Tumor-derived angiogenesis factors from rat Walker 256 carcinoma: an experimental investigation and review

Summary Angiogenesis, the process of developing a hemovascular network, is an essential feature of the growth of solid tumors, and is induced by factors secreted by tumor cells. Assay procedures suitable for the investigation of angiogenesis, and for the screening of angiogenesis factors during purification are reviewed; and a number of reports describing the purification of angiogenesis factors, primarily from the rat Walker 256 carcinoma as starting material, are discussed. Work from the authors' laboratory is also presented. Walker 256 cells grown in large-scale culture were the source of a reproducible and homogeneous source of angiogenic material. Factors secreted by these cells were isolated by a series of chromatographic steps. Ion exchange chromatography on carboxymethyl-Sephadex produced two active fractions, one of which was fractionated into several macromolecular species by lectin affinity and hydrophobic adsorption chromatography. The other gave a high mol.wt, active fraction that was resolved into a low mol.wt, active component and a non-angiogenic but possibly carrier molecule with a mol.wt of 140,000. While none of the angiogenic factors were identified chemically, the results demonstrate the existence of both high and low mol.wt tumor-secreted angiogenic substances, confirming the hypothesis for tumor-induced angiogenesis and predicting potential means to interfere with the process of tumor growth.This work was supported by funds from the Monsanto Company under Agreements with Harward University and from the U.S. Public Health Service, Contract No. N01-CB-43942. We are particularly indebted to Bernard S. Wildi and Monte C. Throdahl for their advice, cooperation, and encouragement. We also thank Dr Judah Folkman for the initial supply of male mouse submaxillary glands and rat Walker carcinoma conditioned medium, for help with the CAM assays, assays in the rabbit cornea, and many helpful discussions; and R. Bretton, A. Ehrlich, B. Evans, F. Fu, N. Hay and B. Tsokanis for excellent technical assistance.Author to whom correspondence should be addressed.     

Increased relative growth rate of normal rat cells in vitro with crocetin

Summary The carotenoid crocetin (earlier found to increase relative growth of Walker 256 tumor cells and their radiosensitivity), is shown to increase the relative growth of normal Sprague-Dawley rat muscle derived cells in vitro, presumably by increasing oxygen transport.     

The regulatory function of SPARC in vascular biology

SPARC is a matricellular protein, able to modulate cell/ECM interactions and influence cell responses to growth factors, and therefore is particularly attuned to contribute to physiological processes involving changes in ECM and cell mobilization. Indeed, the list of biological processes affected by SPARC includes wound healing, tumor progression, bone formation, fibrosis, and angiogenesis. The process of angiogenesis is complex and involves a number of cellular processes such as endothelial cell proliferation, migration, ECM degradation, and synthesis, as well as pericyte recruitment to stabilize nascent vessels. In this review, we will summarize current results that explore the function of SPARC in the regulation of angiogenic events with a particular emphasis on the modulation of growth factor activity by SPARC in the context of blood vessel formation. The primary function of SPARC in angiogenesis remains unclear, as SPARC activity in some circumstances promotes angiogenesis and in others is more consistent with an anti-angiogenic activity. Undoubtedly, the mercurial nature of SPARC belies a redundancy of functional proteins in angiogenesis as well as cell-type-specific activities that alter signal transduction events in response to unique cellular milieus. Nonetheless, the investigation of cellular mechanisms that define functional activities of SPARC continue to contribute novel and exciting paradigms to vascular biology.     

The fine structure of walker 256 carcinoma cells

Summary Several ultrastructural features of Walker 256 rat tumor cells which have not been observed before are revealed. Nuclear bodies either singlely or in pairs were found in some cells. Cytofilaments were observed in the cytoplasm and cytoplasmic projections in a few cells. Desmosomes which are usually regarded as a feature of differentiated cells were also observed.     

Oncogenes and angiogenesis: a way to personalize anti-angiogenic therapy?

The acquisition of oncogenic mutations and promotion of angiogenesis are key hallmarks of cancer. These features are often thought of as separate events in tumor progression and the two fields of research have frequently been considered as independent. However, as we highlight in this review, activated oncogenes and deregulated angiogenesis are tightly associated, as mutations in cancer cells can lead to perturbation of the pro- and anti-angiogenic balance thereby causing aberrant angiogenesis. We propose that normalization of the vascular network by targeting oncogenes in the tumor cells might lead to more efficient and sustained therapeutic effects compared to therapies targeting tumor vessels. We discuss how pharmacological inhibition of oncogenes in tumor cells restores a functional vasculature by bystander anti-angiogenic effect. As genetic alterations are tumor-specific, targeted therapy, which potentially blocks the angiogenic program activated by individual oncogenes may lead to personalized anti-angiogenic therapy.     

Angiogenesis and signal transduction in endothelial cells

Endothelial cells receive multiple information from their environment that eventually leads them to progress along all the stages of the process of formation of new vessels. Angiogenic signals promote endothelial cell proliferation, increased resistance to apoptosis, changes in proteolytic balance, cytoskeletal reorganization, migration and, finally, differentiation and formation of a new vascular lumen. We aim to review herein the main signaling cascades that become activated in angiogenic endothelial cells as well as the opportunities of modulating angiogenesis through pharmacological interference with these signaling mechanisms. We will deal mainly with the mitogen-activated protein kinases pathway, which is very important in the transduction of proliferation signals; the phosphatidylinositol-3-kinase/protein kinase B signaling system, particularly essential for the survival of the angiogenic endothelium; the small GTPases involved in cytoskeletal reorganization and migration; and the kinases associated to focal adhesions which contribute to integrate the pathways from the two main sources of angiogenic signals, i.e. growth factors and the extracellular matrix.Received 13 February 2004; received after revision 25 March 2004; accepted 19 April 2004     

The effect of photodynamic therapy on tumor angiogenesis

Photodynamic therapy (PDT), the activation of a photosensitive drug in tumor tissue with light of specific wavelength, has been used effectively to treat certain solid tumors. Though therapeutic responses are encouraging, PDT-mediated oxidative stress can act as an angiogenic switch that ultimately leads to neovascularization and tumor recurrence. This article explores the effect of PDT on angiogenesis in different tumor models. Overexpression of proangiogenic vascular endothelial growth factor, cyclooxygenase-2 and matrix metalloproteases has often been reported post-illumination. Recent clinical studies have demonstrated that inhibiting angiogenesis after chemotherapy and radiotherapy is an attractive and valuable approach to cancer treatment. In this review, we report the effective therapeutic strategy of combining angiogenesis inhibitors with PDT to control and treat tumors.     

Secretogranin III: a diabetic retinopathy-selective angiogenic factor

Secretogranin III (Scg3) is a member of the granin protein family that regulates the biogenesis of secretory granules. Scg3 was recently discovered as an angiogenic factor, expanding its functional role to extrinsic regulation. Unlike many other known angiogenic factors, the pro-angiogenic actions of Scg3 are restricted to pathological conditions. Among thousands of quantified endothelial ligands, Scg3 has the highest binding activity ratio to diabetic vs. healthy mouse retinas and lowest background binding to normal vessels. In contrast, vascular endothelial growth factor binds to and stimulates angiogenesis of both diabetic and control vasculature. Consistent with its role in pathological angiogenesis, Scg3-neutralizing antibodies alleviate retinal vascular leakage in mouse models of diabetic retinopathy and retinal neovascularization in oxygen-induced retinopathy mice. This review summarizes our current knowledge of Scg3 as a regulatory protein of secretory granules, highlights its new role as a highly disease-selective angiogenic factor, and envisions Scg3 inhibitors as “selective angiogenesis blockers” for targeted therapy.     

Elastase digested urokinase (ED-UK)

Summary Elastase digested urokinase (ED-UK) was prepared from human high mol. wt urokinase (HMW-UK). It resembled low mol. wt urokinase (LMW-UK) in its mol. wt, specific activity, and active sites. The steady-state kinetic parameters of each enzyme for the activation of human Glu-plasminogen also resembled each other, as did their amidase parameters (with pyro-Glu-Gly-Arg-pNA).     

Elastase digested urokinase (ED-UK)

Elastase digested urokinase (ED-UK) was prepared from human high mol. wt urokinase (HMW-UK). It resembled low mol. wt urokinase (LMW-UK) in its mol. wt, specific activity, and active sites. The steady-state kinetic parameters of each enzyme for the activation of human Glu-plasminogen also resembled each other, as did their amidase parameters (with pyro-Glu-Gly-Arg-pNA).     

Matriptase and its putative role in cancer

Tumor progression and metastasis are the pathologic effects of uncontrolled or deregulated invasive growth, a process in which proteases play a fundamental role. They mediate the degradation of extracellular matrix components and intercellular cohesive structures to allow migration of the cells into the extracellular environment and activate growth and angiogenic factors. In addition to metalloproteases and the plasminogen activation system, another protease, matriptase, contributes substantially to these processes. Matriptase is a type II transmembrane trypsin-like serine protease that is expressed by cells of epithelial origin and is overexpressed in a variety of human cancers. It has been suggested that this protease not only facilitates cellular invasiveness but may also activate oncogenic pathways. This review summarizes current knowledge about matriptase, its putative role in tumor initiation and progression, and its potential as a novel target in anti-cancer therapy. Received 29 June 2006; received after revision 1 August 2006; accepted 19 September 2006     

Purification of a nuclease from human serum

Summary The purification procedure for a nuclease from human serum is described. It includes ammonium sulfate precipitation, chromatography on DEAE-Sephadex and on Sephacryl-S 200, and preparative electrophoresis. The enzyme, purified about 2000-fold, is homogeneous in a sodium dodecyl sulfate electrophoretic system, where it has a mol. wt of 78,000. The pH optimum lies around pH 6.5; it is a sugar-nonspecific endonuclease.Acknowledgment. This work has been supported by a research grant from Stiftung Volkswagenwerk through the Akademie der Wissenschaften und der Literatur, Mainz. We thank Mrs R. Nehrbass and Mrs C. Wolpert for technical assistance.     

Evaluation of the anti-angiogenic effect of aloe-emodin

The present study identified aloe-emodin (AE, a hydroxyanthraquinone from Aloe vera and other plants) as a new anti-angiogenic compound with inhibitory effects in an in vivo angiogenesis assay and evaluates its effects on specific key steps of the angiogenic process. AE inhibits endothelial cell proliferation, but this effect is not cell specific, since AE also inhibits tumor cell proliferation. Cell migration and invasion are not remarkably affected by AE. On the other hand, AE has different effects on endothelial and tumor cell gelatinases. Two main targets of the pharmacological action of AE as an anti-angiogenic compound seem to be urokinase secretion and tubule formation of endothelial cells. Finally, AE produces a remarkable photocytotoxic effect on tumor cells. Taken together, our data indicate that AE can behave both as an anti-tumor and an anti-angiogenic compound and suggest that AE could be a candidate drug for photodynamic therapy. Received 7 September 2006; received after revision 17 October 2006; accepted 31 October 2006     

Purification of an L-fucose binding lectin fromUlex europeus by affinity column chromatography

Summary An L-fucose binding lectin fromUlex europeus was purified by affinity column chromatography using an L-fucose-starch complex. The lectin thus purified had a mol.wt of 60,000, and consisted of 2 glycoprotein subunit with mol.wt 29,000 and 31,000, respectively.     

Fenofibrate inhibits angiogenesis in vitro and in vivo

Fenofibrate, a peroxisome proliferator-activated receptor (PPAR)-alpha activator, used as a normolipidemic agent, is thought to offer additional beneficial effects in atherosclerosis. Since angiogenesis is involved in plaque progression, hemorrhage, and instability, the main causes of ischemic events, this study was designed to evaluate the action of fenofibrate on angiogenesis. Our results show that fenofibrate (i) inhibits endothelial cell proliferation induced by angiogenic factors, followed at high concentrations by an increase in apoptosis, (ii) inhibits endothelial cell migration in a healing wound model, (iii) inhibits capillary tube formation in vitro, and (iv) inhibits angiogenesis in vivo. Concerning the mechanism of action, the inhibition of endothelial cell migration by fenofibrate can be explained by a disorganization of the actin cytoskeleton. At the molecular level, fenofibrate markedly decreased basic fibroblast growth factor-induced Akt activation and cyclooxygenase 2 gene expression. This inhibition of angiogenesis could participate in the beneficial effect of fenofibrate in atherosclerosis.     

Role of plasminogen activator-plasmin system in tumor angiogenesis

New blood formation or angiogenesis has become a key target in therapeutic strategies aimed at inhibiting tumor growth and other diseases associated with neovascularization. Angiogenesis is associated with important extracellular remodeling involving different proteolytic systems among which the plasminogen system plays an essential role. It belongs to the large serine proteinase family and can act directly or indirectly by activating matrix metalloproteinases or by liberating growth factors and cytokines sequestered within the extracellular matrix. Migration of endothelial cells is associated with significant upregulation of proteolysis and, conversely, immunoneutralization or chemical inhibition of the system reduces angiogenesis in vitro. On the other hand, genetically altered mice developed normally without overt vascular anomalies indicating the possibility of compensation by other proteases in vivo. Nevertheless, they have in some experimental settings revealed unanticipated roles for previously characterized proteinases or their inhibitors. In this review, the complex mechanisms of action of the serine proteases in pathological angiogenesis are summarized alongside possible therapeutic applications.     

Respiratory control in liver mitochondria of rats hosting the walker 256 carcinoma tumor.

The decrease in the respiratory control ratio of mitochondria is a first order process when these organelles are incubated in isotonic sucrose. Furthermore, the initial respiratory control ratios and the rates of loss in respiratory control in liver mitochondria from rats hosting the Walker 256 carcinoma are not significantly different form the same properties of mitochondria from untreated animals.     

De novo hem- and lymphangiogenesis by endothelial progenitor and mesenchymal stem cells in immunocompetent mice

Cellular pro-angiogenic therapies may be applicable for the treatment of peripheral vascular diseases. Interactions between mesenchymal stem cells (MSCs) and endothelial progenitor cells (EPCs) may provide such a treatment option. With the exception of some studies in man, experiments have only been performed in immunodeficient mice and rats. We studied an immunocompetent syngeneic mouse model. We isolated MSCs from bone marrow and EPCs from the lung of adult C57/Bl.6 mice and co-injected them in Matrigel subcutaneously in adult C57/Bl.6 mice. We demonstrate development of both blood vessels and lymphatics. Grafted EPCs integrated into the lining of the two vessel types, whereas MSCs usually did not incorporate into the vessel wall. Injections of each separate cell type did not, or hardly, reveal de novo angiogenesis. The release of VEGF-A by MSCs has been shown before, but its inhibitors, e.g., soluble VEGF receptors, have not been studied. We performed qualitative and quantitative studies of the proteins released by EPCs, MSCs, and cocultures of the cells. Despite the secretion of VEGF inhibitors (sVEGFR-1, sVEGFR-2) by EPCs, VEGF-A was secreted by MSCs at bioavailable amounts (350 pg/ml). We confirm the secretion of PlGF, FGF-1, MCP-1, and PDGFs by EPCs/MSCs and suggest functions for VEGF-B, amphiregulin, fractalkine, CXCL10, and CXCL16 during MSC-induced hem- and lymphangiogenesis. We assume that lymphangiogenesis is induced indirectly by growth factors from immigrating leukocytes, which we found in close association with the lymphatic networks. Inflammatory responses to the cellular markers GFP and cell-tracker red (CMPTX) used for tracing of EPCs or MSCs were not observed. Our studies demonstrate the feasibility of pro-angiogenic/lymphangiogenic therapies in immunocompetent animals and indicate new MSC/EPC-derived angiogenic factors.