Abstract
Despite multiple previous studies in the field of vascular anomalies, the mechanism(s) leading to their development, progression and maintenance has remained unclear. In this study, we have characterized the expression levels of vascular endothelial growth factors and their receptors in 33 human vascular anomalies. Analysis with quantitative real-time PCR and gene-specific assays showed higher expression of neuropilin-2 (NRP2) and VEGF-receptor-3 (VEGFR-3) mRNAs in vascular malformations (VascM) as compared to infantile hemangiomas (Hem). In addition, the expression levels of PlGF and VEGF-C mRNA were significantly higher in venous VascM when compared to the other VascM and Hem. Higher expression of NRP2 and VEGFR-3 were confirmed by immunohistochemistry. To further study the importance of NRP2 and VEGFR-3, endothelial cell (EC) cultures were established from vascular anomalies. It was found that NRP2 and VEGFR-3 mRNA levels were significantly higher in some of the VascM ECs as compared to human umbilical vein ECs which were used as control cells in the study. Furthermore, adenoviral delivery of soluble decoy NRP2 prevented the proliferation of ECs isolated from most of the vascular anomalies. Our findings suggest that NRP2 functions as a factor maintaining the pathological vascular network in these anomalies. Thus, NRP2 could become a potential therapeutic target for the diagnosis and treatment of vascular anomalies.
Similar content being viewed by others
References
Mulliken JB, Glowacki J (1982) Classification of paediatric vascular lesions. Plast Reconstr Surg 70:120–121
Boye E, Olsen BR (2009) Signaling mechanisms in infantile hemangioma. Curr Opin Hematol 16:202–208
Limaye N, Boon LM, Vikkula M (2009) From germline towards somatic mutations in the pathophysiology of vascular anomalies. Hum Mol Genet 18:R65–R74
Roy H, Bhardwaj S, Yla-Herttuala S (2006) Biology of vascular endothelial growth factors. FEBS Lett 580:2879–2887
Zhang L, Lin X, Wang W, Zhuang X, Dong J, Qi Z, Hu Q (2005) Circulating level of vascular endothelial growth factor in differentiating hemangioma from vascular malformation patients. Plast Reconstr Surg 116:200–204
Przewratil P, Sitkiewicz A, Wyka K, Andrzejewska E (2009) Serum levels of vascular endothelial growth factor and basic fibroblastic growth factor in children with hemangiomas and vascular malformations–preliminary report. Pediatr Dermatol 26:399–404
Walter JW, North PE, Waner M, Mizeracki A, Blei F, Walker JWT, Reinisch JF, Marchuk DA (2002) Somatic mutation of vascular endothelial growth factor receptors in juvenile hemangioma. Genes Chromosomes Cancer 33:295–303
Jinnin M, Medici D, Park L, Limaye N, Liu Y, Boscolo E, Bischoff J, Vikkula M, Boye E, Olsen BR (2008) Suppressed NFAT-dependent VEGFR1 expression and constitutive VEGFR2 signaling in infantile hemangioma. Nat Med 14:1236–1246
Lymboussaki A, Partanen TA, Olofsson B, Thomas-Crusells J, Fletcher JDM, Waal RMW, Kaipainen A, Alitalo K (1998) Expression of the vascular endothelial growth factor C receptor VEGFR-3 in lymphatic endothelium of the skin and in vascular tumors. Am J Pathol 153:395–403
Jussila L, Valtola R, Partanen TA, Salven P, Heikkilä P, Matikainen MT, Renkonen R, Kaipainen A, Detmar M, Tschachler E, Alitalo R, Alitalo K (1998) Lymphatic endothelium and Kaposi’s sarcoma spindle cells detected by antibodies against the vascular endothelial growth factor 3. Cancer Res 58:1599–1604
Jaffe EA, Nachman RL, Becker CG, Minick CR (1973) Culture of human endothelial cells derived from umbilical veins. Identification by morphologic and immunologic criteria. J Clin Invest 52:2745–2756
Rossignol M, Gagnon ML, Klagsbrun M (2000) Genomic organization of human neuropilin-1 and neuropilin-2 genes: identification and distribution of splice variants and soluble isoforms. Genomics 70:211–222
Sallinen H, Anttila M, Narvainen J, Koponen J, Hamalainen K, Kholova I, Heikura T, Toivanen P, Kosma VM, Heinonen S, Alitalo K, Yla-Herttuala S (2009) Antiangiogenic gene therapy with soluble VEGFR-1, -2, and -3 reduces the growth of solid human ovarian carcinoma in mice. Mol Ther 17:278–284
Laitinen M, Makinen K, Manninen H, Matsi P, Kossila M, Agrawal RS, Pakkanen T, Luoma JS, Viita H, Hartikainen J, Alhava E, Laakso M, Ylä-Herttuala S (1998) Adenovirus-mediated gene transfer to lower limb artery of patients with chronic critical leg ischemia. Hum Gene Ther 9:1481–1486
Puumalainen AM, Vapalahti M, Agrawal RS, Kossila M, Laukkanen J, Lehtolainen P, Viita H, Paljärvi L, Vanninen R, Ylä-Herttuala S (1998) Beta-galactosidase gene transfer to human malignant glioma in vivo using replication-deficient retroviruses and adenoviruses. Hum Gene Ther 9:1769–1774
Lu L, Bischoff J, Mulliken JB, Bielenberg DR, Fishman SJ, Greene AK (2011) Increased endothelial cells and vasculogenic factors in higher-staged arteriovenous malformations. Plast Reconstr Surg 128:260e–269e
Staton CA, Kumar I, Reed MW, Brown NJ (2007) Neuropilins in physiological and pathological angiogenesis. J Pathol 212:237–248
Pellet-Many C, Frankel P, Jia H, Zachary I (2008) Neuropilins: structure, function and role in disease. Biochem J 411:211–226
Karpanen T, Heckman CA, Keskitalo S, Jeltsch M, Ollila H, Neufeld G, Tamagnone L, Alitalo K (2006) Functional interaction of VEGF-C and VEGF-D with neuropilin receptors. FASEB J 20:1462–1472
Gray MJ, van Buren G, Dallas NA, Xia L, Wang X, Yang AD, Somcio RJ, Lin YG, Lim S, Fan F, Mangala LS, Arumugam T, Logsdon CD, Lopez-Berestein G, Sood AK, Ellis LM (2008) Therapeutic targeting of neuropilin-2 on colorectal carcinoma cells implanted in the murine liver. J Natl Cancer Inst 100:109–120
Dallas NA, Gray MJ, Xia L, Fan F, van Buren G, Gaur P, Samuel S, Lim SJ, Arumugam T, Ramachandran V, Wang H, Ellis LM (2008) Neuropilin-2-mediated tumor growth and angiogenesis in pancreatic adenocarcinoma. Clin Cancer Res 14:8052–8060
Kim WH, Lee SH, Jung MH, Seo JH, Kim J, Kim MA, Lee YM (2009) Neuropilin2 expressed in gastric cancer endothelial cells increases the proliferation and migration of endothelial cells in response to VEGF. Exp Cell Res 315:2154–2164
Geretti E, Klagsbrun M (2007) Neuropilins: novel targets for anti-angiogenesis therapies. Cell Adh Migr 1:56–61
Gokani VJ, Kangesu L, Harper J, Sebire NJ (2011) Venous malformation associated nerve profiles and pain: an immunohistochemical study. J Plast Reconstr Aesthet Surg 64:439–444
Jeltsch M, Kaipainen A, Joukov V, Meng X, Lakso M, Rauvala H, Swartz M, Fukumura D, Jain RK, Alitalo K (1997) Hyperplasia of lymphatic vessels in VEGF-C transgenic mice. Science 276:1423–1425
Partanen TA, Arola J, Saaristo A, Jussila L, Ora A, Miettinen M, Stacker SA, Achen MG, Alitalo K (2000) VEGF-C and VEGF-D expression in neuroendocrine cells and their receptor, VEGFR-3, in fenestrated blood vessels in human tissue. FASEB J 14:2087–2096
Xu Y, Yuan L, Mak J, Pardanaud L, Caunt M, Kasman I, Larrivee B, del Toro R, Suchting S, Medvinsky A, Silva J, Yang J, Thomas JL, Koch AW, Alitalo K, Eichmann A, Bagri A (2010) Neuropilin-2 mediates VEGF-C-induced lymphatic sprouting together with VEGFR3. J Cell Biol 188:115–130
Laakkonen P, Waltari M, Holopainen T, Takahashi T, Pytowski B, Steiner P, Hicklin D, Persaud K, Tonra JR, Witte L, Alitalo K (2007) Vascular endothelial growth factor receptor 3 is involved in tumor angiogenesis and growth. Cancer Res 67:593–599
Tammela T, Zarkada G, Wallgard E, Murtomaki A, Suchting S, Wirzenius M, Waltari M, Hellström M, Schomber T, Peltonen R, Freitas C, Duarte A, Isoniemi H, Laakkonen P, Christofori G, Ylä-Herttuala S, Shibuya M, Pytowski B, Eichmann A, Betsholtz C, Alitalo K (2008) Blocking VEGFR-3 suppresses angiogenic sprouting and vascular network formation. Nature 454:656–660
Goel HL, Bae D, Pursell B, Gouvin LM, Lu S, Mercurio AM (2011) Neuropilin-2 promotes branching morphogenesis in the mouse mammary gland. Development 138:2969–2976
Hillman RT, Feng BY, Ni J, Woo WM, Milenkovic L, Hayden Gephard MG, Teruel MN, Oro AE, Chen JK, Scott MP (2011) Neuropilins are positive regulators of hedgehog signal transduction. Genes Dev 25:2333–2346
Partanen TA, Alitalo K, Miettinen M (1999) Lack of lymphatic vascular specificity of vascular endothelial growth factor receptor 3 in 185 vascular tumors. Cancer 86:2406–2412
Frischer JS, Huang J, Serur A, Kadenhe A, Yamashiro DJ, Kandel JJ (2004) Biomolecular markers and involution of hemangiomas. J Pediatr Surg 39:400–404
Zhao B, Cai J, Boulton M (2004) Expression of placenta growth factor is regulated by both VEGF and hyperglycaemia via VEGFR-2. Microvasc Res 68:239–246
Roy H, Bhardwaj S, Babu M, Jauhiainen S, Herzig KH, Bellu AR, Haisma HJ, Carmeliet P, Alitalo K, Ylä-Herttuala S (2005) Adenovirus-mediated gene transfer of placental growth factor to perivascular tissue induces angiogenesis via upregulation of the expression of endogenous vascular endothelial growth factor-A. Hum Gene Ther 16:1422–1428
Park JE, Chen HH, Winer J, Houck KA, Ferrara N (1994) Placenta growth factor. Potentiation of vascular endothelial growth factor bioactivity, in vitro and in vivo, and high affinity binding to Flt-1 but not to Flk-1/KDR. J Biol Chem 269:25646–25654
Cao Y, Chen H, Zhou L, Chiang MK, Anand-Apte B, Weatherbee JA, Wang Y, Fang F, Flanagan JG, Tsang ML (1996) Heterodimers of placenta growth factor/vascular endothelial growth factor. Endothelial activity, tumor cell expression, and high affinity binding to Flk-1/KDR. J Biol Chem 271:3154–3162
Acknowledgments
This study was supported by Finnish Academy, Antti and Tyyne Soininen Foundation and Kuopio University Foundation. We thank Seija Sahrio for technical assistance in immunohistochemistry, Sari Järveläinen and Tiina Koponen for preparing adenoviral vectors, and Dr. Johanna Lähteenvuo for invaluable advises in endothelial cell isolation.
Conflict of interest
None.
Author information
Authors and Affiliations
Corresponding author
Additional information
Taina A. Partanen, Pia Vuola and Suvi Jauhiainen contributed equally to the work.
Electronic supplementary material
Below is the link to the electronic supplementary material.
Rights and permissions
About this article
Cite this article
Partanen, T.A., Vuola, P., Jauhiainen, S. et al. Neuropilin-2 and vascular endothelial growth factor receptor-3 are up-regulated in human vascular malformations. Angiogenesis 16, 137–146 (2013). https://doi.org/10.1007/s10456-012-9305-x
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s10456-012-9305-x