Involvement of plasmalogens in post-natal retinal vascular development

Objective: Proper development of retinal blood vessels is essential to ensure sufficient oxygen and nutrient supplies to the retina. It was shown that polyunsaturated fatty acids (PUFAs) could modulate factors involved in tissue vascularization. A congenital deficiency in ether-phospholipids, also termed "plasmalogens'', was shown to lead to abnormal ocular vascularization. Because plasmalogens are considered to be reservoirs of PUFAs, we wished to improve our understanding of the mechanisms by which plasmalogens regulate retinal vascular development and whether the release of PUFAs by calcium-independent phospholipase A2 (iPLA2) could be involved. [br/]Methods and Results: By characterizing the cellular and molecular steps of retinal vascular development in a mouse model of plasmalogen deficiency, we demonstrated that plasmalogens modulate angiogenic processes during the early phases of retinal vascularization. They influence glial activity and primary astrocyte template formation, endothelial cell proliferation and retinal vessel outgrowth, and impact the expression of the genes involved in angiogenesis in the retina. These early defects led to a disorganized and dysfunctional retinal vascular network at adult age. By comparing these data to those obtained on a mouse model of retinal iPLA2 inhibition, we suggest that these processes may be mediated by PUFAs released from plasmalogens and further signalling through the angiopoietin/tie pathways. [br/]Conclusions: These data suggest that plasmalogens play a crucial role in retinal vascularization processes

Survey of Transcript Expression in Rainbow Trout Leukocytes Reveals a Major Contribution of Interferon-Responsive Genes in the Early Response to a Rhabdovirus Infection

Virus infections induce changes in the expression of host cell genes. A global knowledge of these modifications should help to better understand the virus/host cell interactions. To obtain a more comprehensive view of the rainbow trout response to a viral infection, we used the subtractive suppressive hybridization methodology in the viral hemorrhagic septicemia model of infection. We infected rainbow trout leukocytes with viral hemorrhagic septicemia virus (VHSV), and total RNA from infected and mock-infected cells was compared at 40 h postinfection. Twenty-four virus-induced genes were ultimately retrieved from the subtracted cDNA library, and their differential expression was further confirmed by semiquantitative reverse transcription-PCR and Northern blot analysis. Among these sequences, three were already described as VHSV-induced genes. Eight sequences with known homologs were extended to full-length cDNA using 5′ and 3′ rapid amplification of cDNA ends, and they were subsequently divided into three functional subsets. Four genes were homologous to mammalian interferon responsive genes, three were similar to chemo-attractant molecules (CXC chemokine, galectin), and two had nucleic acid binding domains. All of the virus-induced genes were also induced by rainbow trout interferon, indicating that the interferon pathway is the predominant component of the anti-VHSV response. They were also expressed in vivo in experimentally infected fish, indicating their biological relevance in natural infection

002 Évaluation de la toxicité des acides gras branchés sur des cellules conjonctivales en culture

Communication orale lors du 113e congrès de la Société Française d'Ophtalmologie.International audienc

Cholesterol metabolism, autophagy and aging

National audienc

La réponse non spécifique des leucocytes de truite arc-en-ciel au virus de la septicémie hémorragique virale : étude systématique des modifications du transcriptome

National audienc

Fructose diet induced short-term impairment of cone sensitivity and gene expression in rat retina

National audienceAbstract: Purpose A high fructose diet has been widely used to trigger insulin resistance in rodent; insulin resistance is one of the major risk factor for the development of type 2 diabetes. Thirty to 40% of diabetic patients develop diabetic retinopathy. In this study, we aimed to evaluate the short-term effect, at 1, 3, 5, 8 days, of a 60% fructose diet, on photoreceptor sensitivity and gene expression in the retina of Brown Norway rats. Methods: Flicker electroretinograms (8Hz) were recorded under anesthesia, from both eyes simultaneously in order to study sensitivity of photoreceptors. Then, rats were euthanized and enucleated. Retinae and posterior poles were collected to analyze gene expression by RT-PCR. We specifically focused on 45 genes involved in cholesterol homeostasis, lipid trafficking, vascular changes and inflammation. Results: Our data showed that a short period of fructose feeding induced early changes in retinal functionality and homeostasis. Interestingly, we reported a partial loss of cone sensitivity after 8 days of feeding rats with the high-fructose diet. No effect was found in rod sensitivity. Conclusion: These findings are consistent with the sensibility and susceptibility of cones to dietary changes. These data deserve further investigations on the cross-talk between cones and rods upon metabolic changes associated with aging

Effects of 24s-hydroxycholesterol on primary glial Müller cells. New insights on Müller cells function and cholesterol homeostasis in the retina

Purpose: Müller cells are macroglial cells in the retina. These cells express various proteins known to undergo glutamate clearance, which dysfunction may be involved in the loss of retinal ganglion cells (RGC) in the course of glaucoma. In neurons and especially in RGC, 24S-hydroxycholesterol (24SOH) is a metabolite produced by conversion of cholesterol by cholesterol 24S-hydroxylase (CYP46A1). This process participates to cholesterol homeostasis by facilitating the removal of cholesterol from neurons. 24SOH might be a signal molecule ensuring the cross-talk between neurons and glia, and putatively between RGC and glia. The aim of our study was to emphasize the response of primary Müller cells to 24SOH.Methods: Retinas from 10-12 day post-natal Long Evans rats were dissected after enzymatic digestion and seeded into culture dishes. Cells were grown in DMEM (5mM glucose and 2mM glutamine) + 10% FCS at 37°C. At passage 4, Müller cells were incubated with 10, 50 and 100µM 24SOH for 24h. By means of comparison, Müller cells were triggered with 2, 10, 20 ng/µL IL1β and 25mM glucose. Cellular viability was assessed by MTT test. Cholesterol and 24SOH were measured in cell pellets and culture supernatants by isotope dilution gas chromatography-mass spectrometry using internal deuterated standard. Cytokines and chemokines were measured by Luminex® in cell pellets and culture supernatants. The expression of genes involved in lipid metabolism and glial activation was analyzed by RT-qPCR.Results: Cholesterol synthesis was reduced by 39% (10µM 24SOH) and 65% (50 and 100µM 24SOH) compared to controls and did not vary in cells incubated with IL1β and high glucose. The analysis of 24SOH in cells and supernatants revealed the capacity of Müller cells to metabolize 24SOH. MCP-1 and VEGF were increased in cells incubated with 24SOH only (from x1.75 at 10µM compared to controls to x4 at 50 and 100µM) but not in culture supernatants. 24SOH up-regulated the expression of CYP46A1, Ccl-2 (MCP-1), Slc1a2 (EAAT2), GFAP and genes involved in cholesterol transport.Conclusions: 24SOH, used in our in vitro study to mimic the stress of RGC, was found to be actively metabolized by Müller cells and to reduce cholesterol biosynthesis therein. This process may contribute to cholesterol homeostasis in the retina and to Müller cells reactivity in the course of RGC loss and glaucoma

Plasmalogens in the regulation of retinal vascular development: design of a mouse model with chemical inhibition of IPLA2

Plasmalogens in the regulation of retinal vascular development: design of a mouse model with chemical inhibition of IPLA2. annual meeting of the association-for-research-in-vision-and-ophthalmology (ARVO

Docosahexaenoic acid protects human RPE cells against oxidative stress via PI3K/Akt m-TOR/p70-p85S6K pathways

Purpose: Oxidative Stress (OS) plays a critical role in the pathogenesis of age-related macular degeneration (AMD), especially by targeting the retinal pigment epithelium (RPE). Dietary habits with high consumption of docosahexaenoic acid (DHA) have been shown to prevent the development and evolution of AMD. Nevertheless, it is still unclear how DHA affects AMD. Our study aimed to investigate the involvement of the PI3K/Akt and m-TOR/p70-p85S6K pathways in human RPE cells after induction of OS, and then to assess the effect of DHA in the signaling pathways and in the protection against RPE cell death. Methods: For this purpose, we used ARPE-19 cells exposed to the prooxidant agent, tert-butyl hydroperoxide (t-BHP). Results: We found that exposing cells to t-BHP (400µM) showed complete inhibition of Akt and p70/p85S6K active forms. However in cells enriched with DHA (20µM) and then exposed to t-BHP (400µM), we demonstrated that Akt and p85S6K, but not p70S6K, remained phosphorylated for a longer time after stress. In addition there was a 2.6-fold decrease in the number of necrotic cells after 48hours of t-BHP treatment, as assessed by flow cytometry. Conclusion: Our study suggests that 1/ PI3K/Akt and m-TOR/P70-p85S6K pathways play an important role in OS, 2/DHA protects RPE cells from apoptosis and necrosis triggered by OS by enhancing the phosphorylation of Akt and p85S6K

Involvement of plasmalogens and iPLA2 in the control of post-natal retinal vascular development in mice

International audiencePurpose: The correct development of retinal blood vessels is essential to ensure vision process through sufficient oxygen and nutrient supplies to the retina. It was shown that polyunsaturated fatty acids (PUFAs) could modulate factors involved in tissue vascularization. A congenital deficiency in ether-phospholipids also termed as "plasmalogens" was shown to lead to abnormal ocular vascularization. Because plasmalogens are considered as reservoirs of PUFAs, we wanted to better understand the mechanisms by which plasmalogens may regulate retinal vascular development through the release of PUFAs by a calcium-independent phospholipase A2 (iPLA2). Methods: Retinal vascular development was followed in a mouse model of plasmalogen deficiency (DAPAT mice) as well as in a mouse model of retinal iPLA2 inhibition. Retinas were collected from birth until the age of 21 days. They were either flat-mounted and immuno-labelled to visualize the cells involved in vessel formation, or used to determine the expression of genes involved in inflammation and angiogenesis. Results: The lack of plasmalogens was associated to defects in angiogenic processes during early phases of retinal vascular development. Particularly, the absence of plasmalogens influenced astroglial activity during the primary astrocyte template formation, promoted endothelial cell proliferation during sprouting angiogenesis, and impacted the expression of genes involved in angiopoietin/tie pathways. These early defects led to a disorganized and dysfunctional retinal vascular network at adult age. As mice in which iPLA2 was inhibited exhibited a similar vascular phenotype when compared to DAPAT mice, we suggest that these processes may be mediated by PUFA release from plasmalogens and further signalling through angiopoietin/tie pathways. Conclusions: These data suggest a crucial role of plasmalogens during the formation of retinal vessels