Impaired tumor angiogenesis and VEG-Finduced pathway in endothelial CD146 knockout mice

Abstract

CD146 is a newly identified endothelial biomarker that has been implicated in angiogenesis. Though in vitro angiogenic function of CD146 has been extensively reported, in vivo evidence is still lacking. To address this issue, we generated endothelial-specific CD146 knockout (CD146^EC-KO) mice using the Tg(Tek-cre) system. Surprisingly, these mice did not exhibit any apparent morphological defects in the development of normal retinal vasculature. To evaluate the role of CD146 in pathological angiogenesis, a xenograft tumor model was used. We found that both tumor volume and vascular density were significantly lower in CD146^EC-KO mice when compared to WT littermates. Additionally, the ability for sprouting, migration and tube formation in response to VEGF treatmentwas impairedinendothelial cells (ECs) of CD146^EC-KO mice. Mechanistic studies further confirmed that VEGFinduced VEGFR-2 phosphorylation and AKT/p38 MAPKs/ NF-κB activation were inhibited in these CD146-null ECs, whichmight present theunderlyingcause for theobserved inhibition of tumor angiogenesis in CD146^EC-KO mice. These results suggest thatCD146 plays a redundant role in physiological angiogenic processes, but becomes essential during pathological angiogenesis as observed in tumorigenesis.

Keywords

CD146 / tumor angiogenesis / VEGF / knockout mice

References

[1] Anfosso F, Bardin N, Frances V, Vivier E, Camoin-Jau L, Sampol J, Dignat-George F (1998) Activation of human endothelial cells via S-endo-1 antigen (CD146) stimulates the tyrosine phosphorylation of focal adhesion kinase p125 (FAK). J Biol Chem273: 26852-26856

[2] Anfosso F, Bardin N, Vivier E, Sabatier F, Sampol J, Dignat-George F (2001) Outside-in signaling pathway linked to CD146 engagement in human endothelial cells. J Biol Chem276: 1564-1569

[3] Armulik A, Abramsson A, Betsholtz C (2005) Endothelial/pericyte interactions. Circ Res97: 512-523

[4] Baker M, Robinson SD, Lechertier T, Barber PR, Tavora B, D’Amico G, Jones DT, Vojnovic B, Hodivala-Dilke K (2012) Use of the mouse aortic ring assay to study angiogenesis. Nat Protoc7: 89-104

[5] Bardin N, Anfosso F, Masse JM, Cramer E, Sabatier F, Le Bivic A, Sampol J, Dignat-George F (2001) Identification of CD146 as a component of the endothelial junction involved in the control of cell-cell cohesion. Blood98: 3677-3684

[6] Brekken RA, Overholser JP, Stastny VA, Waltenberger J, Minna JD, Thorpe PE (2000) Selective inhibition of vascular endothelial growth factor (VEGF) receptor 2 (KDR/Flk-1) activity by a monoclonal anti-VEGF antibody blocks tumor growth in mice. Cancer Res60: 5117-5124

[7] Bu P, Gao L, Zhuang J, Feng J, Yang D, Yan X (2006) Anti-CD146 monoclonal antibody AA98 inhibits angiogenesis via suppression of nuclear factor-kappaB activation. Mol Cancer Ther5: 2872-2878

[8] Carmeliet P, Jain RK (2011) Molecular mechanisms and clinical applications of angiogenesis. Nature473: 298-307

[9] Carmeliet P, Ferreira V, Breier G, Pollefeyt S, Kieckens L, Gertsenstein M, Fahrig M, Vandenhoeck A, Harpal K, Eberhardt C (1996) Abnormal blood vessel development and lethality in embryos lacking a single VEGF allele. Nature380: 435-439

[10] Chan B, Sinha S, Cho D, Ramchandran R, Sukhatme VP (2005) Critical roles of CD146 in zebrafish vascular development. Dev Dyn232: 232-244

[11] Chung AS, Lee J, Ferrara N (2010) Targeting the tumour vasculature: insights from physiological angiogenesis. Nat Rev Cancer10: 505-514

[12] Crisan M, Chen CW, Corselli M, Andriolo G, Lazzari L, Peault B (2009) Perivascular multipotent progenitor cells in human organs. Hematop Stem Cells VII1176: 118-123

[13] Dejana E, Spagnuolo R, Bazzoni G (2001) Interendothelial junctions and their role in the control of angiogenesis, vascular permeability and leukocyte transmigration. Thromb Haemost86: 308-315

[14] Duan HX, Xing S, Luo YT, Feng LQ, Gramaglia I, Zhang Y, Lu D, Zeng QQ, Fan KL, Feng J (2013) Targeting endothelial CD146 attenuates neuroinflammation by limiting lymphocyte extravasation to the CNS. Sci Rep3

[15] Ferrara N, Alitalo K (1999) Clinical applications of angiogenic growth factors and their inhibitors. Nat Med5: 1359-1364

[16] Ferrara N, Gerber HP, LeCouter J (2003) The biology of VEGF and its receptors. Nat Med9: 669-676

[17] Ferrara N, Hillan KJ, Gerber HP, Novotny W (2004) Discovery and development of bevacizumab, an anti-VEGF antibody for treating cancer. Nat Rev Drug Discov3: 391-400

[18] Flamme I, Frolich T, Risau W (1997) Molecular mechanisms of vasculogenesis and embryonic angiogenesis. J Cell Physiol173: 206-210

[19] Gariano RF, Gardner TW (2005) Retinal angiogenesis in development and disease. Nature438: 960-966

[20] Graesser D, Solowiej A, Bruckner M, Osterweil E, Juedes A, Davis S, Ruddle NH, Engelhardt B, Madri JA (2002) Altered vascular permeability and early onset of experimental autoimmune encephalomyelitis in PECAM-1-deficient mice. J Clin Investig109: 383-392

[21] Gratzinger D, Barreuther M, Madri JA (2003) Platelet-endothelial cell adhesion molecule-1 modulates endothelial migration through its immunoreceptor tyrosine-based inhibitory motif. Biochem Biophys Res Commun301: 243-249

[22] Grothey A, Galanis E (2009) Targeting angiogenesis: progress with anti-VEGF treatment with large molecules. Nat Rev Clin Oncol6: 507-518

[23] Hirata K, Ishida T, Penta K, Rezaee M, Yang E, Wohlgemuth J, Quertermous T (2001) Cloning of an immunoglobulin family adhesion molecule selectively expressed by endothelial cells. J Biol Chem276: 16223-16231

[24] Imbert AM, Garulli C, Choquet E, Koubi M, Aurrand-Lions M, Chabannon C (2012) CD146 expression in human breast cancer cell lines induces phenotypic and functional changes observed in Epithelial to Mesenchymal Transition. PLoS ONE7: e43752

[25] Jiang TX, Zhuang J, Duan HX, Luo YT, Zeng QQ, Fan KL, Yan HW, Lu D, Ye Z, Hao JF (2012) CD146 is a coreceptor for VEGFR-2 in tumor angiogenesis. Blood120: 2330-2339

[26] Koch S, Tugues S, Li XJ, Gualandi L, Claesson-Welsh L (2011) Signal transduction by vascular endothelial growth factor receptors. Biochem J437: 169-183

[27] Kohama K, Tsukamoto Y, Furuya M, Okamura K, Tanaka H, Miki N, Taira E (2005) Molecular cloning and analysis of the mouse gicerin gene. Neurochem Int46: 465-470

[28] Lamalice L, Houle F, Huot J (2006) Phosphorylation of Tyr (1214) within VEGFR-2 triggers the recruitment of Nck and activation of Fyn leading to SAPK2/p38 activation and endothelial cell migration in response to VEGF. J Biol Chem281: 34009-34020

[29] Lehmann JM, Riethmuller G, Johnson JP (1989) MUC18, a marker of tumor progression in human melanoma, shows sequence similarity to the neural cell adhesion molecules of the immunoglobulin superfamily. Proc Natl Acad Sci USA86: 9891-9895

[30] Li Q, Yu Y, Bischoff J, Mulliken JB, Olsen BR (2003) Differential expression of CD146 in tissues and endothelial cells derived from infantile haemangioma and normal human skin. J Pathol201: 296-302

[31] Liu WF, Ji SR, Sun JJ, Zhang Y, Liu ZY, Liang AB, Zeng HZ (2012) CD146 expression correlates with epithelial-mesenchymal transition markers and a poor prognosis in gastric cancer. Int J Mol Sci13: 6399-6406

[32] Meadows KN, Bryant P, Pumiglia K (2001) Vascular endothelial growth factor induction of the angiogenic phenotype requires Ras activation. J Biol Chem276: 49289-49298

[33] Petruzzelli L, Takami M, Humes HD (1999) Structure and function of cell adhesion molecules. Am J Med106: 467-476

[34] Shalaby F, Rossant J, Yamaguchi TP, Gertsenstein M, Wu XF, Breitman ML, Schuh AC (1995) Failure of blood-island formation and vasculogenesis in Flk-1-deficient mice. Nature376: 62-66

[35] Shih IM (1999) The role of CD146 (Mel-CAM) in biology and pathology. J Pathol189: 4-11

[36] So JH, Hong SK, Kim HT, Jung SH, Lee MS, Choi JH, Bae YK, Kudoh T, Kim JH, Kim CH (2010) Gicerin/CD146 is involved in zebrafish cardiovascular development and tumor angiogenesis. Genes Cells15: 1099-1110

[37] Solovey AN, Gui L, Chang L, Enenstein J, Browne PV, Hebbel RP (2001) Identification and functional assessment of endothelial P1H12. J Lab Clin Med138: 322-331

[38] Telo P, Lostaglio S, Dejana E (1997) Structure of intercellular junctions in the endothelium. Therapie52: 395-398

[39] Van Cutsem E, Lambrechts D, Prenen H, Jain RK, Carmeliet P (2011) Lessons from the adjuvant bevacizumab trial on colon cancer: what next? J Clin Oncol29: 1-4

[40] Xie S, Luca M, Huang S, Gutman M, Reich R, Johnson JP, Bar-Eli M (1997) Expression of MCAM/MUC18 by human melanoma cells leads to increased tumor growth and metastasis. Cancer Res57: 2295-2303

[41] Yan XY, Lin Y, Yang DL, Shen Y, Yuan M, Zhang ZQ, Li PY, Xia HT, Li L, Luo DD (2003) A novel anti-CD146 monoclonal antibody, AA98, inhibits angiogenesis and tumor growth. Blood102: 184-191

[42] Zachary I (2001) Signaling mechanisms mediating vascular protective actions of vascular endothelial growth factor. Am J Physiol Cell Physiol280: C1375-C1386

[43] Zachary I, Gliki G (2001) Signaling transduction mechanisms mediating biological actions of the vascular endothelial growth factor family. Cardiovasc Res49: 568-581

[44] Zeng Q, Li W, Lu D, Wu Z, Duan H, Luo Y, Feng J, Yang D, Fu L, Yan X (2012) CD146, an epithelial-mesenchymal transition inducer, is associated with triple-negative breast cancer. Proc Natl Acad Sci USA109: 1127-1132

[45] Zhang Y, Zheng C, Zhang J, Yang D, Feng J, Lu D, Yan X (2008) Generation and characterization of a panel of monoclonal antibodies against distinct epitopes of human CD146. Hybridoma (Larchmt)27: 345-352

[46] Zheng C, Qiu Y, Zeng Q, Zhang Y, Lu D, Yang D, Feng J, Yan X (2009) Endothelial CD146 is required for in vitro tumor-induced angiogenesis: the role of a disulfide bond in signaling and dimerization. Int J Biochem Cell Biol41: 2163-2172

[47] Zhuang J, Jiang T, Lu D, Luo Y, Zheng C, Feng J, Yang D, Chen C, Yan X (2010) NADPH oxidase 4 mediates reactive oxygen species induction of CD146 dimerization in VEGF signal transduction. Free Radic Biol Med49: 227-236

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2014 This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.