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.

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.

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.

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.

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.

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.

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.

Angiogenic activity of hyperplastic and neoplastic lymph nodes.

Angiogenic capacity was tested in 14 non-Hodgkin's lymphomas, 7 Hodgkin's lymphomas and 15 cervical lymph nodes nonneoplastic but draining a territory with a laryngeal carcinoma. The objective was to find out whether different groups of lymphomas showed differences in their angiogenic capacity and to compare the ability to induce neovascularization of neoplastic lymphocytes. Frequency and intensity of the angiogenic response were similar for classes of lymphomas different for morphologic and immunologic characteristics.

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.

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.

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.

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.

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.

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.

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.

Ceruloplasmin, copper ions, and angiogenesis.

The ability to induce new formation of capillaries in the cornea was tested for ceruloplasmin, the copper carrier of serum, for fragments of the ceruloplasmin molecule with and without copper, for heparin, and for glycyl-L-histidyl-L-lysine, bound or not bound to copper ions. Male or female 2- to 3-kg New Zealand White rabbits were used. These experiments were prompted by the previous observation of copper accumulation in the cornea during angiogenesis and by the inability of copper-deficient rabbits to mount an angiogenic response.

Angiogenesis and neoplastic progression in vitro.

In vivo cell populations at high risk of neoplastic transformation have been shown to acquire the ability to induce new formation of vessels. The present experiments tested whether the same change occurred during neoplastic transformation in vitro. In four cell populations (human HBL 100 mammary epithelium, BALB/c fibroblasts, C57BL-MG epithelium, and Syrian golden hamster embryo cells), angiogenic capacity appeared during their cultivation in vitro and was evident long before a neoplastic transformation could be recognized.

Role of prostaglandin E1 and copper in angiogenesis.

The interstitial fluid of MTW9A and Walker carcinomas and their ethanol extract induced strong angiogenic response in the rabbit (New Zealand White) corneal test. The fluid collected in vivo was rich in E-type prostaglandins, and prostaglandin E1 (PGE1) in particular was strongly angiogenic at the lowest dose as compared with the angiogenic responses of prostaglandins E2, I2, and F2 alpha. Neoplastic fibroblasts also induced a strong angiogenic response, but in indomethacin-treated rabbits neovascularization failed to occur.

Temperature gradients and local perfusion in a mammary carcinoma.

Temperature gradients of mammary tumors in randombred Sprague-Dawley rats under normothermia, hypothermia, and hyperthermia were determined, and their experimental modifications were utilized to assess differences in perfusion rates within the neoplastic tissue. Normothermic tumors showed a circadian rhythm with zenith at midnight and nadir at midday. Differences between highest and lowest temperatures recorded during the 24-hour period reached up to 3 degrees C.