Background: Retinal pigment epithelium (RPE) is vital for the homeostasis of the subretina including photoreceptors and choroid. Interestingly, our previous results suggested that the recently discovered lactate receptor GPR81 is abundantly expressed in RPE. To date, only one previous study has shown that activating GPR81 could enhance DNA repair by activating HDAC1. Consequently, we investigated whether GPR81 exhibits epigenetic modification in the subretina by using GPR81?/? mice.
Methods: GPR81?/? mice and wide type littermates were generated on a background of C57BL/6J mice. The thicknesses of their choroid were evaluated by immunohistochemistry. Meanwhile, Q-PCR, western blot and choroid sprout assay were performed. In vitro, primary retinal pigment epithelium (pRPE) cells were isolated from mice, and cultured for treatments.
Results: The thickness of choroid was reduced in GPR81?/? mice compared to GPR81+/+ mice, suggesting that GPR81 is important for the integrity of choroid. In the choroid sprout assay, lactate treated RPE/choroid complex showed a significant increase in angiogenesis compared to controls while lactate treated KO RPE/choroid complex showed no difference compared to their controls. For Q-PCR, most of the genes screened elevated their expression in GPR81?/? mice compared to WT mice, suggesting epigenetic modification may exist, which were confirmed by histone acetylation and HDACs activity assay.
Conclusions: Taking together, the lactate receptor GPR81 in RPE is very important for maintaining homeostasis of the subretina. This novel discovery sheds new light on the relationship between metabolism and epigenetic modification.
Background: We investigated the role of beta-adrenergic receptor (B-AR) on choroidal neovascularization (CNV) in an animal model of age-related macular degeneration in mice.
Methods: The angiogenic effect of the B-AR was evaluated in retinal pigment epithelium (RPE)-choroid explants from C57Bl6 mice stimulated with propranolol or isoproterenol (10 μM) (respectively antagonist and agonist of the B-AR) during 24 h. Conversely, a classic choroidal neovascularization (CNV) model induced by laser burn in C57Bl6 mice (8 weeks) was used to assess the anti-angiogenic effect of propranolol. In this experiment, mice were treated with intraperitoneal propranolol (6 mg/kg/d) or vehicle (saline solution) daily for 10 days, starting on day 4 after laser burn and until sacrifice (day 14). Immunostaining analysis on retinal flatmounts and cryosections were performed to determine the surface of CNV, the distribution of B-AR and the number and morphology of microglia/macrophages associated with CNV. To explore if the antiangiogenic effect of propranolol involved the modulation of the inflammatory microenvironment associated with CNV, we used RPE primary cells, J774 macrophages cell line and polarized M1 and M2 bone marrow-derived macrophage (BMDM). Choroidal explants treated with conditioned media (CM) from J774 or polarized M1/M2 BMDM pre-treated with propranolol to confirm the anti-angiogenic effect of propranolol. Expression of angiogenic factors was evaluated by RT PCR and Elisa.
Results: The expression and distribution of the B-1, B-2 and B-3 adrenergic receptors were localized in the choroid and RPE cells. The stimulation of RPE-choroid explants with isoproterenol increased CNV compared to vehicle, while propranolol decreased CNV. In vivo, propranolol inhibited significantly the levels of VEGF and CNV growth in laser burn model compared to the vehicle. Additionally, the treatment with propranolol decremented the number of activated (amoeboid shape) microglia/macrophages but surprisingly, the number of non-activated microglia/macrophages around the CNV was higher than with the vehicle treatment. In vitro, propranolol modulated the angiogenic balance in macrophages promoting anti-angiogenic factors expression, especially with M2 BMDM. CM from macrophages pre-treated with propranolol reduced CNV on choroidal explants.
Background: Diabetic macular edema (DME) is a leading cause of severe visual impairments in older and the working-age population. An important target of current therapy is vascular endothelial growth factor (VEGF), which plays a role in the pathogenesis of DME by inducing angiogenesis and increasing vascular permeability. Currently available anti-VEGF agents include off-label use of Bevacizumab, which has been shown to be effective in the treatment of DME. However, many patients with DME do not respond or demonstrate only a partial response to this agent. As of November 2016, the Canadian Health authorities approved Aflibercept as an anti-VEGF agent for treatment of DME, and the patients who are non-responders to Bevacizumab are switched to this non-off label medication. We aimed to investigate the anatomical and functional visual changes associated with response to Aflibercept in a real-life Canadian population of Bevacizumab non-responders.
Methods: A retrospective review of chronic DME patients refractory to bevacizumab treatment who were switched to Aflibercept was done. Best-corrected visual acuity (BCVA), Intraocular pressure (IOP), central subfield thickness (CST), average macular thickness, and total macular volume were extracted at the visit prior to switching to Aflibercept (baseline) as well as the first, second and third follow-up visits after switching. Anatomical and functional visual changes were compared using Generalized Estimating Equations and the association between variables was tested using Pearson correlation test with significance set at P<0.05.
Results: Twenty-six eyes with mean age of 63 were included. Average CST at baseline was 421.5±116.1 μm and the number of Bevacizumab injections received prior to switching was 15.3±8.0. No significant changes were observed in terms of BCVA and IOP, from baseline to any of the follow-ups. Switching to Aflibercept significantly improved CST, average macular thickness, and total macular volume. From baseline to the first follow-up visit, CST decreased from 421.5±116.1 to 333.0±91.2 μm (P=0.001) and average macular thickness reduced from 344.6±74.9 to 322.2±60.5 μm (P=0.008). Similarly, total macular volume decreased from 12.4±2.7 to 11.6±2.2 μm3, measured at baseline and the first follow-up (P=0.007). No further improvements were observed from the first follow-up to the subsequent ones. The median CST value at baseline (378 μm) was used to classify the patients into low and high CST groups. We observed that those with higher CST at baseline (>378 μm) showed a trend for improvements in visual acuity (P=0.058). Pearson correlation test confirmed the association between higher CST at baseline and better visual outcomes in response to switching to Aflibercept (P=0.018).
Conclusions: Our data evidenced significant anatomical improvements in macula, which did not translate to immediate functional vision improvements. Bevacizumab non-responders with higher CST might also gain visual acuity and benefit functionally from switching to Aflibercept.
Background: Overexpression of inducible nitric oxide synthase (iNOS) has been reported in diabetic retinopathy (DR). The kinin B1 receptor (B1R) is also overexpressed in DR, and can stimulate iNOS via Gαi/ERK/MAPK pathway. We previously showed that the topical administration of a B1R antagonist, LF22-0542, significantly reduces leukocyte infiltration, increased vascular permeability and overexpression of several inflammatory mediators, including iNOS in DR. Thus, the aim of this study was to determine whether the pro-inflammatory effects of B1R are attributed to oxidative stress caused by the activation of iNOS pathway in order to identify new therapeutic targets for the treatment of DR. iNOS and B1R being absent in the normal retina, their inhibition is unlikely to result in undesirable side effects. The approach will be no invasive by eye application of drops.
Methods: Diabetes was induced in male Wistar rats (200–230 g) by a single intraperitoneal injection of streptozotocin (STZ, 65 mg/kg b.w). One week later, rats were randomly divided into four groups (N=5) and treated for one week as follows: Gr 1: control rats treated with the selective iNOS inhibitor (1,400 W, 0.06 μM twice a day by eye-drops ×7 days), Gr 2, STZ-diabetic rats treated with 1,400 W, Gr 3: control rats received a selective B1R agonist [Sar (D-Phe8)-des-Arg9-BK, 100 μg twice a week] by intravitreal injections (itrv) and treated with 1,400 W, Gr 4: STZ-diabetic rats + B1R agonist +1,400 W. At the end of treatment and two weeks post-STZ, three series of experiments were carried out to measure vascular permeability (by Evans blue dye method) and the expression of vasoactive and inflammatory mediators, including iNOS, VEGF-A, VEGF-R2, IL-1β, Cox-2, TNF-α, bradykinin 1 and 2 receptors and carboxypeptidase M/kininase 1 (by Western Blotting and qRT-PCR). The nitrosative stress (nitrosylation of proteins) was also assessed by Western Blotting. One-way Anova test with Bonferroni post hoc was used for statistical analysis.
Results: STZ-diabetic rats showed a significant increase in retinal vascular permeability (22.8 μg/g Evans blue dye per g of fresh retinas, P=0.016) compared with control rats and control treated rats (17.2 and 16.8 μg/g respectively). The injections of B1R agonist amplified the increase of vascular permeability which was normalized by the 1,400 W. The overexpression of inflammatory markers was also normalized by the 1,400 W in STZ-diabetic rats received or not the B1R agonist.
Conclusions: These results support a contribution of iNOS in the deleterious effects of B1R in this model of diabetic retinopathy. Hence, iNOS inhibition by ocular application of 1,400 W may represent a promising and non-invasive therapeutic approach in the treatment of diabetic retinopathy.
Background: Retinol dehydrogenase 8 (RDH8) is a 312-amino acid (aa) protein involved in the visual cycle. Bound to the outer segment disk membranes of photoreceptors, it reduces all-trans-retinal to all-trans-retinol1 as one of the rate-limiting steps of the visual cycle2. RDH8 is a member of the short-chain dehydrogenase/reductase family. Its C-terminal segment allows its membrane-anchoring through the postulated presence of an amphipathic α-helix and of 1 to 3 acyl groups at positions 299, 302 and 3043. The secondary structure and membrane binding characteristics of RDH8 and its C-terminal segment have not yet been described.
Methods: To evaluate the membrane binding of RDH8, the full-length protein (aa 1–312), a truncated form (aa 1–296), its C-terminal segment (aa 281–312 and 297–312) as well as different additional variants of this segment were used. The truncated protein binds membranes less efficiently than the full-length form. Thus, the C-terminal segment of RDH8 is essential for the binding and has thus been further examined. The intrinsic fluorescence of tryptophan residues at positions 289 and 310 of the wild-type C-terminal segment of RDH8 and the mutants W289F, W310F and W310R have thus been used to determine their extent of binding to lipid vesicles and to monitor their local environment. Unilamellar lipid vesicles composed of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) or a mixture of POPC and 1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-L-serine (POPS) were used to mimic the phospholipid content of the outer segment disk membranes of photoreceptors.
Results: An increase in fluorescence intensity and in fluorescence lifetime is observed upon increasing the concentration of lipid vesicles. These data allowed calculating values of partition coefficient of the C-terminal segment of RDH8 varying between Kp =1.1 E6 to 1.7 E6. It is noteworthy that the observation of a more intense shift to lower wavelengths upon membrane binding of the mutant W310R and W310F indicates a deeper incorporation of the remaining tryptophan residue at position 289 into the lipid bilayer. The secondary structure of the C-terminal segment of RDH8 observed by circular dichroism and infrared spectroscopy shows a superposition of α-helical, β-turn and unordered structures.
Conclusions: The peptides derived from the C-terminal segment of RDH8 show a strong binding to lipid vesicles. These strength of binding is independent of the type of lipid and the presence of a mutation.
Background: Retinal endothelial cells are very active and contribute to the integrity of the neurovascular unit. Vascular dysfunction has been proposed to contribute to the pathogenesis of glaucoma. Here, we evaluated the hypothesis that ocular hypertension triggers mitochondrial alterations in endothelial cells impairing the integrity of the blood retinal barrier (BRB).
Methods: Ocular hypertension was induced by injection of magnetic microbeads into the anterior chamber of EndoMito-EGFP mice, a strain expressing green fluorescent protein selectively in the mitochondria of endothelial cells. Capillary density, mitochondrial volume, and the number of mitochondrial components were quantified in 3D-reconstructed images from whole-mounted retinas using Imaris software. Dynamin-related protein (DRP-1), mitofusin-2 (MFN-2) and optic atrophy-1 (OPA-1) expression were assessed by western blot analysis of enriched endothelial cells. Mitochondrial structure was evaluated by transmission electron microscopy (TEM) and oxygen consumption rate was monitored by Seahorse analysis. The integrity of the BRB was evaluated by quantifying Evans blue leakage.
Results: Our data demonstrate that two and three weeks after ocular hypertension induction, the total mitochondria volume in endothelial cells decreased from 0.140±0.002 μm3 from non-injured retinas to 0.108±0.005 and 0.093±0.007 μm3, respectively in glaucomatous eyes (mean ± S.E.M, ANOVA, P<0.001; N=6/group). Frequency distribution showed a substantial increase of smaller mitochondria complexes (<0.5 μm3) in endothelial cells from glaucomatous retinas. Significant upregulation of DRP-1 was found in vessels isolated from glaucomatous retinas compared to the intact retinas, while MFN-2 and OPA-1 expression was not affected. Structural alteration in endothelial cell mitochondria was confirmed by TEM, which were accompanied by a 1.93-fold reduction in the oxygen consumption rate as well as 2.6-fold increase in vasculature leakage in glaucomatous retinas (n=3–6/group). In addition, this model did not trigger changes in the density of the vascular network, suggesting that mitochondrial fragmentation was not due to endothelial cell loss.
Conclusions: This study shows that ocular hypertension leads to early alterations in the dynamic of endothelial cell mitochondria, contributing to vascular dysfunction in glaucoma.
Background: Retinopathy of prematurity (ROP) is the major cause of blindness in children, mainly caused by the retinal neovascularization (NV). Mounting of evidences shown that macrophage plays a pivotal role in the regulation of angiogenesis in ROP. Numerous studies confirmed that the deletion of macrophage significantly reduce the neovascularized areas in the oxygen-induced retinopathy (OIR) model. We have been studied the effect of lymphocyte derived-microparticles (LMPs) over ten years. LMPs are extracellular vesicles derived from apoptotic human CEM T lymphocytes. Our previous studies demonstrated that LMPs possess strong anti-angiogenic effect. Recently we observed that LMPs are capable to switch the phenotype of macrophage, thus to suppress the choroidal neovascularization (CNV). However, the role of LMPs on macrophage in ROP has not been clarified. Thus, my project is to disclose the relationship between LMPs and macrophage in ROP using the OIR model. Hypothesis: LMPs may inhibit retinal NV in the OIR model through targeting at macrophage by affecting the migration of macrophage, thus to inhibit pathological angiogenesis in ROP.
Methods: Cell culture [RAW 264.7 and bone marrow-derived macrophage (BMDM)] for cell migration and viability assay. Generate the OIR model for in vivo detection of macrophage recruitment. Quantification of retinal NV, immunohistostaining of the macrophage in vivo, ex vivo retinal explants for cell migration and qPCR.
Results: LMPs do not affect RAW 264.7 and BMDM cell viability (P>0.05). LMPs significantly decrease the BMDM cell migration indirectly (P<0.05). I successfully generate the OIR model and confirm that more macrophages infiltrate during retinal angiogenesis with counting the F4/80 immunostaining in the retinal flat mount. LMPs exert inhibiting effect on retinal angiogenesis through decreasing the migration of macrophages in vivo.
Conclusions: LMPs have the negative effect on retinal angiogenesis via reducing the infiltrated macrophages to the neovascularized areas in the OIR model.
Background: The aim of this project is to develop a new standardized and cost-efficient method to compare optical coherence tomography (OCT) scans to their corresponding paraffin embedded histopathology sections in post-mortem eyes. This correlation will clarify the interpretation of OCT images, and it will also enable direct immunohistochemical characterization of features observed on OCT.
Methods: Study design: donor eyes were obtained from two separate eye banks. In order to minimize post-mortem change like retinal detachment and vitreous opacification, the eyes were fixed in a previously tested fixative solution. Time between death and fixation has been kept under 6 hours. Methods: Using a customized imaging device, nine post-mortem eyes were imaged with a SD-OCT machine. Subsequently, an 8mm trephine was used to isolate a portion of the posterior pole including the macular area and the optic nerve head for histopathological analysis. Paraffin embedded cross sections of the retina were obtained and visually compared to each OCT image (b-scans).
Results: To facilitate the correlation of OCT images to their histopathological sections, three principle aspects were controlled during tissue processing: rotation, tilt and location. Using markings as well as anatomical landmarks, serial histopathological sections in an orientation comparable to OCT b-scans were obtained, thereby facilitating image pairing.
Conclusions: Compared to other well-established methods using resin and electron microscopy, our standardized Methods allowed us to successfully compare OCT b-scans to serial retinal cross sections of a wider macular area at a lower cost. Our novel approach allows us to translate features observed on OCT images into well-established histopathological images, providing the clinician with additional tools to obtain difficult diagnoses with more confidence.
