Proliferative diabetic retinopathy (PDR) is the most severe form of diabetic retinopathy and, along with diabetic macular edema, is responsible for the majority of blindness in adults below the age of 65. Therapeutic strategies for PDR are ineffective at curtailing disease progression in all cases; however a deeper understanding of the ocular metabolic landscape in PDR through metabolomic analysis may offer new therapeutic targets. Here, global and targeted mass spectrometry-based metabolomics were used to investigate metabolism. Initial analyses on vitreous humor from patients with PDR (n = 9) and non-diabetic controls (n = 11) revealed an increase of arginine and acylcarnitine metabolism in PDR. The oxygen-induced-retinopathy (OIR) mouse model, which exhibits comparable pathological manifestations to human PDR, revealed similar increases of arginine and other metabolites in the urea cycle, as well as downregulation of purine metabolism. We validated our findings by targeted multiple reaction monitoring and through the analysis of a second set of patient samples [PDR (n = 11) and non-diabetic controls (n = 20)]. These results confirmed a predominant and consistent increase in proline in both the OIR mouse model and vitreous samples from patients with PDR, suggesting that over activity in the arginine-to-proline pathway could be used as a therapeutic target in diabetic retinopathy.

Nothing more dramatically captures the imagination of the visually impaired patient or the ophthalmologist treating them than the possibility of rebuilding a damaged retina or vasculature with “stem cells.” Stem cells (SC) have been isolated from adult tissues and represent a pool of cells that may serve to facilitate rescue/repair of damaged tissue following injury or stress. We propose a new paradigm to “mature” otherwise immature neovasculature or, better yet, stabilize existing vasculature to hypoxic damage. This may be possible through the use of autologous bone marrow (BM) or cord blood derived hematopoietic SC that selectively target sites of neovascularization and gliosis where they provide vasculo- and neurotrophic effects. We have demonstrated that adult BM contains a population of endothelial and myeloid progenitor cells that can target activated astrocytes, a hallmark of many ocular diseases, and participate in normal developmental, or injury-induced, angiogenesis in the adult. Intravitreal injection of these cells from mice and humans can prevent retinal vascular degeneration ordinarily observed in mouse models of retinal degeneration; this vascular rescue correlates with functional neuronal rescue as well. The use of autologous adult BM derived SC grafts for the treatment of retinal vascular and degenerative diseases represents a novel conceptual approach that may make it possible to “mature” otherwise immature neovasculature, stabilize existing vasculature to hypoxic damage and/or rescue and protect retinal neurons from undergoing apoptosis. Such a therapeutic approach would obviate the need to employ destructive treatment modalities and would facilitate vascularization of ischemic and otherwise damaged retinal tissue.

Angiostatic therapies designed to inhibit neovascularization associated with multiple pathological conditions have only been partially successful; complete inhibition has not been achieved. We demonstrate synergistic effects of combining angiostatic molecules that target distinct aspects of the angiogenic process, resulting in the complete inhibition of neovascular growth associated with development, ischemic retinopathy, and tumor growth, with little or no effect on normal, mature tissue vasculature. Tumor vascular obliteration using combination angiostatic therapy was associated with reduced tumor mass and increased survival in a rat 9L gliosarcoma model, whereas individual monotherapies were ineffective. Significant compensatory up-regulation of several proangiogenic factors was observed after treatment with a single angiostatic agent. In contrast, treatment with combination angiostatic therapy significantly reduced compensatory up-regulation. Therapies that combine angiostatic molecules targeting multiple, distinct aspects of the angiogenic process may represent a previously uncharacterized paradigm for the treatment of many devastating diseases with associated pathological neovascularization.

Adult bone marrow (BM) contains cells capable of differentiating along hematopoietic (Lin+) or non-hematopoietic (Lin-) lineages. Lin- hematopoietic stem cells (HSCs) have recently been shown to contain a population of endothelial precursor cells (EPCs) capable of forming blood vessels. Here we show that intravitreally injected Lin- BM cells selectively target retinal astrocytes, cells that serve as a template for both developmental and injury-associated retinal angiogenesis. When Lin- BM cells were injected into neonatal mouse eyes, they extensively and stably incorporated into forming retinal vasculature. When EPC-enriched HSCs were injected into the eyes of neonatal rd/rd mice, whose vasculature ordinarily degenerates with age, they rescued and maintained a normal vasculature. In contrast, normal retinal angiogenesis was inhibited when EPCs expressing a potent angiostatic protein were injected. We have demonstrated that Lin- BM cells and astrocytes specifically interact with one another during normal angiogenesis and pathological vascular degeneration in the retina. Selective targeting with Lin- HSC may be a useful therapeutic approach for the treatment of many ocular diseases.

Pathological angiogenesis contributes directly to profound loss of vision associated with many diseases of the eye. Recent work suggests that human tyrosyl- and tryptophanyl-tRNA synthetases (TrpRS) link protein synthesis to signal transduction pathways including angiogenesis. In this study, we show that a recombinant form of a COOH-terminal fragment of TrpRS is a potent antagonist of vascular endothelial growth factor-induced angiogenesis in a mouse model and of naturally occurring retinal angiogenesis in the neonatal mouse. The angiostatic activity is dose-dependent in both systems. The recombinant fragment is similar in size to one generated naturally by alternative splicing and can be produced by proteolysis of the full-length protein. In contrast, the full-length protein is inactive as an antagonist of angiogenesis. These results suggest that fragments of TrpRS, as naturally occurring and potentially nonimmunogenic anti-angiogenics, can be used for the treatment of neovascular eye diseases.