells (HMVEC) and rat neurosensory retinal (R28) cells. ARPE-19, HMVEC and R28 cell cultures were treated with 10(-2) and 10(-4)M nicotine for 24h. R28 cells were also pre-treated for 4h with ALLN and ALLM (calpain inhibitors) or epicatechin, an antioxidant flavonoid compound. Trypan blue dye exclusion assay, caspase-3/7, LDH activity and DNA laddering assays were performed. With 10(-2)M nicotine treatment, R28 cell cultures showed decreased cell viability that was partially reversed by pre-treatment with the antioxidant epicatechin but not with calpain inhibitors. The DNA ladder assay showed a 200bp banding pattern consistent with apoptosis, however, caspase-3/7 activity was not increased. After treatment with 10(-2)M nicotine, HMVEC cultures showed decreased cell viability and increased LDH activity but no DNA banding patterns or caspase-3/7 activity. ARPE-19 cells showed no change in cell viability or caspase-3/7 activity at any of the nicotine concentrations. We conclude of dissimilar responses to nicotine treatment in three different cell lines. Nicotine was toxic to HMVEC and R28 cell cultures but the ARPE-19 cells were unaffected. In R28 cells, the nicotine effects were through an oxidant pathway that is non-caspase, non-calpain mediated while the HMVEC toxicity was via necrosis. Understanding the mechanisms of cell death may have potential therapeutic implications in the treatment of cigarette smoking related retinal diseases such as age-related macular degeneration (AMD).

Effects of Benzo(e)Pyrene on the Retinal Neurosensory Cells and Human Microvascular Endothelial Cells In Vitro

Purpose: To study the effects of benzo(e)pyrene (B(e)P), a toxic component of cigarette smoke, on retinal neurosensory (R28) cells and human microvascular endothelial cells (HMVEC). Materials and Methods: R28 cells and HMVEC were treated for 24 hours with 1000, 400, 200, and 100 μ M of B(e)P. Cell viability was measured by dye exclusion assay. Caspase-3/7, −8, −9, and −12 activities were measured by fluorochrome assays. DNA ladder was run on agarose gel, and lactate dehydrogenase (LDH) release rate was evaluated using a LDH cytotoxicity kit II. Results: R28 cells exposed to B(e)P 1000 and 400 μ M showed a decrease in cell viability but not at lower concentrations of 200 and 100 μ M. At 400, 200, and 100 μ M B(e)P, there was an increase in caspase-3/7 and at 200 and 100 μM B(e)P caspase-12 activities. Caspase-8 activity was increased only at 200 μ M B(e)P. Caspase-9 activity was not increased at any concentration. DNA ladder revealed bands at 200 bp intervals at lower concentrations and LDH activity at higher concentrations. HMVEC cultures exposed to B(e)P 1000, 400, and 200 μ M showed a decrease in cell viability. Caspase-3/7 activity was not increased at any concentration. DNA laddering revealed no bands at 200 bp intervals at any dose. LDH release rates increased at all three concentrations. However, 100 μ M B(e)P was found safe on HMVEC. Conclusions: B(e)P has different mechanisms of action on R28 cells and HMVEC at different concentrations. In R28 cells, 200 and 100 μ M of B(e)P causes activation of caspase-3/7, −8 (200 μ M only) and −12 pathways, leading to apoptotic cell death, but, at higher concentrations, there is non-apoptotic cell death, which could be due to necrosis. In contrast, the HMVEC cell death is through non-caspase-dependent necrosis pathway. The molecular mechanisms of cell death vary with different cell types and concentrations of B(e)P.

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.

Therapeutic benefit of melatonin in refractory central serous chorioretinopathy

PurposeTo evaluate the efficacy and safety of melatonin for the treatment of chronic central serous chorioretinopathy (CSCR).MethodsProspective comparative case series. A total of 13 patients with chronic CSCR were treated for 1 month: 8 patients were treated orally with 3 mg melatonin t.i.d., and 5 with placebo. All patients had 20/40 or worse Early Treatment Diabetic Retinopathy Study (ETDRS) best-corrected visual acuity (BCVA) in the affected eye or presented an incapacitating scotoma. Most of the patients had previous failed treatments for their condition. Observational procedures included ETDRS BCVA, and complete ophthalmic examination. Optical coherence tomography (OCT) was performed at day 1 and week 4. Fluorescein angiography was performed at baseline only for diagnostic purposes. Data were subjected to two-sample t-test statistical analysis. P-values of <0.05 were considered statistically significant.ResultsAt 1-month follow-up, BCVA significantly improved in 87.5% of patients treated with melatonin (7 of 8 patients, P<0.05). All patients showed a mean significant reduction (P<0.01) of central macular thickness (CMT) when compared with the baseline, with 3 patients (37.5%) exhibiting complete resolution of subretinal fluid at 1-month follow-up. No significant side effects were observed. No changes in BCVA or CMT were noted in the control group.ConclusionsThese results suggest that melatonin is safe, well tolerated, and effective in the treatment of chronic CSCR, as it significantly improved BCVA and CMT in patients with this pathology. Further evaluations with longer follow-up and a larger patient population are desirable.

Self-medication in Ophthalmology: A Questionnaire-based Study in an Argentinean Population

Purpose: The aim of this study was to identify practices of self-medication in the treatment of ocular conditions and to identify a profile of patients who self-medicate. Methods: We conducted a cross-sectional descriptive survey of patients, over the age of 17 years seen in our ophthalmology practice in Cordoba, Argentina. Self-medication was defined as the use of ophthalmic medicines which had not been prescribed by a health care specialist in the previous year. Results: The sample included 379 subjects, 162 males (43%) and 217 females (57%); mean age 46.8 years. Prior to looking for medical attention in our institution, 97 patients (25.6%) reported self-medicating. The most frequently employed products included non-steroidal anti-inflammatory drops in combination with a vasoconstrictive agent (32%) followed by a combination of antibiotics and steroids (9%), however, 14% of patients did not remember the name or type of medication applied. A total of 31% of patients used drugs recommended by a pharmacist; 25% used drugs of their own choosing and 24% followed suggestions from a friend or family member. Only 12% of patients knew the drug’s components and only 3% were aware of any possible side effects. There was no difference in behavior patterns related to educational level or age, however, there was a significant difference related to gender, with males misusing ophthalmic drops more frequently than women (P = 0.004). Conclusions: Patients commonly attempt to treat conditions that require ophthalmologic care by self-medicating with over-the-counter eye drops. Educational efforts to inform patients of the consequences of self-medication are necessary.

Effect of bevacizumab (Avastin TM ) on mitochondrial function of in vitro retinal pigment epithelial, neurosensory retinal and microvascular endothelial cells

Purpose: To evaluate the effect of bevacizumab on the mitochondrial function of human retinal pigment epithelial (ARPE-19), rat neurosensory retinal (R28) and human microvascular endothelial (HMVEC) cells in culture. Materials and Methods: ARPE-19 and R28 cells were treated with 0.125, 0.25, 0.50 and 1 mg/ml of bevacizumab. The HMVEC cultures were treated with 0.125, 0.25, 0.50 and 1 mg/ml of bevacizumab or 1 mg/ml of immunoglobulin G (control). Mitochondrial function assessed by mitochondrial dehydrogenase activity (MDA) was determined using the WST-1 assay. Results: Bevacizumab doses of 0.125 to 1 mg/ml for 5 days did not significantly affect the MDA of ARPE-19 cells. Bevacizumab treatment at 0.125 and 0.25 mg/ml (clinical dose) did not significantly affect the MDA of R28 cells; however, 0.50 and 1 mg/ml doses significantly reduced the R28 cell mitochondrial function. All doses of bevacizumab significantly reduced the MDA of proliferating and non-proliferating HMVEC. Conclusion: Bevacizumab exposure for 5 days was safe at clinical doses in both ARPE-19 and R28 retinal neurosensory cells in culture. By contrast, bevacizumab exposure at all doses show a significant dose-dependent decrease in mitochondrial activity in both the proliferating and non-proliferating HMVEC in vitro. This suggests a selective action of bevacizumab on endothelial cells at clinical doses.

Galectin-1 expression imprints a neurovascular phenotype in proliferative retinopathies and delineates responses to anti-VEGF

Neovascular retinopathies are leading causes of irreversible blindness. Although vascular endothelial growth factor (VEGF) inhibitors have been established as the mainstay of current treatment, clinical management of these diseases is still limited. As retinal impairment involves abnormal neovascularization and neuronal degeneration, we evaluated here the involvement of galectin-1 in vascular and non-vascular alterations associated with retinopathies, using the oxygen-induced retinopathy (OIR) model. Postnatal day 17 OIR mouse retinas showed the highest neovascular profile and exhibited neuro-glial injury as well as retinal functional loss, which persisted until P26 OIR. Concomitant to VEGF up-regulation, galectin-1 was highly expressed in P17 OIR retinas and it was mainly localized in neovascular tufts. In addition, OIR induced remodelling of cell surface glycophenotype leading to exposure of galectin-1-specific glycan epitopes. Whereas VEGF returned to baseline levels at P26, increased galectin-1 expression persisted until this time period. Remarkably, although anti-VEGF treatment in P17 OIR improved retinal vascularization, neither galectin-1 expression nor non-vascular and functional alterations were attenuated. However, this functional defect was partially prevented in galectin-1-deficient (Lgals1−/−) OIR mice, suggesting the importance of targeting both VEGF and galectin-1 as non-redundant independent pathways. Supporting the clinical relevance of these findings, we found increased levels of galectin-1 in aqueous humor from patients with proliferative diabetic retinopathy and neovascular glaucoma. Thus, using an OIR model and human samples, we identified a role for galectin-1 accompanying vascular and non-vascular retinal alterations in neovascular retinopathies.

Lens opacity based modelling of the age-related straylight increase.

This work studies ethnic and geographical differences in the age-related straylight increase by means of a stochastic model and unpublished lens opacity data of 559 residents of Villa Maria (Argentina), as well as data of 912 Indonesian subjects published previously by Husain et al. For both cohorts the prevalence of each type and grade of lens opacity was determined as a function of age, from which a stochastic model was derived capable of simulating the lens opacity prevalence for both populations. These simulated lens opacity data were then converted to estimated straylight by means of an equation derived from previously recorded data of 107 eyes with varying degrees of cataract. Based on these opacity templates 2500 random sets of subject age and lens opacity data were generated by the stochastic model for each dataset, from which estimated straylight could be calculated. For the Argentinian data the estimated straylight was found to closely resemble the published models for age-related straylight increase. For younger eyes the straylight variation of the model was the same as what was previously published (in both cases ±0.200logunits), which doubled in size for older eyes. For the Indonesian data, however, this age-related straylight increase was found to be fundamentally different from the published age model. This suggests that current normative curves for age-related straylight increase may not always be appropriate for non-European populations, and that the inter-individual straylight variations in young, healthy eyes may possibly be due to variations in lens opacities.

Perfluorocarbon liquid vitreous delamination and wide-angle viewing system in the management of complicated diabetic retinal detachment.

Purpose The authors used perfluorocarbon liquid (PFCL) and a wide-angle viewing system (WAVS) to evaluate their efficacy on tractional and combined tractional/rhegmatogenous retinal detachment (RD) secondary to proliferative diabetic retinopathy (PDR). Methods In a prospective, noncomparative, interventional study, 76 consecutive cases of severe PDR with tractional and combined tractional/rhegmatogenous RD were submitted to vitrectomy en bloc excision technique using a WAVS and delamination with PFCL between July 1999 and December 2003. None of the patients had had previous retinal photocoagulation treatment. Preoperative characteristics, intraoperative findings, and procedures as well as postoperative results were recorded. Main outcome measures included visual acuity (VA) and rates of retinal reattachment and complications. Results After 1 to 4 years of follow-up (mean 34.3 months), the number of patients changed from 3 (3.95%) to 11 patients (14.47%) in the ≥ 20/40 VA range, from 12 (15.79%) to 7 (9.21%) in the 20/50 to 20/200 group, and from 61 (80.26%) to 58 (76.31%) in the ≤ 20/400 group, preoperatively and postoperatively, respectively. The mean final VA improved from 1.2 logMAR before surgery to 0.89 after vitrectomy (p=0.001). This modified technique resulted in less bleeding during surgery, a better identification of intraocular structures, faster retinal reattachment, subretinal fluid reabsorption, and easier dissection of fibrovascular membranes, among other benefits. Conclusions PFCL and WAVS appear to reduce intraoperative complication rates in the management of complicated cases of tractional and combined tractional/rhegmatogenous RD secondary to PDR. Retinal reattachment and functional vision rates improved after this technique.