The long-term objectives of this project are to elucidate the biological functions of vascular permeability factor (VPF) and, in particular, to define the significance of VPF for tumor biology.
The Specific Aims of this proposal are 1) to further define VPF structure, including complete amino acid sequence (as deduced from cDNA sequence), and to derive antibodies to synthetic peptides which block VPF activity; 2) to study expression of VPF and VPF mRNA by a wide variety of tumor and normal cells in vivo and in vitro; and 3) to study the long-term effects of VPF on tumor growth, fluid accumulation and endothelium in vivo. cDNA inserts encoding human and guinea pig VPF from lambda-gt11 clones (identified with antibodies to VPF N-terminal peptide) will be subcloned and sequenced, and from these nucleotide sequences, complete VPF amino acid sequence will be derived. Specific peptides (approximately 25 amino acids), representing limited portions of the entire VPF sequence, will be used as immunogens to obtain antibodies which recognize epitopes throughout the VPF molecule. Anti-peptide antibodies which neutralize VPF activity will allow for identification of important functional sites within the molecule. Expression of VPF and VPF mRNA by a wide variety of tumor and normal cells in vivo and in vitro will be studied with highly sensitive methods. Expression of VPF mRNA in vivo will be detected with in situ hybridization. Expression of VPF activity by cells in vitro will be detected with an endothelial cell/cytosolic calcium assay, which is at least 300-times more sensitive than both the Miles dermal vessel permeability assay and our currently available conventional immunoassays for VPF. Comparisons (in vitro) between expression of VPF activity and cellular VPF mRNA levels will be made in conjunction with Northern blotting. Finally, the biological effects of VPF in vivo will be characterized with particular emphasis on determining the significance of VPF expression for tumor growth and fluid accumulation in the peritoneal cavity. To determine the effects of VPF on tumor growth and fluid accumulation, animals bearing ascites tumors will receive repeated intraperitoneal injections of VPF-neutralizing antibodies. To determine the effects of purified VPF (in the absence of tumor) on the vessels and other tissues of the peritoneal wall, animals will receive frequently-repeated injections of purified VPF. The goal will be to determine if any of the profound changes that commonly occur in the peritoneal wall of ascites tumor-bearing animals (e.g., edema, fibrin deposition, angiogenesis, and fibrosis) occur in response to frequently repeated administration of purified VPF.
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