VEGF-induced HUVEC migration and proliferation are decreased by PDE2 and PDE4 inhibitors

Summary

Migration and proliferation of endothelial cells in response to VEGF play an important role in angiogenesis associated to pathologies such as atherosclerosis, diabetes and tumor development. Elevation of cAMP in endothelial cells has been shown to inhibit growth factor-induced proliferation. Our hypothesis was that inactivation of cAMP-specific phosphodiesterases (PDEs) would inhibit angiogenesis. The purpose of this study was to evaluate the effect of PDE inhibitors on in vitro and in vivo angiogenesis, using human umbilical vein endothelial cell (HUVEC) and chick chorioallantoic membrane (CAM) models respectively. Here, we report that: 1) PDE2, PDE3, PDE4 and PDE5 are expressed in HUVEC; 2) EHNA (20 µM), PDE2 selective inhibitor, and RP73401 (10 µM), PDE4 selective inhibitor, are able to increase the intracellular cAMP level in HUVEC; 3) EHNA and RP73401 are able to inhibit proliferation, cell cycle progression and migration of HUVEC stimulated by VEGF; 4) these in vitro effects can be mimic by treating HUVEC with the cAMP analogue, 8-Br-cAMP (600 µM); 5) only the association of EHNA and RP73401 inhibits in vivo angiogenesis, indicating that both migration and proliferation must be inhibited. These data strongly suggest that PDE2 and PDE4 represent new potential therapeutic targets in pathological angiogenesis.

Keywords

Angiogenesis and inhibitors - cell proliferation - cAMP phosphodiesterase - endothelial cell - VEGF

• References

• 1 Pepper MS. Manipulating angiogenesis. From basic science to the bedside. Arterioscler Thromb Vasc Biol 1997; 17: 605-19.
• 2 Folkman J. Angiogenesis in cancer, vascular, rheumatoid and other diseases. Nat Med 1995; 1: 27-31.
• 3 Leitman DC, Fiscus RR, Murad F. Forskolin, phosphodiesterase inhibitors, and cyclic AMP analogs inhibit proliferation of cultured bovine aortic endothelial cells. J Cell Physiol 1986; 127: 237-43.
• 4 D’Angelo G, Lee H, Weiner RI. cAMP-dependent protein kinase inhibits the mitogenic action of vascular endothelial growth factor and fibroblast growth factor in capillary endothelial cells by blocking Raf activation. J Cell Biochem 1997; 67: 353-66.
• 5 Cook SJ, McCormick F. Inhibition by cAMP of Ras-dependent activation of Raf. Science 1993; 262: 1069-72.
• 6 Vadiveloo PK. et al. G1 phase arrest of human smooth muscle cells by heparin, IL-4 and cAMP is linked to repression of cyclin D1 and cdk2. Atherosclerosis 1997; 133: 61-9.
• 7 Beavo JA. Cyclic nucleotide phosphodiesterases: functional implications of multiple isoforms. Physiol Rev 1995; 75: 725-48.
• 8 Manganiello VC. et al. Diversity in cyclic nucleotide phosphodiesterase isoenzyme families. Arch Biochem Biophys 1995; 322: 1-13.
• 9 Francis SH, Turko IV, Corbin JD. Cyclic nucleotide phosphodiesterases: relating structure and function. Prog Nucleic Acid Res Mol Biol 2001; 65: 1-52.
• 10 Stoclet JC. et al. Cyclic nucleotide phosphodiesterases as therapeutics targets in cardiovascular diseases. Expert Opin Investig Drugs 1995; 4: 1081-110.
• 11 Lugnier C, Schini VB. Characterization of cyclic nucleotide phosphodiesterases from cultured bovine aortic endothelial cells. Biochem Pharmacol 1990; 39: 75-84.
• 12 Lugnier C, Komas N. Modulation of vascular cyclic nucleotide phosphodiesterase by cyclic GMP: role in vasodilatation. Eur Heart J 1993; 14: 141-8.
• 13 Lugnier C. et al. Selective inhibition of cyclic nucleotide phosphodiesterase of human, bovine and rat aorta. Biochem Pharmacol 1986; 35: 1743-51.
• 14 Keravis T, Komas N, Lugnier C. Cyclic nucleotide hydrolysis in bovine aortic endothelial cells in culture: differential regulation in cobblestone and spindle phenotypes. J Vasc Res 2000; 37: 235-49.
• 15 Ilan N, Mahooti S, Madri JA. Distinct signal transduction pathways are utilized during the tube formation and survival phases of in vitro angiogenesis. J Cell Sci 1998; 111: 3621-31.
• 16 Auerbach R. et al. A simple procedure for the long-term cultivation of chicken embryos. Dev Biol 1974; 41: 391-4.
• 17 Follenius A, Gerard D. Fluorescence investigations of calmodulin hydrophobic sites. Biochem Biophys Res Commun 1984; 119: 1154-60.
• 18 Klein-Soyer C. et al. A simple in vitro model of mechanical injury of confluent cultured endothelial cells to study quantitatively the repair process. Thromb Haemost 1986; 56: 232-5.
• 19 Lowry OH. et al. Protein measurement with the folin phenol reagent. J Biol Chem 1951; 193: 265-75.
• 20 Keravis TM, Wells JN, Hardman JG. Cyclic nucleotide phosphodiesterase activities from pig coronary arteries: Lack of interconvertibility of major forms. Biochim Biophys Acta 1980; 613: 116-29.
• 21 Pradelles P, Grassi J, Chabardes D, Guiso N. Enzyme immunoassays of adenosine cyclic 3’,5’-monophosphate and guanosine cyclic 3’,5’-monophosphate using acetylcholinesterase. Anal Chem 1989; 61: 447-53.
• 22 Cory AH. et al. Use of an aqueous soluble tetrazolium/formazan assay for cell growth assays in culture. Cancer Commun 1991; 3: 207-12.
• 23 Soderling SH, Bayuga SJ, Beavo JA. Identification and characterization of a novel family of cyclic nucleotide phosphodiesterase. J Biol Chem 1998; 273: 15553-8.
• 24 Fujishige K. et al. Cloning and characterization of a novel human phosphodiesterase that hydrolyzes both cAMP and cGMP (PDE10A). J Biol Chem 1999; 274: 18438-45.
• 25 Yuasa K. et al. Isolation and characterization of two novel phosphodiesterase PDE11A variants showing unique structur and tissue-specific expresssion. J Biol Chem 2000; 275: 31469-79.
• 26 Fawcett L, Baxendale R, Stacey P. et al. Molecular cloning and characterization of a district human phosphodiesterase gene family: PDE 111. Proc Natl Acad Sci USA 2000; 97: 3702-7.
• 27 Reimund JM, Raboisson P, Pinna G. et al. Anti-TNF-α properties of new 0-benzylade-nine derivatives with selective phosphodiesterase-4- inhibiting properties. Biochem Biophys Res Commun 2001; 288: 427-34.
• 28 Podzuweit T, Nennstiel P, Muller A. Isozyme selective inhibition of cGMP-stimulated cyclic nucleotide phosphodiesterases by erythro-9-(2-hydroxy-3-nonyl) adenine. Cell Signal 1995; 7: 733-8.
• 29 Hordijk PL. et al. cAMP abrogates the p21rasmitogen-activated protein kinase pathway in fibroblasts. J Biol Chem 1994; 269: 3534-8.
• 30 Koyama H. et al. Molecular pathways of cyclic nucleotide-induced inhibition of arterial smooth muscle cell proliferation. J Cell Physiol 2001; 186: 1-10.
• 31 Tani T. et al. Pharmacological manipulation of tissue cyclic AMP by inhibitors. Effects of phosphodiesterase inhibitors on the functions of platelets and vascular endothelial cells. Adv Second Messenger Phosphoprotein Res 1992; 25: 215-27.
• 32 Miro X. et al. Phosphodiesterases 4D and 7A splice variants in the response of HUVEC cells to TNF-alpha. Biochem Biophys Res Commun 2000; 274: 415-21.
• 33 Kessler T, Lugnier C. Rolipram increases cyclic GMP content in L-arginine-treated cultured bovine aortic endothelial cells. Eur J Pharmacol 1995; 290: 163-7.
• 34 Michie AM. et al. Rapid regulation of PDE-2 and PDE-4 cyclic AMP phosphodiesterase activity following ligation of the T cell antigen receptor on thymocytes: analysis using the selective inhibitors erythro-9-(2-hydroxy-3-nonyl)-adenine (EHNA) and rolipram. Cell Signal 1996; 8: 97-110.
• 35 Houslay MD. PDE4 cAMP-specific phosphodiesterases. Prog Nucleic Acid Res Mol Biol 2001; 69: 249-315.
• 36 Schaeffer HJ, Schwender CF. Enzyme inhibitors: Bridging hydrophobic and hydrophilic regions on adenosine deaminase with some 9-(2-hydroxy-3-alkyl) adenines. J Med Chem 1974; 17: 6-8.
• 37 Smolenski RT. et al. Endothelial nucleotide catabolism and adenosine production. Car-diovasc Res 1994; 8: 100-4.
• 38 Sexl V. et al. Stimulation of human umbilical vein endothelial cell proliferation by A2-adenosine and beta 2-adrenoceptors. Br J Pharma-col 1995; 114: 1577-86.
• 39 Rose RJ. et al. Cyclic AMP-mediated regulation of vascular smooth muscle cell cyclic AMP phosphodiesterase activity. Br J Pharma-col 1997; 122: 233-40.
• 40 Perez-Roger I, Ivorra C, Diez A. et al. Inhibition of cellular proliferation by drug targeting of cyclin-dependent kinases. Curr Pharm Biotechnol 2000; 1: 107-16.
• 41 Lugnier C. et al. Characterization of cyclic nucleotide phosphodiesterase isoforms associated to isolated cardiac nuclei. Biochim Biophys Acta 1999; 1472: 431-46.
• 42 Dell’ Acqua ML, Scott JD. Protein kinase A anchoring. J Biol Chem 1997; 272: 12881-4.
• 43 Dodge KL. et al. mAKAP assembles a protein kinase A/PDE4 phosphodiesterase cAMP signaling module. EMBO J 2001; 20: 1921-30.
• 44 Bornfeldt KE, Krebs EG. Crosstalk between protein kinase A and growth factor receptor signaling pathways in arterial smooth muscle. Cell Signal 1999; 11: 465-77.
• 45 O’Connor KL, Mercurio AM. Protein kinase A regulates Rac and is required for the growth factor-stimulated migration of carcinoma Cells. J Biol Chem 2001; 276: 47895-900.
• 46 Hall A. Rho GTPases and the actin cytoskele-ton. Science 1998; 279: 509-14.
• 47 Kim S. et al. Regulation of integrin αvβ3-mediated endothelial cell migration and angio-genesis by integrin α5β1 and protein kinase A. J Biol Chem 2000; 275: 33920-8.
• 48 Kim S. et al. Inhibition of endothelial cell survival and angiogenesis by protein kinase A. J Clin Invest 2002; 110: 933-41.
• 49 Okruhlicova L. et al. Cytochemical distribution of cyclic AMP-dependent 3’,5’-nucleotide phosphodiesterase in the rat myocardium. Histochem J 1996; 28: 165-72.
• 50 Okruhlicova L. et al. Species differences in localization of cardiac cAMP-phosphodiesterase activity: a cytochemical study. Mol Cell Biochem 1997; 173: 183-88.
• 51 Palmer D, Tsoi K, Maurice DH. Synergistic inhibition of vascular smooth muscle cell migration by phosphodiesterase 3 and phospho-dies-terase 4 inhibitors. Circ Res 1998; 82: 852-61.