Di Serio, C.

Pellerito, S.

Tarantini, Francesca

English

Florence Research

Letters to the EditorComments on “Blockade of Angiogenesis by Small MoleculeAntagonists to Protease-Activated Receptor-1: Associationwith Endothelial Cell Growth Suppression and Inductionof Apoptosis”Received September 18, 2007; accepted October 9, 2007This letter is in response to the report by Zania et al.(2006). The authors used in vitro (Matrigel) and in vivo(chick chorioallantoic membrane) models of angiogenesisto demonstrate that small molecule PAR1 antagonists,SCH79797 and RWJ56110, block formation of new bloodvessels through their ability to inhibit PAR1-mediatedsignal transduction. They also showed that SCH79797and RWJ56110 inhibit HUVEC proliferation and induceapoptosis when the cells are grown in the presence of 4%serum (FBS).We would like to point out that SCH79797 is able toinhibit cell proliferation and induce apoptosis in vitro in aPAR1-independent manner. Di Serio et al. (2007) demon-strated that SCH79797 (starting from a concentration of100 nM) interferes with the growth of several human andmouse cell lines, including PAR1 null fibroblasts, when thecells are stimulated to grow with 10% serum, FGF-2 (25ng/ml), or PDGF (20 ng/ml), whereas hirudin has no effecton the ability of these cells to grow in culture. Becauseneither FGF-2 nor PDGF depends on PAR1 receptor acti-vation for its mitogenic effect, the ability of SCH79797 toinhibit cell proliferation under these experimental condi-tions is not likely to depend on its PAR1-blocking activity.Moreover, the ability of this drug to inhibit the growth ofPAR1 null cells validates the hypothesis of a PAR1-inde-pendent biological effect of this molecule. We now havetested the growth inhibitory effect of SCH79797 onHUVEC, stimulated in culture by 10% serum (FBS) orFGF-2 (25 ng/ml), and found a significant inhibition of cellgrowth in both experimental conditions (see Fig. 1). Zaniaet al. (2006) reported that, in HUVEC treated with PAR1inhibitors, phosphorylation of extracellular signal-regu-lated kinase 1/2 stimulated by thrombin and 4% serum isreduced. However, they found no effects on FGF-2, vascularendothelial growth factor (VEGF), and epidermal growth fac-tor-mediated extracellular signal-regulated kinase 1/2 acti-vation. They presented this finding to indicate a specificeffect of the inhibitors on PAR1-mediated mitogen-activatedprotein kinase activation. However, when we characterizedthe growth inhibitory effect of SCH79797, we found that theinhibitor was able to reduce phosphorylation of mitogen-activated protein kinase induced by 10% serum, but notFGF-2 or PDGF, although it was still able to block the abilityof all growth factors to induce cell proliferation (data notshown). It will be of interest to test the ability of PAR1 smallinhibitors to block FGF-2- or VEGF-stimulated angiogenesisin vitro and in vivo.We have demonstrated that higher concentrations of theinhibitor (200 nM) induce apoptosis in all of the cell linestested, including PAR1 null fibroblasts (Di Serio et al.,2007). Zania et al. (2006) reported that the antiangiogeniceffect of SCH79797 is evident in fast-growing cultures ofHUVEC (50–60% confluency) but less pronounced in qui-escent cells (100% confluency), which they interpreted toindicate an effect of the inhibitor only on “activated” en-dothelial cells involved in angiogenesis. However, we dem-onstrated that the growth inhibitory effect of SCH79797depends on cell density. Cells seeded at higher density stillrespond to increasing concentrations of the inhibitor(Di Serio et al., 2007).We have little information regarding the cellular effectsof RWJ56110; however, we now have tested this moleculeagainst the ability of PAR1 null fibroblasts to grow inculture and found that, like SCH79797, RWJ56110 signif-icantly impairs the proliferative potential of these cells(Fig. 2).In conclusion, we suggest that the results reported byZania et al. (2006) are not conclusive for an antiangiogeniceffect of SCH79797 solely dependent on its ability to in-hibit PAR1-mediated intracellular signal. Although weagree that SCH79797 and RWJ56110 may be useful asantiangiogenic agents and should be further developed forArticle, publication date, and citation information can be found athttp://jpet.aspetjournals.org.doi:10.1124/jpet.107.131847.ABBREVIATIONS: PAR1, proteinase-activated receptor 1; 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-pyr-rolidinylmethyl)-1H-indol-6-yl]amino]carbonyl]amino]-3,4-difluorobenzenepropanamide]; FGF-2, fibroblast growth factor-2; FBS, fetal bovine se-rum; HUVEC, human umbilical vein endothelial cell(s); PDGF, platelet-derived growth factor.0022-3565/08/3241-404–405$20.00THE JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS Vol. 324, No. 1Copyright © 2008 by The American Society for Pharmacology and Experimental Therapeutics 131847/3289595JPET 324:404–405, 2008 Printed in U.S.A.404 at Univ Studi Di Firenze on February 2, 2012jpet.aspetjournals.orgDownloaded from therapeutic applications, there is data to support thattheir biological effect is, at least in part, independent fromPAR1 inhibition.Department of Critical Care Medicine,Geriatric Medicine Unit,University of FlorenceFlorence, ItalyC. DI SERIO,S. PELLERITO,F. TARANTINIReferencesDi Serio C, Pellerito S, Duarte M, Massi D, Naldini A, Cirino G, Prudovsky I,Santucci M, Geppetti P, Marchionni N, et al. (2007) Protease-activated receptor 1selective antagonist SCH79797 inhibits cell proliferation and induces apoptosis bya protease-activated receptor 1-independent mechanism. Basic Clin PharmacolToxicol 101:63–69.Zania P, Kritikou S, Flordellis CS, Maragoudakis ME, Tsopanoglou NE (2006)Blockade of angiogenesis by small molecule antagonists to protease-activatedreceptor-1: association with endothelial cell growth suppression and induction ofapoptosis. J Pharmacol Exp Ther 318:246–254.0123456n. cells(x104 )day 3048121620n. cells(x104 )day 3p<0.0001p<0.0001NSp<0.02FGF-2 (25ng/ml)+ hirudin (2.5U/ml)+ vehicle (DMSO 50 mM)FGF-2 (25ng/ml)+ hirudin (2.5U/ml)+ SCH79797 (300 nM)FGF-2 (25ng/ml)+ hirudin (2.5U/ml)+ SCH79797 (1 µ M)0% FBS 10% FBSFig. 1. HUVEC cells (2  104 cells/well) were plated in triplicates and grown in M199 containing 10% FBS or no serum in the presence of 25 ng/mlFGF-2, 2.5 U/ml hirudin, and SCH79797 (300 nM and 1 M). Cells were counted at day 3 using an hemocytometer. Data are presented as mean S.D. DMSO, dimethyl sulfoxide.0510152025303540n. cells(x105 ) p<0.02p<0.001p<0.0001day 3vehicle (DMSO 50 mM)RWJ56110 (50 µM)RWJ56110 (1 µM)RWJ56110 (0.1 µM)Fig. 2. PAR1 null fibroblasts (2  104 cells/well)were plated in triplicates and grown in Dulbec-co’s modified Eagle’s medium containing 10%FBS, in the presence of RWJ56110 (0.1, 1, and50 M). Cells were counted at day 3 using anhemocytometer. Data are presented as mean S.D. DMSO, dimethyl sulfoxide.Letters to the Editor 405 at Univ Studi Di Firenze on February 2, 2012jpet.aspetjournals.orgDownloaded from 

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Comments on "Blockade of angiogenesis by small molecule antagonists to protease-activated receptor-1: association with endothelial cell growth suppression and induction of apoptosis".

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Comments on &quot;Blockade of angiogenesis by small molecule antagonists to protease-activated receptor-1: association with endothelial cell growth suppression and induction of apoptosis&quot;.

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Comments on "Blockade of angiogenesis by small molecule antagonists to protease-activated receptor-1: association with endothelial cell growth suppression and induction of apoptosis".