Aptamers recognize their targets with extraordinary affinity and specificity. The aptamer-based therapeutic, Macugen, is derived from a modified 2 fluoro pyrimidine RNA inhibitor to vascular endothelial growth factor (VEGF) and is now being used to treat the wet form of age-related macular degeneration. This VEGF 165 aptamer binds specifically to the VEGF165 isoform, a dimeric protein with a receptor-binding domain and a heparin-binding domain (HBD). To understand the molecular recognition between VEGF and this aptamer, binding experiments were used to show that the HBD contributes the majority of binding energy in the VEGF 165-aptamer complex. A tissue culture-based competition assay demonstrated that the HBD effectively competes with VEGF165 for aptamer binding in vivo. Comparison of NMR spectra revealed that structural features of the smaller HBD-aptamer complex are present in the full-length VEGF 164-aptamer complex. These data show that the HBD provides the binding site for the aptamer and is the primary determinant for the affinity and specificity in the VEGF 165-aptamer complex.age-related macular degeneration ͉ Macugen ͉ RNA ͉ NMR

The longer splice isoforms of vascular endothelial growth factor-A (VEGF-A), including mouse VEGF164, contain a highly basic heparin-binding domain (HBD), which imparts the ability of these isoforms to be deposited in the heparan sulfate-rich extracellular matrix and to interact with the prototype sulfated glycosaminoglycan, heparin. The shortest isoform, VEGF120, lacks this highly basic domain and is freely diffusible upon secretion. Although the HBD has been attributed significant relevance to VEGF-A biology, the molecular determinants of the heparin-binding site are unknown. We used site-directed mutagenesis to identify amino acid residues that are critical for heparin binding activity of the VEGF164 HBD. We focused on basic residues and found Arg-13, Arg-14, and Arg-49 to be critical for heparin binding and interaction with extracellular matrix in tissue samples. We also examined the cellular and biochemical consequences of abolishing heparin-binding function, measuring the ability of the mutants to interact with VEGF receptors, induce endothelial cell gene expression, and trigger microvessel outgrowth. Induction of tissue factor expression, vessel outgrowth, and binding to VEGFR2 were unaffected by the HBD mutations. In contrast, the HBD mutants showed slightly decreased binding to the NRP1 (neuropilin-1) receptor, and analyses suggested the heparin and NRP1 binding sites to be distinct but overlapping. Finally, mutations that affect the heparin binding activity also led to an unexpected reduction in the affinity of VEGF164 binding specifically to VEGFR1. This finding provides a potential basis for previous observations suggesting enhanced potency of VEGF164 versus VEGF120 in VEGFR1-mediated signaling in inflammatory cells.

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The longer splice isoforms of VEGF (vascular endothelial growth factor)-A, including VEGF(164(165)), contain a highly basic HBD (heparin-binding domain). This domain allows these isoforms to interact with and localize to the HS (heparan sulfate)-rich extracellular matrix, and bind to the co-receptor Nrp-1 (neuropilin-1). Heparin-binding VEGF-A isoforms are critical for survival: mice engineered to express exclusively the non-heparin-binding VEGF(120) have diminished vascular branching during embryonic development and die from postnatal angiogenesis defects shortly after birth. Although it is thought that the HBD contributes to the diverse functions of VEGF-A in both physiological and pathological processes, little is known about the molecular features within this domain that enable these functions. In the present paper, we discuss the roles of the VEGF HBD in normal and disease conditions, with a particular focus on the VEGF(164(165)) isoform.

The human leukemic T-cell line Jurkat was used to define the role of the cellular stress pathway with its key player kinase JNK in cancer therapy-induced apoptosis. JNK activity was inhibited by stable transfection with a dominant negative mutant of the upstream kinase JNKK/MKK4 or with the novel, potent and selective JNK1, -2 and -3 inhibitor SP600125. Inhibition of JNK activity delayed the onset of apoptosis induced by cisplatin, doxorubicin, ␥-irradiation and CD95-L but did not prevent apoptosis per se. Early events during apoptosis such as induction of CD95-L, activation of caspase-8 and exposure of phosphatidylserine on the cell surface were strongly inhibited. Also, at early time points of apoptosis, loss of the mitochondrial membrane potential and release of cytochrome c were markedly impaired. However, late signaling events during apoptosis such as cleavage of PARP and DNA fragmentation apoptosis were only marginally affected. These findings are in accordance with the activity of initiator and effector caspases. Whereas activity of the initiator caspase-8 was strongly inhibited early and late after induction, an inhibition of caspase-3 activity was only observed early after induction of apoptosis. We therefore suggest that cellular stress signaling contributes to the initiation of apoptosis, whereas it might be dispensable for the progression of apoptosis. Dysfunction of this pathway under pathological conditions might contribute to therapy resistance of cancer cells.

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Purpose Our research strategy has been to study VEGF‐A and the adaptive response to ischemia, with the aim of better understanding the benefit that VEGF‐A may bring to ischemic tissues, and to characterize the basis for the chaotic and poorly controlled neovascular response that usually accompanies ischemia in retinal diseases such as diabetic retinopathy. Methods Mice genetically engineered with deletion of the VEGF164 isoform were examined during normal vascular development of the retina and following oxygen‐induced retinopathy. Abnormal vascular growth and leukostasis were quantified. To study determinants of VEGF‐A involved in leukostasis, VEGF‐A mutants and VEGF‐A signaling antagonists were injected intraocularly Results Rather than focusing on the abolition of VEGF‐A signaling in ischemic retinal disease, we have tried to find ways to normalize the adaptive response. We previously demonstrated that inflammation was critical for the abnormal vascular response in OIR. Here we found that inflammation was driven by VEGF164, and that a cluster of residues in the heparin‐binding domain of VEGF164 were responsible for its heightened inflammatory activity compared to other VEGF‐A isoforms. This region of VEGF164 imparts high affinity binding to VEGFR1 Conclusion Data suggest that reducing VEGF‐VEGFR1 mediated inflammation, whilst preserving VEGF‐mediated angiogenesis, may be a strategy to help transform the unwanted pathological response to ischemia into a desirable outcome whereby new vessels sprout into the area of need and nurture the ischemic retina. Commercial interest