Angiogenesis, the growth of new blood vessels from extant capillaries, is a continuous process throughout most of the embryonic and mature lifespan of vertebrates. It is also a necessary component of unscheduled remodeling and for the growth of solid tumors. Although angiogenesis is thought to proceed in discrete stages with respect to cellular proliferation, extracellular matrix degradation and synthesis, and directed migration, none of these steps, including that which activates otherwise quiescent endothelial cells, is understood. Our studies on endothelial cells that spontaneously form cords and tubes in vitro (termed angiogenic cultures) have shown that the alpha1 (I) collagen gene (COL1A1) is transcribed at high rates, and that the protein is secreted in fibrillar form during this process, which appears to represent several of the earlier stages of angiogenesis in vivo. The cells align along the fibers and eventually form lumina around them. Angiogenic cultures are stimulated to proliferate by transforming growth factor-beta1 (TGF-beta), whereas nonangiogenic cells exhibit an inhibition of growth. We have proposed that the expression of type I collagen is requisite to the initiation of cord/tube assembly by endothelial cells,a nd that this protein, in the context of other matrix components and a limited number of mitogens/morphogens, directs lumen formation and tubular branching. In five Specific Aims we address the potential relevance of type I collagen to angiogenesis in vivo and in vitro. Since embryonic brain vascularizes exclusively by angiogenesis, we will examine this tissue from staged mouse embryos by immunohistochemistry and in situ hybridization with probes for type I collagen. A similar approach will be taken with mov 13 mice, which as homozygotes fail to transcribe COL1A1. These experiments should reveal whether embryonic angiogenesis requires synthesis of type I collagen. Pulse-chase studies in vitro will address the function of type I collagen during tube formation. Synthesis and fibrillogenesis will be perturbed by specific pharmacologic agents; effects of collagen-binding proteins and genetically altered alpha1(I) on angiogenesis in vitro will also be measured quantitatively by confocal image analysis. Cytokines that generally stimulate matrix production and mitogens with potential angiogenic activity will be tested on angiogenic cultures for regulatory activity of COL1A1. We should then be able to correlate rates of tube formation with the abundance of steady state alpha1(I) mRNA. We will address the regulation of COL1A1 in angiogenic cultures by transfection of plasmid DNA containing various regions of the alpha1(I) promoter, exon 1, and/or intron 1. We predict that the activities of these regulatory regions will be reflected by the matrix with which the cells interact. By these Aims we will attempt to define or clarify early stages of angiogenesis that rely on both instructional and permissive roles for type I collagen.
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