Effects of triamcinolone acetonide on vessels of the posterior segment of the eye

Abstract

Purpose: This study investigates the effects of triamcinolone acetonide (TA) on retinal endothelial cells in vitro and explores the potential vascular toxic effect of TA injected into the vitreous cavity of rats in vivo.

Methods: Subconfluent endothelial cells were treated with either 0.1 mg/ml or 1 mg/ml TA in 1% ethanol. Control cells were either untreated or exposed to 1% ethanol. Cell viability was evaluated at 24 h, 72 h, and five days using the tetrazolium 3-(4,5-dimethylthiazol-2-yl)-2,5 phenyltetrazolium bromide test (MTT) and lactate dehydrogenase (LDH) assays. Cell proliferation was evaluated by 5-bromo-2-deoxyuridine (BrdU) test. Apoptosis was evaluated by terminal deoxynucleotidyl transferase dUTP nick end labeling assay (TUNEL assay), annexin-binding, and caspase 3 activation. Caspase-independent cell deaths were investigated by immunohistochemistry using antibodies against apoptosis inducing factor (AIF), cytochrome C, microtubule-associated protein (MAP)-light chain 3 (MAP-LC3), and Leukocyte Elastase Inhibitor/Leukocyte Elastase Inhibitor-derived DNase II (LEI/L-DNase II). In vivo, semithin and ultrathin structure analysis and vascular casts were performed to examine TA-induced changes of the choroidal vasculature. In addition, outer segments phagocytosis assay on primary retinal pigment epithelium (RPE) cells was performed to assess cyclooxygenase (COX-2) and vascular endothelial growth factor (VEGF) mRNAs upregulation with or without TA.

Results: The inhibitory effect of TA on cell proliferation could not explain the significant reduction in cell viability. Indeed, TA induced a time-dependent reduction of bovine retinal endothelial cells viability. Annexin-binding positive cells were observed. Cytochrome C was not released from mitochondria. L-DNase II was found translocated to the nucleus, meaning that LEI was changed into L-DNase II. AIF was found nuclearized in some cells. LC3 labeling showed the absence of autophagic vesicles. No autophagy or caspase dependent apoptosis was identified. At 1 mg/ml TA induced necrosis while exposure to lower concentrations for 3 to 5 days induced caspase independent apoptosis involving AIF and LEI/L-DNase II. In vivo, semithin and ultrathin structure analysis and vascular casts revealed that TA mostly affected the choroidal vasculature with a reduction of choroidal thickness and increased the avascular areas of the choriocapillaries. Experiments performed on primary RPE cells showed that TA downregulates the basal expression of COX-2 and VEGF and inhibits the outer segments (OS)-dependent COX-2 induction but not the OS-dependent VEGF induction.

Conclusions: This study demonstrates for the first time that glucocorticoids exert direct toxic effect on endothelial cells through caspase-independent cell death mechanisms. The choroidal changes observed after TA intravitreous injection may have important implications regarding the safety profile of TA use in human eyes.

Figure 1

Effect of triamcinolone acetonide on BRECs viability and proliferation. Cell viability was evaluated using an MTT assay. Subconfluent (A) or confluent BRECs (B) were exposed to 0.1 or 1 mg/ml TA in 1% ethanol for 24 h (dark-gray columns), 72 h (red columns), or five days (white columns). Control cells were either exposed to 1% ethanol (C-E) or were left untreated (C). C: Cell proliferation was evaluated using BrdU labeling detection after 24 h of treatment with 0.1 mg/ml or 0.5 µg/ml TA. Control cells were either left untreated (C) or were treated with 1% ethanol (C-E). Results are expressed as mean±standard error; with *p<0.05 for all columns versus control. Four animals were used in each experiment. DAPI staining of untreated control cells are shown in (D), 0.1 mg/ml TA-treated cells in (E) and 1 mg/ml TA-treated cells in (F), all of them after five days of treatment. Scale bar represents 50 µm.

Figure 2

Absence of TUNEL-positive apoptosis in BRECs treated with triamcinolone acetonide. Control cells were treated with the TA vehicle,1% ethanol, (A-C). No TUNEL-positive cells could be observed after exposure to 0.1 mg/ml TA for 72 h (D-F) or five days (D5, G-I). As a positive control for the TUNEL technique, BRECs were treated with 1 μM staurosporine (J-L). Nuclei were stained with DAPI (A, D, G, J). Scale bar represents 60 µm.

Figure 3

Annexin V binding of TA treated BRECs. Annexin-V binding was performed in control cells, treated with 1% ethanol (the TA vehicle; A-C) or with 0.1% TA for 72 h (D-F) or five days (G-I). Condensed nuclei are seen in TA treated cells (D and G, squares) as well as Annexin-V positive cells (E and H, squares). Arrows indicates Annexin-V positive cells. Scale bar represents 50 µm.

Figure 4

Caspase 3 western-blot analysis. As a positive control HL-60 cells were treated with etoposide (Eto). The cleaved, active form of caspase 3 is seen. Expression of pro-caspase 3 is seen en BRECs in all conditions. No expression of activated caspase 3 was detected in any of the treated BRECs, demonstrating that triamcinolone acetonide (TA) did not induce caspase 3 activation.

Figure 5

Cytochrome C release in TA treated BRECs. Absence of cytochrome C release in triamcinolone acetonide (TA)-treated BRECs. Control cells are shown in A to C. TA treated cells for 72 h are shown in D to F and for 5 days in G to I. No cytochrome C release is seen in these cells. Compare to cells treated with staurosporin (Stp) used as positive control (J-L, arrow in K and L). Nuclei were stained with DAPI (A, D, G, J). Scale bar represents 50 µm.

Figure 6

L-DNase II activation in TA treated BRECs. LEI/L-DNase II immuno-labeling was performed in control cells treated with the TA vehicle (1% ethanol). A-C: A cytoplasmic labeling is seen. In cells treated with 0.1 mg/ml TA for 72h (D-F) or 5 days (G-I), cells with condensed nuclei (arrows) present a nuclear staining for LEI/L-DNase II, indicating L-DNase II activation. Nuclei were stained with DAPI (A, D, G). Scale bar represents 50 µm.

Figure 7

Apoptosis Inducing Factor localization in triamcinolone acetonide treated BRECs. Apoptosis inducing factor (AIF) immuno-labeling was performed in control cells treated with the TA vehicle (1% ethanol; A-C). A cytoplasmic labeling is seen. This is also the case in cells treated with 0.1 mg/ml TA for 72h (D-F). In cells treated with TA for 5 days (G-I), cells with condensed nuclei (arrows in H and I) present a nuclear staining for AIF. Nuclei were stained with DAPI (A, D, G). Scale bar represents 50 µm.

Figure 8

Evaluation of autophagy by microtubules-associated protein light chain 3 staining. No intracytoplasmic vacuoles were stained with anti-microtubules-associated protein light chain 3 (MAP-LC3) in control BRECs (A-C). This is also the case for BRECs exposed to 0.1 mg/ml TA for five days (D-F). In the amino acid (AA) deprived medium (positive control), BRECs show multiple MAP-LC3 positive cytoplasmic vacuoles (G-I, arrows in I). Nuclei were stained with DAPI (A, D, G). Scale bar represents 50 µm.

Figure 9

Semithin analysis of the retina-choroid. At 30 days after triamcinolone acetonide (TA) injection, the diameter of retinal vessels was reduced as compared to control PBS injected eyes (B, A, respectively, arrows). The thickness of the choroids was significantly reduced in TA-treated (D) as compared to the control eye (C and E). Vacuoles were observed in retinal pigment epithelial (RPE) cells and outer segment in the TA-treated eyes (D, stars). Results are expressed as mean±standard error, with ***p<0.001. Fours animals were used in this study. Abbreviations: outer nuclear layer (ONL), inner segment (IS), outer segment (OS). Scale bar represents 20 µm.

Figure 10

Choroidal vascular casts. Frontal view of choroidal casts showed that 30 days after injection of triamcinolone acetonide (TA), the avascular areas were increased (B, red double-headed arrow) as compared to the control PBS-treated eyes (A). Cross-sectional cuts of pericentral choroidal corrosion cast (C, D) showed a reduced thickness of the choriocapillaries in TA-treated eyes. Scale bar represents 10 µm. E: Quantification of the avascular areas in the choriocapillaries confirmed a significant increase in TA-treated eyes, and (F) mean vessel diameter was significantly reduced. Results are expressed as mean±standard error with *p<0.05 when consideringTA versus PBS. Six animals were used in this study.

Figure 11

COX-2 and VEGF mRNA expression in RPE cells treated with TA. COX-2 (A) and VEGF (B) mRNA expression (real time quantitative PCR) in untreated control primary rat RPE cells (C), or in RPE cells exposed to triamcinolone acetonide (TA), outer segments (OS) or both (OS-TA). Results are expressed as mean±standard error with *p<0.05, ***p<0.001, when considering all columns versus control in (B) and in (A) *p<0.05 OS-TA versus OS. Four animals were used in each study.