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

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

CD36 plays an important role in the clearance of oxLDL and associated age-dependent sub-retinal deposits

Age-related macular degeneration (AMD) represents the major cause of vision loss in industrialized nations. Laminar deposits in Bruch's membrane (BM) are among the first prominent histopathologic features, along with drusen formation, and have been found to contain oxidized lipids. Increases in concentrations of oxidized LDL (oxLDL) in plasma are observed with age and high fat high (HFHC) cholesterol diet. CD36 is the principal receptor implicated in uptake of oxLDL, and is expressed in the retinal pigment epithelium (RPE). We determined if CD36 participates in oxLDL uptake in RPE and correspondingly in clearance of sub-retinal deposits. Uptake of oxLDL by RPE in vitro and in vivo was CD36-dependent. CD36 deficiency in mice resulted in age-associated accumulation of oxLDL and sub-retinal BM thickening, despite fed a regular diet. Conversely, treatment of HFHC-fed ApoE null mice with a CD36 agonist, EP80317 (300 μg/kg/day), markedly diminished thickening of BM, and partially preserved (in part) photoreceptor function. In conclusion, our data uncover a new role for CD36 in the clearance of oxidized lipids from BM and in the prevention of age-dependent sub-retinal laminar deposits

Delta-like 4 inhibits choroidal neovascularization despite opposing effects on vascular endothelium and macrophages.: DLL4's opposing effects in choroidal neovascularization

International audienceInflammatory neovascularization, such as choroidal neovascularization (CNV), occur in the presence of Notch expressing macrophages. DLL4s anti-angiogenic effect on endothelial cells (EC) has been widely recognized, but its influence on Notch signaling on macrophages and its overall effect in inflammatory neovascularization is not well understood. We identified macrophages and ECs as the main Notch 1 and Notch 4 expressing cells in CNV. A soluble fraction spanning Ser28-Pro525 of the murine extracellular DLL4 domain (sDLL4/28-525) activated the Notch pathway, as it induces Notch target genes in macrophages and ECs and inhibited EC proliferation and vascular sprouting in aortic rings. In contrast, sDLL4/28-525 increased pro-angiogenic VEGF, and IL-1β expression in macrophages responsible for increased vascular sprouting observed in aortic rings incubated in conditioned media from sDLL4/28-525 stimulated macrophages. In vivo, Dll4(+/-) mice developed significantly more CNV and sDLL4/28-525 injections inhibited CNV in Dll4(+/-) CD1 mice. Similarly, sDLL4/28-525 inhibited CNV in C57Bl6 and its effect was reversed by a γ-secretase inhibitor that blocks Notch signaling. The inhibition occurred despite increased VEGF, IL-1β expression in infiltrating inflammatory macrophages in sDLL4/28-525 treated mice and might be due to direct inhibition of EC proliferation in laser-induced CNV as demonstrated by EdU labelling in vivo. In conclusion, Notch activation on macrophages and ECs leads to opposing effects in inflammatory neovascularization in situations such as CNV

CD36 Deficiency Leads to Choroidal Involution via COX2 Down-Regulation in Rodents

Florian Sennelaub and colleagues show that CD36 deficiency leads to choroidal involution, a key feature of "dry" age-related macular degeneration, via COX-2 down-regulation in the retinal pigment epithelium

Implication du récepteur CD36 dans la dégénérescence maculaire liée à l'âge (DMLA)

Mes travaux de thèse avaient pour objectif de déterminer le rôle du récepteur CD36 dans l homéostasie rétinienne et son implication potentielle dans la genèse de la DMLA. J ai utilisé pour cela deux modèles animaux présentant une déficience pour le récepteur CD36 : les souris CD36-/- d une part et les rats SHR (spontaneous hypertensive rats) d autre part. Nous avons montré la production de facteurs proangiogéniques COX-2 et VEGF de façon CD36-dépendante par les cellules de l EPR en conditions physiologiques. La déficience en CD36 menait à une dégénérescence rétinienne associée à une involution choroïdienne COX-2 dépendante dans nos modèles murins vieillissants. Nous avons également observé une augmentation de la concentration des oxLDL dans le sang en fonction de l âge, et montré l existence d une résorption des oxLDL par les cellules de l EPR de façon CD36-dépendante. Nos études histologiques montraient une accumulationd oxLDL et un épaississement de la MB chez les souris CD36-/- vieillissantes. Finalement, dans un contexte plus inflammatoire nous avons montré que le CD36 contribue au développement de NVC et à la dégénérescence rétinienne induite par illumination. Nos expériences montraient une production CD36-dépendante de VEGF par les macrophages péritonéaux et les cellules de l EPR qui pourrait expliquer les différences observées. Ces travaux ont mis en évidence un rôle clé du récepteur CD36 dans la régulation de facteurs angiogénique, comme la COX-2 et le VEGF, dans l EPR. Cette régulation est nécessaire à l homéostasie chorio-rétinienne mais peut avoir un effet négatif dans la néovascularisation choroïdienne et la dégénérescence dans un contexte inflammatoire. Le CD36 pourrait être utilisé comme cible pharmacologique pour maintenir la vascularisation choroïdienne et éliminer des lipides de la membrane de Bruch (agonistes) et inhiber la néovascularisation et dégénérescence dans un contexte inflammatoire (antagonistes)The aim of this work was to evaluate the role of CD36 receptor in retinal homeostasis and its potential involvement in the pathogenesis of AMD disease. I used two different animal models with CD36 deficiency: CD36-/- mice and SHR rats. We demonstrated a CD36-dependant production of angiogenic factor such as COX-2 and VEGF by RPE cells under physiological condition. CD36 deficiency led to retinal degeneration and choroidal involution in aged animals. We also observed an increase in oxLDL concentration in the blood with age and showed a CD36-dependant uptake of oxLDL by RPE cells. Histological studies highlighted oxLDL accumulation and thickening of Bruchs membrane in aged CD36-/- mice. In an inflammatory context, CD36 activation increased CNV and retinal degeneration induced by illumination. We showed a CD36-dependant production of VEGF in RPE cells and macrophages, which might explain the differences observed. These results have thus demonstrated a crucial role of CD36 receptor in the regulation of proangiogenic factors in the RPE. This regulation is necessary for chorio-retinal homeostasis but can have negative effect in choroidal neovascularization in an inflammatory context. In summary, we identified CD36 as a drug target to maintain the choroidal vasculature and evacuate lipids from the Bruchs membrane (agonists) and to inhibit choroidal neovascularization and retinal degeneration in an inflammatory context (antagonists).PARIS-BIUP (751062107) / SudocSudocFranceF

Th1- and Th2-related chemokine and chemokine receptor expression on the ocular surface in endotoxin-induced uveitis.

International audienceTo determine whether the ocular surface inflammation in uveitis mimics or counteracts intraocular inflammatory pathways by directly comparing T-helper (Th) lymphocytes Th1 and Th2 markers in conjunctival and ciliary body expression in endotoxin-induced uveitis (EIU). This study used the following inflammatory markers: chemokine receptor, CC chemokine receptor 4 (CCR4), and its ligand, macrophage-derived chemokine (MDC), to evaluate Th2 participation; chemokine receptor, CCR5, to evaluate the Th1 system; and its ligand, regulated on activation normal T cell expressed and secreted (RANTES), to evaluate both Th1 and Th2 systems