Influence of levamisole and other angiogenesis inhibitors on angiogenesis and endothelial cell morphology in vitro.

Angiogenesis, the formation of new blood vessels from existing vessels is required for many physiological processes and for growth of solid tumors. Initiated by hypoxia, angiogenesis involves binding of angiogenic factors to endothelial cell (EC) receptors and activation of cellular signaling, differentiation, migration, proliferation, interconnection and canalization of ECs, remodeling of the extracellular matrix and stabilization of newly formed vessels. Experimentally, these processes can be studied by several in vitro and in vivo assays focusing on different steps in the process. In vitro, ECs form networks of capillary-like tubes when propagated for three days in coculture with fibroblasts. The tube formation is dependent on vascular endothelial growth factor (VEGF) and omission of VEGF from the culture medium results in the formation of clusters of undifferentiated ECs. Addition of angiogenesis inhibitors to the coculture system disrupts endothelial network formation and influences EC morphology in two distinct ways. Treatment with antibodies to VEGF, soluble VEGF receptor, the VEGF receptor tyrosine kinase inhibitor SU5614, protein tyrosine phosphatase inhibitor (PTPI) IV or levamisole results in the formation of EC clusters of variable size. This cluster morphology is a result of inhibited EC differentiation and levamisole can be inferred to influence and block VEGF signaling. Treatment with platelet factor 4, thrombospondin, rapamycin, suramin, TNP-470, salubrinal, PTPI I, PTPI II, clodronate, NSC87877 or non-steriodal anti-inflammatory drugs (NSAIDs) results in the formation of short cords of ECs, which suggests that these inhibitors have an influence on later steps in the angiogenic process, such as EC proliferation and migration. A humanized antibody to VEGF is one of a few angiogenesis inhibitors used clinically for treatment of cancer. Levamisole is approved for clinical treatment of cancer and is interesting with respect to anti-angiogenic activity in vivo since it inhibits ECs in vitro with a morphology resembling that obtained with antibodies to VEGF.

Influence of angiogenesis inhibitors on endothelial cell morphology in vitro.

Human umbilical vein endothelial cells (HUVEC) propagated in co-culture with fibroblasts form capillary-like networks of tubes. Here we characterize the morphology and ultrastructure of HUVEC in such co-cultures and investigate the influence of different angiogenesis inhibitors on endothelial cell morphology. Addition of angiogenesis inhibitors to the co-culture disrupted endothelial network formation and influenced endothelial cell morphology in two distinct ways. Instead of characteristic capillary-like networks, the endothelial cell morphology appeared as either short cords or compact cell clusters of variable size. Electron microscopy (EM) showed that in co-culture untreated HUVEC formed capillary-like tubes with lumina and retained important ultrastructural and physiological properties of endothelial cells in functional vessels as they contained both Weibel-Palade bodies and transport vesicles. Immuno-EM showed that the endothelial cell marker CD 31 stained endothelial membranes at cell-cell contacts, and at the luminal and abluminal side of the capillary-like tubes, although most abundantly at the luminal membranes. No ultrastructural signs of apoptosis were seen in HUVEC in inhibitor-treated co-cultures. Our results demonstrate that treatment with levamisole or anti-VEGF inhibits endothelial cell differentiation into tubes or instead induces formation of compact endothelial cell clusters. Treatment with platelet factor 4, suramin and TNP-470 results in formation of short endothelial cell cords. We discuss the implications of these findings.

Levamisole inhibits angiogenesis in vitro and tumor growth in vivo.

The synthetic anthelmintic compound Levamisole has previously been used in cancer treatment as an adjuvant in combination with 5-fluorouracil. Its mode of action remains unclear, but an immune-stimulatory effect has been suggested. Here, we show that Levamisole inhibits angiogenesis in vitro and tumor growth in vivo. In vitro, Levamisole specifically inhibits proliferation and differentiation of endothelial cells propagated in co-culture with fibroblasts. In vivo, Levamisole inhibits the growth in nude mice of a transplanted human tumor. The use of nude mice as tumor hosts permits the discrimination between the angiogenesis inhibitory effect of Levamisole and its assumed immune-stimulatory effect. Our findings support a possible therapeutic effect of angiogenesis inhibitors in the treatment of cancer and call for further investigations of the mechanism(s) underlying the anti-angiogenic effect of Levamisole.

A quantitative ELISA-based co-culture angiogenesis and cell proliferation assay.

Since solid tumours and metastases depend on adequate blood supply, much research is focused on inhibition of angiogenesis. Unfortunately, most known angiogenesis inhibitors have serious side effects when used as therapeutic agents in man. It is therefore important to develop methods to identify well-tolerated and efficient angiogenesis inhibitors. As a method for identification of new angiogenesis inhibitors we have further developed the procedure described by Bishop et al. (Angiogenesis 1999;3:335-44) to a quantitative ELISA-based fibroblast and endothelial cell co-culture angiogenesis assay. In each well of a 96-microwell plate, human umbilical vein endothelial cells (HUVEC) are seeded onto normal human dermal fibroblasts (NHDF) and propagated in co-culture for 72 h with or without a potential angiogenesis inhibitor. The effect on total cell proliferation is evaluated by quantitative immunochemical measurement of DNA, and on endothelial tube formation by quantification of CD 31, von Willebrand factor, and collagen IV. After ELISA reading, the morphology of the tubular structures formed by HUVEC is visualised with BCIP/NBT, permitting a quantitative result and a qualitative evaluation of cell morphology from the same well. We have used the assay to demonstrate the effect of well-known angiogenesis inhibitors on HUVEC tube formation.

Immune selection in murine tumors. Ph.d thesis.

It must be assumed that all tumor cells produce proteins which do not belong to a normal cell. These are called tumor-associated or tumor-specific antigens. In the classic immune surveillance theory it is believed that the cellular immune defense (the T-cell system) continuously discovers and eliminates newly arisen tumor cells which express such tumor-specific antigens. Since then it has been shown that one of the preconditions for the T-cell system to be able to recognize antigens is that they are presented by MHC class I histocompatibility antigens. There is a continual processing and presentation of all intracellular proteins in a cell. Thus, a tumor cell which produces an abnormal protein will also present this and thereby expose itself to being killed by cytotoxic T cells. The antigens are presented in the form of short peptides (8-9 aminoacids), which arise as a result of controlled degradation of the original proteins. The peptides thus formed are transported by specialised molecules in the so-called endogenous antigen processing and presentation pathway, and are eventually bound to and presented by MHC class I molecules. It has been shown that many tumors express less MHC class I on their surface compared to the normal tissue from which they have arisen, and also that patients with reduced immune function have an increased incidence of certain forms of cancer. It is therefore widely believed that a low MHC class I level contributes to the ability of tumor cells to avoid the T-cell-mediated immune defense. The aim of the present research project was to confirm the existence of a T-cell-mediated immune selection in primary tumors. Another of its goals was to elucidate the extent to which tumor cells with low MHC class I expression showed poor ability to present antigen, and whether the reason for this could be found in one or more of the molecular systems which participate in antigen processing and presentation. By using the chemical carcinogen 3-methylcholanthrene a total of 144 tumors were induced in immunologically normal and T-cell defective mice, respectively. It was assumed that tumors induced in normal mice would be immune selected, whilst this would not be the case for tumors from T-cell defective mice. This enabled us to work with a tumor-material where the two populations only differed in that the one part had undergone selection by a T-cell system and the other had not. Tumor induction time turned out to be shorter in immune defective than in normal mice, and the tumor frequency was higher, which might be due to the fact that in normal mice tumor growth was inhibited and in certain cases stopped by the T cells. On transplantation of the uncloned cell lines which were established from the primary tumors to immunologically normal congenic recipients, we were able to show that most of the tumors which originated from mice with a functional T-cell system, and which must therefore be assumed to have undergone selection in the primary tumor host, were not immunogenic and were therefore accepted. On the other hand, most tumors which originated from T-cell-defective mice were rejected as a sign that immunogenic tumor cells, assumed to have expressed tumor antigen, had not been eliminated in the primary tumor host. Still, we found that the ability of tumor cells to induce an immune response on transplantation was not reflected in their MHC class I expression. Both tumor lines from immunodeficient and normal mice had highly varying MHC class I levels, and contrary to expectations the highest levels were seen in tumor lines from immunologically normal mice. At the same time we found that the expression levels for the three different MHC class I molecules were the same in the individual tumor lines, which might indicate that the three genes are syn-regulated. The MHC class I mRNA content in tumors from normal mice was generally concordant with the surface level of MHC protein. Among the tumor lines from immunodeficient mice, on the other hand, we found several where there was no such agreement, which was taken to indicate that tumor cells with deviant MHC class I gene transcription had not been eliminated, in contrast to in the immunocompetent tumor hosts. The ability of tumor cells to present antigen was investigated by infecting cells with virus and thereafter assessing their ability to function as target cells for virus-specific T cells in a cytotoxic test. Their ability to do this varied considerably, but showed a correlation with their MHC class I expression. Among the transplanted tumor lines that were not able to present viral antigen, the majority were accepted, while most of the tumor lines which were rejected on transplantation possessed the ability to present virus. Closer analysis of the composition of proteasomes, heat shock protein content and TAP molecule function, which are all involved in the antigen processing system, did not immediately reveal any defects. Treatment with interferon gamma, which is known to upregulate the transcription of MHC class I and a number of other proteins which are involved in antigen presentation, showed that by far the majority of the tumor lines were able to respond normally. This was also true for the tumor lines which had deviant MHC class I gene transcription and the cells which showed poor ability to present viral antigen. We did find, however, three cell lines which did not respond to interferon gamma, and they all had defective interferon gamma-signaling, not because they did not express the interferon-receptor on the surface, but possibly on account of their lacking phosphorylation of an intracellular signal molecule, Stat1.