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

[摘要] Purpose: This study investigates theeffects of triamcinolone acetonide (TA) on retinal endothelial cells invitro and explores the potential vascular toxic effect of TA injectedinto the vitreous cavity of rats in vivo. Methods: Subconfluent endothelial cellswere treated with either 0.1 mg/ml or 1 mg/ml TA in 1% ethanol. Controlcells were either untreated or exposed to 1% ethanol. Cell viabilitywas evaluated at 24 h, 72 h, and five days using the tetrazolium3-(4,5-dimethylthiazol-2-yl)-2,5 phenyltetrazolium bromide test (MTT)and lactate dehydrogenase (LDH) assays. Cell proliferation wasevaluated by 5-bromo-2-deoxyuridine (BrdU) test. Apoptosis wasevaluated by terminal deoxynucleotidyl transferase dUTP nick endlabeling assay (TUNEL assay), annexin-binding, and caspase 3activation. Caspase–independent cell deaths were investigated byimmunohistochemistry using antibodies against apoptosis inducing factor(AIF), cytochrome C, microtubule-associated protein (MAP)-light chain 3(MAP-LC3), and Leukocyte Elastase Inhibitor/Leukocyte ElastaseInhibitor-derived DNase II (LEI/L-DNase II). In vivo, semithin andultrathin structure analysis and vascular casts were performed toexamine 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) andvascular endothelial growth factor (VEGF) mRNAs upregulationwith or without TA. Results: The inhibitory effect of TA oncell proliferation could not explain the significant reduction in cellviability. Indeed, TA induced a time-dependent reduction of bovineretinal endothelial cells viability. Annexin-binding positive cellswere observed. Cytochrome C was not released from mitochondria. L-DNaseII was found translocated to the nucleus, meaning that LEI was changedinto L-DNase II. AIF was found nuclearized in some cells. LC3 labelingshowed the absence of autophagic vesicles. No autophagy or caspasedependent apoptosis was identified. At 1 mg/ml TA induced necrosiswhile exposure to lower concentrations for 3 to 5 days induced caspaseindependent apoptosis involving AIF and LEI/L-DNase II. In vivo,semithin and ultrathin structure analysis and vascular casts revealedthat TA mostly affected the choroidal vasculature with a reduction ofchoroidal thickness and increased the avascular areas of thechoriocapillaries. Experiments performed on primary RPE cells showedthat TA downregulates the basal expression of COX-2 and VEGFand inhibits the outer segments (OS)-dependent COX-2 induction but notthe OS-dependent VEGF induction. Conclusions: This study demonstrates forthe first time that glucocorticoids exert direct toxic effect onendothelial cells through caspase-independent cell death mechanisms.The choroidal changes observed after TA intravitreous injection mayhave important implications regarding the safety profile of TA use inhuman eyes.

[学科分类] 生物化学/生物物理