Effects of light on retinal pigment epithelial cells, neurosensory retinal cells and Müller cells treated with Brilliant Blue G.

BACKGROUND: The aim of this study is to evaluate the safety profile of Brilliant Blue G (BBG) with and without exposure to light (L) on three different retinal cell lines. METHOD: ARPE-19, R28 and MIO-M1 cells were treated with BBG: 0.125 mg/mL (0.5x clinical concentration), 0.25 mg/mL (1x) or 0.5 mg/mL (2x) with or without surgical illumination of halogen light exposure for 10 min, 15 min or 30 min. Cells were further cultured after 24 h and then analysed for cell viability, late stages of apoptosis and mitochondrial damage associated with early apoptosis using assays that measure trypan blue dye exclusion, increases in caspase-3/7 activity or changes in mitochondrial membrane potential (ΔΨm), respectively. RESULT: All three cell lines that were exposed to BBG in the presence or absence of light exposure for 30 min were found to have cell viability and caspase-3/7 activity levels similar to the untreated cultures. The mitochondrial membrane potential (ΔΨm) was decreased significantly at the 2x + L dose and 2x dose in all three retinal cell lines compared to their respective untreated control cells. At the lower doses of BBG, with or without exposure to light, the ΔΨm values were similar to the untreated control cultures. CONCLUSION: Exposure to BBG dye concentrations that are used clinically (0.125 mg/mL and 0.25 mg/mL) in the presence up to 30 min of surgically equivalent light intensity is safe for retinal cells.

Protective effects of lipoic acid on chrysene-induced toxicity on Müller cells in vitro.

PURPOSE: This study evaluates the toxic effects of chrysene (a component from cigarette smoke) on Müller cells (MIO-M1) in vitro and investigates whether the inhibitor lipoic acid can reverse the chrysene-induced toxic effects. METHODS: MIO-M1 cells were exposed to varying concentrations of chrysene with or without lipoic acid. Cell viability was measured by a trypan blue dye exclusion assay. Caspase-3/7 activity was measured by a fluorochrome assay. Lactate dehydrogenase (LDH) release was quantified by an LDH assay. The production of reactive oxygen/nitrogen species (ROS/RNS) was measured with a 2',7'-dichlorodihydrofluorescein diacetate dye assay. Mitochondrial membrane potential (ΔΨm) was measured using the JC-1 assay. Intracellular ATP content was determined by the ATPLite kit. RESULTS: MIO-M1 cells showed significantly decreased cell viability, increased caspase-3/7 activity, LDH release at the highest chrysene concentration, elevated ROS/RNS levels, decreased ΔΨm value, and decreased intracellular ATP content after exposure to 300, 500, and 1,000 µM chrysene compared with the control. Pretreatment with 80 µM lipoic acid reversed loss of cell viability in 500-µM-chrysene-treated cultures (24.7%, p<0.001). Similarly, pretreatment with 80 µM lipoic acid before chrysene resulted in decreased caspase-3/7 activities (75.7%, p<0.001), decreased ROS/RNS levels (80.02%, p<0.001), increased ΔΨm values (86%, p<0.001), and increased ATP levels (40.5%, p<0.001) compared to 500-µM-chrysene-treated cultures. CONCLUSIONS: Chrysene, a component of cigarette smoke, can diminish cell viability in MIO-M1 cells in vitro by apoptosis at the lower concentrations of Chrysene (300 and 500 µM) and necrosis at the highest concentration. Moreover, mitochondrial function was particularly altered. However, lipoic acid can partially reverse the cytotoxic effect of chrysene. Lipoic acid administration may reduce or prevent Müller cell degeneration in retinal degenerative disorders.

Effects of dexamethasone on human trabecular meshwork cells in vitro.

PURPOSE: To study the effects of dexamethasone sodium phosphate (Dex) on human trabecular meshwork (HTM) cells in vitro. METHODS: HTM cells were treated with Dex 2 mg/ml, 1 mg/ml, 0.5 mg/ml, 0.25 mg/ml, 0.1 mg/ml, or 0.05 mg/ml for 24 h. Cell viability was measured by a trypan blue exclusion test. Caspase-3/7, -8, -9 and -12 activities were measured by fluorochrome assays as mean signal intensity (msi) to assess apoptosis. Mitochondrial dehydrogenase activity was determined by a WST assay to quantify mitochondrial damage. RESULTS: Mean cell viabilities of HTM cells exposed to Dex at the higher doses of 2 mg/ml, 1 mg/ml, and 0.5 mg/ml were reduced: 11.9 % ± 3.5 (P < 0.001), 31.2 % ± 3.2 (P < 0.001), and 76.6 % ± 4.4 (P < 0.01). At the lower doses of 0.25 mg/ml, 0.1 mg/ml or 0.05 mg/ml, no significant cell viability reductions were seen: 96.3 % ± 0.7 (P > 0.05), 95.3 % ± 2.5 (P > 0.05) and 93.8 % ± 2.3 (P > 0.05), respectively compared to untreated HTM cells (97.0 % ± 1.9). Caspase-3/7 activity (msi) of HTM cells exposed to Dex 2, 1 or 0.5 mg/ml was 21068 ± 2498 (P < 0.001), 26994 ± 3104 (P < 0.001) and 20416 ± 1150 (P < 0.001) compared to untreated HTM cells 1148 ± 803. Caspase-9 activity (msi) of HTM cells after exposure to Dex 2, 1 or 0.5 mg/ml was 14188 ± 1203 (P < 0.001), 13256 ± 1564 (P < 0.001) and 15041 ± 1584 (P < 0.001) compared to untreated HTM cells 1748 ± 524. The lower doses of Dex did not significantly increase caspase-3/7 or -9 activities. There were no increases for caspase-8 or -12 activities at any of the tested Dex doses. The WST assay showed mitochondrial dehydrogenase activities of 14.3 ± 0.7 (P < 0.001), 9.6 ± 0.3 (P < 0.001) and 56.0 ± 7.6 (P < 0.001) at 2 mg/ml, 1 mg/ml and 0.5 mg/ml Dex compared to untreated HTM cells (186.1 ± 15.0). CONCLUSIONS: Dex at 0.25, 0.1 and 0.05 mg/ml clinical dose did not cause significant reduction in cell viability, increased apoptosis, or mitochondrial dysfunction of HTM cells in vitro. At high doses (2, 1 or 0.5 mg/ml) Dex caused apoptosis via mitochondrial pathways.

Antioxidant Therapy in Oxidative Stress-Induced Neurodegenerative Diseases: Role of Nanoparticle-Based Drug Delivery Systems in Clinical Translation.

Free radicals are formed as a part of normal metabolic activities but are neutralized by the endogenous antioxidants present in cells/tissue, thus maintaining the redox balance. This redox balance is disrupted in certain neuropathophysiological conditions, causing oxidative stress, which is implicated in several progressive neurodegenerative diseases. Following neuronal injury, secondary injury progression is also caused by excessive production of free radicals. Highly reactive free radicals, mainly the reactive oxygen species (ROS) and reactive nitrogen species (RNS), damage the cell membrane, proteins, and DNA, which triggers a self-propagating inflammatory cascade of degenerative events. Dysfunctional mitochondria under oxidative stress conditions are considered a key mediator in progressive neurodegeneration. Exogenous delivery of antioxidants holds promise to alleviate oxidative stress to regain the redox balance. In this regard, natural and synthetic antioxidants have been evaluated. Despite promising results in preclinical studies, clinical translation of antioxidants as a therapy to treat neurodegenerative diseases remains elusive. The issues could be their low bioavailability, instability, limited transport to the target tissue, and/or poor antioxidant capacity, requiring repeated and high dosing, which cannot be administered to humans because of dose-limiting toxicity. Our laboratory is investigating nanoparticle-mediated delivery of antioxidant enzymes to address some of the above issues. Apart from being endogenous, the main advantage of antioxidant enzymes is their catalytic mechanism of action; hence, they are significantly more effective at lower doses in detoxifying the deleterious effects of free radicals than nonenzymatic antioxidants. This review provides a comprehensive analysis of the potential of antioxidant therapy, challenges in their clinical translation, and the role nanoparticles/drug delivery systems could play in addressing these challenges.

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.

Protective Effects of 17ß-Estradiol on Benzo(e) pyrene[B(e)P]-induced Toxicity in ARPE-19 cells.

PURPOSE: The aim of this study was to examine the effect of 17ß-estradiol on Benzo(e)pyrene [B(e)P]-induced toxicity in ARPE-19 cells. METHODS: We pretreated ARPE-19 cells with 20 nM and 40 nM 17ß-estradiol for 6 hours, followed by addition of 300 µM B(e)P for additional 24 hours. Cell viability was measured using Trypan blue dye-exclusion assay. JC-1 assay was performed to measure mitochondrial membrane potential (ΔΨm). For a quantitative estimation of cell death, apoptotic markers such as caspase-3/7, caspase-9, and caspase-12 were measured. RESULTS: Our results demonstrated that when treated with B(e)P, the viability and ΔΨm of ARPE-19 cells declined by 25% and 63%, respectively (P < 0.05). However, pretreating with 17ß-estradiol increased the viability of ARPE-19 cells by 21% (20 nM) and 10% (40 nM) (P < 0.05). Furthermore, the significantly reduced ΔΨm in ßE+B(e)P treated cells ARPE-19 cells was restored by pre-treatment with 17ß-estradiol- ΔΨm was increased by 177% (20 nM) and 158% (40 nM) (P < 0.05). We further observed a significant up-regulation in the activity of Caspases-3/7, -9, and -12 in B(e)P-treated ARPE-19 cells. However, 17ß-estradiol treatment significantly reduced the activity of all apoptotic markers (P < 0.05). CONCLUSION: In conclusion, our results demonstrate that 17ß-estradiol protects ARPE-19 cells against B(e)P-induced toxicity by decreasing apoptosis, preventing cell death, and restoring mitochondrial membrane potential.

Effects of triamcinolone acetonide on human trabecular meshwork cells in vitro.

AIM: To study the effects of triamcinolone acetonide (TA) on cultured human trabecular meshwork (HTM) cells. MATERIALS AND METHODS: HTM cells were cultured and treated with 125, 250, 500 and 1000 µg/mL concentration of TA for 24 h. The cells were treated with both crystalline TA (TA-C) (commercial preparation) and solubilized TA (TA-S). Cell viability was measured by a trypan blue dye exclusion test. The activity of caspse-3/7 was measured by a fluorescence caspase kit and DNA laddering was evaluated by electrophoresis on 3% agarose gel. Levels of lactate dehydrogenase (LDH) were assessed with LDH cytotoxicity assay kit-II. RESULTS: Mean cell viabilities of HTM cells after 24 h exposure to TA-C 125, 250, 500, and 1000 µg/mL were 75.4 ±2.45% (P < 0.0001), 49.43 ± 1.85% (P < 0.0001), 17.07 ± 2.39% (P < 0.0001), and 3.7 ± 0.9% (P < 0.0001), respectively, compared with the untreated HTM cells 92.49 ± 1.21%. The mean cell viabilities with 125, 250, 500, and 1000 µg/mL of TA-S were 94.47 ± 1.60% (P > 0.05), 90.13 ± 0.40% (P < 0.01), 85.57 ± 0.47% (P < 0.001), and 71.67 ± 3.30% (P < 0.0001), respectively, compared to DMSO-equivalent cultures. Untreated HTM control had a cell viability of 96.57 ± 1.98%. DMSO-treated controls of 125, 250, 500, and 1000 µg/mL had a cell viability of 94.73 ± 0.57%, 96.97 ± 1.08%, 93.97 ± 1.85%, and 97.27 ± 1.15%, respectively. There was no increase of caspase-3/7 activity in cultures treated with either TA-C or TA-S. DNA laddering showed no bands in the TA-C or TA-S treated cultures. There were significantly higher LDH release rates at all concentrations of TA-C compared to TA-S. CONCLUSIONS: Results show that the effect of TA-C and TA-S on HTM cells is due to cell death by necrosis at all concentrations except 125 µg/mL of TA-S. Elevated levels of LDH confirmed necrotic cell death. Our study also infers the relative safety of TA-S over TA-C.

Delivery of salmon calcitonin using a microneedle patch.

Peptides and polypeptides have important pharmacological properties but only a limited number have been exploited as therapeutics because of problems related to their delivery. Most of these drugs require a parenteral delivery system which introduces the problems of pain, possible infection, and expertise required to carry out an injection. The aim of this study was to develop a transdermal patch containing microneedles (MNs) coated with a peptide drug, salmon calcitonin (sCT), as an alternative to traditional subcutaneous and nasal delivery routes. Quantitative analysis of sCT after coating and drying onto microneedles was performed with a validated HPLC method. In vivo studies were carried out on hairless rats and serum levels of sCT were determined by ELISA. The AUC value of MNs coated with a trehalose-containing formulation (250 ± 83 ng/mL min) was not significantly different as compared to subcutaneous injections (403 ± 253 ng/mL min), but approximately 13 times higher than nasal administration (18.4 ± 14.5 ng/mL min). T(max) (7.5 ± 5 min) values for MN mediated administration were 50% shorter than subcutaneous injections (15 min), possibly due to rapid sCT dissolution and absorption by dermal capillaries. These results suggest that with further optimization of coating formulations, microneedles may enable administration of sCT and other peptides without the need for hypodermic injections.

Protective effects of memantine and epicatechin on catechol-induced toxicity on Müller cells in vitro.

This study evaluates the toxic effects of catechol (a component from cigarette smoke) on Müller cells (MIO-M1) in vitro, and investigates the inhibitors memantine and epicatechin to determine if they can reverse the catechol toxic effects. MIO-M1 cells were exposed to varying concentrations of catechol with or without memantine or epicatechin. Cell viability (CV) was measured by a trypan blue dye-exclusion assay. Caspase-3/7 activity was measured by fluorochrome assay. The production of reactive oxygen/nitrogen species (ROS/RNS) was measured with 2',7'-dichlorodihydrofluorescein diacetate dye assay. Mitochondrial membrane potential (DeltaPsim) was measured using JC-1 assay. Intracellular ATP content was determined by the ATPLite kit. MIO-M1 cells showed significant decrease in cell viability, increased caspase-3/7 activity, elevated ROS/RNS levels, decreased DeltaPsim value, and decreased intracellular ATP content after exposure to catechol 150, 300, and 600 microM compared with control. Pre-treatment with memantine 10 microM or epicatechin 15 microM reversed loss of cell viability in catechol 150 microM-treated cultures (22.3%, p<0.01 and 17.8%, p<0.05), respectively. Similarly, pre-treatment with memantine 10 microM and epicatechin 15 microM prior to catechol resulted in decreased caspase-3/7 activities (77% and 64.2%, p<0.001), decreased ROS/RNS levels (82.3% and 79%, p<0.001), increased DeltaPsim value (76.4% and 72.2%, p<0.001), and increased ATP levels (46.6% and 40.4%, p<0.001) compared to 150 microM catechol-treated cultures. Catechol, a component of smoking, can diminish cell viability and mitochondrial function in MIO-M1 cells in vitro. However, memantine and epicatechin can partially reverse the cytotoxic effect of catechol. Their administration may reduce or prevent Müller cells degeneration in AMD or other retinal degenerative disorders.

Inhibition of apoptosis in human retinal pigment epithelial cells treated with benzo(e)pyrene, a toxic component of cigarette smoke.

PURPOSE: To study the inhibitory effects of some agents or drugs (inhibitors) on benzo(e)pyrene (B(e)P)-induced cell death and apoptosis on human retinal pigment epithelial (ARPE-19) cells in vitro. METHODS: ARPE-19 cells were pretreated with varying concentrations of different classes of inhibitors (calpain, benzyl isothiocyanate [BITC], simvastatin, epicatechin, genistein, resveratrol, and memantine) before B(e)P exposure. Cell viability (CV) was determined by a trypan blue dye-exclusion assay. Caspase-3/7 and caspase-9 activities were measured by fluorochrome assays. The production of reactive oxygen/nitrogen species (ROS/RNS) was measured with 2',7'-dicholorodihydrofluorescein diacetate dye assay. RESULTS: At 30-microM concentrations, the genistein, resveratrol, and memantine inhibitors were able to reverse significantly the loss of cell viability, the activation of caspase-3/7 and caspase-9, and the production of ROS/RNS in ARPE-19 cell cultures. Memantine was the most potent and genistein was the least effective inhibitor in blocking the B(e)P-induced effects. Calpain, BITC, simvastatin, and epicatechin did not reverse the loss of cell viability in B(e)P-treated ARPE-19 cells. As a matter of fact, at the concentrations studied (15, 30, 45 microM), the BITC plus B(e)P-treated cultures showed significantly lower cell viability than the B(e)P-treated culture alone, suggesting BITC-related toxicity. CONCLUSIONS: Genistein, resveratrol, and memantine can reverse the apoptosis and oxidant production generated by B(e)P, a toxic element of smoking. These inhibitors may be beneficial against retinal diseases associated with the loss of RPE cells.

Intraocular sustained-release delivery systems for triamcinolone acetonide.

Recently, the use of triamcinolone acetonide (TA) injection has increased dramatically in treatment for several ocular diseases. Among them, macular diseases such as macular edema due to diabetic retinopathy, venous occlusive diseases, ocular inflammation and age-related macular degeneration (AMD) are very common vision threatening disorders and are great challenges to treat. In these types of chronic retinal diseases, repeated intraocular injections of TA are often required which increases the likelihood of complications. In order to achieve sustained-release, maintain therapeutic levels of TA over longer times and reduce frequency of intravitreal injections, researchers are investigating different implantable devices or injectable systems. However, as of yet, there is no sustained-release product for TA available on the commercial market. This review discusses and compares different sustained-release devices or injectable systems that are currently being developed.

Oral delivery of mono-PEGylated sCT (Lys18) in rats: regional difference in stability and hypocalcemic effect.

In the in vitro experiment using a luminal, mucosal, and fecal fluid/extract from jejunum and colon of a rat, Lys18-residue modified mono-PEG(2k)-sCT (Lys18-PEG(2K)-sCT) exhibited a longer half-life than salmon calcitonin (sCT) in a colonic fluid and its extract. A physical adsorption study showed that Lys18-PEG(2K)-sCT had lower adsorption in the feces than sCT over an 8-hr period. An absorption study of the sCT and Lys18-PEG(2K)-sCT from the jejunum and colon using an in situ closed-loop technique in anesthetized rats showed a dose-dependent reduction in the plasma Ca2+ level but to a certain limit. Furthermore, the hypocalcemic response by intracolonic administration was significantly higher than the intrajejunal one, demonstrating that the colon had better absorption. In particular, Lys18-PEG(2K)-sCT (5 microg/rats) produced the most pronounced hypocalcemia after the intracolonic administration, which resulted in a sustained reduction in the serum calcium level over an 8-hr period, with a maximum reduction (% max(d)) of 38% after 4 hr. The overall reduction in the serum calcium levels, which was expressed as the net change in the AUC relative to the control over an 8-hr period, was 25.51 +/- 3.38 for Lys18-PEG(2K)-sCT. The relative pharmacological bioavailability of the intracolonically administered Lys18-PEG(2K)-sCT was 2.1-fold higher than sCT and the absolute pharmacological bioavailability was 73.59% of i.v.-injected sCT in an 8-hr period. Overall, this study highlights the feasibility of the oral delivery of Lys18-PEG(2K)-sCT in achieving a sustained calcium-lowering effect.