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Angiogenesis, the generation of new capillaries from existing blood vessels, is rarely observed in the healthy organism, but can present during various paediatric diseases. In this review, we describe recent progress in the understanding of pathological angiogenesis and approaches for an improved therapy of angiogenic childhood diseases.
BACKGROUND: A human tissue-based angiogenesis assay is needed to study the biology of angiogenesis in human tumor tissue and to tailor drug selection for patients. METHODS: Fragments of tumor tissue are embedded in fibrin gels containing medium 199, endothelial growth medium, fetal bovine serum, and epsilon-aminocaproic acid. Tumor implants sprout angiogenic vessels that progressively grow into the fibrin matrix. The differential growth pattern of tumor cells and angiogenic vessels in the fibrin gel matrix separates the angiogenic vessels and the tumor stroma into independently observable regions (vessel and tumor compartments). The reproducibility of the assay was tested by using fresh tissue obtained from human tumor xenografts (IMR-32 [neuroblastoma], MDA-MB-231 [breast cancer], and LNCaP [prostate cancer]) grown in nude mice and from fresh surgical breast and thyroid cancer specimens. RESULTS: All tumor fragments studied showed angiogenic sprouting into the fibrin matrix. This created an angiogenic vessel compartment, which was separate from the tumor fragment. The capillary nature of sprouting was confirmed histologically by factor VIII immunohistochemistry. The angiogenic growth fraction was >80% in all groups studied. CONCLUSIONS: This assay may allow functional assessment of the angiogenic potential of human tumors and simultaneous evaluation of a therapeutic agent's antitumor and antiangiogenic effects by virtue of its dual-compartmental structure.
Experimental and clinical data indicate that in human hepatocellular carcinoma (HCC) tumor progression is associated with angiogenesis and that an increase in microvascular density is associated with a poor prognosis. This review summarizes the literature concerning the relationship between angiogenesis and progression in HCC. It is becoming increasingly evident that agents which interfere with blood vessel formation also block tumor progression. Accordingly, anti-angiogenic tumor therapy has gained much interest in preclinical and clinical assessments. The recent applications of anti-angiogenic agents which interfere or block HCC progression are reviewed.
In this paper, a simple mathematical model developed in H.A. Levine, B.D. Sleeman, M. Nilsen-Hamilton [J. Math. Biol., in press] to describe the initiation of capillary formation in tumor angiogenesis is extended to include the roles of pericytes and macrophages in regulating angiogenesis. The model also allows for the presence of anti-angiogenic (angiostatic) factors. The model is based on the observation that angiostatin can prevent the degradation of fibronectin in the basal lamina by inhibiting the catalytic action of active proteolytic enzyme. That is, it is proposed that the inhibitor 'deactivates' the protease but that it does not reduce the over all concentration of the protease. It consequently explores the possibility of preventing neovascular capillaries from migrating through the extra-cellular matrix toward the tumor by inhibiting protease action. The model is based on the theory of reinforced random walks coupled with Michaelis-Menten mechanisms which view endothelial cell receptors as the catalysts for transforming both tumor and macrophage derived angiogenic factors into proteolytic enzyme which in turn degrade the basal lamina. A simple catalytic reaction is proposed for the degradation of the basal lamina by the active proteases. A mechanism, in which the angiostatin acts as a protease inhibitor is discussed which has been substantiated experimentally. A second mechanism for the production of protease inhibitor from angiostatin by endothelial cells is proposed to be of Michaelis-Menten type. Mathematically, this mechanism includes the former as a subcase.
It is important to survey the molecular targets which are involved in tumor angiogenesis for the development of antiangiogenic agents as one of the cancer therapy. This article is meant to review the recent molecular targets of tumor angiogenesis and the molecular mechanism of antiangiogenic agents in human clinical trials.
The development of new blood vessels (angiogenesis) is necessary to sustain the growth of primary tumor as well as a process of tumor metastasis. Cancer cells activate the quiescent vasculature to produce new blood vessels via an "angiogenic switch". Understanding of molecular mechanisms involved in that process is essential for the development of antiangiogenic drugs. Drugs that inhibit angiogenesis could offer a treatment that is complementary to traditional chemotherapy. Chemotherapy directly targets tumor cells, which are prone to develop acquired drug resistance due to genetic instability. Antiangiogenic therapy is directed against endothelial cells in tumor stroma, which are genetically stabile. First results from animal studies supported the theory that endothelial cells do not develop drug resistance and had excellent results in inducing tumor quiescence. However, recent clinical trials showed that antiangiogenic therapy has limitations but that it can improve conventional therapeutic modalities of disseminated disease.
von Willebrand factor (vWF), a glycoprotein produced uniquely by endothelial cells and megakaryocytes, is routinely used to identify vessels in tissue sections. Vessel density in tumor specimens, as determined by immuno-histochemical staining for vWF or other endothelial cell markers, is a negative prognostic factor for many solid tumors. vWF is heterogeneously distributed throughout the vasculature, transcriptional control in response to the tissue microenvironment being responsible for local variations in endothelial cell levels of vWF. Here, we report that fibroblast growth factor-2 and vascular endothelial growth factor, potent angiogenesis inducers expressed in a variety of tumors, up-regulate expression of vWF mRNA and protein in cultured endothelial cells with a synergistic effect. Our data support the measurement of vWF mRNA in tumors to detect activated endothelium or angiogenesis. For this purpose, we developed a semi-quantitative RT-PCR for vWF mRNA. Preliminary results obtained with specimens from colon carcinoma and the corresponding normal colonic mucosa showed higher vWF mRNA levels in most tumors than in their normal counterparts. The differences in vWF mRNA levels were much larger than the differences in vessel counts between a tumor and the corresponding normal mucosa, indicating that high vWF mRNA levels in tumors may indeed be an early sign of activation of the endothelium. The rapidity, objectivity, sensitivity and specificity of this technique make it suitable for routine clinical application to identify aggressive, highly angiogenic tumors.
The histopathology and the epidemiology of human cancers, as well as studies of animal models of tumorigenesis, have led to a widely accepted notion that multiple genetic and epigenetic changes have to accumulate for progression to malignancy. Formation of new blood vessels (tumor angiogenesis) has been recognized, in addition to proliferative capabilities and ability to down-modulate cell death (apoptosis), as essential for the progressive growth and expansion of solid tumors. Mice overexpressing activated forms of oncogenes or carrying targeted mutations in tumor suppressor genes have proven extremely useful for linking the function of these genes with specific tumor features such as continuous proliferation, escape from apoptosis, invasion and neo-angiogenesis. The interbreeding of these mice allows for studying the extent of cooperativity between different genetic lesions in disease progression, leading to a greater understanding of multi-stage nature of tumorigenesis.
OBJECTIVES: Recently, it was confirmed that angiogenesis is important in the development and spread of a variety of human cancers, including prostate cancer (PCa). Tumor neovascularization is thought to be controlled by chemical signals, known as angiogenic factors (AF). To date, little is known regarding the existence and role of AF in PCa. We previously reported on vascular endothelial growth factor (VEGF) in PCa. Currently, we compare VEGF expression with that of interleukin-8 (IL-8), another putative regulator of angiogenesis. We evaluated the expression of these two important AF in PCa and explored the role of inflammatory cytokines IL-1 and tumor necrosis factor (TNF) in their regulation. METHODS: Ex vivo studies involved previously reported immunohistochemical analysis for VEGF and recent evaluation of IL-8 expression and distribution in archival tissue samples of PCa, benign prostatic hyperplasia (BPH), and normal prostate tissue. In vitro studies used PCa cells (DU-145) grown in culture and stimulated with cytokines thought to induce VEGF and IL-8 (ie, IL-1 alpha, IL-1 beta, TNF-alpha, and TNF-beta). After 24 hours, with or without cytokines, cell culture supernatants were analyzed by enzyme-linked immunosorbent assay or radioimmunoassay for VEGF or IL-8 levels. RESULTS: Immunohistochemical studies of prostate tissue showed that PCa cells stained positively for VEGF and IL-8. Benign prostatic hyperplasia and normal prostate cells displayed little staining for either AF. Low levels of VEGF and IL-8 were produced by unstimulated DU-145 cells. Induction of DU-145 cells with cytokines resulted in differential stimulation whereby TNF was the predominant inducer of VEGF, whereas IL-1 was the predominant inducer of IL-8. CONCLUSIONS: Our results indicate that significant levels of VEGF and IL-8 are present in PCa, but not BPH or normal prostate cells in vivo. In vitro studies suggest that differential regulation of AF expression occurs in PCa. Because IL-1 and TNF are present in the PCa tumor microenvironment, it is likely that differential regulation of AF also occurs in human PCa and contributes to differential tumor growth and metastasis.
Angiogenesis, the process of development of a new microvasculature, is regulated by a balance of positive and negative factors. We show both in vivo and in vitro that the members of the human prolactin/growth hormone family, i.e., human prolactin, human growth hormone, human placental lactogen, and human growth hormone variant are angiogenic whereas their respective 16-kDa N-terminal fragments are antiangiogenic. The opposite actions are regulated in part via activation or inhibition of mitogen-activated protein kinase signaling pathway. In addition, the N-terminal fragments stimulate expression of type 1 plasminogen activator inhibitor whereas the intact molecules have no effect, an observation consistent with the fragments acting via separate receptors. The concept that a single molecule encodes both angiogenic and antiangiogenic peptides represents an efficient model for regulating the balance of positive and negative factors controlling angiogenesis. This hypothesis has potential physiological importance for the control of the vascular connection between the fetal and maternal circulations in the placenta, where human prolactin, human placental lactogen, and human growth hormone variant are expressed.
Previously, the authors cloned and characterized murine brain-specific angiogenesis inhibitor 1 (mBAI1). In this study, the authors cloned mBAI2 and analyzed its functional characteristics. Northern and Western blot analyses demonstrated a unique developmental expression pattern of mBAI2 in the brain. The expression level of mBAI2 appeared to increase as the development of the brain progressed. Reverse transcription-polymerase chain reaction (RT-PCR) analyses demonstrated the existence of alternative splice variants of mBAI2, which were defective in parts of type I repeat of thrombospondin or the third cytoplasmic loop of the seven-span transmembrane domain that were considered essential to the functions of mBAI2. The expressions of spliced variants in the brain were differently regulated compared with wild-type mBAI2 during development and ischemic conditions. In situ hybridization analyses of the brain showed the same localization of BAI2 as BAI1, such as in most neurons of cerebral cortex. In the in vivo focal cerebral ischemia model and the in vitro hypoxic cell culture model with cobalt, BAI2 expression decreased after hypoxia and preceded the increased expression of vascular endothelial growth factor (VEGF). RT-PCR analysis of antisense BAI2 cDNA-transfected SHSY5Y cells showed an increased VEGF expression as well as a decreased BAI2 expression. Immunohistochemical study of focal ischemic cortex showed that the regional localization of decreased BAI2 was related to the formation of new vessels. These results suggest that the brain-specific developmental expression pattern of angiostatic BAI2 is correlated with the decreased neovascularization in the adult brain, and that angiostatic BAI2 participates in the ischemia-induced brain angiogenesis in concert with angiogenic VEGF.
To study angiogenesis in early steps of melanoma progression, 113 cutaneous melanomas 1 mm or less in thickness were stained with Ulex europaeus lectin. Vascular density was determined in the areas of greatest vascularization. To avoid the effect of anatomic location, the quotient between vascular density at the tumor base and in normal skin (vascular ratio) was obtained in each case. Of these melanomas, 46 were immunohistochemically stained for the presence of vascular endothelial growth factor (VEGF). Positivity was scored from 1-4 by comparing staining of melanoma cells with keratinocytes. Vascular ratio values in vertical growth phase melanomas were higher than those in radial growth phase when counting per 200 or 400 magnification (2.29 +/- 1.3 and 2.48 +/- 1.5 for vertical growth phase and 1.34 +/- 0.62 and 1.41 +/- 0.83 for radial growth phase melanomas, respectively). This difference was statistically significant (p < 0.0001 and p < 0.001 at x200 and x400, respectively). Also, VEGF staining was stronger in vertical growth phase melanomas when compared with radial growth phase melanomas (Chi square, p < 0.025). In conclusion, our findings suggest that angiogenesis and VEGF expression are associated with the development of vertical growth phase.
OBJECTIVE: The hematopoietically expressed homeobox (HEX) is transiently expressed in endothelial cells (ECs) during vascular formation in embryo. Here, we investigated whether HEX played any role in angiogenesis-related properties of ECs in vitro. METHODS AND RESULTS: We transiently overexpressed HEX in human umbilical vein ECs (HUVECs). To our surprise, HEX completely abrogated the response of HUVECs to vascular endothelial growth factor (VEGF) with regard to proliferation, migration, and invasion and abolished network formation by HUVECs on Matrigel. cDNA microarray analysis and quantitative real-time reverse transcription-polymerase chain reaction combined with Western blotting revealed that HEX significantly repressed the expression of VEGF receptor-1, VEGF receptor-2, neuropilin-1, tyrosine kinase with Ig and EGF homology domains (TIE)-1, TIE-2, and the integrin alpha(v) subunit, whereas it augmented the expression of endoglin in HUVECs. We established murine embryonic stem cells that were stably transfected with HEX sense cDNA or antisense cDNA, and we examined the in vitro differentiation to ECs. Although the expression of VEGF receptor-2 was decreased in sense transfectants, the number of cells expressing VE-cadherin, a specific marker of ECs, was not altered. CONCLUSIONS: Our present results suggest that HEX may not affect the differentiation of ECs but acts as a negative regulator of angiogenesis.
The objective of the HGF-STAT clinical trial is to determine whether perfusion can be improved by gene transfer with a plasmid DNA containing hepatocyte growth factor (HGF) in the affected limb of patients with unreconstructable critical limb ischemia (CLI). CLI results in a high rate of limb loss and impaired quality of life. The current therapeutic strategies, including bypass surgery and percutaneous interventions, are only successful in treating a subset of patients. Therapeutic angiogenesis is an investigational method that seeks to favorably impact tissue perfusion in CLI. HGF-STAT is a double-blind, parallel-group, placebo-controlled, dose-response study in 100 patients with unreconstructable CLI. Eligible subjects will be randomized 1:1:1:1 to receive saline placebo or one of three dose/regimens of HGF plasmid DNA. The selection of outcome measures, including the primary endpoint, and changes in transcutaneous oxygen pressure (TcPO2) from baseline to 3 months will be discussed. In conclusion, this study will help to determine whether therapeutic angiogenesis with HGF is a viable option in the treatment of patients with CLI.
alpha-Difluoromethylornithine (DFMO), an irreversible inhibitor of ornithine decarboxylase, inhibited B16 melanoma-induced angiogenesis in chick embryo chorioallantoic membrane and subsequently the growth of the tumor on the chorioallantoic membrane. These inhibitions were reversed by exogenous putrescine and spermidine. DFMO also inhibited rapid neovascularization in yolk sac membrane of 4-day-old chick embryos and the inhibition was reversed by exogenous putrescine and spermidine. DFMO strongly inhibited DNA synthesis and proliferation of bovine pulmonary artery endothelial (BPAE) cells in culture and decreased their ornithine decarboxylase activity and intracellular polyamine concentrations. Addition of putrescine to the culture medium of DFMO-treated BPAE cells restored their intracellular putrescine and spermidine concentrations and their DNA synthesis and proliferation. Addition of spermidine to cultures of DFMO-treated BPAE cells restored their intracellular spermidine concentration and their DNA synthesis and proliferation. DFMO inhibited the proliferation of B16 melanoma cells in culture but the inhibitory effect was much less than that on BPAE cells. When one-half the monolayer of confluent cultures of BPAE cells had been peeled off, addition of DFMO to the cultures inhibited the proliferation and extension of the BPAE cells into the vacant area but had no effect on stationary cells in the remaining half of the monolayer, suggesting that it inhibited induction of proliferation of endothelial cells. These findings suggest that the antitumor activity of DFMO against solid tumors is probably due more to its inhibition of tumor-induced angiogenesis by inhibition of proliferation of endothelial cells induced by polyamine depletion than to a direct effect on tumor cell proliferation.
The control of cellular responses on substrate surfaces is essential for logical surface design aiming at endothelialized, vital implant devices. In this paper, the surface property that alters cell adhesion, spreading, migration, and proliferation processes is shown to be a determinant of endothelial cell assembly or angiogenesis in vitro. This was clearly demonstrated on slightly hydrophobic cellulosic surfaces, which induced organized three-dimensional cellular assemblies of bovine thoracic endothelial cells. The results indicated that this was driven by enhanced migratory response and/or retraction or involution of two-dimensional adherent cells, in which cell-cell interaction was enforced in a time dependent fashion. The present study strongly suggests that the mechanism leading to in vitro angiogenesis is primarily due to a weak cell-substrate interaction relative to cell-cell interaction.
The aqueous extracts of 24 herbs traditionally used as curing ischemic heart disease in clinic in China were screened for their in vitro angiogenic activity, another twenty-four traditionally used as anti-tumor or anti-inflammatory remedies in China were screened for their in vitro anti-angiogenic activity. The activity of angiogenesis was determined by quantitation of vessels on chick embryo chorioallantoic membrane (CAM) model and cell proliferation of cultured bovine aortic endothelial cells (BAECs). Among the herbal extracts examined, the aqueous extracts of Epimedium sagittatum, Trichosanthes kirilowii and Dalbergia odorifera showed the strong angiogenetic activity both in CAM and BAECs models; and the aqueous extracts of Berberis paraspecta, Catharanthus roseus, Coptis chinensis, Taxus chinensis, Scutellaria baicalensis, Polygonum cuspidatum and Scrophularia ningpoensis elicited significant inhibition at a concentration of 1g dry herb /ml.
Endothelial-cell-stimulating angiogenesis factor (ESAF) has been shown to activate procollagenase and reactivate complexes of collagenase and gelatinase A with tissue inhibitor of metallo-proteinase (TIMP)-1. In the present paper we show a purification protocol for bovine pineal ESAF and that purified ESAF activates progelatinase A and prostromelysin-1. Unlike the activation of procollagenase by plasmin/plasminogen activator, which requires the presence of stromelysin for full activation, ESAF is able to activate fully all three proenzymes. Purified ESAF is also shown to reactivate the complexes of gelatinase A, collagenase and stromelysin-1 with TIMP-2. Once separated, both enzyme and inhibitor are active; however, ESAF binds to the enzyme in a manner preventing it from further inhibition by TIMP. ESAF is the only physiological molecule able to reactivate the TIMP/enzyme complex.