Effects of triamcinolone acetonide on vessels of the posterior segment of the eye.
OBJECTIVE
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.
Valamanesh
Fatemeh
F
1INSERM, UMRS 872 Physiopathology of Ocular Diseases, Therapeutic innovations Centre de Recherche des Cordeliers 15 rue de L'Ecole de Médecine 75006 Paris France.
Berdugo
Marianne
M
Sennlaub
Florian
F
Savoldelli
Michèle
M
Goumeaux
Cyndie
C
Houssier
Marianne
M
Jeanny
Jean-Claude
JC
Torriglia
Alicia
A
Behar-Cohen
Francine
F
D000818:Animals; D017209:Apoptosis / Q000187:drug effects; D001343:Autophagy / Q000187:drug effects; D020169:Caspases / Q000378:metabolism; D002417:Cattle; D049109:Cell Proliferation / Q000187:drug effects; D002470:Cell Survival / Q000187:drug effects; D002829:Choroid / Q000098:blood supply / Q000187:drug effects; D016319:Corrosion Casting; D051546:Cyclooxygenase 2 / Q000378:metabolism; D042783:Endothelial Cells / Q000166:cytology / Q000187:drug effects / Q000201:enzymology; D004847:Epithelial Cells / Q000166:cytology / Q000187:drug effects / Q000201:enzymology; D005123:Eye / Q000033:anatomy & histology / Q000098:blood supply* / Q000187:drug effects* / Q000648:ultrastructure; D010587:Phagocytosis / Q000187:drug effects; D051381:Rats; D012160:Retina / Q000166:cytology; D013997:Time Factors; D014222:Triamcinolone Acetonide / Q000633:toxicity*; D042461:Vascular Endothelial Growth Factor A / Q000378:metabolism
D042461:Vascular Endothelial Growth Factor A; D051546:Cyclooxygenase 2; C489691:Ptgs2 protein, rat; D020169:Caspases; D014222:Triamcinolone Acetonide