Serine and Lipid Metabolism in Macular Disease and Peripheral Neuropathy.

BACKGROUND: Identifying mechanisms of diseases with complex inheritance patterns, such as macular telangiectasia type 2, is challenging. A link between macular telangiectasia type 2 and altered serine metabolism has been established previously. METHODS: Through exome sequence analysis of a patient with macular telangiectasia type 2 and his family members, we identified a variant in SPTLC1 encoding a subunit of serine palmitoyltransferase (SPT). Because mutations affecting SPT are known to cause hereditary sensory and autonomic neuropathy type 1 (HSAN1), we examined 10 additional persons with HSAN1 for ophthalmologic disease. We assayed serum amino acid and sphingoid base levels, including levels of deoxysphingolipids, in patients who had macular telangiectasia type 2 but did not have HSAN1 or pathogenic variants affecting SPT. We characterized mice with low serine levels and tested the effects of deoxysphingolipids on human retinal organoids. RESULTS: Two variants known to cause HSAN1 were identified as causal for macular telangiectasia type 2: of 11 patients with HSAN1, 9 also had macular telangiectasia type 2. Circulating deoxysphingolipid levels were 84.2% higher among 125 patients with macular telangiectasia type 2 who did not have pathogenic variants affecting SPT than among 94 unaffected controls. Deoxysphingolipid levels were negatively correlated with serine levels, which were 20.6% lower than among controls. Reduction of serine levels in mice led to increases in levels of retinal deoxysphingolipids and compromised visual function. Deoxysphingolipids caused photoreceptor-cell death in retinal organoids, but not in the presence of regulators of lipid metabolism. CONCLUSIONS: Elevated levels of atypical deoxysphingolipids, caused by variant SPTLC1 or SPTLC2 or by low serine levels, were risk factors for macular telangiectasia type 2, as well as for peripheral neuropathy. (Funded by the Lowy Medical Research Institute and others.).

miR-30a-5p inhibition promotes interaction of Fas+ endothelial cells and FasL+ microglia to decrease pathological neovascularization and promote physiological angiogenesis.

Ischemia-induced angiogenesis contributes to various neuronal and retinal diseases, and often results in neurodegeneration and visual impairment. Current treatments involve the use of anti-VEGF agents but are not successful in all cases. In this study we determined that miR-30a-5p is another important mediator of retinal angiogenesis. Using a rodent model of ischemic retinopathy, we show that inhibiting miR-30a-5p reduces neovascularization and promotes tissue repair, through modulation of microglial and endothelial cell cross-talk. miR-30a-5p inhibition results in increased expression of the death receptor Fas and CCL2, to decrease endothelial cell survival and promote microglial migration and phagocytic function in focal regions of ischemic injury. Our data suggest that miR-30a-5p inhibition accelerates tissue repair by enhancing FasL-Fas crosstalk between microglia and endothelial cells, to promote endothelial cell apoptosis and removal of dead endothelial cells. Finally, we found that miR-30a levels were increased in the vitreous of patients with proliferative diabetic retinopathy. Our study identifies a role for miR-30a in the pathogenesis of neovascular retinal disease by modulating microglial and endothelial cell function, and suggests it may be a therapeutic target to treat ischemia-mediated conditions.

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 (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.

Serine biosynthesis defect due to haploinsufficiency of PHGDH causes retinal disease.

Macular telangiectasia type 2 (MacTel) is a progressive, late-onset retinal degenerative disease linked to decreased serum levels of serine that elevate circulating levels of a toxic ceramide species, deoxysphingolipids (deoxySLs); however, causal genetic variants that reduce serine levels in patients have not been identified. Here we identify rare, functional variants in the gene encoding the rate-limiting serine biosynthetic enzyme, phosphoglycerate dehydrogenase (PHGDH), as the single locus accounting for a significant fraction of MacTel. Under a dominant collapsing analysis model of a genome-wide enrichment analysis of rare variants predicted to impact protein function in 793 MacTel cases and 17,610 matched controls, the PHGDH gene achieves genome-wide significance (P = 1.2 × 10-13) with variants explaining ~3.2% of affected individuals. We further show that the resulting functional defects in PHGDH cause decreased serine biosynthesis and accumulation of deoxySLs in retinal pigmented epithelial cells. PHGDH is a significant locus for MacTel that explains the typical disease phenotype and suggests a number of potential treatment options.

A novel method of screening combinations of angiostatics identifies bevacizumab and temsirolimus as synergistic inhibitors of glioma-induced angiogenesis.

Tumor angiogenesis is critical for the growth and progression of cancer. As such, angiostasis is a treatment modality for cancer with potential utility for multiple types of cancer and fewer side effects. However, clinical success of angiostatic monotherapies has been moderate, at best, causing angiostatic treatments to lose their early luster. Previous studies demonstrated compensatory mechanisms that drive tumor vascularization despite the use of angiostatic monotherapies, as well as the potential for combination angiostatic therapies to overcome these compensatory mechanisms. We screened clinically approved angiostatics to identify specific combinations that confer potent inhibition of tumor-induced angiogenesis. We used a novel modification of the ex ovo chick chorioallantoic membrane (CAM) model that combined confocal and automated analyses to quantify tumor angiogenesis induced by glioblastoma tumor onplants. This model is advantageous due to its low cost and moderate throughput capabilities, while maintaining complex in vivo cellular interactions that are difficult to replicate in vitro. After screening multiple combinations, we determined that glioblastoma-induced angiogenesis was significantly reduced using a combination of bevacizumab (Avastin®) and temsirolimus (Torisel®) at doses below those where neither monotherapy demonstrated activity. These preliminary results were verified extensively, with this combination therapy effective even at concentrations further reduced 10-fold with a CI value of 2.42E-5, demonstrating high levels of synergy. Thus, combining bevacizumab and temsirolimus has great potential to increase the efficacy of angiostatic therapy and lower required dosing for improved clinical success and reduced side effects in glioblastoma patients.

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.

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-1 alpha and HIF-2 alpha, and the negative regulator of HIF alpha, 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 microglia are critical for subretinal neovascular formation.

Abnormal subretinal neovascularization is a characteristic of vision-threatening retinal diseases, including macular telangiectasia (MacTel) and retinal angiomatous proliferation (RAP). Subretinal neovascular tufts and photoreceptor dysfunction are observed in very-low-density lipoprotein receptor (Vldlr-/-) mutant mice. These changes mirror those observed in patients with MacTel and RAP, but the pathogenesis is largely unknown. In this study, we show that retinal microglia were closely associated with retinal neovascular tufts in Vldlr-/- mice and retinal tissue from patients with MacTel; ablation of microglia/macrophages dramatically prevented formation of retinal neovascular tufts and improved neuronal function, as assessed by electroretinography. Vldlr-/- mice with retinal pigmented epithelium-specific (RPE-specific) Vegfa had greatly reduced subretinal infiltration of microglia/macrophages, subsequently reducing neovascular tufts. These findings highlight the contribution of microglia/macrophages to the pathogenesis of neovascularization, provide valuable clues regarding potential causative cellular mechanisms for subretinal neovascularization in patients with MacTel and RAP and suggest that targeting microglia activation may be a therapeutic option in these 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.

Mechanisms of endothelial cell guidance and vascular patterning in the developing mouse retina.

The appropriate guidance and patterning of vessels during vascular development is critical for proper tissue function. The loss of these guidance mechanisms can lead to abnormal vascularization and a number of pathological conditions. The molecular basis of endothelial cell guidance and subsequent tissue specific vascular patterning remains largely unknown in spite of its clinical relevance and biological importance. In this regard, retinal vascular development offers many advantages for studying endothelial cell guidance and the mechanisms by which characteristic vascular patterns are formed. In this review, we will provide an overview of the known mechanisms that mediate vascular patterning during mouse retinal development, synthesizing these data to formulate a model of how growth factors, cellular adhesion molecules, and vascular-associated cells mediate directed endothelial cell migration and appropriate vascular remodeling. Finally, we will discuss the many aspects of retinal vascular development that remain unknown and cite evidence that many of these gaps may be addressed by further studying the guidance cues shared by vascular and neuronal elements in the retina and other parts of the central nervous system.

T2-TrpRS inhibits preretinal neovascularization and enhances physiological vascular regrowth in OIR as assessed by a new method of quantification.

PURPOSE: A carboxyl-terminal fragment of tryptophan tRNA synthetase (T2-TrpRS) has demonstrated potent angiostatic activity during retinal developmental neovascularization in vivo. The effects of T2-TrpRS on pathologic neovascularization were tested and compared with a potent VEGF antagonist using the mouse model of oxygen-induced retinopathy (OIR). METHODS: C57BL/6J mice were transiently exposed to hyperoxic conditions (75% O2) between postnatal day 7 (P7) and P12 and then returned to room air. Retinas were isolated, blood vessels stained with isolectin Griffonia simplicifolia, images of retinal whole-mounts acquired, and the area of vascular obliteration and extent of preretinal neovascularization quantified. This method was compared to the commonly used method of OIR quantification in which the number of pre-inner limiting membrane (ILM) nuclei is counted in serial sections of whole eyes. To assess the angiostatic activity of T2-TrpRS, mice were injected intravitreally at P12 with either T2-TrpRS, a VEGF aptamer, or vehicle (PBS) alone, and the effects on area of obliteration and on preretinal neovascular tuft formation were assessed. RESULTS: Using a modified method of quantification in the mouse OIR model based on images of isolectin-stained retinal wholemounts, we were able to assess reliably and consistently both vascular obliteration and preretinal neovascular tuft formation in the same specimen. T2-TrpRS demonstrated potent angiostatic activity, reducing the appearance of pathologic neovascular tufts by up to 90%. Surprisingly, T2-TrpRS also enhanced physiological revascularization of the obliterated retinal vasculature, reducing these areas by up to 60% compared with PBS-injected eyes. In contrast, the VEGF antagonist, while similarly reducing preretinal neovascular tuft formation, did not enhance revascularization of the obliterated areas. CONCLUSIONS: Use of a rapid, quantifiable method to assess the effect of T2-TrpRS on retinal angiogenesis in the OIR model demonstrates the importance of a quantification system that permits simultaneous analysis of a drug's effect on vascular obliteration as well as on preretinal neovascularization. The results obtained using this method suggest enhanced clinical value for compounds such as T2-TrpRS that not only inhibit pathologic neovascularization, but also facilitate physiological revascularization of ischemic tissue.

Angiogenesis and Eye Disease.

The retina consists of organized layers of photoreceptors, interneurons, glia, epithelial cells, and endothelial cells. The economic model of supply and demand used to appropriately determine cost is highly applicable to the retina, in which the extreme metabolic demands of phototransduction are met by precisely localized and designed vascular networks. Proper development and maintenance of these networks is critical to normal visual function; dysregulation is characteristic of several devastating human diseases, including but not limited to age-related macular degeneration and diabetic retinopathy. In this article, we focus on the lessons learned from the study of retinal vascular development and how these lessons can be used to better maintain adult vascular networks and prevent retinal diseases. We then outline the vasculotrophic contributions from neurons, retinal pigment epithelium (RPE) cells, and glia (specifically microglia) before we shift our focus to pathology to provide molecular contexts for neovascular retinal diseases. Finally, we conclude with a discussion that applies what we have learned about how retinal cells interact with the vasculature to identify and validate therapeutic approaches for neurovascular disease of the retina.

Neurovascular crosstalk between interneurons and capillaries is required for vision.

Functional interactions between neurons, vasculature, and glia within neurovascular units are critical for maintenance of the retina and other CNS tissues. For example, the architecture of the neurosensory retina is a highly organized structure with alternating layers of neurons and blood vessels that match the metabolic demand of neuronal activity with an appropriate supply of oxygen within perfused blood. Here, using murine genetic models and cell ablation strategies, we have demonstrated that a subset of retinal interneurons, the amacrine and horizontal cells, form neurovascular units with capillaries in 2 of the 3 retinal vascular plexuses. Moreover, we determined that these cells are required for generating and maintaining the intraretinal vasculature through precise regulation of hypoxia-inducible and proangiogenic factors, and that amacrine and horizontal cell dysfunction induces alterations to the intraretinal vasculature and substantial visual deficits. These findings demonstrate that specific retinal interneurons and the intraretinal vasculature are highly interdependent, and loss of either or both elicits profound effects on photoreceptor survival and function.

Retinal vascular development is mediated by endothelial filopodia, a preexisting astrocytic template and specific R-cadherin adhesion.

PURPOSE: A neonatal mouse retina developmental model was used to study endothelial cell guidance and subsequent formation of vascular patterns. Since most diseases that cause catastrophic loss of vision do so as a result of abnormal ocular angiogenesis, a better understanding of events regulating normal retinal vascular growth may provide insight into pathologic angiogenesis. METHODS: Development of the retinal vasculature at various postnatal and embryonic time points was analyzed by collagen IV immunohistochemistry and staining with isolectin Griffonia simplicifolia. GFAP-GFP transgenic mice were used to evaluate the relationship between developing vessels and retinal glial cells. Immunolocalization of R-cadherin and intravitreous injection of R-cadherin-specific antibodies was performed to determine the role of R-cadherin during patterning of the superficial and deep retinal vascular plexuses. RESULTS: The characteristic honeycomb pattern of vessel formation observed in the superficial layer is a result of endothelial cell migration over a preexisting astrocytic template. Filopodial extensions associate with underlying astrocytes by protruding from the tips of endothelial cells at the migrating vascular front. Branching of vessels in the primary vascular plexus, as well as appropriate localization of the deep vascular network is mediated by R-cadherin, an adhesion molecule known to be involved in neuronal cell guidance. Injection of antibodies directed against R-cadherin prevents the normally extensive collateralization observed during formation of the superficial network. Injection of anti-R cadherin antibodies also dramatically affects vessels of the deep network. These vessels migrate beyond the normal turning point, penetrating into the deeper photoreceptor layer. CONCLUSIONS: . These studies suggest that angiogenesis and formation of vascular patterns in the retina may use many of the same developmental cues used by neurons in both the central and peripheral nervous systems. Furthermore, retinal vascular endothelial cell guidance mediated by filopodial extensions and neuronal guidance cues may represent a novel conceptual framework within which to study the establishment of vascular patterns in a variety of angiogenic systems.

Global gene expression analysis of the developing postnatal mouse retina.

PURPOSE: Postnatal mouse retinal development involves glial and neuronal differentiation, vascularization, and the onset of vision. In the current study, the gene expression profiles of thousands of genes in the developing postnatal mouse retina were analyzed and compared in a large-scale, unbiased microarray gene expression analysis. METHODS: For each of eight different time points during postnatal mouse retinal development, two separate sets of 30 retinas were pooled for RNA isolation, and gene expression was analyzed by hybridization to gene chips in triplicate (Mu74Av2; Affymetrix, Santa Clara, CA). Genes were sorted into clusters based on their expression profiles and intensities. Validation was accomplished by comparing the microarray expression profiles with real-time RT-PCR analysis of selected genes and by comparing selected expression profiles with predicted profiles based on previous studies. RESULTS: The Mu74Av2 chip contains more than 6000 known genes and 6500 estimated sequence tags (ESTs) from the mouse Unigene database. Of these, 2635 known gene sequences and 2794 ESTs were expressed at least threefold above background levels during retinal development. Expressed genes were clustered based on expression profiles allowing potential functions for specific genes during retinal development to be inferred by comparison to developmental events occurring at each time point. Specific data and potential functions for genes with various profiles are discussed. All data can be viewed online at http://www.scripps.edu/cb/friedlander/gene_expression/. CONCLUSIONS: Expression analysis of thousands of different genes during normal postnatal mouse retinal development as reported in this study demonstrates that such an approach can be used to correlate gene expression with known functional differentiation, presenting the opportunity to infer functional correlates between gene expression and specific postnatal developmental events.

Identifying potential regulators of infantile hemangioma progression through large-scale expression analysis: a possible role for the immune system and indoleamine 2,3 dioxygenase (IDO) during involution.

Hemangiomas are benign endothelial tumors. Often referred to as hemangiomas of infancy (HOI), these tumors are the most common tumor of infancy. Most of these lesions proliferate rapidly in the first months of life, and subsequently slowly involute during early childhood without significant complications. However, they often develop on the head or neck, and may pose a significant cosmetic concern for families. In addition, a fraction of these tumors can grow explosively and ulcerate, bleed, or obstruct vision or airway structures. Current treatments for these tumors are associated with significant side effects, and our knowledge of the biology of hemangiomas is limited. The natural evolution of these lesions creates a unique opportunity to study the changes in gene expression that occur as the endothelium of these tumors proliferates and then subsequently regresses. Such information may also increase our understanding of the basic principals of angiogenesis in normal and abnormal tissue. We have performed large-scale genomic analysis of hemangioma gene expression using DNA microarrays. We recently identified insulin-like growth factor 2 as a potentially important regulator of hemangioma growth using this approach. However, little is known about the mechanisms involved in hemangioma involution. Here we explore the idea that hemangioma involution might be an immune-mediated process and present data to support this concept. We also demonstrate that proliferating hemangiomas express indoleamine 2,3 dioxygenase (IDO) and discuss a possible mechanism that accounts for the often slow regression of these lesions.

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.

Chapter 6. Ocular models of angiogenesis.

During normal retinal vascular development, vascular endothelial cells proliferate and migrate through the extracellular matrix in response to a variety of cytokines, leading to the formation of new blood vessels in a highly ordered fashion. However, abnormal angiogenesis contributes to the vast majority of diseases that cause catastrophic loss of vision. During abnormal neovascularization of the iris, retina, or choroid, angiogenesis is unregulated and usually results in the formation of dysfunctional blood vessels. Multiple models of ocular angiogenesis exist which recapitulate particular aspects of both normal and pathological neovascularization. These experimental methods are useful for studying the mechanisms of normal developmental angiogenesis, as well as studying various aspects of pathological angiogenesis including ischemic retinopathies, vascular leak, and choroidal neovascularization. This chapter will outline several protocols used to study ocular angiogenesis, put the protocols into brief historical context, and describe some of the questions for which these protocols are commonly used.

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.

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.