. Novel complexes of guanylate cyclase with heat shock protein 90 and nitric oxide synthase. Am J Physiol Heart Circ Physiol 285: H669-H678, 2003. First published April 3, 2003 10.1152/ajpheart.01025.2002 is an important downstream intracellular target of nitric oxide (NO) that is produced by endothelial NO synthase (eNOS) and inducible NO synthase (iNOS). In this study, we demonstrate that sGC exists in a complex with eNOS and heat shock protein 90 (HSP90) in aortic endothelial cells. In addition, we show that in aortic smooth muscle cells, sGC forms a complex with HSP90. Formation of the sGC/eNOS/HSP90 complex is increased in response to eNOS-activating agonists in a manner that depends on HSP90 activity. In vitro binding assays with glutathione S-transferase fusion proteins that contain the ␣-or ␤-subunit of sGC show that the sGC ␤-subunit interacts directly with HSP90 and indirectly with eNOS. Confocal immunofluorescent studies confirm the subcellular colocalization of sGC and HSP90 in both endothelial and smooth muscle cells. Complex formation of sGC with HSP90 facilitates responses to NO donors in cultured cells (cGMP accumulation) as well as in anesthetized rats (hypotension). These complexes likely function to stabilize sGC as well as to provide directed intracellular transfer of NO from NOS to sGC, thus preventing inactivation of NO by superoxide anion and formation of peroxynitrite, which is a toxic molecule that has been implicated in the pathology of several vascular diseases.smooth muscle cells; endothelium; vascular endothelial growth factor; bradykinin; cGMP accumulation SOLUBLE GUANYLATE CYCLASE (sGC), an ␣/␤-heterodimeric heme protein, catalyzes the conversion of GTP to cGMP in many cells including vascular endothelial cells (ECs) and vascular smooth muscle cells (SMCs). Activation of sGC is by direct binding of nitric oxide (NO) to the sGC heme prosthetic group. Formation of the nitrosyl heme adduct induces a conformational change in sGC that results in an increase in its enzymatic activity (21). The NO that activates sGC in various cells is the product of a reaction that is catalyzed by one of three distinct NO synthase (NOS) molecules, which catalyze the oxidation of L-arginine to produce L-citrulline and NO (1). In ECs, NO production is mediated by the constitutively expressed endothelial NOS (eNOS). Activation of eNOS is by Ca 2ϩ -calmodulin (CaM) after agonist-stimulated elevations in intracellular Ca 2ϩ concentrations. Two signaling pathways exist that involve eNOS and sGC. The first is an intercellular pathway whereby NO, which is produced by eNOS in ECs, diffuses to the underlying SMCs and promotes blood vessel relaxation (16). The second is an intracellular eNOS-sGC pathway that is essential for vascular endothelial growth factor (VEGF)-induced increases in EC permeability and proliferation (24,25,30).Initially, eNOS was thought to function as an isolated homodimer. It is now known, however, that eNOS exists in multiprotein complexes in which it interacts with other proteins. These pr...

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.

1 The involvement of platelets in neovascularization was investigated in the matrigel tube formation assay, an in vitro model of angiogenesis. 2 Platelets promoted the formation of capillary-like structures (expressed as relative tube area) numberand time-dependently. Relative tube area increased from 0.98+0.02 (n=8) in the presence of 6.25610 4 , to 3.21+0.12 (n=8) in the presence of 10 6 platelets/well compared to 0.54+0.04 (n=8) in their absence. This increase was unaected by acetyl salicylic acid (ASA), apyrase, and hirudin. Photographs from representative experiments, showed that platelets adhered along the dierentiating endothelium. 3 Addition of a-thrombin (0.1 ± 1 i.u. ml 71 ), the nitric oxide (NO) donor sodium nitroprusside (SNP; 1 ± 100 mM) or the NO synthase inhibitor, L-NG-arginine-methylester (L-NAME, 30 ± 300 mM) to the assay, had no eect on tube formation compared to that seen with platelets alone. 4 Neuraminidase (0.01 i.u./10 7 platelets), which strips sialic acid residues from membrane glycoproteins, abolished the promoting eect of platelets on tube formation. The relative tube area in the presence of neuraminidase-treated platelets was 0.81+0.03 (n=8), in the presence of untreated platelets 1.69+0.09, P50.001 (n=8) and in the absence of platelets, 0.80+0.04 (n=8). The tetrapeptide Arg-Gly-Asp-Ser (RGDS; 20 ± 200 mM) which inhibits von Willebrand factor, ®brinogen and ®bronectin-mediated adhesion, had no eect on the promoting eect of platelets on tube formation. 5 These results indicate that platelets promote angiogenesis in vitro. This eect is largely independent from activation by a-thrombin, is not modi®ed by manipulating NO and prostaglandin metabolism and proceeds possibly through adhesion of the platelets to the dierentiating endothelium.

The role of thrombin in angiogenesis was investigated in the chick chorioallantoic membrane (CAM) system. alpha-Thrombin promoted angiogenesis in a dose-dependent fashion and at 8.4 pmol/disk reached a maximum of 78% above the control. At a higher dose of alpha-thrombin (25 pmol/disk) the angiogenic effect declines and this can be explained by desensitization of the thrombin receptor. The promotion of angiogenesis by alpha-thrombin is specific as evidenced by the reversal of this effect by hirudin, which binds both the catalytic and the anion-binding exosite of thrombin or by heparin, which binds thrombin and accelerates its inactivation by antithrombin III. gamma-Thrombin, which is catalytically active but lacks the anion-binding exosite required for clotting activity, promotes angiogenesis in the CAM in the same fashion and to the same extent as alpha-thrombin, at doses up to 130 pmol/disk. Phenylalanyl-propyl-arginine chloromethyl ketone (P-PACK)-thrombin, the catalytically inactive analogue of alpha-thrombin that retains the anion-binding exosite, had no significant effect on angiogenesis in the CAM. When combined with alpha-thrombin, P-PACK-thrombin abolished the angiogenesis-promoting effect of alpha-thrombin. These results suggest that alpha-thrombin can promote angiogenesis in the CAM through interaction with its catalytic site without the requirement for fibrin formation.

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.

. 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...

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.

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