Tumor growth requires a competent vascular supply and angiogenesis has been considered as a potential target for the treatment of several cancers. The two clinically approved taxanes, paclitaxel and docetaxel, are novel antimitotic agents that are under extensive investigation in clinical trials. Both taxanes have demonstrated significant activity against many solid tumors, but little is known about the effect of paclitaxel and docetaxel on endothelial cell function and angiogenic processes. The purpose of our study was to examine and compare the effects of these drugs on angiogenic processes in vitro and in vivo. These processes include: proliferation, migration and differentiation of cultured human umbilical vein endothelial cells (HUVEC) (in vitro), capillary sprouting of rat aortic ring explants (ex vivo) and HT1080 tumor growth in vivo. Our results demonstrate that endothelial cells are 10–100‐fold more sensitive to these drugs than tumor cells. Additionally, comparison of the taxanes demonstrated that angiogenesis is blocked by both drugs primarily via inhibition of proliferation and differentiation (tube assay) and induction of cell death. Docetaxel, however, appears to be more potent at inhibiting angiogenesis, with an IC50 concentration 10× less than that of paclitaxel. We conclude that these important findings should be taken in account in clinical trials where tumor angiogenesis is being targeted. © 2002 Wiley‐Liss, Inc.

Tumor growth and angiogenesis are interdependent. Cyclooxygenase (COX) catalyzes the synthesis of prostaglandins from arachidonic acid. Nonsteroidal antiinflammatory drugs (NSAIDs) inhibit COX-mediated synthesis of prostaglandins. COX-1 is constitutively expressed in a wide range of tissues, whereas COX-2 is cytokine inducible. Enhanced COX-2 expression has been attributed a key role in the development of inflammation and related processes observed in pathologically altered disease states. Two specific COX-2 inhibitors, namely rofecoxib (Vioxx) and celecoxib (Celebrex), both oral agents and U.S. Food and Drug Administration approved, have been shown preclinically and clinically to have efficacy comparable to that of NSAIDs for relief of pain and inflammation in osteoarthritis, with decreased risk of gastrointestinal damage. Little is known about how angiogenesis is affected by the combination of rofecoxib and radiation. We have evaluated the combination of rofecoxib, at various concentrations, and radiation on cytokine-induced angiogenesis in vitro. We have found that rofecoxib inhibited endothelial cell proliferation, migration, and tube formation (differentiation) at clinically relevant doses. In combination with radiation, inhibition of endothelial cell function further increased twofold. The combination of rofecoxib and radiation suggests a complementary strategy with clinical ramifications to target angiogenesis- dependent malignancies.

Purpose: Two specific COX-2 inhibitors, rofecoxib (VioxxTM) and celecoxib (CelebrexTM), are FDA approved. To date, there is no published data regarding the ability of rofecoxib to impact on endothelial cell function. The purpose of this study was to examine the effects rofecoxib on endothelial cell processes involved in angiogenesis, at clinically relevant doses, at, or below, the steady-state concentrations achieved at for use in the treatment of arthritis in combination with ionizing radiation. These include the effect of rofecoxib on the proliferation, attachment, and differentiation of cultured human umbilical vein endothelial cells (HUVEC) in-vitro, capillary sprouting of rat aortic ring explants embedded in collagen (ex-vivo) and Matrigel-induced angiogenesis (in-vivo) and COX-2 expression (Immunohistochemical). Materials and Methods: Single-donor human umbilical vein endothelial cells (HUVECs) were isolated from freshly delivered umbilical cords and used at passage 3-5. Cells were incubated at a subconfluent density with different concentrations of rofecoxib (0.5-2.0 μM/ml) and the effect of the drug +/- radiation (2 Gy) on cell proliferation, migration (modified Boyden chamber assay), tube differentiation (3D matrigel tube formation assay). In addition, a modified method of the rat aortic ring explant embedded in Matrigel (ex-vivo) assay of Nicosia was used. The aorta capillary sprouts represent all phases of angiogenesis (invasion, proliferation, migration and tube formation). An in-vivo Matrigel plug assay was used to evaluate the effect in nu/nu mice. Tunnel assay was used for apoptosis. Results: Proliferating HUVECs had a high baseline expression of COX-2. Rofecoxib inhibited endothelial cell proliferation, migration, and tube formation (differentiation) in a dose dependent manner. The half-maximal inhibition dose was 0.5 μM (p <0.001). In combination with low dose radiation, inhibition of sprouting and tube formation increased by 50-100%. Inhibition of angiogenic processes suggests at least an additive effect at low doses of both drug and radiation. Combination therapy increased the percentage of cells undergoing apoptosis. In-vivo, rofecoxib inhibited Matrigel induced angiogenesis. Conclusion: In-vitro, clinically relevant concentrations of rofecoxib inhibited endothelial cell proliferation, migration and tube formation in response to chemotactic and mitogenic growth factors. The addition of low dose ionizing radiation potentiated these antiangiogenic effects in a dose dependent manner. The anti-proliferative effects of rofecoxib-induced radiosensitization of endothelial cells may result from increased induction of apoptosis. A phase II trial using rofecoxib and radiation therapy for the treatment of glioblastoma multiforme by the RTOG is planned. Figure 1 Figure 1View Large Image Figure ViewerDownload (PPT)

Angiogenesis, the process of new vessel growth, is necessary for many normal physiological and pathological processes such as tumor growth, wound healing and ischemia. We have recently examined in vitro and in vivo the ability of two potent angiogenic compounds, SIKVAV (a peptide derived from the alpha chain of laminin-1) and Neuropeptide Y (NPY) to revascularize ischemic tissue. These compounds were tested in an ex vivo capillary sprouting angiogenesis assay that uses rat aortic rings. Both NPY and SIKVAV in the presence of VEGF, stimulated the formation of long sprouts at concentrations of 1 ng NPY (0.2 pmol/L) and 100 micrograms SIKVAV. In comparison very little sprouting occurred in the control rings and 50 ng of VEGF alone was required to induce equivalent number of sprouts as NPY. SIKVAV and NPY were further tested in vivo in a rat hindlimb ischemic model. Both compounds (500 micrograms SIKVAV and 10 ng of NPY) were embedded in the rat hind limb following unilateral ligation of the femoral artery 1 cm proximal to the adductor hiatus. After two weeks control peptides show little or no revascularization of the hindlimb distal to the ligation; however, both SIKVAV and NPY demonstrated a two-fold increase in new vessels in the region proximal to the ligation. Histological sections of latex perfused hindlimb demonstrated that ligated limbs had very few latex-filled dermal capillaries. Limbs treated with SIKVAV and NPY, however, demonstrated normal distribution in the dermal capillary beds. These data indicate that both SIKVAV and NPY are potent angiogenic factors that show promising potential clinical application to the revascularization of ischemic tissue.

Microvascular dysfunction due to endothelial damage is often associated with the ionizing radiation used during cancer therapy. This radiation-induced capillary injury is a major factor in the inhibition of new vessel growth (angiogenesis) and in disease states such as radiation-induced pneumonitis and nephropathy. Many studies have examined the effects of radiation on endothelial cell function; however, little is known regarding the role the basement membrane plays in radiation-induced endothelial cell damage and angiogenesis. Therefore, we examined the effects of gamma radiation on aortic explants, and in vitro on three endothelial cell types (of artery, vein and capillary origin) irradiated with or without the basement membrane glycoprotein laminin-1. As expected, irradiation inhibited angiogenic sprouting of the aortic explants, endothelial cell proliferation, attachment, migration and differentiation in vitro in a dose-dependent manner. However, the effect of radiation on several of these processes in angiogenesis was reduced when the cells were irradiated on laminin-1. To further evaluate the effects of radiation on endothelial cells, we examined the expression of the vascular endothelial cell growth factor (VEGF) kinase domain region receptor in endothelial cells irradiated in the presence and absence of laminin-1. In endothelial cells irradiated on laminin-1, KDR expression increased 2.5-fold over control levels. Therefore, although radiation has a dose-dependent inhibitory effect on processes associated with angiogenesis in vitro, the presence of the basement membrane glycoprotein laminin-1 during irradiation decreases these effects.

Various cell adhesion molecules mediate the diverse functions of the vascular endothelium, such as cell adhesion, neutrophil migration, and angiogenesis. In order to identify cell adhesion molecules important for angiogenesis, we used anin vitromodel (Chalupowicz, Chowdhury, Bach, Barsigian, and Martinez,J. Cell Biol.130, 207–215, 1995) in which human umbilical vein endothelial cell monolayers are induced to form capillary-like tubes when a second gel, composed of either fibrin or collagen, is formed overlying the apical surface. In the present investigation, we observed that a monoclonal antibody directed against the first extracellular domain of human vascular endothelial cadherin (VE-cadherin, cadherin 5) inhibited the formation of capillary tubes formed between either fibrin or collagen gels. Moreover, when added to preformed capillary tubes, this antibody disrupted the capillary network. In contrast, monoclonal antibodies directed against the extracellular domain of N-cadherin, the αvβ3integrin, and PECAM-1 failed to inhibit capillary tube formation. During capillary tube formation, Western blot and RT-PCR analysis revealed no marked change in VE-cadherin expression. Immunocytochemical studies demonstrated that VE-cadherin was concentrated at intercellular junctions in multicellular capillary tubes. Thus, VE-cadherin plays a specific role in fibrin-induced or collagen-induced capillary tube formation and is localized at areas of intercellular contact where it functions to maintain the tubular architecture. Moreover, its function at tubular intercellular junctions is distinct from that at intercellular junctions present in confluent monolayers, since only the former was inhibited by monoclonal antibodies.

Perlecan is a major heparan sulfate proteoglycan of basement membranes and cell surfaces. Because of its strategic location and ability to store and protect growth factors, perlecan has been implicated in the control of tumor cell growth and metastatic behavior. To test the role of perlecan in malignancy, we generated several stably transfected clones of HT-1080, a human fibrosarcoma cell line, harboring a perlecan cDNA in the antisense orientation. Surprisingly, clones with a reduced synthesis of perlecan mRNA and protein core grew faster, formed larger colonies in semisolid agar, and induced faster formation of s.c. tumors in nude mice than the wild-type cells. Their growth properties in vitro were independent of exogenous basic fibroblast growth factor. Reduction of perlecan expression was associated with three distinct properties typical of tumor cells with a more aggressive phenotype: enhanced migration through 8-microm-pore filter, increased invasion in Matrigel-coated filters, and heightened adhesiveness to type IV collagen substrata. These results thus provide the first evidence that perlecan may inhibit the growth and invasiveness of fibrosarcoma cells in a basic fibroblast growth factor-independent pathway and raise the possibility that perlecan may prevent the infiltration of host tissues in mesenchymal neoplasms.

We have tested the effect of thrombin on endothelial cell tube formation in vitro and angiogenesis in vivo. Thrombin induces the differentiation of endothelial cells into capillary structures in a dose-dependent fashion (0.1-0.3 units thrombin/ml) on Matrigel, a laminin-rich reconstituted basement membrane matrix. At higher thrombin concentrations (1.0 unit/ml), a suppression of tube formation is evident, probably due to downregulation (desensitization) of the thrombin receptor. D-Phe-Pro-Arg-CH2Cl-thrombin is without effect when used alone, but it abolishes the tube-promoting effect of thrombin when used in combination with thrombin, indicating the involvement of the catalytic site of thrombin. Activation of protein kinase C (PKC) seems to be the transduction mechanism involved in the stimulation of tube formation by thrombin. Ro-318220 (3 micrograms/ml), a specific inhibitor of PKC, completely abolishes the stimulatory effect of thrombin. In the in vivo Matrigel system of angiogenesis, there is a 10-fold increase in endothelial cell infiltration in response to thrombin. These results provide evidence for the angiogenesis-promoting effect of thrombin in vivo and the induction by thrombin of the angiogenic phenotype of endothelial cells in vitro in the absence of other cell types such as smooth muscle cells, pericytes, and inflammatory cells.

The process of angiogenesis (vessel formation) and the resulting stabilization of the mature vessel are complex events that are highly regulated and require signals from both serum and the extracellular matrix. Endothelial cells rest on a specialized thin extracellular matrix known as the basement membrane. Endothelial cells lining normal blood vessels are usually quiescent. When a proper stimulus is present, angiogenesis beings when endothelial cells degrade their basement membrane and invade the surrounding extravascular matrix. Formation of new vessels involves the migration and proliferation of cells. To assist the cells in their migration, the extravascular matrix provides an environment rich in stromal collagen fibers, fibrin, hyaluoronic acid, vitronectin and fibronectin. Once the endothelial cells assemble to form a new vessel, the cells secrete a basement membrane that helps to stabilize and maintain the vessel wall. The basement membrane adheres tightly to cells comprising the vessel wall, provides inductive signals, and plays a important role in the homeostasis of new vessels. We have demonstrated that two major components of the basement membrane, laminin and collagen IV, possess endothelial cell binding sites which regulate vessel stability. In this chapter, we will define the role of these molecules in endothelial cell behavior.

Here we review the role of angiogenesis as it pertains to the interactions between the epithelium and the mesenchyme, especially during tumor growth and metastasis. We illustrate and discuss several models of angiogenesis including endothelial tube formation on Matrigel. Finally, we examine angiogenic factors using the Matrigel model and investigate several other matrix molecules for their importance in angiogenesis and epithelial/stromal interactions.

D. S. Grant1,*, J. L. Kinsella2, M. C. Kibbey1, S. LaFlamme1, P. D. Burbelo1, A. L. Goldstein2 and H. K. Kleinman1 1Laboratory of Developmental Biology, National Institute of Dental Research, National Institutes of Health, Bldg 30, Rm 430, Bethesda, MD 20892, USA 2Laboratory of Cardiovascular Science, Gerontology Reseach Center, NIA, Baltimore, MD, USA 3Department of Biochemistry and Molecular Biology, The George Washington University Medical School, Washington, DC, USA

BACKGROUND:Angiogenesis is a critical event in wound healing, tumor growth, and the inflammatory vasculitides. Since women have a higher incidence of many vasculitic diseases, we examined the effects of female sex steroids, particularly estradiol, on human umbilical vein endothelial cell (HUVEC) behavior in vitro and on angiogenesis in vivo.METHODS AND RESULTS:HUVECs were grown in estrogen-free medium before each assay. Exogenous 17 beta-estradiol (1 to 5 nmol/L) increased cell attachment to laminin, types I and IV collagen, and fibronectin, as well as to tissue culture plastic. After a confluent monolayer of cells was "wounded" by scraping, estradiol-treated (10(-8) mol/L) cells migrated into the wound three times faster than untreated cells. Cell proliferation on plastic and on laminin increased threefold to fivefold, respectively, in the presence of estradiol. Estradiol also enhanced the ability of HUVECs to organize into tubular networks when plated on a reconstituted basement membrane, Matrigel. Estradiol effects on both the "wounding" assay and tube formation were blocked by the specific estrogen receptor antagonist ICI 182,780. Ovariectomy markedly decreased in vivo vascularization of Matrigel plugs coinjected with basic fibroblast growth factor in mice. With estrogen replacement, angiogenesis was increased to the levels observed in nonovariectomized mice.CONCLUSIONS:These studies demonstrate that, in vitro and in vivo, estradiol enhances endothelial cell activities important in neovascularization and suggest a promoting influence of estrogens on angiogenesis.

We performed differential cDNA hybridization using RNA from endothelial cells cultured for 4 hours on either plastic or basement membrane matrix (Matrigel), and identified early genes induced during the morphological differentiation into capillary-like tubes. The mRNA for one clone, thymosin beta 4, was increased 5-fold. Immunostaining localized thymosin beta 4 in vivo in both growing and mature vessels as well as in other tissues. Endothelial cells transfected with thymosin beta 4 showed an increased rate of attachment and spreading on matrix components, and an accelerated rate of tube formation on Matrigel. An antisense oligo to thymosin beta 4 inhibited tube formation on Matrigel. The results suggest that thymosin beta 4 is induced and likely involved in differentiating endothelial cells. Thymosin beta 4 may play a role in vessel formation in vivo.

The molecular mechanisms involved in the dynamic interaction of human breast carcinoma cells with the endothelial cell lining of lymphatic vessels and post‐capillary blood venules are largely unknown. In the present study, laminar flow assays were used to investigate the ability of various normal breast cells and of breast‐ and colon‐tumor cells to adhere to human umbilical cord endothelial cell monolayers. MCF‐10A breast, MCF‐7 and T‐47D breast‐carcinoma and clone A, RKO, and HT‐29 colon‐carcinoma cells accumulated and rolled, in the presence of flow, on tumor necrosis factor (TNF)‐stimulated but not on unstimulated endothelial cell monolayers. Non‐tumor and tumor cells continued to form transient adhesions with TNF‐stimulated endothelial cells even when the flow rate was increased to levels found in arteries. Incubation of TNF‐stimulated endothelial cells with an E‐selectin‐specific monoclonal antibody (MAb) partially or completely inhibited dynamic interactions and diminished adhesion strength, whereas integrin β1‐ and integrin α6‐specific MAbs had no effect. A set of highly invasive breast‐carcinoma cells (MDA‐231, BT‐549, HS‐578t) neither adhered to nor rolled on resting or TNF‐stimulated endothelial cell monolayers. However, after 5 min of static incubation, a fraction of these cells attached strongly to resting and TNF‐stimulated endothelial cells and this static adhesion could not be blocked by an E‐selectin‐specific monoclonal antibody. Our results suggest that E‐selectin is a major homing receptor in the metastasis of some breast and colon cancers. © 1995 Wiley‐Liss, Inc.

BACKGROUND:The formation of a basement membrane is the last step in the development of a new blood vessel. Matrigel, a laminin-rich reconstituted basement membrane matrix induces the differentiation of endothelial cells into capillary-like structures.EXPERIMENTAL DESIGN:The effect of inhibitors of basement membrane collagen synthesis, tricyclodecan-9-yl xanthate (D609) and 8,9-dihydroxy-7-methyl-benzo[b] quinolizinium bromide (GPA 1734), was investigated on endothelial cell tube formation on Matrigel in vitro and in an angiogenesis assay in C57 black mice in vivo.RESULTS:D609 and GPA 1734 caused a dose-dependent decrease in tube formation in vitro with complete inhibition at 50 micrograms/ml for D609 and 15 micrograms/ml for GPA 1734. The inhibitory effect on capillary tube formation by both agents was reversible. Tube formation correlated well with collagenous protein biosynthesis. Parallel studies on endothelial cells cultured on plastic indicate that cell viability, proliferation, attachment, and morphology were not affected by the presence of these collagen inhibitors at doses that blocked tube formation and collagen biosynthesis. D609 and GPA 1734 also inhibited endothelial cell infiltration in response to SIKVAV in an in vivo angiogenesis model system.CONCLUSIONS:These results indicate that newly synthesised collagen is a prerequisite for expression of the endothelial cell phenotype for tube formation and that prevention of collagenous protein biosynthesis inhibits tube formation and angiogenesis in vivo.

Adhesion of leukocytes to endothelial cells is a critical step in the development of acute and chronic inflammatory lesions. We report here that estradiol treatment of cultured human umbilical vein endothelial cells stimulated up to a twofold increase in TNF-induced adhesion of both polymorphonuclear leukocytes and PMA-activated peripheral blood mononuclear cells. This effect was more evident (threefold increase) when endothelial cells were cultured on the basement membrane glycoprotein laminin. Progesterone, but not testosterone, had a similar stimulatory effect. Estradiol also promoted a slight increase in interferon gamma-stimulated endothelial cell adherence for peripheral blood mononuclear cells, but no effect of estradiol was observed when adhesion of leukocytes to endothelial cells was stimulated with IL-1 or IL-4. The estradiol-induced increase in leukocyte binding to human umbilical vein endothelial cells was partially blocked by antibodies to the adhesion molecules E-selectin, intercellular adhesion molecule type 1 (ICAM-1), and vascular cell adhesion molecule type 1 (VCAM-1). Indirect immunofluorescence techniques showed that estradiol produces an increase in TNF-induced cell surface expression of these molecules. Northern blot analysis demonstrated a transient increase in TNF-induced expression of mRNA for E-selectin, ICAM-1, and VCAM-1 in endothelial cells treated with estradiol. Our data demonstrate that estradiol has important regulatory functions in promoting leukocyte-endothelial cell interactions that might contribute to the observed predominance in females of some autoimmune inflammatory diseases.