Inhibition of angiogenesis by interleukin 4.

Interleukin (IL)-4, a crucial modulator of the immune system and an active antitumor agent, is also a potent inhibitor of angiogenesis. When incorporated at concentrations of 10 ng/ml or more into pellets implanted into the rat cornea or when delivered systemically to the mouse by intraperitoneal injection, IL-4 blocked the induction of corneal neovascularization by basic fibroblast growth factor. IL-4 as well as IL-13 inhibited the migration of cultured bovine or human microvascular cells, showing unusual dose-response curves that were sharply stimulatory at a concentration of 0.01 ng/ml but inhibitory over a wide range of higher concentrations. Recombinant cytokine from mouse and from human worked equally well in vitro on bovine and human endothelial cells and in vivo in the rat, showing no species specificity. IL-4 was secreted at inhibitory levels by activated murine T helper (TH0) cells and by a line of carcinoma cells whose tumorigenicity is known to be inhibited by IL-4. Its ability to cause media conditioned by these cells to be antiangiogenic suggested that the antiangiogenic activity of IL-4 may play a role in normal physiology and contribute significantly to its demonstrated antitumor activity.

CD36 mediates the In vitro inhibitory effects of thrombospondin-1 on endothelial cells.

Thrombospondin-1 (TSP-1) is a naturally occurring inhibitor of angiogenesis that is able to make normal endothelial cells unresponsive to a wide variety of inducers. Here we use both native TSP-1 and small antiangiogenic peptides derived from it to show that this inhibition is mediated by CD36, a transmembrane glycoprotein found on microvascular endothelial cells. Both IgG antibodies against CD36 and glutathione-S-transferase-CD36 fusion proteins that contain the TSP-1 binding site blocked the ability of intact TSP-1 and its active peptides to inhibit the migration of cultured microvascular endothelial cells. In addition, antiangiogenic TSP-1 peptides inhibited the binding of native TSP-1 to solid phase CD36 and its fusion proteins, as well as to CD36-expressing cells. Additional molecules known to bind CD36, including the IgM anti-CD36 antibody SM, oxidized (but not unoxidized) low density lipoprotein, and human collagen 1, mimicked TSP-1 by inhibiting the migration of human microvascular endothelial cells. Transfection of CD36-deficient human umbilical vein endothelial cells with a CD36 expression plasmid caused them to become sensitive to TSP-1 inhibition of their migration and tube formation. This work demonstrates that endothelial CD36, previously thought to be involved only in adhesion and scavenging activities, may be essential for the inhibition of angiogenesis by thrombospondin-1.

Pigment epithelium-derived factor: a potent inhibitor of angiogenesis.

In the absence of disease, the vasculature of the mammalian eye is quiescent, in part because of the action of angiogenic inhibitors that prevent vessels from invading the cornea and vitreous. Here, an inhibitor responsible for the avascularity of these ocular compartments is identified as pigment epithelium-derived factor (PEDF), a protein previously shown to have neurotrophic activity. The amount of inhibitory PEDF produced by retinal cells was positively correlated with oxygen concentrations, suggesting that its loss plays a permissive role in ischemia-driven retinal neovascularization. These results suggest that PEDF may be of therapeutic use, especially in retinopathies where pathological neovascularization compromises vision and leads to blindness.

Captopril inhibits angiogenesis and slows the growth of experimental tumors in rats.

Captopril, an inhibitor of angiotensin converting enzyme, is widely used clinically to manage hypertension and congestive heart failure. Here captopril is shown to be an inhibitor of angiogenesis able to block neovascularization induced in the rat cornea. Captopril acted directly and specifically on capillary endothelial cells, inhibiting their chemotaxis with a biphasic dose-response curve showing an initial decrease at clinically achievable doses under 10 microM and a further slow decline in the millimolar range. Captopril inhibition of endothelial cell migration was not mediated by angiotensin converting enzyme inhibition, but was suppressed by zinc. Direct inhibition by captopril of zinc-dependent endothelial cell-derived 72-and 92-kD metalloproteinases known to be essential for angiogenesis was also seen. When used systemically on rats captopril inhibited corneal neovascularization and showed the antitumor activity expected of an inhibitor of angiogenesis, decreasing the number of mitoses present in carcinogen-induced foci of preneoplastic liver cells and slowing the growth rate of an experimental fibrosarcoma whose cells were resistant to captopril in vitro. These data define this widely used drug as a new inhibitor of neovascularization and raise the possibility that patients on long term captopril therapy may derive unexpected benefits from its antiangiogenic activities.

Retinoic acid and interferon alpha act synergistically as antiangiogenic and antitumor agents against human head and neck squamous cell carcinoma.

Head and neck squamous cell carcinoma (HNSCC) is an aggressive malignancy in which multiple independent lesions develop over time throughout the mucosa of the upper aerodigestive tract. Therefore, the comprehensive treatment of this neoplasm must include a chemopreventive arm to hold premalignant lesions in check, a role well-suited to antiangiogenic agents. Retinoic acid (RA) and interferon alpha (IFN-alpha), drugs with known biological activity against HNSCC when used individually, are also inhibitors of angiogenesis. Here we show that they are remarkably synergistic antiangiogenic agents able to inhibit both the growth and the neovascularization of HNSCC injected into the floor of the mouth of nude mice. The mechanism of action of these drugs as antiangiogenic agents was 2-fold. They decreased the angiogenic activity of the tumor cells, and they caused the endothelial cells to become refractory to inducers of angiogenesis. When tumor cells were treated in vitro with IFN-alpha A/D, there was a dramatic drop in their secretion of interleukin-8, the major angiogenic factor produced by these tumors. When combined with RA, which causes tumor cells to secrete an inhibitor of angiogenesis, there was a synergistic inhibition of both tumor cell growth and secreted angiogenic activity. The combination of RA and IFN-alpha also acted synergistically on endothelial cells by reducing their responsiveness to both interleukin-8 and tumor conditioned media. Doses of each drug could be reduced by two logs without loss of activity. When animals bearing human HNSCC tumor cells were treated systemically with a combination of RA and IFN-alpha A/D at doses that were ineffective when used alone, dramatic decreases in both tumor growth and tumor angiogenesis were seen. These data suggest that the use of antiangiogenic mixtures may be a particularly effective way to design future chemoprevention protocols against HNSCC.

Molecular mediators of angiogenesis in bladder cancer.

Bladder tumors are characterized by markedly increased angiogenesis when compared to the normal urothelium (NU) from which they are derived. Here, we use both cultured cells and immunohistochemistry to demonstrate a primary regulatory role for thrombospondin-1 (TSP-1), a potent inhibitor of angiogenesis, in the development of bladder tumor angiogenesis. Secretions from bladder cancer (CA) cells stimulated endothelial cell migration and corneal neovascularization, whereas those from NU cells were inhibitory. The antiangiogenic activity of NU cells was primarily due to secreted TSP-1 because neutralizing antibodies completely relieved the inhibition. Neutralizing antibodies to several putative angiogenesis inducers identified vascular endothelial growth factor (VEGF) and, to a lesser extent, basic fibroblast growth factor as the primary inducers secreted by bladder cancer cells. The secretion of TSP-1 by low- and high-grade cancer cells was reduced >94% when compared to NU cells, and this loss of inhibitory TSP-1 accounted for the development of an angiogenic phenotype because both NU cells and cancer cells secreted similar levels of total stimulatory activity and VEGF. Immunohistochemistry showed that TSP-1 was significantly reduced in all grades of bladder cancer when compared to NU, whereas VEGF staining remained relatively constant. Taken together, these data suggest that down-regulation of TSP-1 secretion is a key event in the switch from an antiangiogenic to an angiogenic phenotype, which occurs early in the development of bladder cancer.

Inhibition of angiogenesis in human glioblastomas by chromosome 10 induction of thrombospondin-1.

Glioblastoma multiforme is distinguished from its less malignant astrocytoma precursors by intense angiogenesis and frequent loss of tumor suppressor genes on chromosome 10. Here we link these traits by showing that when a wild-type chromosome 10 was returned to any of three human glioblastoma cell lines U251, U87, or LG11, they lost their ability to form tumors in nude mice and switched to an antiangiogenic phenotype, as measured by the inhibition of capillary endothelial cell migration and of corneal neovascularization. This change in angiogenesis was directly due to the increased secretion of a potent inhibitor of angiogenesis, thrombospondin-1, because: (a) neutralizing thrombospondin completely relieved the inhibition; (b) the inhibitory activity of thrombospondin was not dependent on transforming growth factor beta; and (c) chromosome 10 introduction did not alter secreted inducing activity. The inducing activity was dependent on vascular endothelial cell growth factor and had an ED50 of 10 microg/ml in media conditioned by parental cells and 9-13 microg/ml in media conditioned by chromosome 10 revertants. Normal human astrocytes were also antiangiogenic due to secreted thrombospondin. The effect of chromosome 10 on thrombospondin production in vitro was reflected in patient material. Normal brain and lower grade astrocytomas known to retain chromosome 10 stained strongly for thrombospondin, but 12 of 13 glioblastomas, the majority of which lose chromosome 10, did not. These data indicate that the loss of tumor suppressors on chromosome 10 contributes to the aggressive malignancy of glioblastomas in part by releasing constraints on angiogenesis that are maintained by thrombospondin in lower grade tumors.

The distal region of the long arm of human chromosome 1 carries tumor suppressor activity for a human fibrosarcoma line.

Loss or inactivation of tumor suppressor genes has been implicated by indirect methods in the etiology of most human cancers. In the functional studies presented here, tumor suppressors on human chromosome 1 were investigated using microcell-mediated chromosome transfer. Translocated chromosomes from normal human cells representing most of 1q, or all of 1p and a small portion of 1q translocated onto the region of the X chromosome encoding HPRT, were transferred into human fibrosarcoma cell line HT1080. Analysis of HT1080 microcell hybrids showed a tumor suppressor activity associated with 1q. All HT1080 cells carrying transferred 1q in a ratio of 1:1 with the HT1080 genome showed a more flattened morphology and a reduced ability to form tumors in nude mice compared to parental HT1080 cells. Diploid HT1080 cells carrying a single extra 1q also had a longer population doubling time and showed a loss of ability to clone in soft agar. Tumors arose from 1q-containing clones with a longer latency period, and a large majority of the cells comprising these tumors had lost the transferred chromosome. These results indicate the presence on chromosome 1q23-qter of a tumor suppressor gene or genes that can act to suppress transformation of a human fibrosarcoma cell line.

Influence of a hamster tumor suppressor gene on transformation by viral and cellular oncogenes.

To determine if the tumor suppressor gene active in BHK hamster cells acts to maintain the normal phenotype by influencing oncogene transformation, careful, quantitative transfections with a variety of oncogenes were performed on four closely related BHK subclones. Two of the clones had an active suppressor gene (sup+ clones) and two of them had lost the suppressor (sup- clones) yet remained anchorage dependent. Both sup+ and sup- clones could be transformed to anchorage independence by ras, src, mos, neu, polyoma mT and SV40 suggesting that neither the presence nor the absence of the suppressor gene in BHK limits the transforming ability of these common oncogenes. All lines were resistant to transformation by N-myc, E1A and c-sis, oncogenes that may perform redundant functions in the immortal, fast growing BHK cell. SV40 small t antigen which has previously been considered unable to transform cultured cells by itself, was nevertheless able to transform sup+ BHK lines to anchorage independence in the absence of the viral large T antigen. Clones of sup- cells expressing high levels of small t antigen protein could be isolated, but they remained anchorage dependent and in tumorigenicity assays retained the long latent period characteristic of normal BHK cells. Such lines should enable the identification of cellular targets vital to the transforming function of SV40 small t.

Regulation of the activity of a new inhibitor of angiogenesis by a cancer suppressor gene.

An inhibitor has been identified in the conditioned medium of hamster cells and hamster-human hybrids that suppresses neovascularization in vivo in the rat cornea. Inhibitory activity was tightly linked to the presence of an active cancer suppressor gene in transformants and revertants, in segregating hybrids, and in temperature-limited transformants. It copurified with a approximately 140 kd glycoprotein. Polyclonal antiserum raised against the purified preparation recognized a 140 kd protein in conditioned medium and was able to adsorb out all antiangiogenic activity. These results define the control of the activity of an inhibitor of neovascularization as one function of the cancer suppressor gene active in BHK21/cl13 cells and simultaneously identify a new inhibitor of angiogenesis, a process vital to the growth of solid tumors.

Inhibition of squamous cell carcinoma angiogenesis by direct interaction of retinoic acid with endothelial cells.

Retinoic acid (RA) is a multifunctional drug that is particularly effective at preventing the development of multiple primary oral squamous cell carcinomas. A portion of this activity is due to the inhibition of tumor angiogenesis. It has been thought that RA influences tumor angiogenesis only via its interactions with the tumor cells themselves. Here, we test the hypothesis that the drug can also block neovascularization by directly inhibiting the angiogenic activity of normal endothelial cells. Clinically achievable doses of RA rapidly caused large- and small-vessel endothelial cells to become refractory to stimulation of migration either by tumor-conditioned media or purified angiogenic factors (a-fibroblast growth factor (aFGF), bFGF, vascular endothelial GF, platelet-derived GF, TGF beta-1, and IL-8). However, RA had little effect on their proliferation. Inhibition of migration was complete within 3 hours and was reversed 36 hours after drug removal. The migration of human oral keratinocytes was not sensitive to RA, whereas the migration of fibroblasts and vascular smooth muscle cells was inhibited. To determine if systemic RA affected neovascularization, rats were given 1 mg/kg/day of all-trans RA and their angiogenic potential was tested by implanting pellets of tumor-conditioned media into their avascular corneas. This treatment rendered the rats unable to mount a neovascular response in their corneas. These data demonstrate that RA directly affects endothelial cells, rapidly and reversibly inhibiting their ability to migrate toward a variety of stimuli in vitro and halting the formation of new vessels in vivo. These direct effects on vascular cells seem likely to contribute to the success of RA as a chemopreventive agent for oral squamous cell carcinoma.

A human fibrosarcoma inhibits systemic angiogenesis and the growth of experimental metastases via thrombospondin-1.

Concomitant tumor resistance refers to the ability of some large primary tumors to hold smaller tumors in check, preventing their progressive growth. Here, we demonstrate this phenomenon with a human tumor growing in a nude mouse and show that it is caused by secretion by the tumor of the inhibitor of angiogenesis, thrombospondin-1. When growing subcutaneously, the human fibrosarcoma line HT1080 induced concomitant tumor resistance, preventing the growth of experimental B16/F10 melanoma metastases in the lung. Resistance was due to the production by the tumor cells themselves of high levels of thrombospondin-1, which was present at inhibitory levels in the plasma of tumor-bearing animals who become unable to mount an angiogenic response in their corneas. Animals carrying tumors formed by antisense-derived subclones of HT1080 that secreted low or no thrombospondin had weak or no ability to control the growth of lung metastases. Although purified human platelet thrombospondin-1 had no effect on the growth of melanoma cells in vitro, when injected into mice it was able to halt the growth of their experimental metastases, providing clear evidence of the efficacy of thrombospondin-1 as an anti-tumor agent.

Retinoic acid induces cells cultured from oral squamous cell carcinomas to become anti-angiogenic.

Retinoids have shown great promise as chemopreventive against the development of squamous cell carcinomas of the upper aerodigestive tract. However, the exact mechanism by which they block new tumors from arising is unknown. Here, we report that 13-cis- and all-trans-retinoic acid, used at clinically achievable doses of 10(-6) mol/L or less, can directly and specifically affect cell lines cultured from oral squamous cell carcinomas, inducing them to switch from an angiogenic to an anti-angiogenic phenotype. Although retinoic-acid-treated and untreated tumor cells make the same amount of interleukin-8, the major inducer of neovascularization produced by such tumor lines, they vary in production of inhibitory activity. Only the retinoic-acid-treated cells produce a potent angio-inhibitory activity that is able to block in vitro migration of endothelial cells toward tumor cell conditioned media and to halt neovascularization induced by such media in the rat cornea. Anti-angiogenic activity is induced in the tumor cells by low doses of retinoids in the absence of toxicity with a kinetics that suggest that it could be contributing to the effectiveness of the retinoids as chemopreventive agents.

Localization of the angiogenesis inhibitor thrombospondin in human synovial tissues.

Recently, we have shown that a macrophage subpopulation isolated from the synovial tissue of patients with rheumatoid arthritis was potently angiogenic and that a secreted inhibitor of angiogenesis, which is controlled by a tumor suppressor gene in hamster cells, was similar to thrombospondin. In order to investigate the potential role of thrombospondin in human arthritic disorders, we employed immunohistochemistry to examine frozen synovial tissue sections from normal controls (n = 3), patients with rheumatoid arthritis (n = 14) and with osteoarthritis (n = 5). The synovial tissues were stained with monoclonal antibody (mAb) A2.5, which reacts with the heparin-binding domain of thrombospondin, mAb A6.1, which reacts with the epidermal growth factor repeat motif of thrombospondin, and with mAb A4.1, which reacts with the properdin-repeat domain of thrombospondin. In rheumatoid synovial tissues the anti-thrombospondin mAbs reacted with vascular endothelial cells, and to a lesser extent with vascular smooth muscle. Pericytes were stained, particularly with mAb 6.1. Reactivity was also found with isolated macrophages and with the macrophage-derived synovial lining layer in over half the tissues. In osteoarthritis synovial tissues, mAb A2.5 stained fewer macrophages than in rheumatoid arthritis synovial tissues. Slightly fewer blood vessels reacted with mAb A2.5 in normal compared to diseased synovia. The mAbs reacted with capillaries, venules and arterioles in all synovial tissues. We conclude that mAbs to thrombospondin react primarily with blood vessels and macrophages in synovial tissues. Perhaps thrombospondin may function as an adhesive glycoprotein mediating cellular interactions, or it may serve to counteract the effects of the angiogenic factors produced by cells within diseased synovial tissues.

Diagnostic ultrasound is unable to enhance the rate of neoplastic transformation in cultured mammalian cells.

The ability of diagnostic pulsed ultrasound to induce heritable genetic damage of the type that could result in neoplasia was assayed using BHK21/cl 13 hamster cells or normal human fibroblasts as targets. Using an exposure apparatus carefully designed to minimize beam attenuation and reflection, cavitation, and heating, cells were exposed from 20 seconds to 40 minutes either to clinical machines operating at maximum power, or to a highly focused nonclinical transducer at 2900 W/cm2, or to 200 shocks from a lithotripter. No evidence of an increase in the frequency of neoplastically transformed BHK cells or in the frequency of mutant human cells was seen over those found in matched sham-exposed controls.

Transformation of NIH/3T3 to anchorage independence by H-ras is accompanied by loss of suppressor activity.

Despite their familiar sensitivity to transformation by dominant-acting ras oncogenes, NIH/3T3 cells carry a ras suppressor. When tested by cell fusion they were able to suppress the anchorage-independent phenotype of both mouse and human cells transformed by activated H-ras or N-ras. This suppression occurred without a decrease in expression of the activated ras oncogene. Ras-transformed NIH/3T3 clones cured of their oncogene by benzamide treatment reverted to a nontransformed phenotype, but had lost the ability to suppress other ras transformants, indicating that their initial transformation was accompanied by suppressor loss. In hamster cells an active ras oncogene increased the rate of chromosome segregation by > 100-fold. These results suggest that in vitro transformation of NIH/3T3 cells by ras may be more similar to multistep in vivo tumor development than previously suspected, involving not only expression of an active oncogene but also loss of a suppressor activity, perhaps induced by the clastogenic oncogene.

Method of cavitation-suppressed exposure of cells and explant mouse embryos to clinical real-time and pulsed Doppler ultrasound.

An apparatus and procedure for well-controlled exposure of cells or explant mouse embryos to clinical real-time ultrasound are described. Cells or embryos to be exposed are suspended in media made sufficiently viscous through inclusion of methylcellulose that cavitation is suppressed but thermal effects remain negligible. During exposure, the scanning beam is precisely centered in a 2 mm x 20 mm slot in a 20 cm diameter agar disc containing the suspension. The high viscosity causes the cells to remain distributed uniformly throughout the exposure; this fact, along with precision beam alignment, ensures that exposure is well defined. Exposure data are acquired with a 0.6 mm diameter hydrophone.

Effects of thrombospondin-1 on disease course and angiogenesis in rat adjuvant-induced arthritis.

Leukocyte extravasation into the synovium is important in rheumatoid arthritis (RA). Thrombospondin (TSP)-1 mediates cell adhesion and migration and inhibits angiogenesis, and it has been implicated in RA. However, little information is available on the role of TSP-1 in arthritis-associated inflammation and neovascularization. Therefore, we analyzed the effects of TSP-1 in adjuvant-induced arthritis (AIA), a rat model for RA. Hydron pellets containing TSP-1 were implanted in one ankle of AIA rats post-adjuvant injection, while the contralateral ankle received sham implants. Body weight loss and joint swelling were determined in comparison to nonimplanted AIA controls. In addition, synovial vessel counts were obtained in TSP-1-versus sham-implanted ankles of the same rat. The implantation of TSP-1 pellets into one ankle resulted in an enhancement of swelling in both ankles. Furthermore, TSP-1 exhibited a biphasic modulatory effect on synovial vessel counts (P < 0.05). In conclusion, TSP-1 implanted into one ankle of AIA rats may augment the severity of the disease. One possible explanation, among others, for the modulating effect of TSP-1 on inflammation may be its effect on arthritis-related angiogenesis.

Keratinocyte growth factor induces angiogenesis and protects endothelial barrier function.

Keratinocyte growth factor (KGF), also called fibroblast growth factor-7, is widely known as a paracrine growth and differentiation factor that is produced by mesenchymal cells and has been thought to act specifically on epithelial cells. Here it is shown to affect a new cell type, the microvascular endothelial cell. At subnanomolar concentrations KGF induced in vivo neovascularization in the rat cornea. In vitro it was not effective against endothelial cells cultured from large vessels, but did act directly on those cultured from small vessels, inducing chemotaxis with an ED50 of 0.02-0.05 ng/ml, stimulating proliferation and activating mitogen activated protein kinase (MAPK). KGF also helped to maintain the barrier function of monolayers of capillary but not aortic endothelial cells, protecting against hydrogen peroxide and vascular endothelial growth factor/vascular permeability factor (VEGF/VPF) induced increases in permeability with an ED50 of 0.2-0.5 ng/ml. These newfound abilities of KGF to induce angiogenesis and to stabilize endothelial barriers suggest that it functions in microvascular tissue as it does in epithelial tissues to protect them against mild insults and to speed their repair after major damage.