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CHEMOTHERAPY, ANTIBIOTICS, AND GENE THERAPY
Department of Pharmacology, Medical School, University of Patras, Patras, Greece
Received November 30, 2005; accepted March 31, 2006.
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Abstract |
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S)-N-[(1S)-3-amino-1-[[(phenylmethyl)amino]carbonyl]propyl]--[[[[[1-(2,6-dichlorophenyl)methyl]-3-(1-pyrrolidinylmethyl)-1H-indol-6-yl]amino]carbonyl]amino]-3,4-difluorobenzenepropanamide], in the angiogenic cascade. These antagonists suppressed both the basic angiogenesis and that stimulated by thrombin in the chick chorioallantoic membrane model in vivo. PAR-1 antagonists also abrogated tube formation in the in vitro Matrigel system. These inhibitory effects were dose-dependent and well correlated with the inhibitory effects of SCH79797 and RWJ56110 on primary endothelial cell proliferation and on the initiation of apoptosis. PAR-1 blockage resulted in inhibition of endothelial cell growth by increasing the sub-G0/G1 fraction and reducing the percentage of cells in the S phase. Consistent with this, PAR-1 antagonists reduced incorporation of [3H]thymidine in endothelial cells and blocked the phosphorylation of extracellular signal-regulated kinases in a fashion depending specifically on PAR-1 activation. Analysis by annexin V/propidium iodide staining and poly(ADP-ribose) polymerase cleavage revealed that PAR-1 blockage increased apoptotic cell death by a mechanism involving caspases. These results provide further evidence that PAR-1 is a key receptor that mediates angiogenesis and suggest PAR-1 as target for developing antiangiogenic agents with potential therapeutic application in cancer and other angiogenesis-related diseases.
). PAR-1, the first member of this family to be cloned and the first receptor for which this unique mechanism of activation was described previously (Vu et al., 1991), has been shown to respond to a select group of serine protease that include thrombin, plasmin (Kuliopulos et al., 1999), factor Xa (Riewald et al., 2001), activated protein C (Riewald et al., 2002), and matrix metalloprotease-1 (Boire et al., 2005). Synthetic peptides (thrombin receptor agonist peptides) of five or more amino acid residues with the identical sequence as the new amino terminus of PAR-1 are also able to activate the receptor (Ossovskaya and Bunnett, 2004).
Interest in PAR-1 was initiated from its involvement in thrombin-induced activation of platelets. Subsequently, PAR-1 activation was found to mediate several processes in vascular biology, inflammation, and tissue remodeling in normal development (Coughlin, 2005). Recently, we have shown that thrombin is a potent angiogenic factor, and this effect is independent of fibrin formation and dependent on PAR-1 activation (Tsopanoglou and Maragoudakis, 2004). The proangiogenic action of thrombin was shown to have synergistic effect with vascular endothelial growth factor (VEGF), the specific endothelial cell angiogenic factor. PAR-1 activators up-regulate the expression of VEGF receptors of endothelial cells and potentiate the mitogenic effects of VEGF on endothelial cells (Tsopanoglou and Maragoudakis, 1999). The importance of thrombin and its receptor in embryonic development and angiogenesis is also supported by the findings of Griffin et al. (2001), who showed that the expression of PAR-1 by endothelial cells rescues the fetal vessel fragility and bleeding of mouse embryo engineered to lack PAR-1. More recently, Ma et al. (2005) have shown that PAR-1 activation in human platelets stimulates VEGF release and leads to suppression of endostatin, which is a potent endogenous inhibitor of angiogenesis. These findings point to a central role of PAR-1 in the angiogenic cascade.
Despite the presence of functional PAR-1 on the surface of many cell types, there are formidable obstacles in understanding its role in health and disease. A major difficulty was that effective PAR-1 antagonists were not available. PAR-1-deficient mice were used in elucidating the roles of PAR-1, but mice are not always the optimal species to model human diseases. Another complication is the apparent redundancy of the PAR system. Cells express several PARs that are activated by the same protease, and several proteases can activate the same receptor. To date, few PAR-1-targeting agents have been developed and tested in limited disease models such as thrombosis, intestinal inflammatory disorders, and experimental liver fibrosis (Derian et al., 2003; Fiorucci et al., 2004; Vergnolle et al., 2004). The effect of PAR-1 antagonists on angiogenesis has not been reported. In the present study, we show that SCH79797 and RWJ56110, two well characterized specific and potent PAR-1 antagonists (Andrande-Gordon et al., 1999; Ahn et al., 2000), are capable of blocking angiogenesis in the chick embryo angiogenesis model in vivo and in the Matrigel in vitro system. We also show that these PAR-1 antagonists abrogate endothelial cell growth by induction of apoptosis.
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Materials and Methods |
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-thrombin was kindly provided by Dr. J. Fenton II (New York State Department of Health, Albany, NY). VEGF and basic fibroblast growth factor (bFGF) were obtained from Chemicon International Inc. (Temecula, CA). [3H]Thymidine and [14C]proline were purchased from ICN Biomedicals, Inc (Irvine, CA). Fresh fertilized chicken eggs were obtained locally (Ioannina, Greece) and were kept at 10°C until incubation at 37°C. Matrigel was kindly provided by Dr. H. Kleinman (National Institutes of Health, Bethesda, MD). Human umbilical vein endothelial cells (HUVECs) were obtained from freshly delivered umbilical cords from caesarean births. Cells were cultured as described previously (Tsopanoglou and Maragoudakis, 1999) and were used for experiments from passages 3 to 5.
Chick Chorioallantoic Membrane Assay. The in vivo chick chorioallantoic membrane (CAM) angiogenesis model was used as described previously (Maragoudakis et al., 1995). In brief, biochemical evaluation of angiogenesis was performed by determining the extent of collagenous protein biosynthesis in the CAM lying directly under the pellets applied at day 9 of chick embryo development. Both control and test pellets contained [14C]proline (0.5 µCi/pellet), and test pellets also contained thrombin or PAR-1 antagonists or the combination. After 48 h, the tissue under the pellets was subjected to collagenase digestion. The resulting radiolabeled tripeptides, corresponding to basement membrane collagen and other collagenous materials synthesized by the CAM, were counted and expressed as disintegrations per minute per milligram of protein. For each egg, collagenous protein biosynthesis under the test pellet was expressed as a percentage of that under the control pellet in the same egg. It was used in at least 21 eggs for each group. Results are mean ± S.E. expressed as percentage of control. Statistical analysis was performed with Student's t test.
In morphological evaluation of angiogenesis, eggs were treated as above in the absence of radiolabeled proline. At day 11, the eggs were flooded with 10% buffered formalin, the plastic pellets were removed, and the eggs were kept at 37°C until dissection. A large area around the pellet was removed and placed on a glass slide. Representative specimens were mounted on a stereoscope and photographed.
Matrigel Tube Assay. Matrigel is a mixture of basement membrane components extracted from the Englebreth-Holm-Swarm tumor, and it has been demonstrated that endothelial cells attach, migrate, and assemble to form tube-like structures resembling capillaries within 18 h. The tube formation assay was performed as described previously (Grant et al., 1989). In brief, Matrigel was used to coat 24-well plates (0.25 ml/well). After polymerization of Matrigel at 37°C for 1 h, indicated concentrations of PAR-1 antagonists were added in 0.5 ml of serum-free medium. Cells (40,000 cells/well) were then added suspended in 0.5 ml of medium containing fetal bovine serum (FBS) making 4 or 10% final concentration in FBS. After 18 h of incubation, the medium was removed, and the cells were fixed and stained. Thereafter, the tube-like structures formed were quantitated in triplicates with a computerized digital image analyzer. Each experiment was repeated at least twice. Results are given as mean percentage change of control ± S.E. Statistical analysis was performed with Student's t test.
Cell Proliferation Assay. Cell proliferation was evaluated by, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT; Sigma-Aldrich, St. Louis, MO) assay. In brief, endothelial cells (10,000/well) were seeded in 24-well tissue culture plates previously coated with 1% gelatin (Sigma) and incubated with growth medium for 6 h. Cells were then treated with the indicated concentrations of PAR-1 antagonists for 1 to 4 days. After 24, 48, 72, or 96 h of exposure to compounds (SCH79797 or RWJ56110), MTT (50 µl) solution (5 mg/ml) was added to each well to incubated for 3 h at 37°C. The blue formazan crystals were solubilized by addition of DMSO (200 µl). Absorbance at 450 nM was recorded using a 96-well plate reader. Results are expressed as mean ± S.E. of number from cells and presented as -fold increased of cell number at the beginning of experiments. Each experiment included three wells in each condition tested and was repeated at least twice. Statistical analysis was performed with Student's t test.
[3H]Thymidine Incorporation Assay. HUVECs were seeded into gelatin-coated 24-well tissue culture plates and incubated with growth medium until 50 to 60% confluence. After changing the medium to 4% FBS, cells were treated with the indicated concentration of PAR-1 antagonists for 18 h. In some experiments, HUVECs were seeded sparsely into a gelatin-coated well and incubated with growth medium until confluence. After washes, confluent cells were then treated with PAR-1 antagonists for 18 h in medium containing 4% FBS. All cells were pulsed with 0.5 µCi/ml [3H]thymidine for an additional 6 h. DNA synthesis was stopped by removing the radioactive media, washing the cells with PBS, and fixing them with ice-cold methanol (300 µl/well). Cells were then washed twice with 5% trichloroacetic acid, and the acid-insoluble fractions were lysed in 0.5 N NaOH (300 µl/well). The radioactivity was determined in a liquid scintillation counter. Each experiment included three wells in each condition tested and was repeated at least twice. Results are expressed as mean change of control ± S.E. Statistical analysis was performed with Student's t test.
Western Blot Analysis. Endothelial cells were cultured in 35-mm tissue culture plates. After reaching confluency, cells were growth factor-starved and subsequently stimulated for 10 min with thrombin or bFGF or VEGF or epidermal growth factor (EGF). In experiments with PAR-1 antagonists, endothelial cells were pretreated with the indicated concentrations of SCH79797 or RWJ56110 for 15 min and subsequently were stimulated by growth factors. Attached cells were lysed with Laemmli sample lysis buffer, and lysates were resolved with 10% SDS-polyacrylamide gel electrophoresis and transferred to nitrocellulose membranes. Membranes were incubated with primary antibodies using the following antibodies concentration: antiphospho p42/44 mitogen-activated protein kinases (MAPKs; Erk1/2) (1:3000; New England Biolabs, Beverly, MA) that detects p42 and p44 MAPK only when catalytically activated by phosphorylation at Thr 202 and Tyr 204, anti-p42/44 MAPK (1:3000; New England Biolabs). Membranes were then probed with secondary antibodies horseradish peroxidase-conjugated, and proteins were visualized by chemiluminescent detection.
For analysis of poly(ADP-ribose) polymerase (PARP), endothelial cells were cultured in gelatin-coated 60-mm tissue culture plates. After reaching 50 to 60% confluence, cells were treated with the indicated concentrations of PAR-1 antagonists for 24 h or 4 days or staurosporine for 10 h. Attached and suspended cells were lysed with RIPA lysis buffer, and pooled lysates were resolved with 8% SDS-polyacrylamide gel electrophoresis and transferred to nitrocellulose membranes. Membranes were incubated with primary antibodies using the following concentration: mouse anti-PARP monoclonal (1: 3000; BD Biosciences, San Jose, CA) that detects both the 116-kDa intact and 85-kDa cleaved forms of PARP, anti--tubulin monoclonal (1:2000; Sigma).
Assessment of Apoptosis by Flow-Cytometry Analysis. HUVECs were grown in gelatin-coated 100-mm tissue culture plates until approximately 50% confluence. In some experiments, HUVECs were seeded sparsely into gelatin-coated plates and incubated until confluency. In cell cycle analysis, cells were treated in the absence (control) or presence of SCH79797 (3 µM) or RWJ56110 (30 µM) for 24 h. After treatment, attached cell were collected by trypsinization, washed, and fixed in methanol for 1 h at -20°C. Fixed cells were then incubated with RNase (100 µg/ml; Sigma) for 30 min and stained with propidium iodide (50 µg/ml; Sigma) for 20 min at 4°C in the dark. Flow cytometry was performed on a fluorescence-activated cell sorting flow cytometer (EPICS XL-MCL; Beckman Coulter, Fullerton, CA). The propidium iodide-stained cell populations in sub-G0/G1, G1, S, and G2M phases were represented by distinct and quantified peaks in the fluorescence histograms obtained using the WinMDI logiciel program. Experiments were run in triplicate and repeated twice. Results are expressed as mean percentage of cell population in each phase ± S.E. Statistical analysis was performed with Student's t test.
To assess cell death specific to apoptosis, we used the annexin V-FITC assay kit (BD Biosciences PharMingen, San Diego, CA). Endothelial cells were treated with the indicated concentrations of both PAR-1 antagonists for 96 h or with 3 µM SCH79797 for 12, 24, 48, and 72 h. After treatment, attached cells were collected by trypsinization, pooled with suspended cells, and washed in PBS. Cells (1 x 106) were then resuspended in 100 µl of the kit reaction buffer containing propidium iodide and annexin V-FITC, according to the manufacturer's instructions. After mixing, cells were incubated for 15 min in the dark at room temperature and analyzed on the fluorescence-activated cell sorting flow cytometer within 1 h after staining.
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). As shown in Fig. 1A, PAR-1 antagonists, SCH79797 and RWJ56110, were very potent antiangiogenic agents. SCH79797 and RWJ56110, at concentrations of 30 and 100 µg/pellet, respectively, completely blocked the formation of new vessels. This inhibitory effect was dose-dependent and not toxic for the chick embryo, even at as high concentration as 300 µg/pellet. The antiangiogenic effect of PAR-1 antagonists was evident both for basic angiogenesis and that stimulated by thrombin (Fig. 1B).
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PAR-1 Antagonists Inhibit Growth of Endothelial Cells. To investigate the effect of PAR-1 antagonists on endothelial cell proliferation, HUVECs were plated on gelatin-coated 24-well plates and grown with complete culture medium for 96 h in the absence or presence of various concentrations of SCH79797 and RWJ56110. On each of 4 consecutive days, cells were washed and fixed, and cell growth was determined indirectly by MTT assays. As shown in Fig. 3A, endothelial cell number doubled every 18 to 26 h over the 96-h period. In the presence of SCH79797-selective PAR-1 antagonist, the rate of endothelial cell growth was significantly decreased (Fig. 3A). HUVECs growth was essentially blocked by SCH79797 at 3 µM. This inhibitory effect of SCH79797 was dose-dependent with half-maximal inhibitory concentration at approximately 0.3 µM. Similar results were also obtained using the RWJ56110-specific PAR-1 antagonist with half-maximal inhibitory concentration of 10 µM (data not shown).
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The ability of PAR-1 antagonists to inhibit DNA synthesis of endothelial cells was assessed in thymidine incorporation assays. Endothelial cells, which were in fast-growing state (50-60% confluence), were cultured in medium containing 4% FBS for 18 h in the presence or absence of PAR-1 antagonists and subsequently were pulsed with [3H]thymidine for an additional 6 h. As shown in Fig. 3B, SCH79797 and RWJ56110 reduced the DNA synthesis in HUVECs in a dose-dependent manner. However, when the DNA synthesis experiments were repeated with cells that were in the quiescent state (100% confluent), the inhibitory effect of PAR-1 antagonists was much less pronounced (data not shown).
Several studies have shown that the activation of PAR-1 by thrombin or agonist peptides results in MAPK activation. The MAPK (Erk1/2, p42/44) cascade is usually mitogenic since cell cycle progression has been shown to depend on sustained activation of the Erk signal transduction pathway (Roovers and Assoian, 2003). Considering the antimitogenic and antiproliferative effects of PAR-1 antagonists, we examined a possible inhibition of Erk1/2 activity by SCH79797 and RWJ56110 in endothelial cells. As shown in Fig. 4A, SCH79797 inhibits thrombin-induced Erk1/2 activation in a concentration-dependent manner (0.1-3 µM). Similar inhibitory effect was evident using RWJ56110 (Fig. 4B). This effect was specific for thrombin-mediated activation since PAR-1 antagonists did not have any effect on bFGF-, VEGF-, or EGF-induced Erk1/2 phosphorylation (Fig. 4C; data not shown). However, when endothelial cells were stimulated by FBS (final concentration 4%), the presence of both PAR-1 antagonists reduced partially the activated levels of Erk1/2, providing evidence that the serum contains proteases that are able to activate PAR-1 (Fig. 4D).
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Growth Inhibition of Endothelial Cells Is Associated with Induction of Apoptosis. Flow-cytometric cell cycle analysis was performed to determine whether the results of the proliferation and thymidine incorporation were a reflection of cytostatic or cytotoxic effects due to cell cycle arrest or apoptosis. HUVECs at approximately 50% confluence were treated with 3 µM SCH79797 or 30 µM RWJ56110. After 24 h of drug treatment, cells were fixed and resuspended in propidium iodide, and DNA content was measured in comparison with untreated cells. The sub-G0/G1 fraction (subdiploid region on the DNA content histogram) was used as a measure of the percentage of apoptotic cells. SCH79797 induced a 19-fold increase in the subdiploid endothelial cell population (Fig. 5). In addition, SCH79797 reduced the percentage of cells in the S phase (DNA synthesis) by more than 4-fold. This is consistent with the results of the effect of PAR1 antagonists on endothelial cell proliferation and thymidine incorporation. Alterations in the percentages of G1 and G2/M cells were less pronounced (Fig. 5). Similar results were obtained when endothelial cells were treated with 30 µM RWJ56110 (Fig. 5). When the cell cycle experiments were repeated with cells that were in the steady-state condition (100% confluent), the effect of PAR-1 antagonists on cell populations was much less pronounced (data not shown).
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Apoptosis triggered by PAR-1 blockage was also detected by annexin V binding assay. Early in the apoptotic process, phospholipid asymmetry in the plasma membrane is disrupted, leading to exposure of phosphatidylserine on the outer leaflet of the plasma membrane. Annexin V binds preferentially to negatively charged phospholipids and can be used to identify apoptosis. Cells were treated with 0.1, 0.3, 1, and 3 µM SCH79797 for 96 h and were double stained with annexin V and propidium iodide to further assess cell death. Annexin V binds to cells in early apoptosis, and the propidium iodide stains cells in late stages of apoptosis or cells that are already dead. Annexin V-positive and propidium iodide-negative cells were interpreted as early apoptotic cells. The data presented in Fig. 6A revealed that SCH79797 increased the percentage of endothelial cells in early apoptotic state in a concentration-dependent manner. In addition, the percentage of HUVECs staining positive for both annexin V and propidium iodide increased up to 11-fold (39.5% compared with control 3.73%) as the concentration of SCH79797 increased to 3 µM (Fig. 6A), indicating that PAR-1 blockage induces apoptotic cell death. Similar results were also obtained with 30 µM RWJ56110 (Fig. 6A). To further establish that the cytotoxicity achieved by PAR-1 blockage is due to apoptosis, we examined the effect of SCH79797 (3 µM) on endothelial cells after 12, 24, 48, and 72 h. As shown in Fig. 6B, treatment with SCH79797 increased cell population in early apoptotic stage (annexin V staining) by 14.7% as early as 24 h compared with the number of late apoptotic or dead cells (both annexin V and propidium iodide staining), which represented only a small cell population (3%).
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). Western blotting for PARP cleavage was performed on cell lysates from HUVECs after culture with or without SCH79797 (3 µM) for 24 h. PAR-1 antagonist induced PARP cleavage to its signature 85-kDa fragment in all samples assayed (Fig. 7). Staurosporine was used as positive control. Similar results were obtained using RWJ56110 (Fig. 7). Furthermore, endothelial cells were treated with lower concentrations (0.1 and 0.3 µM) of SCH79797, and cell lysates were immunoblotted for PARP after 96 h of drug treatment (Fig. 7). PARP fragments were detected in antagonist-treated samples in a dose-dependent manner. These data suggest that PAR-1 blockage increased apoptosis in growing endothelial cells and provide evidence that the cytotoxicity observed is due at least in part to caspase activation.
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). In addition, a recent study by Arisato et al. (2003) demonstrated that release of VEGF from human smooth muscle cells could be stimulated by PAR-1 agonists.
Thrombin has been shown to activate PAR-1 in vitro and is considered the endogenous activator of PAR-1 on platelets (Coughlin, 2005). Because thrombin is rapidly inactivated in plasma, its temporal and local effect depends on the presence of tissue factor. Tissue factor activates factor X, which leads to cleavage of circulating prothrombin, thereby generating thrombin. Tissue factor production has been shown to be up-regulated in endothelial cells by serum (Faucette et al., 1993) as well as by the angiogenic factors VEGF and bFGF (Zucker et al., 1998; Kaneko et al., 2003).
In addition, PAR-1 can be activated by other proteases from the coagulation cascade such as factor Xa or VIIa and serine proteases that include plasmin and activated protein C (Ossovskaya and Bunnett, 2004). Recently, Boire et al. (2005) have identified matrix metalloprotease-1 to be a novel nonserine protease agonist that cleaves PAR-1 at the proper site for receptor activation and generates PAR-1-dependent intracellular signals. Indeed, matrix metalloprotease-1 is one the dominant matrix metalloprotease secreted by endothelial cells and seems to play an important role during angiogenesis (Fisher et al., 1994; Sato et al., 2000). We have shown that PAR-1 antagonists suppressed basic angiogenesis (in the absence of thrombin). Therefore, in view of these findings, the possibility exists that proteases other than thrombin might be implicated in PAR-1 activation and may initiate angiogenesis. This may also explain the inhibitory effect of PAR-1 antagonists on tube formation, proliferation, DNA synthesis, and MAPK activation by endothelial cells, which were performed in serum-containing medium. In addition, these findings point to the fact that PAR-1 can be activated in endothelial cell cultures. The majority of cells express more than one PAR for the same protease, and several proteases can activate the same PAR. It is possible that PAR-1 acts as a high-gain sensor of various extracellular proteases and allows the cell to react to a proteolytically altered environment. This unique ability to sense proteases can be used by cells for migration and differentiation or growth and survival.
Endothelial cell proliferation was inhibited after exposure of PAR-1 antagonists in a concentration- and time-dependent manner, and this inhibition does not seem to be the result of a nonspecific cytotoxic mechanism. SCH79797 and RWJ56110 treatment of HUVECs inhibited the thrombin-induced activation of MAPK. This effect was specific since the levels of activated MAPK triggered by other growth factors such as VEGF, bFGF, or EGF were not affected by PAR-1 antagonists. The ras-Raf-MAPK pathway is one of the major downstream pathways that is initiated as a consequence of PAR-1 activation (Ossovskaya and Bunnett, 2004). MAPKs phosphorylate a large number of targets, which eventually initiate and regulate various cellular processes including proliferation, differentiation, and development. We have seen in HUVECs that PAR-1 activation results in elevated phosphorylated levels of MAPK through a mechanism involving EGF receptor (EGFR) transactivation (P. Zania and N. E. Tsopanoglou, unpublished data). Previous work has shown that EGFR signaling plays a role in angiogenesis because inhibition of EGFR decreases the production of proangiogenic molecules and inhibits neoangiogenesis (Hirata et al., 2002). It is intriguing that EGFR antagonists readily induce apoptosis in cells dependent solely on EGFR signaling for cell growth and survival (Moyer et al., 1997). Likewise, in our experiments, exposure of cells to SCH79797 and RWJ56110 had an effect on cell cycle progression as early as 24 h. PAR-1 antagonists increased the percentage of apoptotic cells (sub-G0/G1 fraction) and reduced the number of cells in S phase. These results are in agreement with data showing that PAR-1 antagonists inhibited DNA synthesis on endothelial cells. Furthermore, our experiments with annexin V binding assay clearly showed that SCH79797 and RWJ56110 induced apoptotic cell death. It is of interest that these apoptotic effects of PAR-1 antagonists were evident at concentrations comparable with the concentrations that were sufficient to inhibit PAR-1 activation (P. Zania and N. E. Tsopanoglou, personal observations; Andrande-Gordon et al., 1999; Ahn et al., 2000) and endothelial cell growth. In addition, the proapoptotic effect of PAR-1 antagonists was evident only in fast-growing endothelial cells. On the contrary, when confluent cell were exposed to PAR-1 antagonists, the induction of apoptosis was much less pronounced. Therefore, it is anticipated that PAR-1 antagonists will not have an effect on the normally quiescent endothelial cells but only on the activated endothelial cells involved in angiogenesis. The observed endothelial cell growth-inhibitory effect of PAR-1 antagonists is most likely due to induction of apoptosis. Although the precise molecular mechanisms involved require further investigation, our results showed that the cytotoxicity observed by PAR-1 blockage was due at least in part to caspase activation.
Activation of PAR-1 has been found to induce or inhibit apoptosis in a variety of cells, depending on the dosage of thrombin or that of synthetic receptor activators (Flynn and Buret, 2004). It has been shown that thrombin or thrombin receptor agonist peptide (SFLLRN) prevent apoptosis of CC139 fibroblast cells and promote survival of prostate cancer cells through inhibition of Bim (Bcl-2-interacting mediator of cell death) expression and nuclear factor 
B activation, respectively (Chalmers et al., 2003; Tantivejkul et al., 2005). However, Zain et al. (2000) have shown that thrombin has a bimodal effect on PAR-1-dependent growth of melanoma, colon, and prostate carcinomas. Low thrombin concentrations enhance the growth of these cancer cells, whereas high thrombin impairs growth and induces apoptosis. PAR-1-mediated effects after exposure to high concentrations of thrombin may be related to desensitization and down-regulation of PAR-1 by thrombin, and this inactivation of PAR-1 could lead to initiation of apoptosis and inhibition of angiogenesis.
PAR-1 was identified as an oncogene that actively induced the loss of anchorage-dependent growth and focus-forming activity in NIH-3T3 cells (Martin et al., 2001) and has long been proposed to be involved in the invasive and metastatic processes of cancers of the breast, colon, lung, pancreas, prostate, and melanoma (Ossovskaya and Bunnett, 2004). These aforementioned findings and the results from this study point to the pivotal role of PAR-1 activation in tumor biology and angiogenesis. Furthermore, our results validate the concept that PAR-1 antagonists are effective antiangiogenic agents and as such have the potential therapeutic application in cancer and other disease states where angiogenesis is deranged. The fact that PAR-1 antagonists also function as antithrombotic agents (Derian et al., 2003) may present another beneficial action for the thrombosis-prone cancer patient.
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Acknowledgements |
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Article, publication date, and citation information can be found at http://jpet.aspetjournals.org.
ABBREVIATIONS: PAR, protease-activated receptor; VEGF, vascular endothelial growth factor; SCH79797, N-3-cyclopropyl-7-{[4-(1-methylethyl)phenyl]methyl}-7H-pyrrolo[3,2-f]quinazoline-1,3-diamine; RWJ56110, (S)-N-[(1S)-3-amino-1-[[(phenylmethyl)amino]carbonyl]propyl]--[[[[[1-(2,6-dichlorophenyl)methyl]-3-(1-pyrrolidinylmethyl)-1H-indol-6-yl]amino]carbonyl]amino]-3,4-difluorobenzenepropanamide; DMSO, dimethyl sulfoxide; bFGF, basic fibroblast growth factor; HUVEC, human umbilical vein endothelial cell; CAM, chorioallantoic membrane; FBS, fetal bovine serum; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; EGF, epidermal growth factor; MAPK, mitogen-activated protein kinase; Erk, extracellular signal-regulated kinase; PARP, poly(ADP-ribose) polymerase; FITC, fluorescein isothiocyanate; EGFR, epidermal growth factor receptor; PI, propidium iodide.
Address correspondence to: Nikos E. Tsopanoglou, Department of Pharmacology, Medical School, University of Patras, 26500, Patras, Greece. E-mail: ntsopan{at}med.upatras.gr
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References |
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TopAbstractMaterials and MethodsResultsDiscussionReferences |
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Ahn H-S, Foster C, Boykow G, Stamford A, Manna M, and Graziano M (2000) Inhibition of cellular action of thrombin by N3-cyclopropyl-7-{[4-(1-methylethyl-)phenyl]methyl}-7H-pyrrolo[3,2-f]quinazoline-1,3-diamine (SCH79797), a nonpeptide thrombin receptor antagonist. Biochem Pharmacol 60: 1425-1434.[CrossRef][Medline]
Andrande-Gordon P, Maryanoff BE, Derian CK, Zhang H-C, Addo MF, Darrow AL, Eckardt AJ, Hoekstra J, McComsey DF, Oksenberg D, et al. (1999) Design, synthesis and biological characterization of a peptide-mimetic antagonist for a tethered-ligand receptor. Proc Natl Acad Sci USA 96: 12257-12262.
Arisato T, Sarker KP, Kawahara K, Nakata M, Hashiguchi T, Osame M, Kitajima I, and Maruyama I (2003) The agonist of the protease-activated receptor-1 (PAR-1) but not PAR-3 mimics thrombin-induced vascular endothelial growth factor release in human smooth muscle cells. Cell Mol Life Sci 60: 1716-1724.[CrossRef][Medline]
Boire A, Covic L, Agarwal A, Jacques S, Sherifi S, and Kuliopulos A (2005) PAR1 is a matrix metalloprotease-1 receptor that promotes invasion and tumorigenesis of breast cancer cells. Cell 120: 303-313.[CrossRef][Medline]
Chalmers CJ, Balmanno K, Hadfield K, Ley R, and Cook SJ (2003) thrombin inhibits Bim (Bcl-2-interacting mediator of cell death) expression and prevents serum-withdrawal-induced apoptosis via protease-activated receptor-1. Biochem J 375: 99-109.[CrossRef][Medline]
Coughlin SR (2005) Protease-activated receptors in haemostasis, thrombosis and vascular biology. J Thromb Haemost 3: 1800-1814.[CrossRef][Medline]
Derian CK, Damiano BP, Addo MF, Darrow AL, D'Andrea MR, Nedelman M, Zhang H-C, Maryanoff BE, and Andrande-Gordon P (2003) Blockage of the thrombin receptor protease-activated receptor-1 with a small-molecule antagonist prevents thrombus formation and vascular occlusion in nonhuman primates. J Pharmacol Exp Ther 304: 855-861.
Faucette KJ, Fitzgerald LA, Liu L, Parker CJ, and Rodgers GM (1993) Characterization of the interaction between procoagulant albumin and human endothelial cells. Blood 82: 2684-2692.
Fiorucci S, Antonelli E, Distrutti E, Severino B, Fiorentina R, Baldoni M, Galiando G, Santagada V, Morelli A, and Cirino G (2004) PAR1 antagonism protects against experimental liver fibrosis: role of proteinase receptors in stellate cell activation. Hepatology 39: 365-375.[CrossRef][Medline]
Fisher C, Gilbertson-Beadling S, Powers EA, Petzold G, Poorman R, and Mitchell MA (1994) Interstitial collagenase is required for angiogenesis in vitro. Dev Biol 162: 499-511.[CrossRef][Medline]
Flynn AN and Buret AG (2004) Proteinase-activated receptor 1 (PAR-1) and cell apoptosis. Apoptosis 9: 729-737.[CrossRef][Medline]
Grant DS, Tashiro KI, Segui-Real B, Yamada Y, Martin GR, and Kleinman HK (1989) Two different laminin domains mediate the differentiation of human endothelial cells into capillary-like structures in vivo. Cell 58: 933-943.[CrossRef][Medline]
Griffin CT, Srinivasa Y, Zheng YW, Huang W, and Coughlin SR (2001) A role for thrombin receptor signaling in endothelial cells during embryonic development. Science (Wash DC) 293: 1666-1670.
Hirata A, Ogawa S, Kometani T, Kuwano T, Naito S, Kuwano M, and Ono M (2002) ZD1839 (Iressa) induces antiangiogenic effects through inhibition of epidermal growth factor receptor tyrosine kinase. Cancer Res 62: 2554-2560.
Kaneko T, Fujii S, Matsumoto A, Goto D, Makita N, Hamada J, Moriuchi T, and Kitabatake A (2003) Induction of tissue factor expression in endothelial cells by basic fibroblast growth factor and its modulation by fenofibric acid. Thromb J 1: 6.[CrossRef][Medline]
Kuliopulos A, Covic L, Seeley SK, Sheridan PJ, Helin J, and Costello CE (1999) Plasmin desensitization of the PAR1 thrombin receptor: kinetics, sites of truncation and implications for thrombolytic therapy. Biochemistry 38: 4572-4585.[CrossRef][Medline]
Ma L, Perini R, McKnight W, Dicay M, Klein A, Hollenberg MD, and Wallace JL (2005) Proteinase-activated receptors 1 and 4 counter-regulate endostatin and VEGF release from human platelets. Proc Natl Acad Sci USA 102: 216-220.
Maragoudakis ME, Haralabopoulos G, Tsopanoglou NE, and Pipilli-Synetos E (1995) Validation of collagenous protein synthesis as an index for angiogenesis with the use of morphological methods. Microvasc Res 50: 215-222.[CrossRef][Medline]
Martin CB, Mahon GM, Klinger MB, Kay RJ, Symons M, Der CJ, and Whitehead IP (2001) The thrombin receptor, PAR-1, causes transformation by activation of Rho-mediated signaling pathways. Oncogene 20: 1953-1963.[CrossRef][Medline]
Moyer JD, Barbacci EG, Iwata KK, Arnold L, Boman B, Cunningham A, DiOrio C, Doty J, Morin MJ, Moyer MP, et al. (1997) Induction of apoptosis and cell cycle arrest by CP-358,774, an inhibitor of epidermal growth factor receptor tyrosine kinase. Cancer Res 57: 4838-4848.
Oliver FJ, Menissier-de Murcia J, and de Murcia G (1999) Poly(ADP-ribose) polymerase in the cellular response to DNA damage, apoptosis and disease. Am J Hum Genet 64: 1282-1288.[CrossRef][Medline]
Ossovskaya VS and Bunnett NW (2004) Protease-activated receptors: contribution to physiology and disease. Physiol Rev 84: 579-621.
Riewald M, Kravchenko VV, Petrovan RJ, O'Brien PJ, Prass LF, Ulevitch RJ, and Ruf W (2001) Gene induction by coagulation factor Xa is mediated by activation of protease-activated receptor 1. Blood 97: 3109-3116.
Riewald M, Petrovan RJ, Donner A, Mueller BM, and Ruf W (2002) Activation of endothelial cell protease-activated receptor 1 by the protein C pathway. Science (Wash DC) 296: 1880-1882.
Roovers K and Assoian RK (2003) Effects of Rho kinase and actin stress fibers on sustained extracellular signal-regulated kinase activity and activation of G([1]) phase cyclin-dependent kinases. Mol Cell Biol 23: 4283-4294.
Sato Y, Kanno S, Oda N, Abe M, Ito M, Shitara K, and Shibuya M (2000) properties of two VEGF receptors, Flt-1 and KDR, in signal transduction. Ann NY Acad Sci 902: 201-205.
Tantivejkul K, Loberg RD, Mawocha SC, Day LL, St. John L, Pienta BA, Rubin MA, and Pienta KJ (2005) PAR1-mediated NFB activation promotes survival of prostate cancer cells through a Bcl-xL-dependent mechanism. J Cell Biochem 96: 641-652.[CrossRef][Medline]
Tsopanoglou NE and Maragoudakis ME (1999) On the mechanism of thrombin-induced angiogenesis: potentiation of vascular endothelial growth factor activity on endothelial cells by up-regulation of its receptors. J Biol Chem 274: 23969-23976.
Tsopanoglou NE and Maragoudakis ME (2004) Role of thrombin in angiogenesis and tumor progression. Sem Thromb Haemost 30: 63-69.[CrossRef][Medline]
Vergnolle N, Cellar L, Mercarelli A, Rizzo G, Swaminathan S, Beck P, Steinhoff M, Andrande-Gordon P, Bunnett NW, Hollenberg MD, et al. (2004) A role for proteinase-activated receptor-1 in inflammatory bowel diseases. J Clin Investig 114: 1444-1456.[CrossRef][Medline]
Vu T-KH, Hung DT, Wheaton VI, and Coughlin SR (1991) Molecular cloning of a functional thrombin receptor reveals a novel proteolytic mechanism of receptor activation. Cell 64: 1057-1068.[CrossRef][Medline]
Zain J, Huang Y, Feng XS, Nierodzik ML, Li J-J, and Karpatkin S (2000) Concentration-dependent dual effects of thrombin on impaired growth/apoptosis or mitogenesis in tumor cells. Blood 95: 3133-3138.
Zucker S, Mirza H, Conner CE, Lorenz AF, Drews MH, Bahou WF, and Jesty J (1998) Vascular endothelial growth factor induces tissue factor and matrix metalloproteinase production in endothelial cells: conversion of protrombin to thrombin results in progelatinase A activation and cell proliferation. Int J Cancer 75: 780-786.[CrossRef][Medline]
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