Neuroprotection and regeneration of adult lesioned retinal ganglion cells by the modulation of fibroblast growth factor-2 and receptor tyrosine phosphatase-sigma

Thèse numérisée par la Direction des bibliothèques de l'Université de Montréal

Evaluation of the vitreous microbial contamination rate in office-based three-port microincision vitrectomy surgery using Retrector technology

Background: To perform a microbiological contamination analysis of the vitreous during office-based micro-incision vitrectomy surgery (MIVS) assessing whether the bacteria detected correlated with patient's ocular conjunctival flora. Methods: This is a prospective, interventional, nonrandomized case series of patients undergoing office-based MIVS, anti-VEGF, and dexamethasone intravitreal injections (triple therapy) for the treatment of wet age-related macular degeneration (AMD) and diabetic macular edema (DME). All patients were operated at a small procedure room in an ambulatory clinic of the Department of Ophthalmology, University of Montreal, Quebec, Canada. Conjunctival samples were done before placing the sclerotomies. The MIVS was done with a 23-gauge retractable vitrector, a 27-gauge infusion line, and a 29-gauge chandelier. Undiluted and diluted vitreous were collected for aerobic, anaerobic and fungal cultures. Outcomes measured were bacterial species identification within samples collected from the conjunctiva and the vitreous. Results: Thirty-seven patients (37 eyes) were recruited and completed over 17 months of follow-up. Twenty-eight had wet AMD and nine had DME. There were 13 men and 24 women, with a mean age of 78 years. Eighteen patients (46%) had culture positive conjunctival flora. Twenty-six bacterial colonies were tabulated in total from the conjunctival swabs. All bacteria detected were gram-positive bacteria (100%), most commonly: Staphylococcus epidermitis in 11 (42%) and Corynebacterium sp. in 6 (23%). Only 1/18 patients had more than 3 species isolated, 6/18 patients had 2 species and 11/18 patients had 1 species identified on the conjunctival swab. Only 1 of the 37 undiluted midvitreous samples was culture positive, equating to a contamination rate of 2.7%. None of the diluted vitreous samples were culture positive. All cultures were negative for fungus. No serious postoperative complications occurred, including bacterial endophthalmitis, choroidal detachment, and retinal detachment. Conclusion: This preliminary study of office-based MIVS gives us insights on the ocular surface microbial profile and vitreous contamination rate of performing such procedures outside the OR-controlled environment. Our initial results seem to indicate that there is little risk of bacterial translocation and contamination from the conjunctiva into the vitreous. Therefore, if endophthalmitis occurs post-operatively, the source may likely arise after the procedure. Larger studies are needed to confirm our data

Anti-proliferative and anti-tumour effects of lymphocyte-derived microparticles are neither species- nor tumour-type specific

Background: Unregulated cell proliferation or growth is a prominent characteristic of cancer. We have previously demonstrated that LMPs (cell membrane microparticles derived from apoptotic human CEM T lymphoma cells stimulated with actinomycin D) strongly suppress the proliferation of not only human endothelial cells but also mouse Lewis lung carcinoma cells. Methods: LMPs were generated either from CEM T cells using different stimuli or from 3 different types of lymphocytes. The effects of LMPs on cancer cell proliferation were examined using cell lines from different species and tissues. The cell cycle kinetics was evaluated by FACS and the expression of cell cycle-related genes was determined using quantitative RT-PCR. The in vivo anti-tumor effect of LMPs was investigated using xenografts and allografts. Results: LMPs at doses far above physiological levels dramatically suppressed the proliferation of cancer cells in a non species-specific manner. LMPs selectively target high proliferating cells and their anti-proliferative effect is not dependent on parental cell origin or stimuli. The anti-proliferative effect of LMPs was due to induction of cell-cycle arrest in G0/G1, with associated increases in expression of the cyclin-dependent kinase inhibitors p15INK4b, p16INK4a, and p21Cip1. In vivo, LMPs significantly suppressed tumor growth in animal tumor models. Conclusion: These results highlight the potential role of LMPs in modulating the growth of high proliferating cells. Given that cell-based therapies are considered less toxic than pharmacologic approaches and have the potential to target multiple pathways in a synergistic manner, LMPs may serve as a veritable option for cancer treatment

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

Neuron-Derived Semaphorin 3A Is an Early Inducer of Vascular Permeability in Diabetic Retinopathy via Neuropilin-1

SummaryThe deterioration of the inner blood-retinal barrier and consequent macular edema is a cardinal manifestation of diabetic retinopathy (DR) and the clinical feature most closely associated with loss of sight. We provide evidence from both human and animal studies for the critical role of the classical neuronal guidance cue, semaphorin 3A, in instigating pathological vascular permeability in diabetic retinas via its cognate receptor neuropilin-1. We reveal that semaphorin 3A is induced in early hyperglycemic phases of diabetes within the neuronal retina and precipitates initial breakdown of endothelial barrier function. We demonstrate, by a series of orthogonal approaches, that neutralization of semaphorin 3A efficiently prevents diabetes-induced retinal vascular leakage in a stage of the disease when vascular endothelial growth factor neutralization is inefficient. These observations were corroborated in TgCre-Esr1/Nrp1flox/flox conditional knockout mice. Our findings identify a therapeutic target for macular edema and provide further evidence for neurovascular crosstalk in the pathogenesis of DR

MicroRNA signatures in vitreous humour and plasma of patients with exudative AMD

Age-related macular degeneration (AMD) is a leading cause of blindness worldwide affecting individuals over the age of 50. The neovascular form (NV AMD) is characterized by choroidal neovascularization (CNV) and responsible for the majority of central vision impairment. Using non-biased microRNA arrays and individual TaqMan qPCRs, we profiled miRNAs in the vitreous humour and plasma of patients with NV AMD. We identified a disease-associated increase in miR-146a and a decrease in miR-106b and miR-152 in the vitreous humour which was reproducible in plasma. Moreover, miR-146a/miR-106b ratios discriminated patients with NV AMD with an area under the Receiver Operating Characteristic curve (ROC AUC) of 0,977 in vitreous humour and 0,915 in plasma suggesting potential for a blood-based diagnostic. Furthermore, using the AMD Gene Consortium (AGC) we mapped a NV AMD-associated SNP (rs1063320) in a binding site for miR-152-3p in the HLA-G gene. The relationship between our detected miRNAs and NV AMD related genes was also investigated using gene sets derived from the Ingenuity Pathway Analysis (IPA). To our knowledge, our study is the first to correlate vitreal and plasma miRNA signatures with NV AMD, highlighting potential future worth as biomarkers and providing insight on NV AMD pathogenesis

Choroid Sprouting Assay: An Ex Vivo Model of Microvascular Angiogenesis

Angiogenesis of the microvasculature is central to the etiology of many diseases including proliferative retinopathy, age-related macular degeneration and cancer. A mouse model of microvascular angiogenesis would be very valuable and enable access to a wide range of genetically manipulated tissues that closely approximate small blood vessel growth in vivo. Vascular endothelial cells cultured in vitro are widely used, however, isolating pure vascular murine endothelial cells is technically challenging. A microvascular mouse explant model that is robust, quantitative and can be reproduced without difficulty would overcome these limitations. Here we characterized and optimized for reproducibility an organotypic microvascular angiogenesis mouse and rat model from the choroid, a microvascular bed in the posterior of eye. The choroidal tissues from C57BL/6J and 129S6/SvEvTac mice and Sprague Dawley rats were isolated and incubated in Matrigel. Vascular sprouting was comparable between choroid samples obtained from different animals of the same genetic background. The sprouting area, normalized to controls, was highly reproducible between independent experiments. We developed a semi-automated macro in ImageJ software to allow for more efficient quantification of sprouting area. Isolated choroid explants responded to manipulation of the external environment while maintaining the local interactions of endothelial cells with neighboring cells, including pericytes and macrophages as evidenced by immunohistochemistry and fluorescence-activated cell sorting (FACS) analysis. This reproducible ex vivo angiogenesis assay can be used to evaluate angiogenic potential of pharmacologic compounds on microvessels and can take advantage of genetically manipulated mouse tissue for microvascular disease research

Retinal Expression of Wnt-Pathway Mediated Genes in Low-Density Lipoprotein Receptor-Related Protein 5 (Lrp5) Knockout Mice

Mutations in low-density lipoprotein receptor-related protein 5 (Lrp5) impair retinal angiogenesis in patients with familial exudative vitreoretinopathy (FEVR), a rare type of blinding vascular eye disease. The defective retinal vasculature phenotype in human FEVR patients is recapitulated in Lrp5 knockout $(Lrp5^{-/-})$ mouse with delayed and incomplete development of retinal vessels. In this study we examined gene expression changes in the developing $Lrp5^{−/−}$ mouse retina to gain insight into the molecular mechanisms that underlie the pathology of FEVR in humans. Gene expression levels were assessed with an Illumina microarray on total RNA from $Lrp5^{−/−}$ and WT retinas isolated on postnatal day (P) 8. Regulated genes were confirmed using RT-qPCR analysis. Consistent with a role in vascular development, we identified expression changes in genes involved in cell-cell adhesion, blood vessel morphogenesis and membrane transport in $Lrp5^{−/−}$ retina compared to WT retina. In particular, tight junction protein claudin5 and amino acid transporter slc38a5 are both highly down-regulated in $Lrp5^{−/−}$ retina. Similarly, several Wnt ligands including Wnt7b show decreased expression levels. Plasmalemma vesicle associated protein (plvap), an endothelial permeability marker, in contrast, is up-regulated consistent with increased permeability in $Lrp5^{−/−}$ retinas. Together these data suggest that Lrp5 regulates multiple groups of genes that influence retinal angiogenesis and may contribute to the pathogenesis of FEVR