Michael I. Dorrell

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.

Rescue of retinal degeneration by intravitreally injected adult bone marrow–derived lineage-negative hematopoietic stem cells

Inherited retinal degenerations afflict 1 in 3,500 individuals and are a heterogeneous group of diseases that result in profound vision loss, usually the result of retinal neuronal apoptosis. Atrophic changes in the retinal vasculature are also observed in many of these degenerations. While it is thought that this atrophy is secondary to diminished metabolic demand in the face of retinal degeneration, the precise relationship between the retinal neuronal and vascular degeneration is not clear. In this study we demonstrate that whenever a fraction of mouse or human adult bone marrow-derived stem cells (lineage-negative hematopoietic stem cells [Lin- HSCs]) containing endothelial precursors stabilizes and rescues retinal blood vessels that would ordinarily completely degenerate, a dramatic neurotrophic rescue effect is also observed. Retinal nuclear layers are preserved in 2 mouse models of retinal degeneration, rd1 and rd10, and detectable, albeit severely abnormal, electroretinogram recordings are observed in rescued mice at times when they are never observed in control-treated or untreated eyes. The normal mouse retina consists predominantly of rods, but the rescued cells after treatment with Lin- HSCs are nearly all cones. Microarray analysis of rescued retinas demonstrates significant upregulation of many antiapoptotic genes, including small heat shock proteins and transcription factors. These results suggest a new paradigm for thinking about the relationship between vasculature and associated retinal neuronal tissue as well as a potential treatment for delaying the progression of vision loss associated with retinal degeneration regardless of the underlying genetic defect.

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.

Insulin-like growth factor 2 and potential regulators of hemangioma growth and involution identified by large-scale expression analysis

Hemangiomas are benign tumors of the vascular endothelium and are the most common tumors of infancy. These tumors are characterized by an initial phase of rapid proliferation, which is followed, in most cases, by spontaneous involution. Although most lesions resolve without complication, there are some cases in which hemangiomas can be life threatening when occurring near a vital structure. Treatment for these aggressive tumors represents an unmet clinical need. In addition, this characteristic progression of hemangiomas through distinct phases provides a unique opportunity for studying endothelial cell biology and angiogenesis. Using DNA microarrays representing approximately 10,000 human genes, we identified insulin-like growth factor 2 (IGF-2) as a potentially important regulator of hemangioma growth. IGF-2 was highly expressed during the proliferative phase and substantially decreased during involution. This finding was confirmed at the message level by quantitative reverse transcription–PCR and at the protein level by immunohistochemistry. IGF-2 protein was localized primarily to tumor vessels or vascular channels. Using a human hemangioma explant model, we show that IGF-2 promotes sprouting from intact hemangioma tissue. In addition, several angiogenesis-related factors, including integrins αvβ3 and α5β1, are present in proliferating hemangiomas. During the involuting phase, an increase in several IFN-induced genes was observed. These studies identify potential regulators of hemangioma growth and involution and provide a foundation on which to build further mechanistic investigations into angiogenesis, using hemangiomas as a model.

A fragment of human TrpRS as a potent antagonist of ocular angiogenesis

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.

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.

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.

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.

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.

Evaluating the binding selectivity of transthyretin amyloid fibril inhibitors in blood plasma

Transthyretin (TTR) tetramer dissociation and misfolding facilitate assembly into amyloid fibrils that putatively cause senile systemic amyloidosis and familial amyloid polyneuropathy. We have previously discovered more than 50 small molecules that bind to and stabilize tetrameric TTR, inhibiting amyloid fibril formation in vitro. A method is presented here to evaluate the binding selectivity of these inhibitors to TTR in human plasma, a complex biological fluid composed of more than 60 proteins and numerous small molecules. Our immunoprecipitation approach isolates TTR and bound small molecules from a biological fluid such as plasma, and quantifies the amount of small molecules bound to the protein by HPLC analysis. This approach demonstrates that only a small subset of the inhibitors that saturate the TTR binding sites in vitro do so in plasma. These selective inhibitors can now be tested in animal models of TTR amyloid disease to probe the validity of the amyloid hypothesis. This method could be easily extended to evaluate small molecule binding selectivity to any protein in a given biological fluid without the necessity of determining or guessing which other protein components may be competitors. This is a central issue to understanding the distribution, metabolism, activity, and toxicity of potential drugs.