ABSTRACT
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.
ABSTRACT
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.