Background: The oxygen induced retinopathy rodent model is widely used, notably for the assessment of developmental dystrophies in preclinical studies of vascular retinal diseases. Typically, the quantification of vessel tufts and avascular regions is computed manually from flat mounted retinas imaged using fluorescent probes that highlight the vascular network. However, such manual measurements are time-consuming and hampered by user variability and bias, thus a rapid and objective alternative is required.

Methods: We employ a machine learning approach to segment and characterize vascular tufts. The proposed quantitative retinal vascular assessment (QuRVA) technique uses quadratic discrimination analysis and morphological techniques to provide reliable measurements of vascular density and pathological vascular tuft regions, devoid of user intervention within seconds. Our algorithms allow also delineating the whole vasculature network, and identifying and analyzing avascular regions.

Results: Our first experiment shows the high degree of error and variability of manual segmentations. In consequence, we developed a set of algorithms to perform this task automatically. We benchmark and validate the results of our analysis pipeline using the consensus of several manually curated segmentations using commonly used computer tools. We describe the method, provide details for reproducing the algorithm, and validate all aspects of the analysis.

Conclusions: Manual and semi-automated procedures for tuft detection present strong fluctuations among users, demonstrating the need for fast and unbiased tools in this highly active research field with tremendous implications for basic research and industry.

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: The bone morphogenetic protein (BMP) family of proteins has a multitude of roles throughout the body. It plays important roles in development and in the adult vascular endothelium, by modulating the angiogenic response. The endothelial-specific receptor BMP receptor Alk1 is of particular importance in the proper remodeling of the vasculature and its ligand BMP9 has been shown to be a potent inhibitor of neovascularization. Dysregulated BMP signaling has been linked to multiple vascular diseases and can lead to the abnormal angiogenesis. We therefore investigated the role of BMP9/Alk1 signaling in retinal angiogenesis, and its therapeutic implications for vascular pathologies of the eye.