Hua, PhiloGene Inc. (IC50, 8 pg/vision). Subcutaneous administration of 100 g Rabbit Polyclonal to FA12 (H chain, Cleaved-Ile20) twice a week also inhibited fluorescein leakage and neovascularization and reduced lesion size. Conclusions. These results show that VEGF-A165b is usually a potent antiangiogenic agent in a mouse model of age-related macular degeneration and suggest that increasing the ratio of antiangiogenic-to-proangiogenic isoforms may be therapeutically effective in this condition. Ocular neovascularization (ONV) is the leading cause of blindness in the western world. In age-related macular degeneration (AMD), choroidal neovascularization (CNV) affects 10% of all patients and is the most severe and rapidly progressing form of the disease. In wet or neovascular AMD, there is rapid, devastating loss of central vision with rarely any symptoms until the later stages. Examination of pathologic specimens has demonstrated that there is an abnormal proliferation of choroidal vessels beneath the retina, which subsequently bleed, resulting in fibrosis and macular scarring. VEGF-A has been shown to be substantially upregulated in AMD, 1 particularly in retinal pigment epithelial cells and fibroblasts.2 ONV seen in AMD is associated with increased levels of VEGF. Angiogenesis is usually a complex process mediated by factors from the VEGF, angiopoietin, and ephrin families.3,4 It is essential in normal physiology, such as in wound healing, endometrial maturation, embryogenesis, and fat deposition. However, it also underlies many disease says in addition to AMD, including retinal and other complications of diabetes, malignancy, atherosclerosis,5 rheumatoid arthritis, and psoriasis.6,7 VEGF-A is the dominant proangiogenic factor in AMD,8,9 stimulating endothelial cell proliferation and migration and increased microvascular permeability by activation of its cognate receptors VEGFR1 (flt-1) and VEGFR2 (KDR/flk1).10 Anti-VEGF therapies that influence new vessel formation and that were shown to be effective in animal models11,12 have successfully completed clinical trials in AMD, and three agents have been widely used: pegaptanib (Macugen; Pfizer, New York, NY),13,14 an RNA aptamer that targets the heparin-binding domain name of VEGF-A, ranibizumab (Lucentis; Genentech, South San Francisco, CA), an antibody fragment to the VEGFR binding domain name of VEGF-A, and bevacizumab (Avastin; Genentech), the full-length antibody equivalent to ranibizumab. Other members of the VEGF family of proteins, (e.g., VEGF-C, VEGF-D) are formed from different gene products and are structurally distinct from VEGF-A (see Ref. 15 for review). Human VEGF-A is usually differentially spliced from 8 exons to form a variety of different mRNAs encoding at least 14 different proteins in two Mephenesin families, the proangiogenic Mephenesin VEGF-Axxx family and the antiangiogenic VEGF-Axxxb family, where xxx denotes the number of amino acids of the secreted isoform, VEGF-A121, VEGF-A165, (the dominant proangiogenic isoform) VEGF-A165b, as well as others.15,16 VEGF-Axxxb isoforms are formed by alternate splice acceptor site selection in exon 8, forming an mRNA containing 18 bases coded by exon 8b in place of the 18 bases of exon 8a.17 This alternative splicing produces proteins of the same length as in the VEGF-Axxx family but with a different C-terminal amino acid sequence.18 Exons 8a and 8b both code for six amino acids, exon 8a for CDKPRR and exon 8b for SLTRKD. Therefore, exon 8b lacks the Cys residue that forms the final disulfide bond19 and the terminal two charged Arg residues postulated to be involved with receptor signaling.20,21 Instead, exon 8b codes for Ser instead of Cys and a less basic C-terminal than exon 8a. The receptor binding domains are still present in VEGF-A165b, which acts as a competitive inhibitor of VEGF-A165 (i.e., it binds the receptors but does not stimulate angiogenesis signaling). VEGF-A165b inhibits the proliferative, migratory, and vasodilator effects of VEGF-A165.17 VEGF-A165b is antiangiogenic in the rabbit cornea, chick chorioallantoic membrane, mouse skin,22 lactating mammary gland,23 and rat mesentery, and it inhibits tumor growth in xenotransplanted tumors in mice.24C26 Unlike angiogenic VEGF isoforms, the antiangiogenic isoform VEGF-A165b is downregulated in renal and colorectal carcinoma and malignant prostate tissue,17,25,27 metastatic melanoma,28 diabetic retinopathy,18 and Denys-Drash syndrome.29 We have also identified VEGF-A165b protein expression in many other human tissues, including the eye.18 To determine whether the antiangiogenic activity of VEGF-A165b was sufficient to inhibit CNV, we used a laser-induced photocoagulation model of CNV in Mephenesin the mouse. We show here that VEGF-A165b dose dependently inhibited CNV as assessed by fluorescein angiography (FA), lectin staining, and lesion size. Materials and Methods Protein Extraction Protein was extracted from human eyes obtained from the Bristol Vision Lender (Bristol, UK) with local ethics.
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