Real-Life Efficacy, Safety, and Use of Dexamethasone Intravitreal Implant in Posterior Segment Inflammation Due to Non-infectious Uveitis (LOUVRE 2 Study).

INTRODUCTION: To evaluate real-life efficacy, safety, and treatment patterns with the dexamethasone intravitreal implant (DEX) in posterior segment inflammation due to non-infectious uveitis (treatment-naïve or not) in French clinics. METHODS: In this prospective, multicenter, observational, non-comparative, post-reimbursement study, consecutive patients with posterior segment inflammation due to non-infectious uveitis were enrolled and evaluated at baseline (day 0). Those who received DEX on day 0 were re-evaluated at months 2, 6, and 18. Retreatment with DEX and/or alternative therapies was allowed during follow-up. PRIMARY OUTCOME: patients (%) with at least a 15-letter gain in best corrected visual acuity (BCVA) at 2 months. Secondary outcomes included patients (%) with at least 15-letter BCVA gains at 6 and 18 months; mean BCVA change from baseline at 2, 6, and 18 months; and patients (%) retreated, mean central retinal thickness (CRT), and adverse events (AEs) at all post-baseline visits. RESULTS: Ninety-seven of 245 enrolled patients with posterior segment inflammation due to non-infectious uveitis (80% previously treated) and disease duration of 5 years (average) received DEX on day 0 and were included in efficacy analyses. At month 2 (n = 91), 20.5% of patients (95% CI 12.0-28.9) gained at least 15 letters from a baseline mean of 60.9 letters; the mean gain was 6.2 letters (95% CI 3.5-8.9). At month 6, 50.0% (n = 38/76) of patients did not receive alternative treatment or DEX retreatment, mostly because inflammation had sufficiently subsided (n = 27/38, 71.1%). Although early study termination prevented efficacy analysis at 18 months (n = 12), CRT reductions persisted throughout follow-up. From baseline to month 18, 21/245 (8.6%) patients had DEX-related AEs; 17/245 (6.9%) had ocular hypertension (most common AE). CONCLUSION: LOUVRE 2 confirms DEX efficacy on visual acuity and CRT in predominantly DEX-pretreated patients with relatively old/stabilized uveitis. DEX tolerability was consistent with known/published data, confirming treatment benefits in posterior segment inflammation due to non-infectious uveitis. GOV IDENTIFIER: NCT02951975.

ATM localization and gene expression in the adult mouse eye.

PURPOSE: High levels of metabolism and oxygen consumption in most adult murine ocular compartments, combined with exposure to light and ultraviolet (UV) radiation, are major sources of oxidative stress, causing DNA damage in ocular cells. Of all mammalian body cells, photoreceptor cells consume the largest amount of oxygen and generate the highest levels of oxidative damage. The accumulation of such damage throughout life is a major factor of aging tissues. Several multiprotein complexes have recently been identified as the major sensors and mediators involved in the maintenance of DNA integrity. The activity of these complexes initially seemed to be restricted to dividing cells, given their ultimate role in major cell cycle checkpoints. However, it was later established that they are also active in post-mitotic cells. Recent findings demonstrate that the DNA damage response (DDR) is essential for the development, maintenance, and normal functioning of the adult central nervous system. One major molecular factor in the DDR is the protein, ataxia telangiectasia mutated (ATM). It is required for the rapid induction of cellular responses to DNA double-strand breaks. These cytotoxic DNA lesions may be caused by oxidative damage. To understand how ATM prevents oxidative stress and participates in the maintenance of genomic integrity and cell viability of the adult retina, we determined the ATM expression patterns and studied its localization in the adult mouse eye. METHODS: Atm gene expression was analyzed by RT-PCR experiments and its localization by in situ hybridization on adult mouse ocular and cerebellar tissue sections. ATM protein expression was determined by western blot analysis of proteins homogenates extracted from several mouse tissues and its localization by immunohistochemistry experiments performed on adult mouse ocular and cerebellar tissue sections. In addition, subcellular localization was realized by confocal microscopy imaging of ocular tissue sections, with a special focus on retinal cells. RESULTS: Using RT-PCR, we detected a band of the expected size, with its sequence matching the amplified Atm cDNA sequence. Atm mRNA was detected in most cell bodies of the adult mouse eye by in situ hybridization of ocular tissue sections with specific digoxigenin-labeled PCR-amplified cDNA probes. Western blotting with different specific antibodies revealed bands corresponding to the expected sizes of ATM and its active forms (ATMp). These bands were not observed in the analysis of protein homogenates from Atm-deficient mouse tissues. ATM immunoreactivity was detected in the nucleus of all adult mice retinal cells and in most non-neuronal ocular cell types. The active phosphorylated form of ATM was also present in the retina as well as in non-neuronal cells of the adult mouse eye. However, its subcellular localization differed as a function of the cell type examined. A major finding of this study was that ATMp immunostaining in photoreceptor cells was exclusively in the cytoplasm, whereas ATM immunostaining was only in the nucleus of these cells. Furthermore, the specific and distinct ATM and ATMp immunolabeling patterns in photoreceptor cells were identical to those observed in the adult mouse cerebellar granule cells. CONCLUSIONS: We report the expression profile of Atm gene and protein in the adult mouse eye. In particular, we observed a difference between the localization patterns of the active and inactive forms of ATM in photoreceptor cells. These localization patterns suggest that ATM and its phosphorylated activated form may be involved in both the protection of cells from oxidative damage and the maintenance of ocular cell structure and function. The protection mechanisms mediated by the two forms of ATM appear to be particularly important in maintaining photoreceptor integrity.

Expression of 8-oxoguanine DNA glycosylase (Ogg1) in mouse retina.

PURPOSE: The retina is highly exposed to oxidative stress due to the high level of oxygen consumption in this tissue and its exposure to light. The main DNA base lesion generated by oxygen free radicals is 8-oxoguanine (8-oxoG). However, its presence in retinal cells and the mechanisms underlying its repair remain undetermined. METHODS: 8-oxoguanine DNA glycosylase (Ogg1) gene expression and messenger localization in adult mouse ocular tissues was analyzed by RT-PCR and in situ hybridization. Using immunohistochemistry, we determined the localization of Ogg1 protein and three base excision repair (BER) enzymes: apurinic/apyrimidic endonuclease (APE1), DNA polymerase beta, and X-ray repair cross-complementation group 1 (XRCC1). Ogg1 and AP-lyase activities in the neuroretina were obtained using double-stranded oligonucleotides harboring either an 8-oxoG residue or a tetrahydrofuran. RESULTS: We report here that 8-oxoG is abundant in the retina. Ogg1, the enzyme responsible for the recognition and excision of the oxidized base, is present in its active form and found mainly in ganglion cells and photoreceptor inner segments. We show that APE1 and DNA polymerase beta, two BER proteins involved in 8-oxoG repair, are also present in these cells. The cellular distribution of these proteins was similar to that of Ogg1. XRRC1 is present in both inner nuclear and ganglion cells layers; however, this protein is absent from photoreceptor inner segments. CONCLUSIONS: This is the first study to demonstrate the presence of a functional 8-oxoG BER pathway in retinal neurons. The study of three BER proteins involved in 8-oxoG elimination demonstrates that XRCC1 localization differs from those of Ogg1, APE1, and DNA polymerase beta. This result suggests that the elimination of 8-oxoG is coordinated through two pathways, which differ slightly according to the cellular localization of the abnormally oxidized guanine.

Expression of SR-BI receptor and StAR protein in rat ocular tissues.

The class-B type-I scavenger receptor (SR-BI) plays a key role in cholesterol homeostasis; it mediates the selective uptake of lipoprotein cholesterol to steroidogenic tissues. We show by RT-PCR, western blot, in situ hybridization and immunohistochemistry analysis that SR-BI is highly expressed in different neuro-retinal and non-neuronal cells types on rat eye. Immunohistochemistry of the steroidogenic acute regulatory protein (StAR) involved in neurosteroid production showed the same expression pattern than SR-BI in rat eye. Our results may suggest a key role of these genes in the ocular cholesterol metabolism for membranes biosynthesis and neurosteroidogenesis.

A novel antiangiogenic and vascular normalization therapy targeted against human CD160 receptor.

Angiogenesis plays an essential role in several diseases of the eye and in the growth of solid tumors, but existing antiangiogenic therapies have limited benefits in several cases. We report the antiangiogenic effects of a monoclonal antibody, CL1-R2, in several animal models of neovascularization. CL1-R2 recognizes human CD160, a membrane receptor which is conserved in various mammal species. We show that CD160 is expressed on the endothelial cells of newly formed blood vessels in human colon carcinoma and mouse B16 melanoma but not in vessels of healthy tissues. CL1-R2 reduced fibroblast growth factor 2-induced neovascularization in the rabbit cornea, in a mouse model of oxygen-induced retinopathy, and in a mouse Matrigel plug assay. Treatment of B16 melanoma-bearing mice with CL1-R2 combined with cyclophosphamide chemotherapy caused regression of the tumor vasculature and normalization of the remaining vessels as shown by Doppler ultrasonography, intravital microscopy, and histology. These studies validate CD160 as a potential new target in cases of human pathological ocular and tumor neoangiogenesis that do not respond or become resistant to existing antiangiogenic drugs.

Sirt1 involvement in rd10 mouse retinal degeneration.

PURPOSE: Sirtuin1 (Sirt1) is an NAD(+)-dependent deacetylase involved in development, cell survival, stress resistance, energy metabolism, and aging. It is expressed in the mammalian central nervous system (CNS) and is activated during processes associated with neuroprotection. The retinal degeneration 10 (rd10) mouse model of retinitis pigmentosa (RP) was used to investigate the possible role of Sirt1 in this type of retinal degeneration. METHODS: Eyes from control and rd10 mice were used. Sirt1 mRNA was detected by in situ hybridization, and its abundance was estimated by semiquantitative RT-PCR. The presence of Sirt1 protein was investigated by immunohistofluorescence and Western blot analysis. The apoptosis of photoreceptor cells was analyzed by terminal dUTP transferase nick-end labeling (TUNEL). Immunolabeling for Sirt1, apoptosis-inducing factor (Aif), and caspase-12 (Casp-12) was performed on retinal tissue sections. RESULTS: Sirt1 mRNA and immunoreactivity were observed in normal adult mouse eyes. In the control retina, Sirt1 was immunolocalized mostly to the nucleus. In rd10 mice with retinal degeneration, changes in Sirt1 immunolabeling were observed only in the retinal outer nuclear layer (ONL). The pathologic pattern of Sirt1 immunoreactivity correlated with the start of retinal degeneration in rd10 mice. CONCLUSIONS: The results suggest a link between Sirt1 production and retinal degeneration in rd10 mice. The anti-apoptotic, neuroprotective role of Sirt1 in the mouse retina is based on the involvement of Sirt1 in double DNA strand-break repair mechanisms and in maintaining energy homeostasis in photoreceptor cells. The results suggest that the neuroprotective properties of Sirt1 may gradually weaken in rd10 mouse photoreceptor cells.

Morphologic and electroretinographic phenotype of SR-BI knockout mice after a long-term atherogenic diet.

PURPOSE: To evaluate functional and ultrastructural changes in the retina of scavenger receptor B1 (SR-BI) knockout (KO) mice consuming a high fat cholate (HFC) diet. METHODS: Three-month-old male KO and wild-type (WT) mice were fed an HFC diet for 30 weeks. After diet supplementation, plasma cholesterol levels and electroretinograms were analyzed. Neutral lipids were detected with oil red O, and immunohistochemistry was performed on cryostat ocular tissue sections. The retina, Bruch's membrane (BM), retinal pigment epithelium (RPE), and choriocapillaris (CC) were analyzed by transmission electron microscopy. RESULTS: Using the WT for reference, ultrastructural changes were recorded in HFC-fed SR-BI KO mice, including lipid inclusions, a patchy disorganization of the photoreceptor outer segment (POS) and the outer nuclear layer (ONL), and BM thickening with sparse sub-RPE deposits. Within the CC, there was abnormal disorganization of collagen fibers localized in ectopic sites with sparse and large vacuolization associated with infiltration of macrophages in the subretinal space, reflecting local inflammation. These lesions were associated with electroretinographic abnormalities, particularly increasing implicit time in a- and b-wave scotopic responses. Abnormal vascular endothelial growth factor (VEGF) staining was detected in the outer nuclear layer. CONCLUSIONS: HFC-fed SR-BI KO mice thus presented sub-RPE lipid-rich deposits and functional and morphologic alterations similar to some features observed in dry AMD. The findings lend further support to the hypothesis that atherosclerosis causes retinal and subretinal damage that increases susceptibility to some forms of AMD.