We have studied the behavior of cloned capillary endothelial cells grown inside a three dimensional collagen matrix. Cell monolayers established on the surface of collagen gels were covered with a second layer of collagen. This induced the monolayers of endothelial cells to reorganize into a network of branching and anastomosing capillary-like tubes. As seen by electron microscopy, the tubes were formed by at least two cells (in transverse sections) delimiting a narrow lumen. In addition, distinct basal lamina material was present between the abluminal face of the endothelial cells and the collagen matrix. These results showed that capillary endothelial cells have the capacity to form vessel-like structures with well-oriented cell polarity in vitro. They also suggest that an appropriate topological relationship of endothelial cells with collagen matrices, similar to that occurring in vivo, has an inducive role on the expression of this potential. This culture system provides a simple in vitro model for studying the factors involved in the formation of new blood vessels (angiogenesis).

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In vitro rapid organization of endothelial cells into capillary-like networks is promoted by collagen matrices.

Tumor-promoting phorbol esters induce angiogenesis in vitro

This study was designed to identify the set of functions activated in cultured endothelial cells by the hematopoietic growth factors, granulocyte colony-stimulating factor (G-CSF) and granulocyte macrophage-colony-stimulating factor (GM-CSF), and to compare them with those elicited by prototypic cytokines active on these cells. Moreover, indications as to the in vivo relevance of in vitro effects were obtained. G-CSF and GM-CSF induced endothelial cells to proliferate and migrate. In contrast, unlike appropriate reference cytokines (IL-1 and tumor necrosis factor, IFN-gamma), G-CSF and GM-CSF did not modulate endothelial cell functions related to hemostasis-thrombosis (production of procoagulant activity and of platelet activating factor), inflammation (expression of leukocyte adhesion molecule-1 and production of platelet activating factor), and accessory function (expression of class II antigens of MHC). Other colony-stimulating factors (IL-3 and macrophage-colony-stimulating factor) were inactive on all functions tested. In comparison to basic fibroblast growth factor (bFGF), G-CSF and GM-CSF induced lower maximal proliferation of endothelial cells, whereas migration was of the same order of magnitude. G-CSF and GM-CSF stimulated repair of mechanically wounded endothelial monolayers. Exposure to both cytokines induced shape changes and cytoskeletal reorganization consistent with a migratory phenotype. To explore the in vivo relevance of the in vitro effects of these cytokines on endothelium, we studied the angiogenic activity of human G-CSF in the rabbit cornea. G-CSF, but not the heat-inactivated molecule, had definite angiogenic activity, without any sign of inflammatory reactions. G-CSF was less active than bFGF. However, the combination of a nonangiogenic dose of bFGF with G-CSF resulted in an angiogenic response higher than that elicited by either individual cytokines. Thus, G-CSF and GM-CSF induce endothelial cells to express an activation/differentiation program (including proliferation and migration) related to angiogenesis.

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In vitro and in vivo activation of endothelial cells by colony-stimulating factors.

Substance P stimulates neovascularization in vivo and proliferation of cultured endothelial cells.

Human cDNAs coding for angiogenin, a human tumor derived angiogenesis factor, were isolated from a cDNA library prepared from human liver poly(A) mRNA employing a synthetic oligonucleotide as a hybridization probe The largest cDNA insert (697 base pairs) contained a short 5'-noncoding sequence followed by a sequence coding for a signal peptide of 24 (or 22) amino acids, 369 nucleotides coding for the mature protein of 123 amino acids, a stop codon, a 3'-noncoding sequence of 175 nucleotides, and a poly(A) tail The gene coding for human angiogenin was then isolated from a genomic lambda Charon 4A bacteriophage library employing the cDNA as a probe The nucleotide sequence of the gene and the adjacent 5'- and 3'-flanking regions (4688 base pairs) was then determined The coding and 3'-noncoding regions of the gene for human angiogenin were found to be free of introns, and the DNA sequence for the gene agreed well with that of the cDNA The gene contained a potential TATA box in the 5' end in addition to two Alu repetitive sequences immediately flanking the 5' and 3' ends of the gene The third Alu sequence was also found about 500 nucleotides downstream from the Alu sequence at the 3' end of the gene The amino acid sequence of human angiogenin as predicted from the gene sequence was in complete agreement with that determined by amino acid sequence analysis It is about 35% homologous with human pancreatic ribonuclease, and the amino acid residues that are essential for the activity of ribonuclease are also conserved in angiogenin This provocative finding is thought to have important physiological implications

Sequence of the cDNA and gene for angiogenin, a human angiogenesis factor.

New formation of vessels occurs during embryonal development and throughout the lifespan of an organism following repair processes or under certain pathologic conditions. In particular, neoplastic cell populations can grow to form a clinically evident tumor only if the host produces a vascular network sufficient to sustain tumor growth (Folkman, 1974 a, 1975; Brem et al., 1976). The hypothesis that angiogenesis is due to the production of a factor(s) by cells involved in the process of neovascularization arose from several kinds of observations: During an inflammatory response, new formation of vessels appeared when certain types of cells invaded the area; a neoplastic cell population induced the formation of a vascular network even when the tumor fragment was separated from the host by a Millipore filter; (Greenblatt and Shubik, 1968); and cell-free fluid sampled in vivo from the extracellular compartment of a growing tumor elicited an angiogenic response (Gullino, unpublished observation), as does the media of cultured neoplastic cells (Folkman, 1974 b).

P. M. Gullino

Papers

Angiogenesis Factor(s)