Edith Aguilar

Regulation of angiogenesis by tissue factor cytoplasmic domain signaling

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 (TFΔCT 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.

Myeloid progenitors differentiate into microglia and promote vascular repair in a model of ischemic retinopathy

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 1alpha (HIF-1alpha) 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.

Targeted deletion of Vegfa in adult mice induces vision loss

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.

Combination angiostatic therapy completely inhibits ocular and tumor angiogenesis

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.

Astrocyte hypoxic response is essential for pathological but not developmental angiogenesis of the retina.

Vascular/parenchymal crosstalk is increasingly recognized as important in the development and maintenance of healthy vascularized tissues. The retina is an excellent model in which to study the role of cell type‐specific contributions to the process of blood vessel and neuronal growth. During retinal vascular development, glial cells such as astrocytes provide the template over which endothelial cells migrate to form the retinal vascular network, and hypoxia‐regulated vascular endothelial growth factor (VEGF) has been demonstrated to play a critical role in this process as well as pathological neovascularization. To investigate the nature of cell‐specific contributions to this process, we deleted VEGF and its upstream regulators, the hypoxia‐inducible transcription factors HIF‐1α and HIF‐2α, and the negative regulator of HIFα, von Hippel–Lindau protein (VHL), in astrocytes. We found that loss of hypoxic response and VEGF production in astrocytes does not impair normal development of retinal vasculature, indicating that astrocyte‐derived VEGF is not essential for this process. In contrast, using a model of oxygen‐induced ischemic retinopathy, we show that astrocyte‐derived VEGF is essential for hypoxia‐induced neovascularization. Thus, we demonstrate that astrocytes in the retina have highly divergent roles during developmental, physiological angiogenesis, and ischemia‐driven, pathological neovascularization.

Retinal vascular permeability suppression by topical application of a novel VEGFR2/Src kinase inhibitor in mice and rabbits

Retinal and choroidal vascular diseases, with their associated abnormalities in vascular permeability, account for the majority of patients with vision loss in industrialized nations. VEGF is upregulated in ischemic retinopathies such as diabetes and is known to dramatically alter vascular permeability in a number of nonocular tissues via Src kinase-regulated signaling pathways. VEGF antagonists are currently in clinical use for treating the new blood vessels and retinal edema associated with neovascular eye diseases, but such therapies require repeated intraocular injections. We have found that vascular leakage following intravitreal administration of VEGF in mice was abolished by systemic or topical delivery of what we believe is a novel VEGFR2/Src kinase inhibitor; this was confirmed in rabbits. The relevance of Src inhibition to VEGF-associated alterations in vascular permeability was further substantiated by genetic studies in which VEGF injection or laser-induced vascular permeability failed to augment retinal vascular permeability in Src-/- and Yes-/- mice (Src and Yes are ubiquitously expressed Src kinase family members; Src-/- and Yes-/- mice lacking expression of these kinases show no vascular leak in response to VEGF). These findings establish a role for Src kinase in VEGF-mediated retinal vascular permeability and establish a potentially safe and painless topically applied therapeutic option for treating vision loss due to neovascular-associated retinal edema.

Antioxidant or neurotrophic factor treatment preserves function in a mouse model of neovascularization-associated oxidative stress

In several disease states, abnormal growth of blood vessels is associated with local neuronal degeneration. This is particularly true in ocular diseases such as retinal angiomatous proliferation (RAP) and macular telangiectasia (MacTel), in which, despite the absence of large-scale leakage or hemorrhage, abnormal neovascularization (NV) is associated with local neuronal dysfunction. We describe here a retinal phenotype in mice with dysfunctional receptors for VLDL (Vldlr-/- mice) that closely resembles human retinal diseases in which abnormal intra- and subretinal NV is associated with photoreceptor cell death. Such cell death was evidenced by decreased cone and, to a lesser extent, rod opsin expression and abnormal electroretinograms. Cell death in the region of intraretinal vascular abnormalities was associated with an increased presence of markers associated with oxidative stress. Oral antioxidant supplementation protected against photoreceptor degeneration and preserved retinal function, despite the continued presence of abnormal intra- and subretinal vessels. What we believe to be novel, Müller cell-based, virally mediated delivery of neurotrophic compounds specifically to sites of NV was also neuroprotective. These observations demonstrate that neuronal loss secondary to NV can be prevented by the use of simple antioxidant dietary measures or cell-based delivery of neurotrophic factors, even when the underlying vascular phenotype is not altered.

Maintaining retinal astrocytes normalizes revascularization and prevents vascular pathology associated with oxygen-induced retinopathy

Astrocytes are well known modulators of normal developmental retinal vascularization. However, relatively little is known about the role of glial cells during pathological retinal neovascularization (NV), a leading contributor to vision loss in industrialized nations. We demonstrate that the loss of astrocytes and microglia directly correlates with the development of pathological NV in a mouse model of oxygen‐induced retinopathy (OIR). These two distinct glial cell populations were found to have cooperative survival effects in vitro and in vivo. The intravitreal injection of myeloid progenitor cells, astrocytes, or astrocyte‐conditioned media rescued endogenous astrocytes from degeneration that normally occurs within the hypoxic, vaso‐obliterated retina following return to normoxia. Protection of the retinal astrocytes and microglia was directly correlated with accelerated revascularization of the normal retinal plexuses and reduction of pathological intravitreal NV normally associated with OIR. Using astrocyte‐conditioned media, several factors were identified that may contribute to the observed astrocytic protection and subsequent normalization of the retinal vasculature, including vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF). Injection of VEGF or bFGF at specific doses rescued the retinas from developing OIR‐associated pathology, an effect that was also preceded by protection of endogenous glia from hypoxia‐induced degeneration. Together, these data suggest that vascular‐associated glia are also required for normalized revascularization of the hypoxic retina. Methods developed to target and protect glial cells may provide a novel strategy by which normalized revascularization can be promoted and the consequences of abnormal NV in retinal vascular diseases can be prevented.

Differential Macrophage Polarization Promotes Tissue Remodeling and Repair in a Model of Ischemic Retinopathy

Diabetic retinopathy is the leading cause of visual loss in individuals under the age of 55. Umbilical cord blood (UCB)–derived myeloid progenitor cells have been shown to decrease neuronal damage associated with ischemia in the central nervous system. In this study we show that UCB-derived CD14+ progenitor cells provide rescue effects in a mouse model of ischemic retinopathy by promoting physiological angiogenesis and reducing associated inflammation. We use confocal microscopy to trace the fate of injected human UCB-derived CD14+ cells and PCR with species-specific probes to investigate their gene expression profile before and after injection. Metabolomic analysis measures changes induced by CD14+ cells. Our results demonstrate that human cells differentiate in vivo into M2 macrophages and induce the polarization of resident M2 macrophages. This leads to stabilization of the ischemia-injured retinal vasculature by modulating the inflammatory response, reducing oxidative stress and apoptosis and promoting tissue repair.

Progenitor cells and retinal angiogenesis.

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.