Hemostasis initiates angiogenesis-dependent wound healing, and thrombosis is frequently associated with advanced cancer. Although activation of coagulation generates potent regulators of angiogenesis, little is known about how this pathway supports angiogenesis in vivo. Here we show that the tissue factor (TF)-VIIa protease complex, independent of triggering coagulation, can promote tumor and developmental angiogenesis through protease-activated receptor-2 (PAR-2) signaling. In this context, the TF cytoplasmic domain negatively regulates PAR-2 signaling. Mice from which the TF cytoplasmic domain has been deleted (TF Delta CT mice) show enhanced PAR-2-dependent angiogenesis, in synergy with platelet-derived growth factor BB (PDGF-BB). Ocular tissue from diabetic patients shows PAR-2 colocalization with phosphorylated TF specifically on neovasculature, suggesting that phosphorylation of the TF cytoplasmic domain releases its negative regulatory control of PAR-2 signaling in angiogenesis. Targeting the TF-VIIa signaling pathway may thus enhance the efficacy of angiostatic treatments for cancer and neovascular eye diseases.

Current therapies directed at controlling vascular abnormalities in cancers and neovascular eye diseases target VEGF and can slow the progression of these diseases. While the critical role of VEGF in development has been well described, the function of locally synthesized VEGF in the adult eye is incompletely understood. Here, we show that conditionally knocking out Vegfa in adult mouse retinal pigmented epithelial (RPE) cells, which regulate retinal homeostasis, rapidly leads to vision loss and ablation of the choriocapillaris, the major blood supply for the outer retina and photoreceptor cells. This deletion also caused rapid dysfunction of cone photoreceptors, the cells responsible for fine visual acuity and color vision. Furthermore, Vegfa deletion showed significant downregulation of multiple angiogenic genes in both physiological and pathological states, whereas the deletion of the upstream regulatory transcriptional factors HIFs did not affect the physiological expressions of angiogenic genes. These results suggest that endogenous VEGF provides critical trophic support necessary for retinal function. Targeting factors upstream of VEGF, such as HIFs, may be therapeutically advantageous compared with more potent and selective VEGF antagonists, which may have more off-target inhibitory trophic effects. IntroductionPhototransduction by rod and cone photoreceptors in the retina converts light into electrical energy. This process, forming the basis for vision, is facilitated by the vascular plexus (the choriocapillaris) and retinal pigmented epithelial (RPE) cells, both of which underlie and are intimately associated with the photoreceptors. Degeneration of photoreceptors, RPE cells, and the choriocapillaris is associated with age-related macular degeneration (AMD), the leading cause of vision loss in industrialized nations. While VEGF clearly plays a role in physiological vascular development (1-4) and pathological neovascularization (5, 6) in the retina and the choroid, there is increasing evidence that it is also important for the trophic maintenance of neurons (7-9). Inhibition of VEGF has become the mainstay of therapy for treating AMD (10-12), and increasingly potent VEGF antagonists have recently been introduced into the clinics. In order to better understand the trophic activity of VEGF in the adult retina and choriocapillaris and to assess the effect of deleting its expression by RPE cells, we utilized inducible Cre/loxP technology to genetically delete floxed Vegf alleles in vivo with an RPE-specific VMD2-Cre transgenic line (13).

Tissues with high metabolic rates often use lipid as well as glucose for energy, conferring a survival advantage during feast and famine.1 Current dogma suggests that high-energy consuming photoreceptors depend on glucose.2,3 Here we show that retina also uses fatty acids (FA) β-oxidation for energy. Moreover, we identify a lipid sensor Ffar1 that curbs glucose uptake when FA are available. Very low-density lipoprotein receptor (VLDLR), expressed in tissues with a high metabolic rate, facilitates the uptake of triglyceride-derived FA.4,5 Vldlr is present in photoreceptors.6 In Vldlr−/− retinas, Ffar1, sensing high circulating lipid levels despite decreased FA uptake5, suppresses glucose transporter Glut1. This impaired glucose entry into photoreceptors results in a dual lipid/glucose fuel shortage and reduction in the Krebs cycle intermediate α-ketoglutarate (KG). Low α-KG levels promote hypoxia-induced factor-1α (Hif1a) stabilization and vascular endothelial growth factor (Vegfa) secretion by starved Vldlr−/− photoreceptors, attracting neovessels to supply fuel. These aberrant vessels invading normally avascular photoreceptors in Vldlr−/− retinas are reminiscent of retinal angiomatous proliferation (RAP), a subset of neovascular age-related macular degeneration (AMD)7, associated with high vitreous VEGF levels in humans. Dysregulated lipid and glucose photoreceptor energy metabolism may therefore be a driving force in neovascular AMD and other retinal diseases.

Vision loss associated with ischemic diseases such as retinopathy of prematurity and diabetic retinopathy are often due to retinal neovascularization. While significant progress has been made in the development of compounds useful for the treatment of abnormal vascular permeability and proliferation, such therapies do not address the underlying hypoxia that stimulates the observed vascular growth. Using a model of oxygen-induced retinopathy, we demonstrate that a population of adult BM-derived myeloid progenitor cells migrated to avascular regions of the retina, differentiated into microglia, and facilitated normalization of the vasculature. Myeloid-specific hypoxia-inducible factor 1α (HIF-1α) expression was required for this function, and we also demonstrate that endogenous microglia participated in retinal vascularization. These findings suggest what we believe to be a novel therapeutic approach for the treatment of ischemic retinopathies that promotes vascular repair rather than destruction.

Photoreceptors are the most numerous and metabolically demanding cells in the retina. Their primary nutrient source is the choriocapillaris, and both the choriocapillaris and photoreceptors require trophic and functional support from retinal pigment epithelium (RPE) cells. Defects in RPE, photoreceptors, and the choriocapillaris are characteristic of age-related macular degeneration (AMD), a common vision-threatening disease. RPE dysfunction or death is a primary event in AMD, but the combination(s) of cellular stresses that affect the function and survival of RPE are incompletely understood. Here, using mouse models in which hypoxia can be genetically triggered in RPE, we show that hypoxia-induced metabolic stress alone leads to photoreceptor atrophy. Glucose and lipid metabolism are radically altered in hypoxic RPE cells; these changes impact nutrient availability for the sensory retina and promote progressive photoreceptor degeneration. Understanding the molecular pathways that control these responses may provide important clues about AMD pathogenesis and inform future therapies.DOI: http://dx.doi.org/10.7554/eLife.14319.001