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: The neovascular aged-related macular degeneration (AMD) is the leading cause of legal blindness in the elderly. It is presently treated by anti-VEGF intravitreal injection in order to stop the neovascularization. In seeking of more efficient treatments to prevent retinal damage, it has been proposed that the kinin-kallikrein system (KKS), a key player in inflammation, could be involved in AMD etiology. However, the role of kinin receptors and their interaction with VEGF in AMD is poorly understood.
Methods: In order to address this question, choroidal neovascularization (CNV) was induced in the left eye of Long-Evans rat. After laser induction, anti-VEGF or IgG control were injected into the vitreal cavity. Gene expression was measured by qRT-PCR, retinal adherent leukocytes were labelled with FITC-Concanavalin A lectin, vascular leakage by the method of Evans blue and cellular localisation by immunohistochemistry.
Results: The number of labelled adherent leucocytes was significantly increased in laser-induced CNV compared to the control eye. This was significantly reversed by one single injection of anti-VEGF. Extravasation of Evans blue dye was significantly increased in laser-induced CNV eyes compared to control eyes and partially reversed by one single injection of anti-VEGF or by R954 treatment. The mRNA expression of inflammatory mediators was significantly increased in the retina of CNV rats. Immunodetection of B1R was significantly increased in CNV eyes. B1R immunolabeling was detected on endothelial and ganglion cells.
Conclusions: This study is the first to highlight an effect of the kinin/kallikrein system in a model of CNV that could be reduced by both anti-VEGF therapy and topically administered B1R antagonist R-954.
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.
