Abstract-Endothelial adherens junctions (AJ) promote intercellular adhesion and may contribute to the control of vascular permeability. These structures are formed by a transmembrane and cell-specific adhesive protein, vascular endothelial (VE)-cadherin, which is linked by its cytoplasmic tail to intracellular proteins called catenins (␣-catenin, ␤-catenin, and plakoglobin) and to the actin cytoskeleton. Little is known about the functional regulation of AJ in endothelial cells. In this study, we analyzed the effect of histamine on AJ organization in cultured endothelial cells. We first observed that histamine induced detectable intercellular gaps only in loosely-confluent cells, whereas this effect was strongly reduced or absent in long-confluent cultures. Despite this difference, in vitro permeability was augmented by histamine in both conditions. In resting conditions, tyrosine phosphorylation of AJ components and permeability values were higher in recently-confluent cells as compared with long-confluent cells. Histamine did not affect the phosphorylation state of AJ in recently-confluent cells but strongly increased this parameter in long-confluent cultures. In addition, in long-confluent cells, histamine caused dissociation of VE-cadherin from the actin cytoskeleton measured by a decrease of the amount of the molecule in the detergent-insoluble fraction of the cell extracts. Dibutyryl cAMP was able to prevent the effect of histamine on both tyrosine phosphorylation of AJ components and on endothelial permeability. The effect of histamine was specific for VE-cadherin because the phosphorylation state of neural (N)-cadherin, the other major endothelial cadherin, was unchanged by this agent. Hence AJ components are a target of histamine activation cascade; we suggest that induction of tyrosine phosphorylation of VE-cadherin and catenins contributes to the histamine effect on permeability, even in absence of frank intercellular gaps and cell retraction. (Arterioscler Thromb Vasc Biol. 1999;19

The involvement of nitric oxide (NO) in the regulation of angiogenesis was examined in the in vivo system of the chorioallantoic membrane (CAM) of the chick embryo and in the matrigel tube formation assay. Sodium nitroprusside (SNP) (0.37–28 nmol/disc), which releases NO spontaneously, caused a dose‐dependent inhibition of angiogenesis in the CAM in vivo and reversed completely the angiogenic effects of α‐thrombin (6.7 nmol/disc) and the protein kinase C (PKC) activator 4‐β‐phorbol‐12‐myristate‐13‐acetate (PMA) (0.97 nmol/disc). In addition, SNP (28 × 10−6 m) stimulated the release of guanosine 3′‐5′‐cyclic monophosphate (cyclic GMP) from the CAM in vitro. In the matrigel tube formation assay, an in vitro assay of angiogenesis, both SNP (1–3 × 10−6 m) and the cell permeable cyclic GMP analogue, Br‐cGMP (0.3–1.0 × 10−3 m) reduced tube formation. The inhibitors of NO synthase, NG‐monomethyl‐l‐arginine (l‐NMMA) (3.8–102 nmol/disc) and NG‐nitro‐l‐arginine methylester (l‐NAME) (1.3–34.2 nmol/disc) stimulated angiogenesis in the CAM in vivo, in a dose‐dependent fashion. d‐NMMA and d‐NAME on the other hand had no effect on angiogenesis in this system. l‐Arginine (10.9 nmol/disc), although it had a modest antiangiogenic effect by itself, was capable of abolishing the angiogenic effects of l‐NMMA (34.2 nmol/disc) and of l‐NAME (3.8 nmol/disc). Dexamethasone, an inhibitor of the induction of NO synthase, at 0.2–116.1 nmol/disc, stimulated angiogenesis in the CAM, whereas at 348.4–1161 nmol/disc it inhibited this process. Combination of 38.7 nmol/disc dexamethasone with l‐NAME (9.3 nmol/disc) resulted in a potentiation of the angiogenic effect of the former. It appears therefore that both the constitutive and the inducible NO synthase may contribute to the NO‐mediated inhibition of angiogenesis. Superoxide dismutase (SOD), which prevents the destruction of NO, at 300 i.u./disc had a modest antiangiogenic effect in the CAM, by itself. In addition, SOD, prevented α‐thrombin (6.7 nmol/disc) and PMA (0.97 nmol/disc) from stimulating angiogenesis in the CAM. These results suggest that NO may be an endogenous antiangiogenic molecule of pathophysiological importance.

Clinical, laboratory, histopathological and pharmacological evidence support the notion that a systemic activation of blood coagulation is often present in cancer patients. Additionally, thrombin was shown to promote tumour progression and metastasis in animals, and epidemiological studies suggest an increased risk of cancer diagnosis after primary thromboembolism. We have proposed that the aforementioned results may be related to our finding that thrombin is a potent activator of angiogenesis. This is a thrombin receptor-mediated event (the receptor is referred to as protease-activate receptor) and is independent of fibrin formation. Many cellular effects of thrombin on endothelial cells can contribute to the angiogenic action of thrombin. (i) Exposure of endothelial cells to thrombin cause a time- and dose-dependent decrease in the attachment of these cells to basement membrane components, with a concomitant increase in matrix metalloproteinase 2 activation. (ii) Thrombin upregulates the expression of integrin alphavbeta3, the marker of the angiogenic phenotype of endothelial cells. (iii) Thrombin has chemotactic and aptotactic effects on endothelial cells and upregulates the expression of the vascular endothelial growth factor (VEGF) receptors (KDR and Flt1). Thus, thrombin synergizes with the key angiogenic factor VEGF in endothelial cell proliferation. Furthermore, thrombin enhances the secretion of VEGF and matrix metalloproteinase 9 of PC3 prostate cancer cells. These results can explain the angiogenic and tumour-promoting effect of thrombin and provide the basis for development of thrombin receptor mimetics or antagonists for therapeutic application.

. On the mechanism of thrombin-induced angiogenesis: involvement of ␣v␤3-integrin. Am J Physiol Cell Physiol 283: C1501-C1510, 2002. First published July 17, 2002 10.1152/ajpcell.00162.2002Thrombin has been reported to be a potent angiogenic factor both in vitro and in vivo, and many of the cellular effects of thrombin may contribute to activation of angiogenesis. In this report we show that thrombin-treatment of human endothelial cells increases mRNA and protein levels of ␣v␤3-integrin. This thrombin-mediated effect is specific, dose dependent, and requires the catalytic site of thrombin. In addition, thrombin interacts with ␣v␤3 as demonstrated by direct binding of ␣v␤3 protein to immobilized thrombin. This interaction of thrombin with ␣v␤3-integrin, which is an angiogenic marker in vascular tissue, is of functional significance. Immobilized thrombin promotes endothelial cells attachment, migration, and survival. Antibody to ␣v␤3 or a specific peptide antagonist to ␣v␤3 can abolish all these ␣v␤3-mediated effects. Furthermore, in the chick chorioallantoic membrane system, the antagonist peptide to ␣v␤3 diminishes both basal and the thrombin-induced angiogenesis. These results support the pivotal role of thrombin in activation of endothelial cells and angiogenesis and may be related to the clinical observation of neovascularization within thrombi. attachment; migration; apoptosis; reverse transcription-polymerase chain reaction THE FREQUENCY OF BLOOD COAGULATION in cancer patients, known for more than 130 years, is supported by clinical, laboratory, and histopathological evidence. This is explained at the molecular and cellular level by the thromboplastic activity of circulating tumor cells, the existence of "a cancer coagulative factor," the activation of factor X, the generation of prothrombinase by tumor cells, and the encircling of cancerous tissue by fibrin deposits (38,50). In addition, the possibility of a relation between blood clotting mechanisms and tumor progression and development of metastases was postulated as early as 1878 by Billroth (7) on the basis of the observation that cancer cells exist within thrombi. This finding was interpreted as evidence that tumor cells spread by thromboembolism. More recently, large epidemiological studies have provided evidence that the standardized incidence ratio for certain types of cancer is as high as 6.7 within a year following a thromboembolic episode (3, 41). These clinical data are in line with animal experiments where thrombin-treated B16 melanoma cells show a dramatic increase in their metastatic potential in the lung of rats (37). These observations have led to experimental use of heparin, aspirin, and warfarin for the prevention and treatment of tumors in animal models and humans (23, 50).We proposed earlier (33, 47) that the tumor-promoting effect of thrombin/thrombosis may be related to our finding that thrombin is a potent promoter of angiogenesis, a process essential for tumor growth and metastasis. The angiogenic action of thrombin was shown to b...

The anthracycline antibiotics, daunorubicin, doxorubicin, and epirubicin, which are widely used for treatment of malignancies, have been evaluated for their effect on angiogenesis in relation to the inhibition of collagenase type IV reported previously. In the chick chorioallantoic membrane (CAM) system of angiogenesis, anthracyclines inhibited vascular density at doses of 5-20 micrograms/disc as well as collagenous protein biosynthesis, which is a reliable index of angiogenesis. Similarly, all three anthracyclines inhibited tube formation in the in vitro system of angiogenesis using human umbilical vein endothelial cells (HUVECs) plated on Matrigel. The inhibition was dose-dependent and caused 50% inhibition at concentrations of 2.5-15 micrograms/mL. At concentrations of anthracyclines which prevented tube formation and angiogenesis, there were no cytotoxic effects, as evidenced by methylene blue uptake, and the growth of these endothelial cells was not inhibited. The experimental antitumor agent titanocene dichloride inhibited collagenase type IV from Walker 256 carcinosarcoma with IC50 approximately 0.2 mM. Titanocene also prevented angiogenesis in the CAM and tube formation by HUVECs on Matrigel at concentrations that were without effect on growth or cytotoxicity of endothelial cells or Walker 256 cells in culture. The antiangiogenic effect of the aforementioned antitumor agents at therapeutically attainable concentrations may explain, at least in part, their antitumor properties because angiogenesis is an essential process for tumor growth and metastasis. The antiangiogenic effect is, however, unrelated to metalloproteinase inhibition because higher concentrations are required for that effect than for inhibition of angiogenesis.