Angiogenic and angiostatic microenvironment in tumors--role of gangliosides.

Gangliosides are important components of the cell membrane that are usually shed in the surrounding microenvironment by neoplastic cells. Gangliosides can also modulate the angiogenic response of microvessels stimulated by angiogenic factors. The experiments reported here make a contribution to the assessment of the nature of this angiogenic modulation, by demonstrating that a) GM3 gangliosides can block the proliferation of endothelium induced by neoplastic cells from human tumors of five different origins; b) this block also occurs when the endothelial cells are preincubated with GM3 and disappears when the cells are returned to a medium poor in GM3; c) in the presence of GM3 the capacity of the endothelial cells to bind to fibronectin and to collagen types I and IV was sharply reduced; d) concentrations of GM3 able to block endothelial cell growth are counteracted by addition to the medium of GT1b ganglioside. The data suggest that the prevalence of a microenvironment rich in GM3 prevents proliferation of vascular endothelium, but the appropriate presence of another ganglioside, such as GT1b, nullifies the effect. Modulation of the angiogenic response of vascular endothelium to angiogenic factors released by tumors is probably dependent on the distribution and activity of growth factor receptors on the endothelial cell surface. The nature and concentration of the gangliosides in the endothelial microenvironment have a decisive influence on this event and possibly on the progression of tumor-induced angiogenesis.

Prostaglandins and gangliosides of tumor microenvironment: their role in angiogenesis.

In the solid tumor the microenvironment is the space limited by the basement membrane of the microvessels and the neoplastic cells membrane. It includes the stroma and a liquid phase, the tumor interstitial fluid (TIF). We developed a method to sample TIF in vivo and found it rich in prostaglandins. In the rabbit cornea PGE1 induces neovascularization and acts as an angiogenesis factor. Before angiogenesis appears the ganglioside content of the cornea doubles with sharp reduction of the GM3/GD3 ratio. These gangliosides are not angiogenic but they influence the endothelial cell behavior. In particular when the PGE1 dose is insufficient to induce angiogenesis, enrichment of corneal tissue with GD3 or GM1 stimulates angiogenesis. However, when the corneal tissue is enriched with GM3, doses of PGE1, normally angiogenic fail to do so. The same was observed when bFGF substituted PGE1 as an angiogenesis trigger. The gangliosides tested acted as modulators of the angiogenic response by promoting the angiogenic capacity of molecules, such as PGE1 or bFGF, normally present in the tissue microenvironment. Several neoplastic cells, especially melanomas, shed gangliosides in the microenvironment. Their modulatory effect on angiogenesis may influence metastatic and/or primary tumor growth.

Angiogenesis can be stimulated or repressed in vivo by a change in GM3:GD3 ganglioside ratio.

BACKGROUND: We had previously observed that rabbit cornea stimulated by an angiogenic factor 1) became richer in total gangliosides and 2) reduced the GM3:GD3 ganglioside ratio. Moreover, experimentally induced global enrichment of corneal gangliosides favors angiogenesis. EXPERIMENTAL DESIGN: The objective of this work was to explain the possible relationship between angiogenic response and changes in the GM3:GD3 ratios observed in vivo. Cornea was utilized because it is avascular and transparent; i.e., the onset of opacity permitted exclusion of angiogenesis produced by a generic inflammatory response. Prostaglandin E1 or basic fibroblast growth factor were applied as angiogenesis triggers. Angiogenesis in vivo and mobilization and growth of microvascular endothelium in vitro were taken as parameters to indicate whether differences in GM3:GD3 ratios could modify the extent of the angiogenic response. RESULTS: In vivo angiogenesis, whether prostaglandin E1 or basic fibroblast growth factor induced, was repressed by GM3 and enhanced by GD3 or GM1 enrichment of the cornea. In vitro growth and motility of microvascular endothelium were reduced by GM3 addition to the medium and returned to normal levels by addition of GD3. CONCLUSIONS: Formation of new vessels induced by two different angiogenic factors could be stimulated or repressed in the cornea by reduction or enhancement of the GM3:GD3 ratio of tissue gangliosides. Changes in the relative proportion of molecules normally present in adult tissues, like prostaglandin E1, basic fibroblast growth factor, GM3, GD3, were sufficient to modulate or even block angiogenesis.

Growth and motility of microvascular endothelium are modulated by the relative concentration of gangliosides in the medium.

The data reported were obtained as an attempt to understand whether the change in total concentration and relative ratios of the 3 major corneal gangliosides (GM3, GM2, GD3) previously observed in corneas stimulated by an angiogenic molecule (Ziche et al., 1989) was a relevant event in the angiogenic response of the tissue. The effect on endothelial cell growth was tested for the 3 corneal gangliosides added singly to the culture medium, and GM3 was found to possess a substantial growth inhibitory effect as compared to GM2 and GD3. The growth-limiting effect of GM3 was counteracted to a different degree by the addition of a second ganglioside to the culture medium. A mixture of GM3 + GM2 + GD3 in the proportion similar to that found in the cornea stimulated by an angiogenic molecule was able to sustain a sharp increment in cell growth and motility when added to cultures of capillary endothelium. On the contrary, when the 3 components of the mixture were in the proportion present in the normal cornea, the increment in growth or motility did not occur. A simple change in the relative proportions of the 3 gangliosides was sufficient to trigger or prevent an increment in growth and motility of the endothelial cells. These data in vitro suggest that the changes in concentration and relative ratios of the 3 major corneal gangliosides observed in vivo when the cornea was stimulated by an angiogenic molecule were an event targeted to favour growth and mobilization of the capillary endothelium located within the limbal vessels at the periphery of the cornea.

Gangliosides, copper ions and angiogenic capacity of adult tissues.

The report summarizes the work of our laboratory aimed at improving the understanding of the angiogenic response of adult tissues, an event that transforms a micro-embolus of neoplastic cells into a growing metastasis. Attention has been focused on tumor-induced angiogenesis. The following aspects of the subject are discussed: (a) relationship between size of vascular network and tumor growth rate or tumor cell population; (b) angiogenic capacity of tumors and role that prostaglandin E1 may have as an angiogenesis factor; (c) relationship between acquisition of angiogenic capacity and neoplastic transformation of a cell population; (d) modification of tissue composition at the onset of angiogenesis; (e) behaviour of copper ions and copper carriers in the course of the angiogenic response; (f) the influence of gangliosides on endothelial cell motility, survival and growth in vitro; (g) modulation of the angiogenic response by gangliosides (GM1, GT1b) in vivo.

Synergism between gangliosides and basic fibroblastic growth factor in favouring survival, growth, and motility of capillary endothelium.

The experiments reported were motivated by the observation that in vivo gangliosides promoted angiogenesis when the dose of the angiogenic factor was too low to be effective (Ziche et al.: Laboratory Investigation 61:629-634, 1989). As an approach to understanding the mechanism of this modulatory effect, we analysed the influence that gangliosides have on survival, growth, and migration of capillary endothelium when an angiogenesis factor like basic fibroblast growth factor (bFGF) was present in the culture medium. Clones of bovine capillary endothelium were cultivated in media unable to sustain survival over a 72 h period. With this experimental approach, cell survival was evaluated after addition of either bFGF or gangliosides or both to the medium. The Boyden chamber procedure was utilized to measure the influence of bFGF or gangliosides on cell mobilization across a micropore filter. Low doses of both molecules, ineffective when added singly to the culture media, improved all three parameters when added in combination. A synergic effect between bFGF and the gangliosides (GM1, GD1b, GT1b) was observed for the improvement of survival or growth and for the acceleration of endothelial cell migration. The removal of sialic acid from the ganglioside molecule prevented any effect on all three parameters. The addition of sialic acid alone to cultures was also totally ineffective. In the adult organism most angiogenic events occur under conditions of tissue damage. The synergism between gangliosides and bFGF can be interpreted as the initial phase of a process for which endothelial cell survival is the indispensable first step in the formation of a new vascular network.

Gangliosides promote the angiogenic response.

Formation of new capillaries was induced in rabbit corneas by optimal doses of prostaglandin E1 (PGE1) or basic fibroblastic growth factor. Suboptimal doses of either of these angiogenesis inducers were unable to elicit corneal angiogenesis. However, the addition of gangliosides (GM1 or GT1b) to the insufficient dose of the angiogenic inducer, not only promoted neovascularization but strongly enhanced the number and growth rate of the newly formed capillaries as compared with the effect of an optimal dose of the angiogenesis inducer alone. Removal of the ganglioside stimulation led to the disappearance of the newly formed capillaries. Re-establishment of the ganglioside stimulation renewed the appearance of a conspicuous vascular network. Removal of sialic acid from the ganglioside molecule nullified its stimulatory effect on angiogenesis. GMI or GT1b or sialic acid were not angiogenic by themselves. Corneas ready to be invaded by newly formed capillaries induced by an optimal dose of an angiogenesis factor such as PGE1 had a ganglioside content about twice that of unstimulated corneas. The results are interpreted to indicate that ganglioside molecules influence capillary formation not as angiogenesis inducers but as promoters of vessel morphogenesis. The data support the hypothesis that in vivo the level of gangliosides in the tissue milieu could influence the evolution of pathologic processes in which new formation of capillaries is a critical event.

Retinyl acetate-induced arthritis in C3H-A(vy) mice.

Severely impaired musculoskeletal mobility in C3H-A(vy) mice was noted during a pharmacologic trial evaluating the antitumorigenic properties of retinyl acetate (RAc). To determine the etiology of this impairment, we studied 103 female C3H-A(vy) mice that were fed RAc in daily doses of 75-300 micrograms or placebo and were killed after 3-16 months. Whole-body radiographs and histologic sections of the hindlimbs were scored for presence and severity of arthritis. C3H-A(vy) mice treated with RAc in any dose had a significantly higher incidence of arthritis than placebo-treated mice. Histologic evidence of enthesopathic disease closely paralleled the radiographic changes and ranged from small enthesophytes at tendinous and capsular insertions to complete periarticular bony bridging. Articular cartilage was not grossly affected. The incidence and severity of arthritis were significantly correlated with the total dose of RAc administered. The bony metaplasia induced by RAc was similar to the pathologic changes caused by other retinoids. This model may be useful for studying the pathogenesis of periarticular bone formation in diffuse idiopathic skeletal hyperostosis and related syndromes.

Influence of gangliosides on primary and metastatic neoplastic growth in human and murine cells.

The influence of gangliosides on tumor growth and frequency of metastasis in vivo as well as on growth and motility of neoplastic cells in vitro was tested utilizing human and rodent cell populations. In mice receiving injections of a ganglioside mixture twice daily the tumor volume, the number of spontaneous metastases per animal, and the number of mice with metastasis was approximately double that of controls. Preincubation of neoplastic cells with the ganglioside mixture doubled the number of metastatic foci in the lungs of mice receiving the cells by i.v. injection. Addition of a ganglioside mixture to the culture medium enhanced motility of neoplastic cells about 3-fold. This finding was similar to that observed for capillary endothelium. The presence of gangliosides in the culture media for a 48-h incubation period about doubled the number of neoplastic cells as compared to controls; the same was observed for capillary endothelium. The data are interpreted to indicate that gangliosides improve growth and mobilization of capillary endothelium and neoplastic cells. Both events may concur in enhancing tumor growth in vivo, the first by improving angiogenesis, the second by direct action on the neoplastic cell population.

Considerations on the mechanism of the angiogenic response.

The principal objective of our work is to sufficiently understand the mechanism of angiogenesis in the adult organism to allow interference with the process on a rational basis. It is apparent that several "factors" can trigger angiogenesis. To test these, we used the rabbit cornea mostly because it is avascular (i.e., the background is zero) and transparent (i.e., the newly formed capillaries that invade the cornea are easily visible in the unanaesthetized animal). Under these conditions, it was found that the cornea ready to be colonized by capillaries under the action of an angiogenesis effector becomes rich in copper ions and sialic acid. Motility of bovine capillary endothelium was utilized to analyze the angiogenesis process on the ground that mobilization of capillary endothelium is the first morphological event observed during angiogenesis in vivo and the methods to measure cell motility are reasonably accurate. With this approach it was found that heparin, fibronectin, and gangliosides are involved in the mobilization of capillary endothelium. The precise interaction among these three components is not yet clear.

Interaction of gangliosides with fibronectin in the mobilization of capillary endothelium. Possible influence on the growth of metastasis.

Mobilization of the capillary endothelium is one of the first events observed during angiogenesis, and the study of conditions that control or influence the mobilization of the endothelium in vitro has been assumed to offer information relevant to the understanding of angiogenesis in vivo. In vitro mobilization of the bovine capillary endothelium was substantially enhanced by addition of gangliosides to the culture medium. Optimal mobilization was obtained when the endothelium incorporated the gangliosides first and was then seeded on fibronectin anchored to collagen type I. Preincubation of the capillary endothelium with gangliosides, trisialoganglioside in particular, doubled the amount of fibronectin bound to the cells and enhanced the migration about 5-fold. 'Blockage' of ganglioside binding with cholera toxin or gamma-interferon substantially reduced migration. Rabbit corneas, treated in vivo with a variety of angiogenesis effectors to induce neovascularization, consistently showed an increase in sialic acid content just prior to the time the tissue would be penetrated by the capillaries. This finding was interpreted to indicate that an increment of the ganglioside content of the capillary endothelial cell membranes may play a determinant role in the mobilization of the capillary endothelium in vivo as shown here to take place in vitro. Since the formation of a tumor from a micrometastasis requires formation of new capillaries and highly metastasizing tumors very frequently have high levels of sialic acid on the cell surface, it is hypothesized that production and shedding of gangliosides from the surface of neoplastic cells may be a factor in promoting angiogenesis and metastatic growth.

Effect of pregnancy and nursing on the growth of metastases from N-nitroso-N-methylurea-induced mammary carcinomas.

Pregnancy increased the survival times of inbred BUF/N rats bearing occult metastasis of a mammary carcinoma at the time of conception. Nursing following delivery nullified the effect of pregnancy. The similarity of the controlled experimental data with the limited clinical observations is noted. An experimental model of rat mammary carcinoma has been described that possesses a highly metastasizing capacity and can be utilized to study the behavior of clinically silent metastasis.

Cooperation between fibronectin and heparin in the mobilization of capillary endothelium.

Angiogenesis is indispensable to sustain promotion and growth of metastases. As a contribution to the understanding of the angiogenesis process, the experiments reported showed that: (a) fibronectin is involved in the mobilization of capillary endothelium which is the first event in angiogenesis; (b) antifibronectin serum can block the mobilization, and neutralization of the antiserum can restore it; (c) the combination of fibronectin + heparin is a powerful mobilizer of capillary endothelium, and (d) fragments of the fibronectin and heparin molecules in combination can mobilize capillary endothelium as effectively as the intact molecules. The results are interpreted to indicate that molecules normally present in the extracellular matrix like heparin and fibronectin, may act as angiogenesis effectors when the physiological structure of the tissue is altered, for instance by lytic enzymes released by metastatic neoplastic cells.

Expression of kappa-casein in normal and neoplastic rat mammary gland is under the control of prolactin.

An 820-nucleotide-long cDNA clone for the kappa-casein (the casein micelle-stabilizing protein) from rat mammary gland was isolated, and its nucleotide sequence was determined. The deduced amino acid sequence from the nucleotide sequence revealed a signal peptide, 21 amino acids long, and a mature protein of 157 amino acids. The signal peptide of rat kappa-casein was highly homologous to that of the precursor to ovine kappa-casein. However, little homology was apparent when the mature kappa-casein protein sequences from ovine or bovine sources were compared with rat kappa-casein. The kappa-casein mRNA content of the mammary tissue was found to increase during its functional differentiation. Prolactin appears to modulate the production of kappa-casein mRNA. Mammary glands of virgin females had no detectable kappa-casein mRNA; however, a marked induction of kappa-casein mRNA was obtained by intravenous infusion of prolactin. Mammary carcinomas did not follow the same pattern. 7,12-Dimethylbenz[a]anthracene-induced mammary carcinomas had normally low levels of kappa-casein mRNA, but intravenous prolactin infusion increased the levels by 2-fold. The MTW9 mammary carcinoma that grows only in the presence of high levels of mammotropic hormones had kappa-casein mRNA content equivalent to that in 10-day lactating rat mammary gland. Continuous venous infusion of prolactin to MTW9 mammary carcinoma did not modify the kappa-casein mRNA levels. Nitrosomethylurea-induced mammary carcinomas had no detectable kappa-casein mRNA, and intravenous prolactin infusion was unable to induce it.

Angiogenesis in vivo and selective mobilization of capillary endothelium in vitro by heparin-copper complex.

The objective of this work was to contribute to the interpretation of the mechanisms of capillary formation in the adult tissues. We have observed that the heparin-copper complex is angiogenic in vivo and stimulates migration of capillary endothelium in vitro. This effect is specific for capillary endothelium since aortic endothelium or fibroblasts from the rabbit cornea or human skin were unresponsive.

Characterization of a chemoattractant for endothelium induced by angiogenesis effectors.

The mechanism of neovascularization was further explored by the use of chemically defined angiogenesis effectors. The vascularization of the rabbit cornea was selected as an experimental approach that permits comparison of one cornea treated by the angiogenesis effector with the contralateral cornea of the same subject treated by the same molecule deprived of angiogenic capacity. Under these conditions, we observed that neovascularization was initiated by the appearance of a chemoattractant for the bovine capillary endothelium only in the cornea treated by the angiogenesis effector. The chemoattractant was purified about 150-fold by a single-step procedure, using gelatin:Sepharose affinity chromatography. Chemoattraction resulted from the combined effect of a chemotactic factor(s) and an activating factor(s). The association of the two enhanced 5- to 8-fold the motility of the capillary endothelium in a concentration-dependent manner with optimum at 0.2 mg/ml. The activating factor(s) does not have chemotactic capacity, but without it, chemotaxis is reduced to about one half. The chemotactic complex was present in the cornea regardless of the nature of the angiogenesis effector used as the triggering device. Heat and proteases eliminated chemotaxis and destroyed the chemotactic complex. Thus, neovascularization may be triggered by effectors able to induce in the cornea proteins, normally not present, that influence angiogenesis via mobilization of capillary endothelium.

Mobilization of capillary endothelium in vitro induced by effectors of angiogenesis in vivo.

An assay to measure endothelial cell mobilization on a gelatin substratum has been developed. Utilization of the gelatin-agarose and Boyden chamber assays established that: (a) fragments or extracts of corneas treated with several effectors of angiogenesis in vivo acquired the capacity to mobilize the capillary endothelium in vitro; (b) this mobilization was selective for the capillary endothelium; endothelium from aorta and fibroblasts from human skin or rabbit cornea were unresponsive; and (c) among the effectors of angiogenesis utilized alone; i.e., without the intermediary action of the cornea, none were able to mobilize the capillary endothelium in vitro, except for the heparin-copper complex. The data are interpreted to indicate that new formation of capillaries in vivo is the end result of a cascade of events of which heparin and copper are important components.