Memantine, Simvastatin, and Epicatechin Inhibit 7-Ketocholesterol-induced Apoptosis in Retinal Pigment Epithelial Cells But Not Neurosensory Retinal Cells In Vitro.

PURPOSE: 7-ketocholesterol (7kCh), a natural byproduct of oxidation in lipoprotein deposits is implicated in the pathogenesis of diabetic retinopathy and age-related macular degeneration (AMD). This study was performed to investigate whether several clinical drugs can inhibit 7kCh-induced caspase activation and mitigate its apoptotic effects on retinal cells in vitro. METHODS: Two populations of retinal cells, human retinal pigment epithelial cells (ARPE-19) and rat neuroretinal cells (R28) were exposed to 7kCh in the presence of the following inhibitors: Z-VAD-FMK (pan-caspase inhibitor), simvastatin, memantine, epicatechin, and Z-IETD-FMK (caspase-8 inhibitor) or Z-ATAD-FMK (caspase-12 inhibitor). Caspase-3/7, -8, and -12 activity levels were measured by fluorochrome caspase assays to quantify cell death. IncuCyte live-cell microscopic images were obtained to quantify cell counts. RESULTS: Exposure to 7kCh for 24 hours significantly increased caspase activities for both ARPE-19 and R28 cells (P < 0.05). In ARPE cells, pretreatment with various drugs had significantly lower caspase-3/7, -8, and -12 activities, reported in % change in mean signal intensity (msi): Z-VAD-FMK (48% decrease, P < 0.01), memantine (decreased 47.8% at 1 µM, P = 0.0039 and 81.9% at 1 mM, P < 0.001), simvastatin (decreased 85.3% at 0.01 µM, P < 0.001 and 84.8% at 0.05 µM, P < 0.001) or epicatechin (83.6% decrease, P < 0.05), Z-IETD-FMK (68.1% decrease, P < 0.01), and Z-ATAD-FMK (47.7% decrease, P = 0.0017). In contrast, R28 cells exposed to 7kCh continued to have elevated caspase-3/7, -8, and -12 activities (between 25.7% decrease and 17.5% increase in msi, P > 0.05) regardless of the pretreatment. CONCLUSION: Several current drugs protect ARPE-19 cells but not R28 cells from 7kCh-induced apoptosis, suggesting that a multiple-drug approach is needed to protect both cells types in various retinal diseases.

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.

Long-term hypotony because of accidental break of a 23 g microcannula after transconjunctival sutureless vitrectomy.

PURPOSE: To report a case of long-lasting hypotony because of accidental break, with scleral tunnel entrapment, of a 23-gauge microcannula during transconjunctival sutureless vitrectomy. METHODS: Interventional case report. An 80-year-old Spanish woman who underwent 23-gauge transconjunctival sutureless vitrectomy presented at the postoperative ocular examination with irreversible, refractory low intraocular pressure of unknown cause. Two weeks after surgery, a piece of the microcannula was found at the inferotemporal sclerotomy site during a scheduled medical appointment. Surgical intervention was indicated to explore and remove the foreign body. RESULTS: The day after foreign body extraction, the patient's pressure rose to normal levels. However, her visual acuity did not improve until 3 weeks later. CONCLUSION: Transient postoperative hypotony is unsurprising after 23-gauge vitrectomy because of leakage of small-diameter open sclerotomies. However, when long-term low intraocular pressure fails to return to normal levels because of an unidentified condition, breaking of the microcannula piece with scleral tunnel entrapment may be contemplated.

Stenotrophomonas maltophilia endophthalmitis caused by surgical equipment contamination: an emerging nosocomial infection.

PURPOSE: We report three cases of Stenotrophomonas maltophilia endophthalmitis after uneventful extracapsular cataract extraction with intraocular lens implantation-related to surgical equipment contamination. CASE REPORT: All patients developed acute, culture-positive endophthalmitis in a period ranging from 2 to 13 days. Cultures from vitreous tap, as well as those obtained from the hand-piece of the irrigation-aspiration system, revealed S. maltophilia as the causing infectious agent. All patients received intravitreal antibiotic treatment as initial therapy, nevertheless, visual disturbance continued to be present, hence pars plana vitrectomy was required. CONCLUSION: Contamination of surgical-reusable equipment should be considered in addition to the well-known risk factors associated with development of endophthalmitis by S. maltophilia.

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.

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.

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.

Delayed corneal epithelial healing after intravitreal bevacizumab: a clinical and experimental study.

PURPOSE: To report corneal epithelial defects (CEDs) and delayed epithelial healing after intravitreal bevacizumab (IVB) injection and to describe delayed corneal epithelial healing with topical administration of bevacizumab in an experimental rabbit model. METHODS: A retrospective chart review was performed on 850 eyes of 850 patients with neovascular eye disease and diabetic macular edema who had received 1.25 to 2.5 mg IVB. In the experimental arm of the study, photorefractive keratectomy was used to create a 3 mm CED in the right eyes of 18 New Zealand rabbits which were then randomized to three equal groups. All rabbits received topical antibiotics, additionally those in group A received topical bevacizumab and animals in group B were treated with topical corticosteroids. The rate of epithelial healing was assessed at different time points using slitlamp photography. RESULTS: In the clinical study, seven eyes of seven subjects developed CEDs the day after IVB injection. All of these eyes had preexisting corneal edema. The healing period ranged from 3 to 38 days (average 11 days) despite appropriate medical management. In the experimental study, topical bevacizumab and corticosteroids both significantly hindered corneal epithelial healing at 12 and 24 hours. CONCLUSION: Bevacizumab was demonstrated to cause CEDs in clinical settings. Moreover, corneal epithelial healing was delayed by topical application of bevacizumab, in the experimental model. These short-term results suggest that corneal edema may be considered as a risk factor for epithelial defects after IVB.

Mitochondrial DNA damage induced by 7-ketocholesterol in human retinal pigment epithelial cells in vitro.

PURPOSE: To assess oxysterol-induced mitochondrial DNA (mtDNA) damage and mitochondrial dysfunction in cultured human retinal pigment epithelial cells (ARPE- 19). METHODS: ARPE-19 cultures were exposed for 6 and 24 hours to 40 microg/mL 7-ketocholesterol (7kCh), and total DNA was extracted. Long-extension polymerase chain reaction was performed to amplify the full-length mtDNA genome. The products were separated by electrophoresis on a 0.8% agarose gel stained with ethidium bromide. Superoxide and reactive oxygen/nitrogen species (ROS/RNS; hydrogen peroxide, peroxynitrite anions, and peroxyl radicals) were measured with an amine-reactive green-dye assay and 2',7'-dicholorodihydrofluorescein diacetate (H(2)DCFDA) dye assay, respectively. The changes in mitochondrial membrane potential (DeltaPsim) were measured with a cationic (green) dye assay. Western blot analysis was used to assess porins, a structural protein of the mitochondrial membranes. RESULTS: The 7kCh-treated cultures showed significant increase in ROS/RNS production (P < 0.001) compared with untreated controls, but the superoxide levels were unchanged. The 7kCh-treated ARPE-19 cultures had diminished levels of the full-length 16.2-kb mtDNA band, a 2.2-fold decrease of the DeltaPsim compared with control cultures (P < 0.001), and decreased levels of porins. CONCLUSIONS: 7kCh causes significant damage to the full-length intact mtDNA and mitochondrial dysfunction in ARPE-19 cells. These observations suggest that the mitochondria and its DNA may be targets for oxysterol-induced oxidative stress and may play a role in the pathogenesis of retinal diseases.

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 micro 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 micro M showed a decrease in cell viability but not at lower concentrations of 200 and 100 micro M. At 400, 200, and 100 micro M B(e)P, there was an increase in caspase-3/7 and at 200 and 100 microM B(e)P caspase-12 activities. Caspase-8 activity was increased only at 200 micro 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 micro 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 micro 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 micro M of B(e)P causes activation of caspase-3/7, -8 (200 micro 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.

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).

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 > or =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 < or =20/400 group, preoperatively and postoperatively, respectively. The mean final VA improved from 1.2 log-MAR 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.

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.

7-Ketocholesterol activates caspases-3/7, -8, and -12 in human microvascular endothelial cells in vitro.

7-Ketocholesterol (7kCh) is a major oxysterol found associated with vascular diseases. Human microvascular endothelial cells (HMVECs) were cultured with different concentrations of 7kCh with and without inhibitors. Cell viabilities and caspase activities were assessed. 7kCh caused loss of cell viability in a dose-dependent manner. Caspases-8, -12, and -3/7 but not caspase-9 were activated by 7kCh treatment. The 7kCh-induced caspase-8 activity was blocked partially by pre-treatment with z-VAD-fmk and z-IETD-fmk, a caspase-8 inhibitor. However, pre-treatment with z-ATAD-fmk, a caspase-12 inhibitor, followed by 7kCh exposure lead to significantly increased caspase-8 activity. This suggests that caspase-8 and caspase-12 pathways have unique inhibition patterns and that caspase-12 is likely not upstream and feeding into caspase-8 but the pathways may function in parallel to each other. Caspase-3/7 activation was inhibited partially by low density lipoprotein (LDL), high density lipoprotein (HDL), z-VAD-fmk (pan-caspase inhibitor), and low doses (0.01 and 0.001 microM) of the cholesterol lowering drug, simvastatin. However, only LDL partially protected against 7kCh-induced loss of cell viability suggesting that caspase-independent pathways also contributed to the cell loss and that protection from oxysterol damage may require inhibition of multiple pathways. Moreover, our data suggest that oxysterols such as 7kCh can damage HMVECs cells in part via caspase-dependent apoptosis and may play a role in vascular and retinal diseases.

Effect of retinal laser photocoagulation on the activity of metalloproteinases and the alpha(2)-macroglobulin proteolytic state in the vitreous of eyes with proliferative diabetic retinopathy.

Panretinal photocoagulation (PRP) reduces the incidence of severe visual loss in proliferative diabetic retinopathy (PDR). The aim of the study was to determine the effect of PRP on the activity of matrix metalloproteinase-2 (MMP-2) and MMP-9, and also on the alpha(2)-Macroglobulin (alpha(2)M) proteolytic state in the vitreous of eyes with PDR. Vitreous samples were obtained from patients undergoing vitrectomy for the treatment of retinal diseases: 17 with PDR and eight with idiopathic macular hole (MH). Qualitative evaluation of the MMP-2 and MMP-9 activation status was performed by gelatin zymography and quantitative assay was carried out for vitreous total protein content and alpha(2)M. The proteolytic state of alpha(2)M was evaluated by Western blotting. Although all vitreous samples contained proMMP-2, increased proMMP-9 and active MMP-9 were detected in PDR samples without PRP. In addition, after PRP the proMMP-9 activity was significantly decreased, whereas the proMMP-2 activity was not affected. Enhanced total protein and alpha(2)M concentrations were observed in all vitreous samples from PDR patients with and without previous PRP compared with samples from patients with MH. However, a differential proteolytic state of alpha(2)M, expressed as 180/85-90kDa ratio, was detected among PDR patients with and without PRP treatment. Whereas a low 180/85-90kDa ratio of alpha(2)M in vitreous of PDR patients without PRP was observed, a high proportion of 180kDa subunit was principally detected in PDR with PRP. These results demonstrate that PDR occurs with an enhanced activity of MMP-9 and activation of alpha(2)M by proteinases, which is reversed after PRP. In addition, we suggest that alpha(2)M plays a key role in the control and regulation of the ocular neovascularization involved in the pathogenesis of ischemic retinal diseases such as PDR.

Caspase-8, -12, and -3 activation by 7-ketocholesterol in retinal neurosensory cells.

PURPOSE: To determine the caspase pathways involved with 7-ketocholesterol (7kCh)-induced apoptosis in rat R28 cells. METHODS: R28 cells were exposed to 7kCh with or without low-density lipoprotein (LDL) and z-VAD-fmk, a pan-caspase inhibitor. Cell viability was measured by a trypan blue dye exclusion assay. Caspase-3, -8, -9, and -12 activities were measured by fluorochrome caspase assays. ARPE-19 cells were used as control for caspase-3 inhibition experiments. RESULTS: R28 cultures showed decreased cell viability on 7kCh exposure compared with controls (P < 0.001), and this was reversed with LDL and LDL + z-VAD-fmk (P < 0.001). The 7kCh-treated R28 cultures had increased caspase-8 activity compared with controls (P < 0.001). This activity was blocked partially with LDL (P < 0.01) or LDL + z-VAD-fmk (P < 0.001) but not with z-VAD-fmk alone. Caspase-12 activity was increased after 7kCh treatment compared with controls (P < 0.01), and this activity was increased further with the addition of LDL. Caspase-3 activity in R28 cultures increased with 7kCh treatment compared with controls (P < 0.001). In R28 cultures, the z-VAD-fmk treatment did not blocked 7kCh-induced caspase-3 activity but did block activity in ARPE-19 cultures (P < 0.001). Caspase-9 was not activated by 7kCh treatment. CONCLUSIONS: In R28 cells, 7kCh-induced apoptosis involves the caspase-3 along with the caspase-8 and caspase-12 pathways. LDL partially blocked 7kCh-induced caspase-8 activity but increased caspase-12 activities, suggesting that caspase-8 and caspase-12 pathways are independent of each other. The z-VAD-fmk inhibitor blocked caspase-3 activities in the homogeneous ARPE-19 cultures but not in the heterogeneous R28 cultures.

Evaluation of in vitro effects of bevacizumab (Avastin) on retinal pigment epithelial, neurosensory retinal, and microvascular endothelial cells.

PURPOSE: To evaluate the short-term in vitro safety of bevacizumab (Avastin) in human retinal pigment epithelial (ARPE-19), rat neurosensory retinal (R28), and human microvascular endothelial (HMVECad) cells. METHODS: ARPE-19 and R28 cells were treated with 0.125 mg/mL, 0.25 mg/mL, 0.50 mg/mL, and 1 mg/mL of bevacizumab for 2, 6, and 24 hours. HMVECad cells were treated with 5 ng/mL of vascular endothelial growth factor (VEGF) and 0.125 mg/mL, 0.25 mg/mL, 0.50 mg/mL, and 1 mg/mL of either bevacizumab for 2, 6, and 24 hours or a nonspecific human purified immunoglobulin (IgG) for 24 hours. Cell viability was measured using trypan blue dye exclusion assay. RESULTS: The cell viabilities of ARPE-19 cells, R28 cells, and HMVECad cells treated with bevacizumab were not significantly different (P > 0.05) from that of untreated controls. There was no significant difference (P > 0.05) between viabilities of HMVECad cells treated with bevacizumab and IgG. CONCLUSION: This study suggests that bevacizumab, at concentrations at or above the dose normally used in clinical practice, is not toxic to human retinal pigment epithelial, rat neurosensory retinal, or human microvascular endothelial cells in vitro. This report is consistent with the recent report of lack of toxicity of intravitreal bevacizumab in rabbits as well as the lack of apparent toxicity in clinical use.