Background: Sight-threatening diabetic macular edema (DME) is caused by increased microvascular permeability. While few direct vascular targeting strategies are available, VEGF pathway inhibition has shown to be effective in reducing retinal vascular leakage but is associated with non-negligible side effects. Thus, more options are needed. Vascular specific Activin-like kinase receptor type I (ALK1) pathway and its circulating ligand Bone morphogenetic protein-9 (BMP9) is known for its potent quiescent and stabilizing effect on the vasculature. However, little is known about this pathway in the context of microvascular permeability associated with diabetes. We hypothesize that BMP9/ALK1 pathway is inhibited in diabetic (DB) retinas leading to vascular destabilization and leakage and that its activation could re-establish proper vascular endothelial barrier functions (EBF).

Methods: The effect of hyperglycemia (i.e., HG >10 mM of D-glucose) on Alk1 signaling was evaluated in vitro by subjecting endothelial cells (EC) to increasing concentrations of D-glucose (5, 11, 25 mM) and in vivo using DB mice (Streptozotocin-induced diabetes). The contribution of Alk1 signaling on EBF was evaluated using Evans Blue permeation in inducible endothelial specific Alk1 KO mice. To evaluate the potential protective effects of BMP9/Alk1 signaling on EBF, BMP9 overexpression was achieved using adenoviral delivery in DB mice. Statistical-One-Way ANOVA or Student’s t-test was used.

Results: Endothelial tissue from DB mice showed a significant inhibition of BMP9/ALK1-canonical Smad1,5,8 quiescence signaling (DB n=5; CTL n=4; P<0.01), which was associated with reduced expression of target genes (JAG1, Id1,3, Hey1,2 & HES). Moreover, we showed that retinal hyperpermeability associated with diabetes was exacerbated in Alk1 heterozygote mice (n=4–9/group; P<0.0001). Finally, we demonstrated that activation of Alk1 signaling in ECs prevented vascular permeability induced by HG, both in vitro (n=3; P=0.009) and in vivo (n=4–9/group; P<0.0001).

Conclusions: Consistent with our hypothesis, vascular stability and quiescence induced by BMP9-ALK1 signaling is inhibited in the DB/HG endothelium which could be an important factor in vascular leakage leading to DME. Our results show that activation of this pathway could offer a therapeutically interesting future option to slow down the onset of DME.

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: Pathological retinal neovascularization is the hallmark of primary blinding diseases across all age groups, yet surprisingly little is known about the causative factors. These diseases include diabetic retinopathy and retinopathy of prematurity where progressive decay of retinal vasculature yields zones of neural ischemia. These avascular zones and the hypoxic neurons and glia that reside in them are the source of pro-angiogenic factors that mediate destructive pre-retinal angiogenesis. Central neurons such as retinal ganglion cells (RGCs), which are directly apposed to degenerating vasculature in ischemic retinopathies, require stable metabolic supply for proper function. However, we unexpectedly found that RGCs are resilient to hypoxia/ischemia and a generally compromised metabolic supply and instead of degenerating, trigger protective mechanisms of cellular senescence. Paradoxically, while potentially favoring neuronal survival, the senescent state of RGCs is incompatible with vascular repair as they adopt a senescence-associated secretory phenotype (SASP) that provokes release of a secretome of inflammatory cytokines that drives paracrine senescence and further exacerbates pathological angiogenesis. The mechanisms that lead to retinal cellular senescence and dormancy as well as the therapeutic potential of targeting these pathways will be discussed.

Abstract: Subretinal inflammation plays a critical role in retinal degenerative diseases. Although activated macrophages have been shown to play a key role in the progression of retinopathies and specifically in age-related macular degeneration, little is known about the mechanisms involved in the loss of photoreceptors leading to vision impairment. In our study on retinal damages induced by photo-oxidative stress, we have observed that CD36-deficient mice featured less subretinal macrophage accumulation with attenuated photoreceptor degeneration compared to wild-type (WT) mice. Treatment with CD36-selective azapeptide ligand (labelled MPE-001) as modulator of the inflammatory environment of the retina reduced subretinal macrophage/activated microglia accumulation with preservation of photoreceptor layers and function assessed by ERG in WT, in a CD36-dependent manner. The azapeptide modulated the transcriptome of subretinal macrophage/activated microglia by reducing pro-inflammatory markers. In isolated macrophages, the CD36-selective azapeptide induced dissociation of the CD36-TLR2/6 heterodimer complex (using FRET) altering the TLR2 signaling pathway, thus decreasing NF-KB activation and inflammasome activity. The azapeptide also incurred cytoprotection against photoreceptor apoptosis elicited by activated macrophages. These findings suggest that the azapeptide as ligand of co-receptor CD36 decreases the inflammatory response by modulating CD36-TLR2/6 complex signaling pathway in macrophages, and suggests its potential application in the treatment of retinal degenerative diseases.

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.

Abstract: Ocular vessel networks develop in a highly stereotyped fashion. Abnormal ocular angiogenesis is associated with major diseases including age-related macular degeneration and diabetic retinopathy. Better understanding of mechanisms driving angiogenesis is expected to uncover novel targets to prevent vision loss. Capillary growth is driven by endothelial tip cells, which are selected by dynamic interplay between VEGF, Notch and BMP signaling, with VEGF acting as a positive regulator, and Notch and the BMP receptor Alk1 acting as negative regulators of tip cell formation. The concerted interplay between these molecules ensures that appropriate tip cell numbers leading new vessel branches are formed. In addition, guidance receptors including Neuropilins and Roundabout receptors contribute to vascular patterning by regulating VEGF and BMP signaling. Possibilities to target these pathways during pathological ocular neovascularization will be discussed.

Abstract: Disorders of lipid metabolism and macrophage function have been implicated in tissue aging and in diseases such as age-related macular degeneration (AMD). Genetic studies and expression profiling have identified widespread abnormalities in cholesterol metabolism in the aging macrophage. In addition, the molecular pathways that regulate the transition from aging to disease have not been elucidated. The current status regarding the mechanisms that regulate macrophage aging and the molecular mechanisms of transition to disease in the context of AMD will be presented with a special focus on factors that influence pathologic angiogenesis and neurodegeneration.

Abstract: Pediatric neuro-ophthalmology is a subspecialty within neuro-ophthalmology. Pediatric neuro-ophthalmic diseases must be considered separate from their adult counterparts, due to the distinctive nature of the examination, clinical presentations, and management choices. This manuscript will highlight four common pediatric neuro-ophthalmic disorders by describing common clinical presentations, recommended management, and highlighting recent developments. Diseases discussed include pediatric idiopathic intracranial hypertension (IIH), pseudopapilledema, optic neuritis (ON) and optic pathway gliomas (OPG). The demographics, diagnosis and management of common pediatric neuro-ophthalmic disease require a working knowledge of the current research presented herein. Special attention should be placed on the differences between pediatric and adult entities such that children can be appropriately diagnosed and treated.