Tumor Angiogenesis Factor Research Articles

Excessive production of transforming growth-factor β1 can play an important role in the development of tumorigenesis by its action for angiogenesis: validity of neutralizing antibodies to block tumor growth

Angiogenesis is an important part of tumor growth in vivo. We used the transfected Chinese hamster ovary (CHO) cells that overproduced recombinant transforming growth-factor β1 (TGF-β1) to examine the possible role of this factor in tumor growth and angiogenesis in a nude mouse model. The in-vitro proliferation of TGF-β1-transfected CHO cells was unaffected by the treatment of either recombinant TGF-β1 or an anti-TGF-β1 antibody. The TGF-β1-transfected cells grew more rapidly than the parental CHO cells when injected subcutaneously into nude mice. The tumors derived from the TGF-β1-transfected cells showed prominent tumor-associated angiogenesis, whereas the parental cells produced tumors without such angiogenesis. In addition, an anti-TGF-β1 neutralizing antibody was able to inhibit both growth and angiogenesis in the tumors derived from TGF-β1-transfected 0ells. These findings suggest that the overproduction of TGF-β1 by tumor cells can contribute to neovacularization and may help promote tumor development in vivo.

Vascular endothelial growth factor is a potential tumour angiogenesis factor in human gliomas in vivo.

Clinical and experimental studies suggest that angiogenesis is a prerequisite for solid tumour growth. Several growth factors with mitogenic or chemotactic activity for endothelial cells in vitro have been described, but it is not known whether these mediate tumour vascularization in vivo. Glioblastoma, the most common and most malignant brain tumour in humans, is distinguished from astrocytoma by the presence of necroses and vascular proliferations. Here we show that expression of an endothelial cell-specific mitogen, vascular endothelial growth factor (VEGF), is induced in astrocytoma cells but is dramatically upregulated in two apparently different subsets of glioblastoma cells. The high-affinity tyrosine kinase receptor for VEGF, flt, although not expressed in normal brain endothelium, is upregulated in tumour endothelial cells in vivo. These observations strongly support the concept that tumour angiogenesis is regulated by paracrine mechanisms and identify VEGF as a potential tumour angiogenesis factor in vivo.

A Mathematical Model for the Diffusion of Tumour Angiogenesis Factor into the Surrounding Host Tissue

Unless they are furnished with an adequate blood supply and a means of disposing of their waste products by a mechanism other than diffusion, solid tumours cannot grow beyond a few millimetres in diameter. It is now a well-established fact that, in order to accomplish this neovascularization, solid tumours secrete a diffusable chemical compound known as tumour angiogenesis factor (TAF) into the surrounding tissue. This stimulates nearby blood vessels to migrate towards and finally penetrate the tumour. Once provided with the new supply of nutrient, rapid growth takes place. In this paper, a mathematical model is presented for the diffusion of TAF into the surrounding tissue. The complete process of angiogenesis is made up of a sequence of several distinct events and the model is an attempt to take into account as many of these as possible. In the diffusion equation for the TAF, a decay term is included which models the loss of the chemical into the surrounding tissue itself. A threshold distance for the TAF is incorporated in an attempt to reflect the results from experiments on corneal implants in test animals. By formulating the problems in terms of a free boundary problem, the extent of the diffusion of TAF into the surrounding tissue can be monitored. Finally, by introducing a sink term representing the action of proliferating endothelial cells, the boundary of the TAF is seen to recede, and hence the position and movement of the capillaries can be indirectly followed. The changing concentration gradient observed as the boundary recedes may offer a possible explanation for the initiation of anastomosis. Several functions are considered as possible sink terms and numerical results are presented. The situation where the tumour (i.e. the source of TAF) is removed is also considered.

Effect of tumour angiogenesis factor on proliferation of endothelial cell and tube formation

The effect of a tumour angiogenesis factor on proliferation of various kinds of cells was examined in vitro. The factor (TAF) a polypeptide of 14000 molecular weight, was extracted and purified from the conditioned medium of ovarian clear cell carcinoma cell line (HUOCA-II). TAF at concentrations of 10 ng/ml and 100 ng/ml promoted proliferation of the endothelial cells and induced tube formation. However, it had no stimulatory effect on the proliferation of fibroblasts, endometrial columnar cells, squamous epithelial cells or cancer cells.

Amino Acid Sequence of a Lectin from Japanese Frog (Rana japonica) Eggs1

The complete amino acid sequence and the location of disulfide bonds of a lectin from Japanese frog (Rana japonica) eggs, which specifically agglutinates transformed cells, are presented. The sequence was determined by analysis of peptides generated by digestion of the S-carboxyamidomethylated protein with Achromobacter protease I, or chymotrypsin, and by chemical cleavage with BNPS-skatole or cyanogen bromide. The lectin is a single-chain protein consisting of 111 residues, with a pyroglutamyl residue at the amino terminus. Four disulfide bonds link half-cystinyl residue 19 to 72, 34 to 82, 52 to 97, and 94 to 111. The sequence and the location of the disulfide bonds are highly homologous to those of bull frog (Rana catesbeiana) egg S-lectin. They are also homologous to human angiogenin, a tumor angiogenesis factor, and a family of pancreatic ribonucleases.

A Mathematical Model for the Production and Secration of Tumour Angiogenesis Factor in Tumours

Solid tumour growth was hypothesized by Folkman (1976) to take place in two phases: the avascular phase and the vascular phase. In the first (avascular) phase, the tumour obtains its nutrients and disposes of its metabolic wastes by diffusion transport processes alone. Since the mechanism for growth is diffusion-limited, these tumours cannot expand indefinitely, but grow to a dormant state in which they have ceased expanding. The second (vascular) phase involves the eliciting of new blood vessels from the surrounding tissue and there is now firm evidence that tumour cells produce a chemical compound which triggers this process. The compound has been termed tumour angiogenesis factor (TAF) and considerable research has been carried out to try and isolate it and identify its biological structure as well as to elucidate its effects on the endothelial cells which form the lining of the blood vessels. In this second phase, the tumour grows rapidly and can spread to other parts of the body via blood-borne metastases. In this paper, the authors present a theoretical model for the production of the TAF within the tumour while in its diffusion-limited state and prior to its release into the surrounding host tissue. Using experimental results on vascularized tumours in conjunction with the findings of Oosaki et al. (1987), it is assumed that the profile of the TAF concentration within the tumour prior to secretion is qualitatively the same as that of the blood vessels found in neovascularized tumours. The TAF concentration c(x, t) is taken to satisfy the diffusion equation and the TAF production is accounted for either by the inclusion of a production term phi(c) in the diffusion equation itself or via inclusion in the boundary conditions. Taking phi(c) to be of the form 1/(1-c) produces the desired TAF profile and also leads to the possibility of a critical level being reached. It is shown that if the tumour is small enough this critical level can never be attained. However, if the tumour exceeds a certain size, then the critical level is attained and the TAF is subsequently secreted into the external tissue. This is described mathematically by the phenomenon of quenching, that is, the solution c(x, t) remains finite while some derivative becomes unbounded in finite time.

Tumor-related angiogenesis

In recent years, tumor-related angiogenesis has become an important field of research in oncology. It could be stated that growth of solid tumors is completely dependent on neovascularization to provide the tumor with all required nutrients. Special compounds (tumor angiogenesis factor[s]) are released by tumor cells into the environment to stimulate different types of normal cells to become active for the tumor. In particular, endothelial cells of neighboring capillaries are induced to react. They disintegrate their own basal lamina, detach from their neighbors, enter the extracellular matrix, and migrate toward the tumor mass. Cell divisions occur within such sprouts, thereby increasing the number of migrating endothelial cells. Strands of such cells are formed, and inter- and intracellular lumina develop. Loops of these hollow strands anastomose to form a network of new vessels which become connected with the blood circulation. The tumor mass thus becomes vascularized and can continue to grow. The prevention of neoangiogenesis has an enormous impact on cancer treatment by inhibiting the growth of the tumor. In this review, all important aspects of tumor-related angiogenesis are presented.

Fibroblast growth factors

The existence of fibroblast growth factors (FGFs) was proposed over 40 years ago to account for the ability of tissue extracts to stimulate fibroblast proliferation. In the 1970s it became clear that preparations containing FGF activity were in fact pleiotropic, affecting the growth and function of a wide variety of mesenchymal, endocrine and neural cells. Their angiogenic effects have promoted research in cardiology and neurology because of their proposed role in stimulating collateral vascularisation and recovery from ischemia. Their identity with a component of tumour angiogenesis factor activity has stimulated research in oncology and their capacity to enhance wound healing, nerve regeneration and cartilage repair has affected research in neurology, orthopaedic medicine and pathology. The potential therapeutic value of FGFs is just beginning to be realized and will be dependent on a concerted effort to establish their function in the regulation of normal cell homeostasis and the pathophysiology of disease.

Extraction of angiogenesis factor from chronic subdural haematomas. Significance in capsule formation and haematoma growth

The extraction of angiogenesis factor (AGF) from the contents of chronic subdural haematomas was attempted in order to explain the angiogenesis in the capsule. AGF was extracted from eight patients using the modified Phillip's method, which has previously been used for the extraction of tumour angiogenesis factor. The thickness of the haematoma capsule was measured immediately after removal. The Hounsfield units were evaluated as the average value of three areas in the haematoma on CT scans. Chromatographic separation of the treated contents gave five fractions. The highest activity was observed in the fourth fraction on bioassay by the air sac method. The intensities of AGF activity varied from patient to patient. Although no correlation existed between the degree of AGF activity and thickness of the capsule, a positive correlation was seen between the activity and the density of the contents as indicated by Hounsfield's units on a CT scan. This may indicate the existence of a sequence of cause and effect between increase in the AGF activity and haemorrhage in the capsule of the haematoma. AGF activity increases after haemorrhage in the haematoma capsule, leading to acceleration of angiogenesis in the capsule, which promotes the haemorrhagic cause in the haematoma. Thus, a vicious circle between AGF in the haematoma, angiogenesis in the capsule and haemorrhage in the haematoma is established. It is considered that this gives rise to growth of the haematoma.

Tumor angiogenic activity of gynecologic tumor cell lines on the chorioallantoic membrane

Tumor angiogenic activity (TAA) from tumor angiogenesis factor (TAF), produced by 24 cell lines of various kinds of gynecologic tumors, was assayed onto chorioallantoic membranes (CAMs) of chick embryos. Methylcellulose (1%) pellets containing 1 × 10 7 cells were placed on 8-day-old postfertilized CAMs, and the grade of neovascularization was assayed 3 days after inoculation. Neovascularization occurred prominently in such cell lines, as HTBOA (poorly differentiated ovarian carcinoma), HUOCA-II (poorly differentiated clear cell adenocarcinoma), HWUA (poorly differentiated endometrial adenocarcinoma), and in HKUS (uterine cervical small cell carcinoma); however, neovascularization did not occur in SNK (uterine leiomyosarcoma line). The cell lines which secreted TAF showed high heterotransplantability in the nude mice and produced rapidly growing tumors which were rich in blood vessels. However, the SKN line which did not secret TAF was not transplantable. These results suggested that there was a close relationship among TAA, transplantability, and tumor growth rate.

New Aspects of Blood Vessel Growth: Tumor and Tissue-Derived Angiogenesis Factors

Angiogenesis factors have been recently purified from both tumors and nonneoplastic tissues. The biological properties shown by these factors in vivo and on cultured endothelial cells will be discussed.

Establishment of HUOCA-II, a Human Ovarian Clear Cell Adenocarcinoma Cell Line, and Its Angiogenic Activity

A cell line designated "HUOCA-II" was established from a human ovarian clear cell adenocarcinoma. The HUOCA-II cells, which were oval, spindle, or polygonal and had neoplastic and pleomorphic features, grew in multiple layers without contact inhibition. The cell line grew fast (population doubling time, 24 hr), and 55 serial passages were carried out within 11 months. The chromosomal number ranged around 46, and no karyological abnormality was found in G-band karyotyping. When heterotransplanted into the subcutis of BALB/c nude mice, HUOCA-II cells produced a poorly differentiated clear cell adenocarcinoma. The tumor angiogenesis factor (TAF) of a molecular weight of about 14,000 was purified from the conditioned medium of HUOCA-II cells, and neovascularization was detected by bioassay with the use of the chorioallantoic membrane of a chick embryo. This TAF also stimulated the growth of endothelial cells in an in vitro culture system.

Tumours acquire their vasculature by vessel incorporation, not vessel ingrowth

This study was designed to investigate the early development of tumour circulation using a transplantable mouse mammary adenocarcinoma. Tumour cells (10(6] were injected subcutaneously into the flank and groups of treated mice were killed at 24 h intervals up to 12 days; the tumours and surrounding tissues being processed by standard histological methods. Sections of tumour were sub-divided into neoplastic tissue, vessels and connective tissue using computer-assisted morphometric techniques: the host tissue around the tumour was similarly quantified for vessels and connective tissue. With increasing tumour size, the proportion of vessels within tumours rapidly increased to reach a plateau of approximately 1.5 per cent of tumour volume, a 400 per cent increase on the vascular density of normal subcutaneous tissue. Within tumours, vascular density was always higher at the periphery than the centre. The most pronounced increase in vascular density affected the host tissue around the tumour. It is not clear why vascular development is most prominent outwith the tumour when postulated angiogenic factors, such as tumour angiogenesis factor, are presumably released within. Our results imply, however, that tumours acquire their vasculature by infiltration into, and expansion between, a network of newly formed vessels in the surrounding connective tissue.

Tumor angiogenesis activity of human choriocarcinoma cells grown in vitro

The growth of five different kinds of human Choriocarcinoma cell lines in vitro was quite alike. However, two distinct types of rapid and slow growth were observed in tumors grown in the hamster cheek pouch. The rapidly grown tumor tissues (BeWo, JEG-3, and NUC-1) involved significantly large numbers of blood vessels per microscopic field, as compared to slowly grown ones (SCH and HM). The vascular response was assayed using cell-free crude tumor angiogenesis factors (TAF) on chorioallantoic membrane (CAM) of the chick embryo. In all cell lines TAF activity correlated well with the extent of tumor growth in vivo. Gel filtration analysis showed that relatively high-molecular-weight (more than 10,000) factors could induce neovascularization in the both assays using CAM and rabbit cornea. These results suggest that a heterogeneity of TAF activity is present among human Choriocarcinoma cell lines and tumor growth in xenograft depends on the secretion of TAF by the tumor cells themselves.

VASCULAR CHANGES IN CUTANEOUS NEUROFIBROMAS

A variety of vascular changes has been associated with neurofibromatosis, and morphological alterations of arteries have been described previously. However, little attention has been paid to structural modifications of the microvasculature in neurofibromas themselves. Small vessels of 10 cutaneous neurofibromas, excised from patients with neurofibromatosis, were studied by using transmission electron microscopy and compared with normal skin vessels. The major alterations were: (1) increased thickness of the cytoplasm of endothelial cells which send numerous long processes to the lumen and, in some instances, obliterate it; (2) larger numbers of endothelial cell processes on the abluminal surface which are apposed to pericyte processes; (3) increased amounts of filaments and free ribosomes in the cytoplasm of endothelial cells, and (4) abundant pericytes with bundles of cytoplasmic filaments and many small subplasmalemmal densities. Mitotic figures were not seen in any of the vascular elements. These changes were interpreted to be the result of a generalized malformative process of blood vessels in neurofibromas rather than a consequence of the action of a tumor angiogenesis factor.

Detection of tumor angiogenesis factor in adenocarcinoma of kidney

Implantation of human renal adenocarcinoma in the rabbit cornea has resulted in new vascular growth from the limbus toward the tumor implant. This suggests that renal adenocarcinoma elaborates tumor angiogenesis factor (TAF) which stimulates endothelial cell growth. Such a substance could conceivably be responsible for the luxuriant vascularity of most renal adenocarcinomas. Conversely, absence or diminished secretion of TAF may be responsible for the hypovascular papillary renal adenocarcinomas and their recognized relatively benign clinical behavior.

Controlled release of macromolecules: Biological studies

Controlled release polymers composed of ethylene—vinyl acetate have served as an integral part of in vivo bioassays for substances which both induce and inhibit tumor neovascularization. These substances include partially purified extracts of tumor angiogenesis factor, and inhibitors from calf and shark cartilage as well as a heparin—cortisone combination. This combination has also been incorporated into bioerodible polyanhydrides which dissolved over a 3 week period and completely inhibited tumor neovascularization in the rabbit cornea. The application of these polymer systems to other bioassays as well as potential clinical applications is also discussed.

A mathematical model of tumour-induced capillary growth

The corneal limbal vessels of an animal host respond to the presence of a source of Tumour Angiogenesis Factor (TAF) implanted in the cornea by the formation of new capillaries which grow towards the source. This neovasculature can be easily seen and studied and this paper describes a mathematical model of some of the important features of the growth. The model includes the diffusion of TAF, the formation of sprouts from pre-existing vessels and models the movement of these sprouts to form new capillaries as a chemotactic response to the presence of TAF. Numerical results are produced for various values of the parameters which characterize the model and it is suggested that the model might form the framework for further theoretical work on related phenomena such as wound healing or to develop strategies for the investigation of anti-angiogenesis.

Effect of tumour angiogenesis factors on the membrane of capillary and aortic endothelial cells: an e.s.r. spin-label study.

Two distinct factors have been separated from an angiogenic extract of a rat Walker 256 carcinoma, one inducing proliferation and the other migration of capillary endothelial cells in vitro, but having no detectable effect on aortic endothelial cells. The influence of these factors on the order of plasma membranes of these cells was examined by electron spin resonance, using the lipophilic spin label 5-doxyl stearic acid. No detectable effect was observed on treating whole cells or isolated membranes with proliferation factor. In contrast, exposure of capillary endothelial cell membranes to migration factor caused a reduction of membrane order, particularly at temperatures above 30 degrees C. The migration factor had no detectable effect on membrane order of aortic endothelial cells.

Interrelationship between differentiation and malignancy-associated properties in glioma

The phenotypic expression of cells derived from human anaplastic astrocytomas, rat glioma, normal human adult and foetal brain tissue have been examined for differentiated and malignancy-associated properties. Glial fibrillary acidic protein (GFAP), high affinity glutamate and gamma-amino butyric acid (GABA) uptake and glutamine synthetase were used as indicators of astroglial differentiation. Plasminogen activator and tumour angiogenesis factor were the malignancy-associated markers. The normal adult brain-derived lines showed some differentiated astroglial features and expressed low levels of the malignancy-associated properties. The foetal cultures contained highly differentiated astroglia while the glioma lines showed considerable phenotypic heterogeneity from highly differentiated to undifferentiated. The least differentiated glioma cells exhibited the highest plasminogen activator activities. The density-dependent control of phenotypic expression was also investigated. High affinity GABA uptake, and GFAP in rat C6 glioma cultures, increased with increasing monolayer cell density, events probably mediated by an increase in the formation of cell-cell contacts at confluence. Plasminogen activator activity decreased with increasing cell density.