Vascular endothelial growth factor (VEGF) is a highly specific mitogen for vascular endothelial cells. Five VEGF isoforms are generated as a result of alternative splicing from a single VEGF gene. These isoforms differ in their molecular mass and in biological properties such as their ability to bind to cell-surface heparan-sulfate proteoglycans. The expression of VEGF is potentiated in response to hypoxia, by activated oncogenes, and by a variety of cytokines. VEGF induces endothelial cell proliferation, promotes cell migration, and inhibits apoptosis. In vivo VEGF induces angiogenesis as well as permeabilization of blood vessels, and plays a central role in the regulation of vasculogenesis. Deregulated VEGF expression contributes to the development of solid tumors by promoting tumor angiogenesis and to the etiology of several additional diseases that are characterized by abnormal angiogenesis. Consequently, inhibition of VEGF signaling abrogates the development of a wide variety of tumors. The various VEGF forms bind to two tyrosine-kinase receptors, VEGFR-1 (flt-1) and VEGFR-2 (KDR/flk-1), which are expressed almost exclusively in endothelial cells. Endothelial cells express in addition the neuropilin-1 and neuropilin-2 coreceptors, which bind selectively to the 165 amino acid form of VEGF (VEGF165). This review focuses on recent developments that have widened considerably our understanding of the mechanisms that control VEGF production and VEGF signal transduction and on recent studies that have shed light on the mechanisms by which VEGF regulates angiogenesis.

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Vascular endothelial growth factor (VEGF) was discovered 10 years ago as a growth factor that can regulate angiogenesis and in addition the permeability of blood vessels. Numerous studies have revealed that it is essential for normal embryonic development and that it plays a major role in physiological and pathological events of angiogenesis in adults. It is unique in that its expression is regulated directly by hypoxia. These properties are now being exploited in attempts aimed at the induction of new blood vessels in pathological situations such as ischemic heart disease. Five VEGF forms of 121 to 206 aminoacids are produced from a single gene by alternative splicing. Cells expressing VEGF usually express several forms simultaneously. VEGF121 does not contain exons 6 and 7 of the gene and consequently lacks a heparin binding ability. However, this form is fully active as an inducer of angiogenesis, and as a blood vessel permeabilizing agent. Exon 6 and 7 contain 2 independent heparin binding domains. The VEGF form containing exon 7 (VEGF165) and the vascular endothelial growth factor form containing exon 6 (VEGF145) display similar biological potencies raising the question of why so many VEGF forms are required. It was found that VEGF121 diffuses better because it does not bind to heparan-sulfate proteoglycans. In contrast, VEGF145 binds to extracellular matrix and is released from it slowly. When the receptor binding properties of VEGF121 and VEGF165 were compared it was found that VEGF165 binds to a class of VEGF receptors that is not recognized by VEGF121. These receptors are encoded by the neuropilin-1 gene, and we have recently found that the related neuropilin-2 gene also encodes a VEGF165 receptor. We have recently found evidence indicating the neuropilins form complexes with another VEGF receptor, VEGFR-1. However, the biological function of this complex remains to be elucidated.

Recombinant human interleukin-12 (rHuIL-12, HemaMaxTM) is being developed for mitigation of HSARS under the FDA Animal Rule using a NHP model of HSARS for proof of efficacy and clinical studies in healthy human subjects to demonstrate safety. We have shown previously that a single injection of rHuIL-12 administered 24-25 hours after lethal total body irradiaton (TBI), in the absence of antibiotics, fluids or blood products, resulted in improved survival while filgrastim (G-CSF) did not provide any survival benefit in our NHP HSARS model (Basile et al 2012, Gluzman-Poltorak et al 2014, Gluzman-Poltorak et al 2014). The mechanism by which IL-12 mitigates HSARS following TBI appears to involve multiple effects of IL-12 on hematopoieses. In our previous studies, animals treated with rHuIL-12 showed statistically significant reductions in the occurrence of severe neutropenia and severe thrombocytopenia, as well as attenuated nadirs for lymphocytes, neutrophils, platelets, and reticulocytes. To characterize further relationship between survival and hematological nadirs (lymphocytes, neutrophils, platelets, RBCs and reticulocytes) in the rhesus model of HSARS and to better understand the effects of rHuIL-12 versus G-CSF on blood cell nadirs, we undertook a meta-analysis analysis across three studies in irradiated rhesus monkeys. Animals used in this analysis were irradiated (700 cGy) and treated with a single subcutaneous injection of vehicle (n=64) or rHuIL-12 50-500ng/kg (n=108) 24-25 hours after irradiation, or daily subcutaneous injections of G-CSF at 10μg/kg/d for 18 days starting 24-25 hours after irradiation (n=26). Males and females were equal in each group. Lymphocytes, neutrophils and platelets were significantly lower in decedents versus monkeys that survived to day 60 overall and this was true in each treatment group (p<0.001, Wilcoxon rank-sum test). The mean RBC nadir was significantly higher in survivors compared to non-survivors in the rHuIL-12 group (p = 0.008), but not in the control group or the G-CSF group. Lymphocytes nadir appears to be the strongest and most consistent predictor of death followed by neutrophils and platelets (Spearman’s rank correlation). RBCs and reticulocytes are less informative in terms of predicting survival status. In addition, the receiver operating characteristic (ROC) curve area under the curve (AUC) by nadir value was assessed. The operating characteristics for lymphocytes, neutrophils and platelets allow for relatively large positive predictive values (PPV) of death with relatively high sensitivity. A cutoff value for lymphocytes nadir of 0.08x109/L (values less than or equal predicting death and higher values predicting life) allows for largest PPVs (97.2% and 92.5%) with 76.1% and 62.7% sensitivities for control and rHuIL-12 treatments, respectively. A cutoff value for neutrophils nadir of 0.03x109/L permits for 84% and 71.1% PPVs with sensitivities of 91.3% and 91.5 % for control and rHuIL-12 treatments, respectively. A cutoff value for platelets nadir of 9x109/L permits for 84.1% and 76.8% PPVs with sensitivities of 80.4% and 72.9 % for control and rHuIL-12 treatments, respectively. RBCs and reticulocytes were found less informative. To conclude, in the rhesus model of HSARS we have observed that an augmented hematological nadirs generally predicts an increased potential for survival as this effect reflects early bone marrow regeneration. The nadir for lymphocytes appears to be the strongest and most consistent predictor of death. Decrease of lymphocyte counts has been established as a best marker of bone marrow damage in a large database of human victims of acute radiation (METREPOL, Fliedner et al 2001). Thus, the correlation of our results with the human data supports the validity of our animal model as an accurate representation of human HSARS and its ability to predict effectiveness in humans exposed to lethal radiation. These data also suggest that the significant increase in early bone marrow regeneration seen in our studies, resulting in increases in nadir values for all major blood cell types, may be the main mechanism of action by which rHuIL-12 mitigates the lethality of HSARS. This project has been entirely funded with Federal funds from BARDA/ASPR/DHHS under Contract No. HHSO100201100037C. Disclosures Gluzman Poltorak: Neumedicines Inc.: Employment, Equity Ownership. Vladimir:Neumedicines Inc.: Consultancy, Employment, Equity Ownership. Basile:Neumedicines Inc.: Employment, Equity Ownership, Neumedicines Inc. Patents & Royalties.