Honokiol,a small molecular weight natural product,inhibits angiogenesis in vitro and tumor growth in vivo

Natural products comprise a major source of small molecular weight angiogenesis inhibitors. We have used the transformed endothelial cell line SVR as an effective screen of natural product extracts to isolate anti-angiogenesis and anti-tumor compounds. Aqueous extracts of Magnolia grandiflora exhibit potent activity in our SVR proliferation assays. We found that the small molecular weight compound honokiol is the active principle of magnolia extract. Honokiol exhibited potent anti-proliferative activity against SVR cells in vitro. In addition, honokiol demonstrated preferential inhibition of primary human endothelial cells compared with fibroblasts and this inhibition was antagonized by antibodies against TNF alpha-related apoptosis-inducing ligand. In vivo, honokiol was highly effective against angiosarcoma in nude mice. Our preclinical data suggests that honokiol is a systemically available and non-toxic inhibitor of angiogenesis and should be further evaluated as a potential chemotherapeutic agent.     

A PlGF-1 Derived Peptide Inhibits Angiogenesis via HIF-1β/VEGF Pathway

Many diseases are associated with angiogenesis. Therefore, inhibition of angiogenesis is deemed as a treatment for those diseases. To date, many angiogenesis inhibitors are from large plasma proteins. In this study we identified a 21-aa peptide (named peptide ZY1) from human placenta growth factor-1 and it may serve as a potent angiogenesis inhibitor. Our study demonstrated ZY1 inhibited angiogenesis in vitro by suppressing proliferation, migration and tube formation of HUVECs. The in vivo inhibition activity of ZY1 was observed in chicken chorioallantoic membrane assays and tumor-bearing mouse models. Moreover, we found ZY1 inhibited angiogenesis by decreasing the expression of HIF-1β and subsequently reducing its downstream molecule VEGF. In conclusion, peptide ZY1 can inhibit angiogenesis and may serve as an anti-angiogenesis drug candidate.     

Antiangiogenic effect of carnosic acid and carnosol, neuroprotective compounds in rosemary leaves

Carnosic acid, a diterpene in rosemary, is considered to be beneficial in the prevention of chronic neurodegenerative diseases. Recently, it has been found that drugs with antiangiogenic activity lower the risk of neurodegenerative diseases. Thus it is of interest whether carnosic acid has antiangiogenic activity. In this study, carnosic acid suppressed microvessel outgrowth on ex vivo angiogenesis assay using a rat aortic ring at higher than 10 μM. The antiangiogenic effect of carnosic acid was found in angiogenesis models using human umbilical vein endothelial cells with regard to tube formation on reconstituted basement membrane, chemotaxis and proliferation. Although the carnosol in rosemary also suppressed angiogenesis, its effect was not more potent than that of carnosic acid in the ex vivo model. These results suggest that carnosic acid and rosemary extract can be useful in the prevention of disorders due to angiogenesis, and that their antiangiogenic effect can contribute to a neuroprotective effect.     

High efficacy and minimal peptide required for the anti‐angiogenic and anti‐hepatocarcinoma activities of plasminogen K5

Kringle 5(K5) is the fifth kringle domain of human plasminogen and its anti‐angiogenic activity is more potent than angiostatin that includes the first four kringle fragment of plasminogen. Our recent study demonstrated that K5 suppressed hepatocarcinoma growth by anti‐angiogenesis. To find high efficacy and minimal peptide sequence required for the anti‐angiogenic and anti‐tumour activities of K5, two deletion mutants of K5 were generated. The amino acid residues outside kringle domain of intact K5 (Pro452‐Ala542) were deleted to form K5mut1(Cys462‐Cys541). The residue Cys462 was deleted again to form K5mut2(Met463‐Cys541). K5mut1 specifically inhibited proliferation, migration and induced apoptosis of endothelial cells, with an apparent two‐fold enhanced activity than K5. Intraperitoneal injection of K5mut1 resulted in more potent tumour growth inhibition and microvessel density reduction than K5 both in HepA‐grafted and Bel7402‐xenografted hepatocarcinoma mouse models. These results suggested that K5mut1 has more potent anti‐angiogenic activity than intact K5. K5mut2, which lacks only the amino terminal cysteine of K5mut1, completely lost the activity, suggesting that the kringle domain is essential for the activity of K5. The activity was enhanced to K5mut1 level when five acidic amino acids of K5 in NH₂ terminal outside kringle domain were replaced by five serine residues (K5mut3). The shielding effect of acidic amino acids may explain why K5mut1 has higher activity. K5, K5mut1 and K5mut3 held characteristic β‐sheet spectrum while K5mut2 adopted random coil structure. These results suggest that K5mut1 with high efficacy is the minimal active peptide sequence of K5 and may have therapeutic potential in liver cancer.     

Synthesis and anti-angiogenic activity of 6-(1,2,4-thiadiazol-5-yl)-3-amino pyridazine derivatives

General screening for inhibitors of microvessel growth in vitro in the rat aortic ring assay led to the discovery of a novel series of thiadiazole pyridazine compounds with potential anti-angiogenic activity. Chemical optimization produced orally active compounds with potent in vitro and in vivo anti-angiogenesis and anti-tumor activities.     

Angiogenic inhibitor protein fractions derived from shark cartilage

Development of therapies based on the growth inhibition of new blood vessels is among the most intensively studied approaches to the treatment of cancer and other angiogenesis-related diseases. Shark cartilage has been proven to have inhibitory effects on the endothelial cell angiogenesis, metastasis, cell adhesion and MMP (matrix metalloprotease) activity. In the present study, we have used a chromatography-based procedure for the isolation and partial purification of a shark cartilage protein fraction containing anti-angiogenesis activity. Proteins were extracted in 4 M guanidinium chloride, followed by sequential anion- and cation-exchange column chromatography. Angiogenesis assays were performed using the rat aortic ring and chick CAM (chorioallantoic membrane) assay models. The results show that the final fraction contains two proteins with molecular masses of 14.7 and 16 kDa. The protein fraction is able to block microvessel sprouting in the collagen-embedded rat aortic ring assay in vitro and inhibition of capillary sprouting in the CAM assay in vivo. It is suggested that these are partially purified anti-angiogenesis proteins, which have further biotechnological or biomedical applications.     

Multiple signaling pathways are involved in endothelin-1-induced brain endothelial cell migration

We have observed that the vasoactive peptide endothelin-1 is a potent inducer of migration of primary human brain-derived microvascular endothelial cells. By blocking signal transduction pathways with specific inhibitors, and using dominant negative mutant infections, we have demonstrated that multiple pathways are involved in endothelin-1-induced migration. Absolutely required for migration are protein tyrosine kinase Src, Ras, protein kinase C (PKC), phosphatidylinositol 3-kinase, ERK, and JNK; partial requirements were exhibited by cAMP-activated protein kinase and p38 kinase. Partial elucidation of the signal transduction sequences showed that the MAPKs ERK, JNK, and p38 are positioned downstream of both PKC and cAMP-activated protein kinase in the signal transduction scheme. The results show that human brain endothelial cell migration has distinct characteristics, different from cells derived from other vascular beds, or from other species, often used as model systems. Furthermore, the results indicate that endothelin-1, secreted by many tumors, is an important contributor to tumor-produced proangiogenic microenvironment. This growth factor has been associated with increased microvessel density in tumors and is responsible for endothelial cell proliferation, migration, invasion, and tubule formation. Because many signal transduction pathways investigated in this study are potential or current targets for anti-angiogenesis therapy, these results are of critical importance for designing physiological antiangiogenic protocols. signal transduction; angiogenesis; microvessels; vasoactive peptides     

Tamoxifen and trifluoroperazine (Stelazine) potentiate cytostatic/cytotoxic effects of P-30 protein, a novel protein possessing anti-tumor activity

P-30 protein, a novel protein isolated in our laboratory from fertilized Rana pipiens eggs, has been shown to possess significant anti-proliferative and cytotoxic activity against a variety of human tumour cell lines. This protein also shows a potent anti-tumour activity in vivo in animal tumour models and is currently undergoing Phase I human clinical trials in cancer patient volunteers. The present study describes the in vitro effects of the concerted action of this protein and two other agents which affect the cell proliferative cycle. A significant potentiation of the P-30 protein-induced cell growth inhibition by tamoxifen as well as trifluoroperazine (Stelazine) in both the human A-549 lung carcinoma and the ASPC-1 pancreatic adenocarcinoma systems at wide ranges of drug concentrations was observed. The effect was apparently due to the synergistic action of P-30 protein and the agents tested. This data may provide clues that can be useful in explaining the mechanism of its anti-tumour activity. The results are also helpful for the designing in vivo animal and, perhaps eventually, human studies, whereby the combination therapies utilizing P-30 protein with agents of relatively low toxicity such as tamoxifen and/or Stelazine could offer a promising treatment(s) for these notoriously refractory types of human cancer.     

Tamoxifen and trifluoroperazine (Stelazine) potentiate cytostatic/cytotoxic effects of P-30 protein, a novel protein possessing anti-tumour activity

Abstract. P-30 protein, a novel protein isolated in our laboratory from fertilized Rana pipiens eggs, has been shown to possess significant anti-proliferative and cytotoxic activity against a variety of human tumour cell lines. This protein also shows a potent anti-tumour activity in vivo in animal tumour models and is currently undergoing Phase I human clinical trials in cancer patient volunteers. The present study describes the in vitro effects of the concerted action of this protein and two other agents which affect the cell proliferative cycle. A significant potentiation of the P-30 protein-induced cell growth inhibition by tamoxifen as well as trifluoroperazine (Stelazine) in both the human A-549 lung carcinoma and the ASPC-1 pancreatic adenocarcinoma systems at wide ranges of drug concentrations was observed. The effect was apparently due to the synergistic action of P-30 protein and the agents tested. This data may provide clues that can be useful in explaining the mechanism of its anti-tumour activity. The results are also helpful for the designing in vivo animal and, perhaps eventually, human studies, whereby the combination therapies utilizing P-30 protein with agents of relatively low toxicity such as tamoxifen and/or Stelazine could offer a promising treatment(s) for these notoriously refractory types of human cancer.     

Endostatin inhibits microvessel formation in the ex vivo rat aortic ring angiogenesis assay

Endostatin has demonstrated potent antiangiogenic and antitumor activity in mouse models. We have investigated the ex vivo rat aortic ring assay and a human vein model to assess the biological activity of murine and human endostatin. Rat aortic rings were exposed to recombinant murine endostatin (Spodoptera frugipera; Calbiochem, San Diego, CA) or recombinant human endostatin (Pichia pastoris; EntreMed, Rockville, MD). After 5 days, murine endostatin (500 microgram/ml) demonstrated inhibition of microvessel outgrowth with dose-dependent effects (down to 16 microgram/ml). No significant inhibition was observed with human endostatin in the rat assay. Human endostatin at 250 and 500 microgram/ml inhibited outgrowths from human saphenous vein rings after a 14-day incubation. Electron microscopy assessed the formation of basal lamina, confirming that the microvessels were progenitors of patent vessels. Immunostaining for Factor VIII or CD34 demonstrated that the microvessel cells were endothelial. BrdU incorporation assays supported the presence of proliferating endothelial cells, correlating with neovascularization from the aortic wall. We conclude that the rat aortic ring assay confirms the antiangiogenic activity of murine but not human endostatin, suggesting that the model may have species specificity. However, the human form shows biological activity against human vascular tissue.     

Inhibition of angiogenesis- and inflammation-inducing factors in human colon cancer cells <Emphasis Type="Italic">in vitro</Emphasis> and <Emphasis Type="Italic">in ovo</Emphasis> by free and nanoparticle-encapsulated redox dye,DCPIP

Background  

The redox dye, DCPIP, has recently shown to exhibit anti-melanoma activity in vitro and in vivo. On the other hand, there is increasing evidence that synthetic nanoparticles can serve as highly efficient carriers of drugs and vaccines for treatment of various diseases. These nanoparticles have shown to serve as potent tools that can increase the bioavailability of the drug/vaccine by facilitating absorption or conferring sustained and improved release. Here, we describe results on the effects of free- and nanoparticle-enclosed DCPIP as anti-angiogenesis and anti-inflammation agents in a human colon cancer HCT116 cell line in vitro, and in induced angiogenesis in ovo.     

AL101, a gamma-secretase inhibitor,has potent antitumor activity against adenoid cystic carcinoma with activated NOTCH signaling

Adenoid cystic carcinoma (ACC) is an aggressive salivary gland malignancy with limited treatment options for recurrent or metastatic disease. Due to chemotherapy resistance and lack of targeted therapeutic approaches, current treatment options for the localized disease are limited to surgery and radiation, which fails to prevent locoregional recurrences and distant metastases in over 50% of patients. Approximately 20% of patients with ACC carry NOTCH-activating mutations that are associated with a distinct phenotype, aggressive disease, and poor prognosis. Given the role of NOTCH signaling in regulating tumor cell behavior, NOTCH inhibitors represent an attractive potential therapeutic strategy for this subset of ACC. AL101 (osugacestat) is a potent γ-secretase inhibitor that prevents activation of all four NOTCH receptors. While this investigational new drug has demonstrated antineoplastic activity in several preclinical cancer models and in patients with advanced solid malignancies, we are the first to study the therapeutic benefit of AL101 in ACC. Here, we describe the antitumor activity of AL101 using ACC cell lines, organoids, and patient-derived xenograft models. Specifically, we find that AL101 has potent antitumor effects in in vitro and in vivo models of ACC with activating NOTCH1 mutations and constitutively upregulated NOTCH signaling pathway, providing a strong rationale for evaluation of AL101 in clinical trials for patients with NOTCH-driven relapsed/refractory ACC.Subject terms: Head and neck cancer, Targeted therapies     

Evaluation of angiogenesis in normal and lichen planus skin by CD34 protein immunohistochemistry: preliminary findings

Angiogenesis is a critical component of both neoplastic and chronic inflammatory disorders, but whether angiogenesis also occurs in inflammatory skin diseases (such as lichen planus) has yet to be established. This study tests the hypothesis that the development of cutaneous lichen planus is associated with alterations of dermal vascularization (microvessel density, MVD). Thirty cases of cutaneous lichen planus and 40 cases of normal skin were studied. Dermal microvessels were immunostained for CD34 and counted in 10 areas with the highest numbers of microvessels; the mean value represented the final MVD. Compared with normal skin, dermal microvessel density was increased in cutaneous lichen planus [mean, 2.50 (SEM, 0.09) versus 1.39 (SEM, 0.12)]. The microvessel number was higher in the dermal inflammatory infiltrate (intralichenoid infiltrate) and at dermoepidermal junction (below Max-Josef space) compared with adventitial dermis [mean, 2.50 (SEM, 0.09) versus 1.6 (SEM, 0.10)]. The higher MVD values in cutaneous lichen planus suggest a possible link between angiogenesis, and development of these cutaneous lesions. These results provide a morphological evidence of potent angiogenic activity in cutaneous lichen planus, indicating that the local microvasculature may undergo an intense process of inflammation-dependent angiogenesis.     

Effect of thalidomide affecting VEGF secretion, cell migration, adhesion and capillary tube formation of human endothelial EA.hy 926 cells

Angiogenesis, new blood vessel formation, is a multistep process, precisely regulated by pro-angiogenic cytokines, which stimulate endothelial cells to migrate, proliferate and differentiate to form new capillary microvessels. Excessive vascular development and blood vessel remodeling appears in psoriasis, rheumatoid arthritis, diabetic retinopathy and solid tumors formation. Thalidomide [alpha-(N-phthalimido)-glutarimide] is known to be a potent inhibitor of angiogenesis, but the mechanism of its inhibitory action remains unclear. The aim of the study was to investigate the potential influence of thalidomide on the several steps of angiogenesis, using in vitro models. We have evaluated the effect of thalidomide on VEGF secretion, cell migration, adhesion as well as in capillary formation of human endothelial cell line EA.hy 926. Thalidomide at the concentrations of 0.01 microM and 10 microM inhibited VEGF secretion into supernatants, decreased the number of formed capillary tubes and increased cell adhesion to collagen. Administration of thalidomide at the concentration of 0.01 microM increased cell migration, while at 10 microM, it decreased cell migration. Thalidomide in concentrations from 0.1 microM to 10 microM did not change cell proliferation of 72-h cell cultures. We conclude that anti-angiogenic action of thalidomide is due to direct inhibitory action on VEGF secretion and capillary microvessel formation as well as immunomodulatory influence on EA.hy 926 cells migration and adhesion.     

A novel peptide isolated from a phage display library inhibits tumor growth and metastasis by blocking the binding of vascular endothelial growth factor to its kinase domain receptor

Vascular endothelial growth factor (VEGF), one of the most important angiogenic factors, plays an essential role in both physiological and pathological angiogenesis. The VEGF receptor KDR/Flk-1 (a kinase domain receptor) mediates various biological activities of VEGF related to proliferation, differentiation, and migration of endothelial cells. Here we present a novel peptide designated K237-(HTMYYHHYQHHL), which was isolated from a phage-displayed peptide library, binding to KDR with high affinity and specificity. By interfering with the VEGF-KDR interaction, the peptide K237 inhibited proliferation of cultured primary human umbilical vein endothelial cells induced by recombinant human VEGF(165) in a dose-dependent and cell type-specific manner. The peptide also exerted an anti-angiogenesis activity in vivo as revealed using the chick embryo chorioallantoic membrane angiogenesis assay. Moreover, the peptide K237 significantly inhibited the growth of solid tumors implanted beneath the breasts and their metastases to lungs in severe combined immunodeficient mice. Taken together, these findings suggest that the peptide K237 can functionally disrupt the interaction between VEGF and the KDR receptor and cause potent biological effects that include the inhibition of angiogenesis and tumor growth. As a consequence, this peptide (and its future derivatives) may have use as a potential cancer therapy.     

Tumor angiogenesis factor. Speculations on an approach to cancer chemotherapy

Tumor angiogenesis factor (TAF) and its importance in determining a strategy for cancer chemotherapy are discussed. It is suggested that inhibition of RNA synthesis or increased RNA catabolism might interfere with the metabolism of solid tumor cells more so than in normal cells, and thus hinder angiogenesis and pursuant tumor growth by preventing the synthesis of the RNA component of TAF. An attempt is made to indicate potential models for anti-angiogenesis agents of this type. The drugs offered as initial prototypes for investigations along these lines are actinomycin D (which likely has antimetabolite and anti-angiogenesis activities), polyriboinosinic-polyribocytidylic acid (which likely has adjuvant and anti-angiogenesis activities) and ribonuclease (which in theory might be a purely anti-angiogenetic agent). It is noted that these models may turn out to be less than ideal as therapeutic agents due to problems of toxicity, metabolism, potency, or distribution, but nonetheless might serve to yield insights into the design of new cancer chemotherapeutic drugs. In addition, some evidence is cited suggesting that actinomycin D may be more effective against certain tumors when employed in lower, chronic dosages rather than its present use in “loading” dosages.The concept of anti-angiogenesis agents as fundamentally “tumoristatic” therapies is discussed, and the likelihood that such agents might be effectively “tumoricidal” in immunocompetent hosts is mentioned. The main promise of an anti-angiogenetic strategy is efficacy against presently intractable slowly growing human cancers when used in combination with other treatment modalities. In summary, a strategy of cancer chemotherapy predicated upon interference with RNA synthesis or increase in RNA catabolism is offered as a potential mechanism for establishing anti-angiogenesis, and as a promising alternative and adjunct to present methods.     

The quest for surrogate markers of angiogenesis: a paradigm for translational research in tumor angiogenesis and anti-angiogenesis trials

Inhibition of tumor angiogenesis suppresses tumor growth and metastatic spreading in many experimental models, suggesting that anti-angiogenic drugs may be used to treat human cancer. During the past decade more than eighty molecules that showed anti-angiogenic activity in preclinical studies were tested in clinical cancer trials, but most of them failed to demonstrate any measurable anti-tumor activity and none have been approved for clinical use. Recent results stemming from trials with anti-VEGF antibodies, used alone or in combination with chemotherapy, suggest that systemic anti-angiogenic therapy may indeed have a measurable impact on cancer progression and patient survival. From the clinical studies it became nevertheless clear that the classical endpoints used in anti-cancer trials do not bring sufficient discriminative power to monitor the effects of anti-angiogenic drugs. It is therefore necessary to identify and validate molecular, cellular and functional surrogate markers of angiogenesis to monitor activity and efficacy of anti-angiogenic drugs in patients. Availability of such markers will be instrumental to re-evaluate the role of tumor angiogenesis in human cancer, to identify new molecular targets and drugs, and to improve planning, monitoring and interpretation of future studies. Future anti-angiogenesis trials integrating biological endpoints and surrogate markers or angiogenesis will require close collaboration between clinical investigators and laboratory-based researchers.     

Expression of recombinant kringle 1-5 domains of human plasminogen by a prokaryote expression system

Kringle1-5 (K1-5), a proteolytic fragment containing five kringle domains of human plasminogen generated by plasmin-mediated proteolysis, has been already identified by Cao et al. with relation to anti-angiogenesis and proliferation of endothelial cells. To investigate anti-angiogenesis activity of recombinant human K1-5 (rhK1-5) expressed in Escherichia coli BL21, the cDNA of human K1-5 obtained from cloning vector pUC57-K1-5 by PCR, was inserted into an expression vector pET30(+) to construct a prokaryotic expression vector pET-K1-5. Recombinant K1-5 efficiently expressed in E. coli BL21 after IPTG induction was monitored by SDS-PAGE and Western blotting with an anti-angiostatin monoclonal antibody and an anti-hexahistidine tag antibody. The expressed K1-5 accounted for approximately 32% of the total bacterial proteins as estimated by densitometry, and existed mainly as inclusion bodies. The inclusion bodies were washed, lysed, purified, and refolded to a purity of 96% as estimated by capillary electrophoresis and the final purification yield of K1-5 in E. coli system was approximately 5.8 mg/L. Purified K1-5 protein was tested on chicken embryo chorioallantoic membranes (CAMs), and a large number of newly formed blood vessels were significantly regressed. In the present study, we demonstrated that bacterial-expressed K1-5 effectively inhibited angiogenesis of the chicken embryo in a dose-dependent manner through CAM assay. In addition, the rhK1-5 potently inhibited endothelial cell proliferation but not non-endothelial cells. For the first time, these findings demonstrate that the rhK1-5 produced by a prokaryote expression system effectively inhibited angiogenesis of the chicken embryo in a dose-dependent manner and specially suppressed in vitro the proliferation of human umbilical vein endothelial cells. This fact derived from the present study further suggests the rhK1-5 can be used for anti-angiogenesis therapy of cancer.     

Tumours can adapt to anti-angiogenic therapy depending on the stromal context: lessons from endothelial cell biology

It has long been recognized that interference with the blood supply of a tumour is an effective way to halt tumour progression, and even induce tumour regression. This can be accomplished by anti-angiogenic treatment which prevents the formation of a tumour neovasculature, or anti-vascular treatment, which aims at destruction of existent tumour vessels. The latter has received relatively little attention because there is a lack of specific tumour-endothelial markers. Instead, the current detailed knowledge on the factors and mechanisms, involved in angiogenesis, has enabled the development of a variety of angiogenesis inhibitors, especially those that target cellular signalling by vascular endothelial growth factor-A (VEGF-A), the most potent angiogenic factor known. These inhibitors have received lots of attention because they effectively inhibit tumour growth in pre-clinical models. However, in clinical trials these same inhibitors showed very poor anti-tumour activity. In this review we discuss this discrepancy, and we show that the tumour microenvironment is crucial to the sensitivity of tumours to anti-angiogenic therapy.     

Modulation of bovine microvascular endothelial cell proteolytic properties by inhibitors of angiogenesis

A tightly controlled increase in extracellular proteolysis, restricted both in time and space, is an important component of the angiogenic process, while anti-proteolysis is effective in inhibiting angiogenesis. By focussing on the plasminogen activator (PA)-plasmin system, the objective of the present studies was to assess whether previously described inhibitors of angiogenesis modify bovine microvascular endothelial cell proteolytic properties. We demonstrate that although synthetic angiostatic steroids (U-24067 and U-42129), heparin, suramin, interferon alpha-2a, and retinoic acid are all inhibitors of in vitro angiogenesis, each of these agents has distinct effects on the plasminogen-dependent proteolytic system. Specifically, angiostatic steroids and interferon alpha-2a reduce urokinase-type PA (u-PA) and PA inhibitor-1 activity, while heparin and retinoic acid increase u-PA activity. Suramin reduces cell-associated u-PA activity and greatly increases PAI-1 production at doses which induce monolayer disruption. These findings demonstrate that a spectrum of alterations in extracellular proteolysis is associated with anti-angiogenesis, and that anti-angiogenesis and anti-proteolysis are not necessarily correlated. A reduction in extracellular proteolysis would be expected to reduce invasion, whereas an increase in proteolysis might modulate the activity of inhibitory cytokines, which in turn could reduce endothelial cell proliferation and migration and inhibit angiogenesis. The spectrum of effects on different elements of the PA system observed in response to the agents assessed suggests that the role of modulations in extracellular proteolytic activity in anti-angiogenesis is likely to be varied and complex. © 1994 Wiley-Liss, Inc.