Angiogenesis depends on cytokines and vascular cell adhesion events. Two cytokine-dependent pathways of angiogenesis were shown to exist and were defined by their dependency on distinct vascular cell integrins. In vivo angiogenesis in corneal or chorioallantoic membrane models induced by basic fibroblast growth factor or by tumor necrosis factor-alpha depended on alpha v beta 3, whereas angiogenesis initiated by vascular endothelial growth factor, transforming growth factor-alpha, or phorbol ester depended on alpha v beta 5. Antibody to each integrin selectively blocked one of these pathways, and a cyclic peptide antagonist of both integrins blocked angiogenesis stimulated by each cytokine tested. These pathways are further distinguished by their sensitivity to calphostin C, an inhibitor of protein kinase C that blocked angiogenesis potentiated by alpha v beta 5 but not by alpha v beta 3.

Angiogenesis depends on both cell adhesion and proteolytic mechanisms. In fact, matrix metalloproteinase 2 (MMP-2) and integrin alphavbeta3 are functionally associated on the surface of angiogenic blood vessels. A fragment of MMP-2, which comprises the C-terminal hemopexin-like domain, termed PEX, prevents this enzyme binding to alphavbeta3 and blocks cell surface collagenolytic activity. PEX blocks MMP-2 activity on the chick chorioallantoic membrane where it disrupts angiogenesis and tumor growth. Importantly, a naturally occurring form of PEX can be detected in vivo in conjunction with alphavbeta3 expression in tumors and during developmental retinal neovascularization. Levels of PEX in these vascularized tissues suggest that it interacts with endothelial cell alphavbeta3 where it serves as a natural inhibitor of MMP-2 activity, thereby regulating the invasive behavior of new blood vessels.

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.

Aminoacyl-tRNA synthetases catalyze the first step of protein synthesis. It was shown recently that human tyrosyl-tRNA synthetase (TyrRS) can be split into two fragments having distinct cytokine activities, thereby linking protein synthesis to cytokine signaling pathways. Tryptophanyl-tRNA synthetase (TrpRS) is a close homologue of TyrRS. A natural fragment, herein designated as mini TrpRS, was shown by others to be produced by alternative splicing. Production of this fragment is reported to be stimulated by IFN-␥, a cytokine that also stimulates production of angiostatic factors. Mini TrpRS is shown here to be angiostatic in a mammalian cell culture system, the chicken embryo, and two independent angiogenesis assays in the mouse. The full-length enzyme is inactive in the same assays. Thus, protein synthesis may be linked to the regulation of angiogenesis by a natural fragment of TrpRS.

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).