Angiogenesis in cancer, vascular, rheumatoid and other disease.

Recent discoveries of endogenous negative regulators of angiogenesis, thrombospondin, angiostatin and glioma-derived angiogenesis inhibitory factor, all associated with neovascularized tumours, suggest a new paradigm of tumorigenesis. It is now helpful to think of the switch to the angiogenic phenotype as a net balance of positive and negative regulators of blood vessel growth. The extent to which the negative regulators are decreased during this switch may dictate whether a primary tumour grows rapidly or slowly and whether metastases grow at all.

Dormancy of micrometastases: balanced proliferation and apoptosis in the presence of angiogenesis suppression.

In cancer patients, dormant micrometastases are often asymptomatic and clinically undetectable, for months or years, until relapse. We have studied dormant lung metastases under angiogenesis suppression in mice. The metastases exhibited rapid growth when the inhibition of angiogenesis was removed. Tumour cell proliferation, as measured by bromodeoxyuridine incorporation and immunohistochemical staining proliferating cell nuclear antigen, was not significantly different in dormant and growing metastases. However, tumour cells of dormant metastases exhibited a more than threefold higher incidence of apoptosis. These data show that metastases remain dormant when tumour cell proliferation is balanced by an equivalent rate of cell death and suggest that angiogenesis inhibitors control metastatic growth by indirectly increasing apoptosis in tumour cells.

Angiostatin induces and sustains dormancy of human primary tumors in mice.

There is now considerable direct evidence that tumor growth is angiogenesis-dependent. The most compelling evidence is based on the discovery of angiostatin, an angiogenesis inhibitor that selectively instructs endothelium to become refractory to angiogenic stimuli. Angiostatin, which specifically inhibits endothelial proliferation, induced dormancy of metastases defined by a balance of apoptosis and proliferation. We now show that systemic administration of human angiostatin potently inhibits the growth of three human and three murine primary carcinomas in mice. An almost complete inhibition of tumor growth was observed without detectable toxicity or resistance. The human carcinomas regressed to microscopic dormant foci in which tumor cell proliferation was balanced by apoptosis in the presence of blocked angiogenesis. This regression of primary tumors without toxicity has not been previously described. This is also the first demonstration of dormancy therapy, a novel anticancer strategy in which malignant tumors are regressed by prolonged blockade of angiogenesis.

Inhibition of tumor angiogenesis mediated by cartilage.

Capillary proliferation induced by tumor is shown to be inhibited by neonatal scapular cartilage. Using the rabbit cornea as an assay, the cartilage implant decreased the rate of capillary growth, induced by tumor, by an average of 75%. Vascularization was prevented completely in 28% of tumors. The inhibitory effect of small cartilage implants operates over distances of up to 2.0 mm and displays a gradient from the cartilage source. The experiments suggest that the cartilage inhibitor does not antagonize tumor angiogenesis factor, but appears to inhibit capillary proliferation directly. The inhibitory material does not elicit an inflammatory response in either the rabbit cornea or in the chick chorioallantoic membrane. Thus with further purification, it may prove useful as a means of maintining tumor dormancy by "antiangiogenesis."

Self-regulation of growth in three dimensions.

Multi-cell spheroids were grown in soft agar. When each spheroid was cultured in a large volume of medium, frequently renewed, all spheroids eventually reached a dormant phase at a diameter of approximately 3-4 mm and a population of approximately 10(6) cells. In the dormant spheroid, newly generated cells at the periphery balanced those lost by necrosis in the center. We propose that this dormant phase is due to a gradual reduction in the ratio of surface area to volume: a size is achieved beyond which there is insufficient surface area for the spheroid to eliminate catabolites and absorb nutrients. Thus, in the face of unlimited space and of new medium, three-dimensional cell populations become self-regulating. This phenomenon contrasts with standard tissue culture in which cell populations, living on a flat plane in two dimensions, will not stop growing in the face of unlimited space and new medium because the ratio of surface area to volume remains constant. These experiments provide a mechanism for our observations in vivo: before vascularization, solid tumors live by simple diffusion as three-dimensional spheroids or ellipsoids. They become dormant at a diameter of only a few millimeters; once vascularized, they are released from this dormant phase and begin exponential growth. Thus, tumor dormancy resulting from absence of angiogenesis in vivo, may operate by the same mechanism responsible for dormancy of spheroids in vitro.

Isolation of a tumor factor responsible for angiogenesis.

Human and animal solid tumors elaborate a factor which is mitogenic to capillary endothelial cells. This factor has been called tumor-angiogenesis factor. The important components of TAF are RNA and protein. It is suggested that blockade of this factor (inhibition of angiogenesis) might arrest solid tumors at a tiny diameter of a few millimeters.

gro-beta, a -C-X-C- chemokine, is an angiogenesis inhibitor that suppresses the growth of Lewis lung carcinoma in mice.

We have found that two chemokines, recombinant gro-alpha and gro-beta, specifically inhibit growth factor-stimulated proliferation of capillary endothelial cells in a dose-dependent manner, whereas gro-gamma has no inhibitory effect. In vivo, gro-beta inhibits blood vessel formation in the chicken chorioallantoic membrane assay. It is sufficiently potent to effectively suppress basic fibroblast growth factor-induced corneal neovascularization after systemic administration in mice. Further, gro-beta significantly inhibits the growth of murine Lewis lung carcinoma in syngeneic C57B16/J and immunodeficient nude mice without toxicity. In vitro, Lewis lung carcinoma cells are completely insensitive to recombinant gro-beta at high concentrations that significantly inhibit endothelial cell proliferation. This finding supports the conclusion that gro-beta inhibits Lewis lung tumor growth by suppression of tumor-induced neovascularization.

Mast cell heparin stimulates migration of capillary endothelial cells in vitro.

Migration of capillary endothelial cells is an important component of angiogenesis in vivo. Increased numbers of mast cells have been associated with several types of angiogenesis. We have used a quantitative assay in vitro to demonstrate that mast cells release a factor that significantly increases bovine capillary endothelial cell migration. The factor is present in medium conditioned by mast cells as well as lysates of mast cells. The stimulatory effect of mast cells on migration is specific for capillary endothelial cells. Furthermore, mast cells have no mitogenic activity for capillary endothelial cells. Of all the secretory products of mast cells tested, only heparin stimulated capillary endothelial cell migration in vitro. Heparin preparations from a variety of sources stimulated capillary endothelial cell migration to the same degree but did not stimulate migration of several other cell types. The migration activity of heparin and mast cell conditioned medium was blocked by specific antagonists of heparin (protamine and heparinase), but not by chondroitinase ABC. The migration activity of mast cell conditioned medium was resistant to heat (100 degrees C) and incubation with proteolytic enzymes. These results suggest that the role of mast cells in angiogenesis may be to enhance migration of the endothelial cells of growing capillaries.

Tumor dormancy in vivo by prevention of neovascularization.

Dormant solid tumors were produced in vivo by prevention of neovascularization. When small fragments of anaplastic Brown-Pearce carcinoma were implanted directly on the iris in susceptible rabbits, they always vascularized. A characteristic growth pattern, consisting of prevascular, vascular, and late phases, was observed, which terminated with destruction of the eye within 2 wk. The beginning of exponential volume increase was shown to coincide with vascularization of the implant, as demonstrated by perfusion with intravenous fluorescein and by histologic sections. In contrast, implants placed in the anterior chamber, at a distance from the iris, did not become vascularized. After initial growth into spheroids, they remained arrested at a small size comparable to prevascular iris implants, for periods as long as 6 wk. Although dormant in terms of expansion, these avascular tumors contained a population of viable and mitotically active tumor cells. When reimplanted on the iris, vascularization was followed by rapid, invasive growth. These observations suggest that neovascularization is a necessary condition for malignant growth of a solid tumor. When a small mass of tumor cells is prevented from eliciting new vessel ingrowth from surrounding host tissues, population dormancy results. These data suggest that the specific blockade of tumor-induced angiogenesis may be an effective means of controlling neoplastic growth.

Mechanochemical switching between growth and differentiation during fibroblast growth factor-stimulated angiogenesis in vitro: role of extracellular matrix.

The angiogenic factor, basic fibroblast growth factor (FGF), either stimulates endothelial cell growth or promotes capillary differentiation depending upon the microenvironment in which it acts. Analysis of various in vitro models of spontaneous angiogenesis, in combination with time-lapse cinematography, demonstrated that capillary tube formation was greatly facilitated by promoting multicellular retraction and cell elevation above the surface of the rigid culture dish or by culturing endothelial cells on malleable extracellular matrix (ECM) substrata. These observations suggested to us that mechanical (i.e., tension-dependent) interactions between endothelial cells and ECM may serve to regulate capillary development. To test this hypothesis, FGF-stimulated endothelial cells were grown in chemically defined medium on bacteriological (nonadhesive) dishes that were precoated with different densities of fibronectin. Extensive cell spreading and growth were promoted by fibronectin coating densities that were highly adhesive (greater than 500 ng/cm2), whereas cell rounding, detachment, and loss of viability were observed on dishes coated with low fibronectin concentrations (less than 100 ng/cm2). Intermediate fibronectin coating densities (100-500 ng/cm2) promoted cell extension, but they could not completely resist cell tractional forces. Partial retraction of multicellular aggregates resulted in cell shortening, cessation of growth, and formation of branching tubular networks within 24-48 h. Multicellular retraction and subsequent tube formation also could be elicited on highly adhesive dishes by overcoming the mechanical resistance of the substratum using higher cell plating numbers. Dishes coated with varying concentrations of type IV collagen or gelatin produced similar results. These results suggest that ECM components may act locally to regulate the growth and pattern-regulating actions of soluble FGF based upon their ability to resist cell-generated mechanical loads. Thus, we propose that FGF-stimulated endothelial cells may be "switched" between growth, differentiation, and involution modes during angiogenesis by altering the adhesivity or mechanical integrity of their ECM.

Protein kinase C activators suppress stimulation of capillary endothelial cell growth by angiogenic endothelial mitogens.

The intracellular events regulating endothelial cell proliferation and organization into formalized capillaries are not known. We report that the protein kinase C activator beta-phorbol 12,13-dibutyrate (PDBu) suppresses bovine capillary endothelial (BCE) cell proliferation (K50 = 6 +/- 4 nM) and DNA synthesis in response to human hepatoma-derived growth factor, an angiogenic endothelial mitogen. In contrast, PDBu has no effect on the proliferation of bovine aortic endothelial cells and is mitogenic for bovine aortic smooth muscle and BALB/c 3T3 cells. Several observations indicate that the inhibition of human hepatoma-derived growth factor-stimulated BCE cell growth by PDBu is mediated through protein kinase C. Different phorbol compounds inhibit BCE cell growth according to their potencies as protein kinase C activators (12-O-tetradecanoylphorbol 13-acetate greater than PDBu much greater than beta-phorbol 12,13-diacetate much much greater than beta-phorbol; alpha-phorbol 12,13-dibutyrate; alpha-phorbol 12,13-didecanoate). PDBu binds to a single class of specific, saturable sites on the BCE cell with an apparent Kd of 8 nM, in agreement with reported affinities of PDBu for protein kinase C in other systems. Specific binding of PDBu to BCE cells is displaced by sn-1,2-dioctanoylglycerol, a protein kinase C activator and an analog of the putative second messenger activating this kinase in vivo. The weak protein kinase C activator, sn-1,2-dibutyrylglycerol, does not affect PDBu binding. A cytosolic extract from BCE cells contains a calcium/phosphatidylserine-dependent protein kinase that is activated by sn-1,2-dioctanoylglycerol and PDBu, but not by beta-phorbol. These findings indicate that protein kinase C activation can cause capillary endothelial cells to become desensitized to angiogenic endothelial mitogens. This intracellular regulatory mechanism might be invoked during certain phases of angiogenesis, for example when proliferating endothelial cells become differentiated to organize into nongrowing tubes.

Human vascular endothelial cells in culture. Growth and DNA synthesis.

Human endothelial cells, obtained by collagenase treatment of term umbilical cord veins, were cultured using Medium 199 supplemented with 20% fetal calf serum. Small clusters of cells initially spread on plastic or glass, coalesced and grew to form confluent monolayers of polygonal cells by 7 days. Cells in primary and subcultures were identified as endothelium by the presence of Weibel-Palade bodies by electron microscopy. A morphologically distinct subpopulation of cells contaminating some primary endothelial cultures was selectively subcultured, and identified by ultrastructural criteria as vascular smooth muscle. Autoradiography of endothelial cells after exposure to [(3)H]thymidine showed progressive increases in labeling in growing cultures beginning at 24 h. In recently confluent cultures, labeling indices were 2.4% in central closely packed regions, and 53.2% in peripheral growing regions. 3 days after confluence, labeling was uniform, being 3.5 and 3.9% in central and peripheral areas, respectively. When small areas of confluent cultures were experimentally "denuded," there were localized increases in [(3)H]thymidine labeling and eventual reconstitution of the monolayer. Liquid scintillation measurements of [(3)H]thymidine incorporation in primary and secondary endothelial cultures in microwell trays showed a similar correlation of DNA synthesis with cell density. These data indicate that endothelial cell cultures may provide a useful in vitro model for studying pathophysiologic factors in endothelial regeneration.

Tumor angiogenesis. Rapid induction of endothelial mitoses demonstrated by autoradiography.

Walker ascites tumor cells and an extract derived from such cells (tumor angiogenesis factor, TAF) were injected into the subcutaneous tissue of rats by using a dorsal air sac technique. At intervals thereafter, thymidine-(3)H was injected into the air sac and the tissues were examined by autoradiography and electron microscopy. Autoradiographs of 1micro thick Epon sections showed thymidine-(3)H labeling in endothelial cells of small vessels 1-3 mm from the site of implantation, as early as 6-8 hr after exposure to live tumor cells At this time interval endothelial cells appeared histologically normal. DNA synthesis by endothelium subsequently increased and within 48 hr new blood vessel formation was detected. The presence of thymidine-(3)H-labeled endothelial nuclei, endothelial mitoses, and regenerating-type endothelium was confirmed by electron microscopy. TAF also induced neovascularization and endothelial cell DNA synthesis after 48 hr. A similar response was not evoked in saline controls. Formic acid, which elicited a more intense inflammatory response, was associated with less endothelial labeling and neovascularization at the times studied. Pericytes and other connective tissue cells were also stimulated by live tumor cells and TAF. The mechanism of new blood vessel formation induced by tumors is still unknown but our findings argue against cytoplasmic contact or nonspecific inflammation as prerequisites for tumor angiogenesis.

Oligomerization-dependent regulation of motility and morphogenesis by the collagen XVIII NC1/endostatin domain.

Collagen XVIII (c18) is a triple helical endothelial/epithelial basement membrane protein whose noncollagenous (NC)1 region trimerizes a COOH-terminal endostatin (ES) domain conserved in vertebrates, Caenorhabditis elegans and Drosophila. Here, the c18 NC1 domain functioned as a motility-inducing factor regulating the extracellular matrix (ECM)-dependent morphogenesis of endothelial and other cell types. This motogenic activity required ES domain oligomerization, was dependent on rac, cdc42, and mitogen-activated protein kinase, and exhibited functional distinction from the archetypal motogenic scatter factors hepatocyte growth factor and macrophage stimulatory protein. The motility-inducing and mitogen-activated protein kinase-stimulating activities of c18 NC1 were blocked by its physiologic cleavage product ES monomer, consistent with a proteolysis-dependent negative feedback mechanism. These data indicate that the collagen XVIII NC1 region encodes a motogen strictly requiring ES domain oligomerization and suggest a previously unsuspected mechanism for ECM regulation of motility and morphogenesis.

Angiogenic factors.

Within the past 2 years, several angiogenic factors have been fully purified, their amino acid sequences determined, and their genes cloned. These polypeptides include acidic and basic fibroblast growth factor, angiogenin, and transforming growth factors alpha and beta. Other less well characterized angiogenesis factors have also been isolated, some of which are lipids. This article traces the discovery of the angiogenic factors and describes their possible significance in understanding growth regulation of the vascular system. When evaluated according to their putative targets, they appear to fall into two groups: those that act directly on vascular endothelial cells to stimulate locomotion or mitosis, and those that act indirectly by mobilizing host cells (for example, macrophages) to release endothelial growth factors. In addition to their presence in tumors undergoing neovascularization, the same angiogenic peptides are found in many normal tissues where neovascularization is not occurring. This suggests that physiological expression of angiogenic factors is tightly regulated. In addition to the persistent angiogenesis induced by tumors, it now appears that a variety of nonneoplastic diseases, previously thought to be unrelated, can be considered as "angiogenic diseases" because they are dominated by the pathologic growth of capillary blood vessels.

Silicone rubber: oxygen, carbon dioxide, and nitrous oxide measurement in gas mixtures.

Pressure changes, arising from counter diffusion of gases through a sealed silicone rubber tube, may be used to measure tension of gases in a mixture. When the tube is initially filled with one of the gases, and the mixture surrounds it, the pressure rise is related to the tension of the remaining gas.

Silicone rubber: a new diffusion property useful for general anesthesia.

Ether, nitrous oxide, halothane, and cyclopropane diffuse through silicone rubber. General anesthesia can be produced in dogs by passing the vapors of any of these anesthetic agents through a coil of silicone rubber tubing, each end of which is placed in an artery and vein. Potential applications include a new method for general anesthesia and a simple accurate vaporizer for halothane.

A new class of steroids inhibits angiogenesis in the presence of heparin or a heparin fragment.

Steroids that lack glucocorticoid or mineralocorticoid activity were found to inhibit angiogenesis in the presence of heparin or specific heparin fragments. This newly discovered steroid function appears to be governed by distinct structural configurations of the pregnane nucleus. These compounds are here named angiostatic steroids.

Acrylamide polymerization: new method for determining the oxygen content in blood.

A new principle for determining the oxygen content of 0.1-milliliter blood samples has been developed, based on measurement of the delay in gelation during copolymerization of acrylamide and bisacrylamide initiated by free radicals. The logarithm of this time interval is linearly proportional to the oxygen content of the blood sample over the range 0 to 22 milliliters of oxygen per 100 milliliters of whole blood. Physiological variations of pH and pCO(2) do not affect the sensitivity of the assay.

Isolations of a cartilage factor that inhibits tumor neovascularization.

A cartilage fraction isolated by guanidine extraction and purified by affinity chromatography inhibits tumor-induced vascular proliferation and consequently restricts tumor growth. This fraction contains several different proteins; the major one has a molecular weight of about 16,000. The fraction strongly inhibits protease activity.