Tumor growth beyond minimal size requires generation of new blood vessels and other stroma to provide a lifeline for tumor sustenance and waste disposal. Despite its importance for both primary and metastatic tumor growth, the mechanisms of tumor angiogenesis are poorly understood, though there is general agreement that tumor-secreted products play a central role. Tumor blood vessels are lined by endothelial cells (ECs) that differ from those of normal adult tissues in at least two respects: they are hyperpermeable to circulating maromolecules and they divide frequently. VPF (also known as vascular endothelial growth factor) is a multifunctional cytokine expressed and secreted by many transplantable and autochthonous animal and human tumors. It likely contributes to tumor angiogenesis both by its direct, mitogenic effect on ECs and also indirectly by increasing microvascular permeability to plasma proteins whose extravasation profoundly alters the extracellular matrix. Extravasated plasma fibrinogen clots to deposit an extravascular fibrin gel which is degraded by proteases and invaded by macrophages, fibroblasts and endothelial cells, forming vascular and later fibrous connective tissue. This sequence of events closely mimics normal wound healing; hence, tumors behave in some respects as """"""""wounds that do not heal"""""""". Recent work has identified a new organelle in EC cytoplasm, the vesicular- vacuolar organelle (VVO), comprised of interconnecting, membrane-bound vesicles and vacuoles. VVOs provide the major trans-EC route by which circulating macromolecules extravasate from tumor vessels. Similar appearing VVOs are also found in normal post-capillary venule ECs where they are functionally quiescent. Experiments are proposed to investigate the mechanisms of angiogenesis, and the roles played by VPF, other cytokines, vascular hyperpermeability, and fibrin deposition/degradation in this response. Other experiments will determine whether VPF activates VVOs, elucidate the 3-D structure of VVOS, and investigate long and short term effects of VPF on the biology of cultured ECs. More formally, we will investigate the following aims: l. Role of VPF in chronic microvascular hyperpermeability, angiogenesis and stroma generation in vivo. 2. U1trastructural localization of bound VPF and of VPF receptors in tumor microvessels. Relation to vesicular-vacuolar organelles (VVOs). 3-D structure of VVOs. 3. Properties of cultured microvessel endothelium derived from post-capillary venules and tumors. Responses to VPF, including effects on permeability to macromolecules. 4. Tumor microvessel-associated VPF and VPF receptors (KDR, flt-1) as potential antigens for targeting tumors.
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