Background: Decrease of ocular blood flow has been linked to the pathogenesis of ocular diseases such as glaucoma and age-related macular degeneration. Current methods that measure the pulsatile blood flow have major limitations, including the assumption that ocular rigidity is the same in all eyes. Our group has recently developed a new method to measure the pulsatile choroidal volume change by direct visualization of the choroid with OCT imaging and automated segmentation. Our goal in this study is to describe the distribution of PCBF in a healthy Caucasian population.

Methods: Fifty-one subjects were recruited from the Maisonneuve-Rosemont Hospital Ophthalmology Clinic and underwent PCBF measurement in one eye. The distribution of PCBF in healthy eyes was assessed.

Results: The distribution of PCBF among the healthy eyes was found to be 3.94±1.70 μL with this technique.

Conclusions: This study demonstrates the normal range of PCBF values obtained in a healthy Caucasian population. This technique could be used for further investigation of choroid pulsatility and to study glaucoma pathophysiology.

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.

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.