[Show abstract][Hide abstract] ABSTRACT: Tumor escape from immunity, as well as the failure of several anti-cancer vaccination and cellular immunotherapy approaches, is suggested to be due to the angiogenesis-mediated suppression of endothelial cell (EC) adhesion molecules involved in leukocyte-vessel wall interactions. We hypothesized that inhibition of angiogenesis would overcome this escape from immunity. We investigated this in vivo by means of intravital microscopy and ex vivo by immunohistochemistry in two mouse tumor models. Angiogenesis inhibitors anginex, endostatin, and angiostatin, and the chemotherapeutic agent paclitaxel were found to significantly stimulate leukocyte-vessel wall interactions by circumvention of EC anergy in vivo, i.e., by the up-regulation of endothelial adhesion molecules in tumor vessels. This was confirmed by in vitro studies of cultured EC at the protein and mRNA levels. The new angiostatic designer peptide anginex was most potent at overcoming EC anergy; the enhanced leukocyte-vessel interactions led to an increase in the numbers of tumor infiltrating leukocytes. While anginex inhibited tumor growth and microvessel density significantly, the amount of infiltrated leukocytes (CD45), as well as the number of CD8+ cytotoxic T lymphocytes, was enhanced markedly. The current results suggest that immunotherapy strategies can be improved by combination with anti-angiogenesis.
The FASEB Journal 05/2006; 20(6):621-30. · 5.70 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: To study the relationship between the angiogenic profile and leukocyte infiltration of tumors, single cell suspensions of archival frozen medullary and ductal breast cancer tissues were analyzed by flow cytometry. The amount of leukocytes and endothelial cells was measured, as well as the expression of intercellular adhesion molecule-1 (ICAM-1) on the endothelial cell fraction. A significantly higher number (3.2-fold) of infiltrating leukocytes was observed in medullary carcinoma. The composition of this infiltrate was similar to that seen in ductal carcinomas. The more intense infiltrate was explained by the approximately 3-fold enhanced endothelial ICAM-1 expression in medullary carcinoma. The angiogenic profile of all tumors was assessed by quantitative real-time reverse transcription-PCR analysis. Vascular endothelial growth factor (VEGF)-C and VEGF-D, but not VEGF-A, basic fibroblast growth factor, placental growth factor, and angiopoietins 1, 2, and 3 showed a relatively higher level of expression in ductal carcinoma than in medullary carcinoma. In vitro, both VEGF-C and VEGF-D were found to decrease endothelial ICAM-1 expression in the presence of basic fibroblast growth factor. These data suggest that in vivo angiogenic stimuli prevent the formation of an effective leukocyte infiltrate in tumors by suppressing endothelial ICAM-1 expression.
Clinical Cancer Research 12/2004; 10(21):7171-8. · 7.84 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: It is known that angiogenesis is of pivotal importance for the development of endometriosis. However, in the treatment of endometriosis patients, prevention of endometriosis lesion development only will not be sufficient as a therapy. Treatment options, aimed at interfering with established lesions, have to be developed. In this study we evaluated whether inhibition of angiogenesis by angiostatic therapy is also effective in antagonizing the sustentation of endometriosis. We evaluated the effect of the angiostatic compounds antihuman vascular endothelial growth factor, TNP-470, endostatin, and anginex on the growth of established endometriosis lesions in the nude mouse model. We show that human endometrium in the proliferative endometrium is highly angiogenic and that vascular endothelial growth factor-A is the most important angiogenesis promotory factor. The angiostatic compounds significantly decreased microvessel densities and the number of established endometriosis lesions. In the remaining lesions, the number of pericyte-protected vessels is not different in control and treated mice; however, the number of unprotected vessels was significantly reduced in the groups treated with the angiostatic agents. Our data demonstrate that inhibitors of angiogenesis effectively interfere with the maintenance and growth of endometriosis by inhibiting angiogenesis. This suggests that the use of angiostatic agents may be promising as a therapy for endometriosis.
[Show abstract][Hide abstract] ABSTRACT: The expression of endothelial cell (EC) adhesion molecules involved in leukocyte-vessel wall interactions is suppressed in malignancies. In the present study, we investigated in vivo the regulation of leukocyte-vessel wall interactions by the presence of a tumor. By means of intravital microscopy, tumor necrosis factor alpha-stimulated leukocyte-vessel wall interactions were studied in ear skin microvessels of nude mice bearing small human LS174T colon carcinomas and in C57Bl/6 mice bearing murine B16F10 melanomas. Leukocyte-vessel wall interactions were studied both within and outside small tumors growing in the ear, and in ear microvessels of mice with a large tumor growing on their flank. Tumor-free mice were used as controls. Compared with values measured at the edge of the ear and in the contralateral ear, leukocyte adhesion was found to be diminished significantly in vessels inside the ear tumor in both mouse models. This reduction disappeared with increasing distance from the tumor. Surprisingly, the level of leukocyte adhesion in ear venules of mice with a large flank tumor was also reduced significantly. Leukocyte rolling, i.e., the step preceding adhesion, was not influenced by the presence of a tumor in nude mice, but was down-regulated in immune-competent C57Bl/6 mice. Treatment of mice bearing a small ear tumor with a humanized antivascular endothelial growth factor antibody prevented the down-regulation of leukocyte-vessel wall interactions inside the tumor vessels compared with the nontreated group. Fluorescence-activated cell sorter analysis showed that isolated tumor ECs have suppressed levels of intercellular adhesion molecule 1 as compared with ECs from normal mouse tissues. In cultured b.END5 cells the tumor necrosis factor alpha-induced up-regulation of intercellular adhesion molecule 1 and vascular cell adhesion molecule 1 was reduced in ECs that were preincubated with basic fibroblast growth factor or vascular endothelial growth factor. The current results may have an impact on the effectiveness of clinical immunotherapeutic treatment protocols, because immune effector cells may not be able to enter tumor tissue.
Cancer Research 06/2003; 63(9):2322-9. · 8.65 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The current research aimed to define hypothesis-based anti-angiogenic properties of the vascular targeting agent combretastatin A-4 phosphate (combreAp). The in vitro wound assay indicated that combreAp potently inhibited migration of endothelial cells (EC). A significant inhibition of migration could already be measured after 2 hr of treatment. In a three-dimensional (3D) tube formation assay, combreAp inhibited sprout formation at concentrations that did not inhibit the proliferation of EC. At sub-ng concentrations the half-maximal response was reached. Interestingly, although combreAp is considered a vascular targeting agent, the human tumor cell lines tested were found to be 20-30 times more sensitive for combreAp than the human umbilical vein endothelial cells (HUVEC). A similar response difference between rat EC and R1 rat rhabdomyosarcoma tumor cells was observed. The growth inhibition in EC was only in part mediated by induction of apoptosis. The growth delay results obtained with the in vivo rodent tumor models involving repeat dosing of combreAp can partly be explained by anti-angiogenic activity of the compound. The results obtained with the various in vitro and in vivo assays substantiate an anti-angiogenic profile of combreAp, largely at the level of EC migration. This mechanism may operate to a different extent in different tumor types.
International Journal of Cancer 06/2003; 105(1):20-5. · 6.20 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The de novo designed angiogenesis inhibitor anginex was tested in vitro and in vivo for its mechanism of action and antitumor activity. The data presented here demonstrate that anginex is a powerful antiangiogenic agent with significant antitumor activity. The mechanism of action of anginex was found to be the induction of anoikis leading to apoptosis in angiogenically activated endothelial cells, resulting in an up to 90% inhibition of migration in the wound assay. Anginex inhibited angiogenesis as demonstrated in the in vitro mouse aortic ring assay. In addition, tumor-induced angiogenesis in the chick chorioallantoic membrane was markedly inhibited. Anginex showed profound antitumor activity in the syngeneic mouse B16F10 melanoma model and in a xenograft human tumor model. Microvessel density determination as well as magnetic resonance imaging showed that the antitumor activity in these tumor models resulted from the antiangiogenic activity of anginex. A complete absence of toxicity was observed in these models. The data presented here demonstrate that anginex is a promising agent for further clinical development.
The FASEB Journal 01/2003; 16(14):1991-3. · 5.70 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Angiogenesis, or the formation of new vasculature out of preexisting capillaries, is a sequence of events that is essential in the normal physiological processes of tissue growth and in a broad spectrum of pathologies. The diseases in which angiogenesis plays a key role are divided into diseases that are characterized by hypoxia/ ischemia and diseases that are dependent on neovascularization. The formerpathologies may benefit from therapeutic angiogenesis stimulation. This review concentrates on the different strategies to inhibit angiogenesis in diseases that are characterized by excessive angiogenesis, for example, cancer, arthritis, diabetic retinopathy, and inflammatory diseases. These diseases are dependent on the development of newvasculature, and hence, a large variety of different strategies to inhibit angiogenesis are underwayin laboratories throughout the world. At present, over250 angiogenesis inhibitors are described, and approximately half of them display activity in in vivo models. A large percentage of these molecules are natural, nonnatural, or synthetic so-called small molecules. Others are of protein origin, either endogenous or exogenous by nature. The authors highlight the current knowledge on the development of angiostatic proteins and peptides and their potential in the treatment of disease.