Background: Rods and cones are critical for light detection. Although there has been considerable work done in elucidating the molecular mechanisms involved in rod development, not much is known about how the cone cell fate decision is made by the multipotent retinal progenitor cells during development. Analysis of the promoter regions of Nrl and trβ2, rod and cone differentiation factors respectively, revealed DNA binding motifs of two POU-domain containing transcription factors, Pou2f1 and Pou2f2. Preliminary experiments showed that Pou2f1/2 are expressed during the peak of cone genesis in the embryonic retina. Therefore, we hypothesize that Pou2f1/2 specify cone cell fate in the developing retina.
Methods: We used immunofluorescence and in situ hybridization to establish the spatiotemporal expression of Pou2f1/2 during retinogenesis. We performed in vivo electroporation in post-natal mice to misexpress Pou2f1/2 and used antibodies specific to proteins expressed in cones such as Rxrγ and S-opsin to count cones. Using ex vivo electroporation of embryonic retinal explants, we knocked out Pou2f1 and Pou2f2 using CRISPR/Cas9 gRNAs at the peak of cone production window. Finally, we transfected post-natal retinal explants with a combination of regulatory elements of Nrl or thrb with control backbone vector, Pou2f1 or Pou2f2 using electroporation.
Results: We found that Pou2f1/2 are expressed in retinal progenitor cells in the developing retina and subsequently in the differentiated cones. Pou2f1/2 misexpression outside the cone genesis window led to an increase in cones at the expense of rods. Pou2f1/2 indel knockouts generated by CRISPR/Cas9 gRNAs led to a decrease in cones and a converse increase in rods. Finally, we found that Pou2f1/2 activate the cis-regulatory module (CRM) of the thrb gene and repress the activity of the CRM of Nrl.
Conclusions: These results uncover novel players that establish the complex gene regulatory network for cone photoreceptor fate specification in the retinal progenitor cells. We anticipate that this work should help us devise improved replacement therapies in the future utilizing stem cells for retinal degenerative diseases such as aged-related macular degeneration (AMD) and Stargardt’s disease.
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.
Abstract: Mononuclear phagocytes (MP) comprise a family of cells that include microglial cells (MC), monocytes, and macrophages. The subretinal space, located between the RPE and the photoreceptor outer segments, is physiologically devoid of MPs and a zone of immune privilege mediated, among others, by immunosuppressive RPE signals. Age-related macular degeneration (AMD) is a highly heritable major cause of blindness, characterized by a breakdown of the subretinal immunosuppressive environment and an accumulation of pathogenic inflammatory MPs. Studies in mice and humans suggest that the AMD-associated APOE2 isoform promotes the breakdown of subretinal immunosuppression and increased MP survival. Of all genetic factors, variants of complement factor H (CFH) are associated with greatest linkage to AMD. Using loss of function genetics and orthologous models of AMD, we provide mechanistic evidence that CFH inhibits the elimination of subretinal MPs. Importantly, the AMD-associated CFH402H isoform markedly increased this inhibitory effect on microglial cells, indicating a causal link to disease etiology. Pharmacological acceleration of resolution of subretinal inflammation might be a powerful tool for controlling inflammation and neurodegeneration in late AMD.
Abstract: Genetic studies have revealed that variants in genes that encode regulators of the complement system are major risk factors for the development of age-related macular degeneration (AMD). The biochemical consequences of the common polymorphism in complement factor H (Tyr402His) include increased formation of the membrane attack complex (MAC), which is deposited at the level of the inner choroid and choriocapillaris. Whereas the MAC is normally protective against foreign pathogens, it can also damage resident bystander cells when it is insufficiently regulated. Indeed, human maculas with early AMD show loss of endothelial cells in the choriocapillaris, the principal site of MAC activation. Modeling of MAC injury of choroidal endothelial cells in vitro reveals that these cells are susceptible to cell lysis by the MAC, and that unlysed cells alter their gene expression profile to undergo a pro-angiogenic phenotype that includes increased expression of matrix metalloproteinase-9. Strategies for protecting choriocapillaris endothelial cells from MAC-mediated lysis and for replacing lysed endothelial cells will be discussed.
Abstract: Autophagy recycles intracellular substrate in part to fuel mitochondria during starvation. Deregulated autophagy caused by dyslipidemia, oxidative stress, and aging is associated with early signs of age-related macular degeneration (AMD), such as lipofuscin and perhaps drusen accumulation. Intracellular nutrient sensors for glucose and amino acids regulate autophagy. The role of lipid sensors in controlling autophagy, however, remains ill-defined. Here we will show that abundant circulating lipids trigger a satiety signal through FA receptors that restrain autophagy and oxidative mitochondrial metabolism. In the presence of excess dietary lipids, fatty acid sensors might protect tissues with high metabolic rates against lipotoxicity, favoring their storage, instead, in adipose tissues. However, sustained exposure to lipid reduces retinal metabolic efficiency. In photoreceptors with high metabolic needs, it predisposes to an energy failure and triggers compensatory albeit pathological angiogenesis, leading to blinding neovascular AMD.
