Mycophenolic Acid Is a Potent Inhibitor of Angiogenesis

Abstract

HomeArteriosclerosis, Thrombosis, and Vascular BiologyVol. 26, No. 10Mycophenolic Acid Is a Potent Inhibitor of Angiogenesis Free AccessLetterPDF/EPUBAboutView PDFView EPUBSections ToolsAdd to favoritesDownload citationsTrack citationsPermissionsDownload Articles + Supplements ShareShare onFacebookTwitterLinked InMendeleyReddit Jump toSupplementary MaterialsFree AccessLetterPDF/EPUBMycophenolic Acid Is a Potent Inhibitor of Angiogenesis Xinrong Wu, Hanbing Zhong, Jianbo Song, Robert Damoiseaux, Zhen Yang and Shuo Lin Xinrong WuXinrong Wu From the Liu Hua Qiao Hospital (X.W.), Guangzhou, P.R. China; Shenzhen Graduate School, Peking University (Z.Y., S.L.); and the Department of Molecular, Cell, & Developmental Biology (X.W., H.Z., J.S., S.L.) and MIMG/Pharmacology (R.D.), University of California, Los Angeles. Search for more papers by this author , Hanbing ZhongHanbing Zhong From the Liu Hua Qiao Hospital (X.W.), Guangzhou, P.R. China; Shenzhen Graduate School, Peking University (Z.Y., S.L.); and the Department of Molecular, Cell, & Developmental Biology (X.W., H.Z., J.S., S.L.) and MIMG/Pharmacology (R.D.), University of California, Los Angeles. Search for more papers by this author , Jianbo SongJianbo Song From the Liu Hua Qiao Hospital (X.W.), Guangzhou, P.R. China; Shenzhen Graduate School, Peking University (Z.Y., S.L.); and the Department of Molecular, Cell, & Developmental Biology (X.W., H.Z., J.S., S.L.) and MIMG/Pharmacology (R.D.), University of California, Los Angeles. Search for more papers by this author , Robert DamoiseauxRobert Damoiseaux From the Liu Hua Qiao Hospital (X.W.), Guangzhou, P.R. China; Shenzhen Graduate School, Peking University (Z.Y., S.L.); and the Department of Molecular, Cell, & Developmental Biology (X.W., H.Z., J.S., S.L.) and MIMG/Pharmacology (R.D.), University of California, Los Angeles. Search for more papers by this author , Zhen YangZhen Yang From the Liu Hua Qiao Hospital (X.W.), Guangzhou, P.R. China; Shenzhen Graduate School, Peking University (Z.Y., S.L.); and the Department of Molecular, Cell, & Developmental Biology (X.W., H.Z., J.S., S.L.) and MIMG/Pharmacology (R.D.), University of California, Los Angeles. Search for more papers by this author and Shuo LinShuo Lin From the Liu Hua Qiao Hospital (X.W.), Guangzhou, P.R. China; Shenzhen Graduate School, Peking University (Z.Y., S.L.); and the Department of Molecular, Cell, & Developmental Biology (X.W., H.Z., J.S., S.L.) and MIMG/Pharmacology (R.D.), University of California, Los Angeles. Search for more papers by this author Originally published1 Oct 2006https://doi.org/10.1161/01.ATV.0000238361.07225.fcArteriosclerosis, Thrombosis, and Vascular Biology. 2006;26:2414–2416Angiogenesis is necessary for the vascularization of a tumor, providing essential nourishment for tumor growth, and the progression and metastasis of cancer cells.1Although tremendous efforts and resources have been dedicated to discovering angiogenesis inhibitors, very few candidates have been approved by FDA for therapeutic applications. Currently, Avastin (bevacizumab), a humanized monoclonal antibody, and Macugen (Pegaptanib), a pegylated oligonucleotide aptamer, both directed against vascular endothelial growth factor (VEGF) pathway, are two of the approved drugs for clinical use. There is still a great need to develop small molecule drugs for antiangiogenesis purpose. One approach to broaden the discovery platform is to identify antiangiogenic activities from existing drugs that have well defined toxicity and pharmacokinetics. Once a known drug is demonstrated to inhibit angiogenesis, it would move into the clinic trails more rapidly. Because most known drugs have well defined structures and defined biological targets, it is also possible to further improve the compound structure or select the target as a new entry point for angiogenesis-based studies and therapies. Currently there are more than 2000 known drugs that can be used for re-purposing applications. It would be desirable to have a single in vivo assay allowing rapid screening of a large number of compounds for new activities such as angiogenesis inhibition. Recently, the zebrafish has been shown to be a useful model organism for this type of approach.2,3 Zebrafish blood vessels form by angiogenic sprouting and appear to use the same pathways necessary for blood vessel growth in mammals.4 Using transgenic embryos expressing GFP in vessels, the embryonic blood vessels in live zebrafish can be directly visualized in multi-well plates for rapidly screening compounds affecting blood vessel formation and patterning.5 This approach is highly effective and specific as was shown by the effect of previously known antiangiogenic compounds, such as SU6668 and SU5416, on embryonic blood vessel formation of zebrafish.5To identify clinically applicable drugs for antiangiogenesis we screened a library of 1120 compounds consisting of 85% FDA approved drugs (Prestwick Chemical, Inc) for inhibition of blood vessels. Live transgenic zebrafish embryos expressing GFP in vascular endothelial cells under the control of flk (also known as the VEGF receptor 2 gene) regulatory sequences were directly visualized under a fluorescent microscope to reveal drug-induced alteration of fluorescent blood vessels.5 An initial screen of the drugs (at 10 μmol/L concentration with 3 embryos in 200 μL fish water per well of 96-well duplicate plates) identified several drugs that exhibited certain degree of inhibition of blood vessels but had no other detrimental effects on general embryonic development. Here we report one of the inhibitors, mycophenolic acid (MPA, Figure, A), for its efficacy and the subsequent target validation studies. Download figureDownload PowerPointA, Structure of mycophenolic acid [chemical name: 6-(4-Hydroxy-6-methoxy-7-methyl-3-oxo-5-phthalanyl)-4-methyl-4-hexenoic acid]. B, Untreated transgenic embryo (32 hpf). Blood vessels appear as green fluorescence. C, Embryo (32 hpf) treated with 0.9 μmol/L MPA. Shorter fluorescent ISVs were observed. D, Untreated nontransgenic embryo (48 hpf) analyzed by microangiography with injection of fluorescein dextran (MW=2000000Da; Sigma, Catalogue #FD-2000S) into the common cardinal vein as previously described.4 Green fluorescent dye is seen in circulation, revealing all connected blood vessels. E, Embryo (48 hpf) treated with 0.9 μmol/L MPA followed by microangiography analysis. No circulation in ISVs was observed, although circulation in the aorta and the posterior cardinal vein is seen. F, Transgenic embryo (32 hpf) injected with 5 ng of IMPDH2-MO. Shorter fluorescent ISVs similar to the panel C were observed. G through I, Confocal analysis of head vessels of flk-GFP control embryo (G) and embryo treated with MPA (H) or injected with IMPDH2-MO (I) at 32 hpf. The GFP-positive vessels in the heads of both embryos (H and I) are more dilated and appear wider. J, the inhibition of ISVs by MPA is dose dependent. The ISV/Somite ratio indicates the ratio of length of 4 individual ISVs above the end of yolk extension and height of their corresponding somites. Numbers of embryos from each treatment are larger than 150. Zebrafish embryos at 26 hpf showing expression patterns of IMPDH2 (K), IMPDH1a (L), and IMPDH1b (M) as detected by RNA whole mount in situ hybridization. Only IMPDH2 has major expression in the somites where the ISVs are present. Translation-blocking morpholino oligos were obtained from Gene-Tools, LLC and the sequences are: IMPDH1b 5′-GATCAGGTAATCAGCCATGAGTCTC-3′; IMPDH2 5′-GCTGATTAAATAGTCCGCCATAGT-3′.MPA is an immuno-suppressive drug that is widely used to prevent rejection of transplanted organs.6,7 Transgenic zebrafish embryos treated with 0.9 μmol/L MPA significantly inhibited the sprouting of intersegmental blood vessels (ISV; Figure, B and C). We demonstrated that the circulation of these embryos was also blocked using microangiographic analysis (Figure, D and E), suggesting potent inhibition of angiogenesis. The inhibition of angiogenesis by MPA is dose dependent, and embryos treated with MPA concentration that is higher than 1.5 μmol/L no longer developed any extended ISVs (Figure, J). Consistent with our finding, Huang et al and Chong et al also recently reported an inhibitory effect of MPA on in vitro cultured endothelial cells and anti-tumor activity.8,9 Strengthening their discovery, our independent study established the action of MPA in vivo. Furthermore, because inosine monophosphate dehydrogenase (IMPDH) is known as the molecular target of MPA,6,7 we identified the target genes from zebrafish. Sequence alignment revealed that humans have 2 isoforms of IMPDH10,11 whereas the zebrafish genome contains 3, possibly because of additional gene duplications.12 The amino acid sequences are highly conserved from human to zebrafish (more than 90% identity; see data supplement, available online at http://atvb.ahajournals.org). Expression analysis by RNA whole mount in situ hybridization of the 3 genes in zebrafish embryos showed that IMPDH 2 is mainly expressed in the ventral regions of the developing trunk (Figure, K). This is where we used the ISVs as an angiogenic sprouting assays for new blood vessels. The other two IMPDH isoforms are expressed in the superficial epithelial cells that are distal to the ISVs (Figure, L and M). To study function of IMPDH in angiogenesis, antisense morpholino (MO) oligos against the translational sites of mRNA encoding the IMPDH1b and IMPDH2 were synthesized and injected in the flk-GFP transgenic embryos. As expected we did not observe any effect of IMPDH1b MO on angiogenesis (data not shown). However, injection of IMPDH2 MO (5 ng MO per embryo) significantly inhibited angiogenesis in zebrafish (n>200, >50% showed phenotype whereas MO for IMPDH1b showed less than 2%, serving as a negative control for MO injection; see supplementary data). The phenotypes induced by MPA treatment and IMPDH2 knockdown appear indistinguishable (Figure, C and F). In addition, we observed a synergistic action between IMPDH2-MO knockdown and treatment with MPA. Nearly complete inhibition of ISV sprouting could be achieved by applying combination of 20% of the normal amount of IMPDH2-MO and MPA concentrations that are individually required to have an effect on inhibiting angiogenesis in zebrafish embryos. As an internal control, we analyzed vascular structures in the head region. We noticed that all major vessels were present in the head, although ISVs in the same embryos were blocked by MAP or IMPDH2-MO. However, the head vessels were dilated and appeared wider compared with the control embryos (Figure, G to I). Because the expression of IMPDH2 in the head is less extensive than that in the trunk, the abnormally dilated appearance of head vessels may be caused by localized blockade of angiogenesis. In summary, we established in vivo that treatment of MPA and knockdown of its biological target IMPDH both have antiangiogenic activity in zebrafish. Because the angiogenesis process and IMPDH genes are highly conserved from zebrafish to mammals, together with the other recent independent studies,8,9 it should be a reasonable next step to test clinic use of MPA on inhibiting angiogenesis and to develop new drugs targeting IMPDH for antiangiogenesis therapies in humans.Original received June 7, 2006; final version accepted July 12, 2006. Sources of FundingThis work was supported by grants from the National Institutes of Health (to S.L.; DK54508 and ES012990).DisclosuresNone.FootnotesCorrespondence to Shuo Lin, Department of Molecular, Cell, & Developmental Biology, University of California Los Angeles, 621 Charles E. Young Drive South, LS4325, Los Angeles, CA 90095-1606. E-mail [email protected] References 1 Folkman J. Role of angiogenesis in tumor growth and metastasis. Semin Oncol. 2002; 29: 15–18.CrossrefMedlineGoogle Scholar2 Zon LI, Peterson RT. In vivo drug discovery in the zebrafish. Nat Rev Drug Discov. 2005; 4: 35–44.CrossrefMedlineGoogle Scholar3 Kidd KR, Weinstein BM. Fishing for novel angiogenic therapies. Br J Pharmacol. 2003; 140: 585–594.CrossrefMedlineGoogle Scholar4 Isogai S, Horiguchi M, Weinstein BM. The vascular anatomy of the developing zebrafish: an atlas of embryonic and early larval development. Dev Biol. 2001; 230: 278–301.CrossrefMedlineGoogle Scholar5 Cross LM, Cook MA, Lin S, Chen J-N, Rubinstein AL. Rapid analysis of angiogenesis drugs in a live fluorescent zebrafish assay. Arterioscler Thromb Vasc Biol. 2003; 23: 911–912.LinkGoogle Scholar6 Mitsui A, Suzuki S. Immunosuppressive effect of mycophenolic acid. J Antibiot. 1969; 22: 358–363.CrossrefMedlineGoogle Scholar7 Allison AC, Eugui EM. Mycophenolate mofetil and its mechanisms of action. Immunopharmacology. 2000; 47: 85–118.CrossrefMedlineGoogle Scholar8 Huang Y, Liu Z, Huang H, Liu H, Li L. Effects of mycophenolic acid on endothelial cells. Int Immunopharmacol. 2005; 5: 1029–1039.CrossrefMedlineGoogle Scholar9 Chong CR, Qian DZ, Pan F, Wei Y, Pili R, Sullivan DJ Jr, Liu JO. Identification of type 1 inosine monophosphate dehydrogenase as an antiangiogenic drug target. J Med Chem. 2006; 49: 2677–2680.CrossrefMedlineGoogle Scholar10 Natsumeda Y, Ohno S, Kawasaki H, Konno Y, Weber G, Suzuki K. 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