Investigation of VEGF and PDGF signals in vascular formation by 3D culture models using mouse ES cells


Vascular formation in vivo involves several processes and signal cascades subsequently occurring in the embryo. Several models by ES cells have been reported for analysis in vitro. We show here a 3D culture system using collagen gel (AteloCell) as a simple and useful system for investigating vascular formations and analyzing the roles of factors in vivo. Although VEGF and PDGF are growth factors with multi-potentials for vascular formation, their sequential roles have not been elucidated. We investigated the effects of VEGF and PDGF B signals for vascular formation by a 3D culture system that embedded embryoid bodies (EBs) from ES cells into a collagen gel. After embedding EBs in the collagen gel with a medium containing VEGF, EBs gave off CD105 immunopositive vessels as the initial step of vasculogenesis. When the factor in the culture medium for EBs was switched from VEGF to PDGF B after 5 days of culture, the morphological features of vessels varied, suggesting the occurrence of vascular-type differentiation. After 11 days of 3D culture, vessels in both groups cultured with VEGF alone and switching to VEGF B at day 5 showed Flk-1 immunoreactivity. Some blood vessels cultured with PDGF B after day 5 expressed either EphrinB2 (arteriole marker) or Flt-4 (lymphatic marker) immunoreactivity, but vessels cultured with VEGF alone exhibited neither of them. Vessels cultured with these two factors could not differentiate into a venous type. The present study indicates that VEGF is the initial signal for vasculogenesis, and that PDGF B is probably involved in vascular diversification.

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Hosoe, H. , Yamamoto, Y. , Tanaka, Y. , Kobayashi, M. , Ninagawa, N. and Torihashi, S. (2012) Investigation of VEGF and PDGF signals in vascular formation by 3D culture models using mouse ES cells. Stem Cell Discovery, 2, 70-77. doi: 10.4236/scd.2012.22011.

Conflicts of Interest

The authors declare no conflicts of interest.


[1] Wang, Z., Cohen, K., Shao, Y., Mole, P., Dombkowski, D. and Scadden, D.T. (2004) Ephrin receptor, EphB4, regulates ES cell differentiation of primitive mammalian hemangioblasts, blood, cardiomyocytes, and blood vessels. Blood, 103, 100-109. doi:10.1182/blood-2003-04-1063
[2] Gerecht-Nir, S., Ziskind, A., Cohen, S. and ItskovitzEldor, J. (2003). Human embryonic stem cells as an in vitro model for human vascular development and the induction of vascular differentiation. Laboratory Investigation, 83, 1811-1820. doi:10.1097/01.LAB.0000106502.41391.F0
[3] Nakagami, H., Nakagawa, N., Takeya, Y., Kashiwagi, K., Ishida, C., Hayashi, S., Aoki, M., Matsumoto, K., Nakamura, T., Ogihara, T. and Morishita, R. (2006) Model of vasculogenesis from embryonic stem cells for vascular research and regenerative medicine. Hypertension, 48, 112-119. doi:10.1161/01.HYP.0000225426.12101.15
[4] Boyd, N.L., Dhara, S.K., Rekaya, R., Godbey, E.A., Hasneen, K., Rao, R.R., West III, F.D., Gerwe, B.A. and Stice, S.L. (2007) BMP4 promotes formation of primitive vascular networks in human embryonic stem cellderived embryoid bodies. Experimental Biology and Medicine (Maywood), 232, 833-843.
[5] Feraud, O., Cao, Y. and Vittet, D. (2001) Embryonic stem cell-derived embryoid bodies development in collagen gels recapitulates sprouting angiogenesis. Laboratory Investigation, 81, 1669-1681. doi:10.1038/labinvest.3780380
[6] Lindskog, H., Athley, E., Larsson, E., Lundin, S., Hellstrom, M. and Lindahl, P. (2006) New insights to vascular smooth muscle cell and pericyte differentiation of mouse embryonic stem cells in vitro. Arteriosclerosis, Thrombosis, and Vascular Biology, 26, 1457-1464. doi:10.1161/01.ATV.0000222925.49817.17
[7] Magnusson, P.U., Looman, C., Ahgren, A., Wu, Y., Claesson-Welsh, L. and Heuchel, R.L. (2007) Platelet-Derived growth factor receptor-beta constitutive activity promotes angiogenesis in vivo and in vitro. Arteriosclerosis, Thrombosis, and Vascular Biology, 27, 2142-2149. doi:10.1161/01.ATV.0000282198.60701.94
[8] Yurugi-Kobayashi, T., Itoh, H., Yamashita, J., Yamahara, K., Hirai, H., Kobayashi, T., Ogawa, M., Nishikawa, S., Nishikawa, S-I. and Nakao, K. (2003). Effective contribution of transplanted vascular progenitor cells derived from embryonic stem cells to adult neovascularization in proper differentiation stage. Blood, 101, 2675-2678. doi:10.1182/blood-2002-06-1877
[9] Coultas, L., Chawengsaksophak, K. and Rossant, J. (2005) Endothelial cells and VEGF in vascular development. Nature, 438, 937-945.
[10] Lanner, F., Sohl, M. and Farnebo, F. (2007) Functional arterial and venous fate is determined by graded VEGF signaling and notch status during embryonic stem cell differentiation. Arteriosclerosis, Thrombosis, and Vascular Biology, 27, 487-493. doi:10.1161/01.ATV.0000255990.91805.6d
[11] Ferrari, G., Cook, B.D., Terushkin, V., Pintucci, G. and Mignatti, P. (2009) Transforming growth factor-beta 1 (TGF-beta1) induces angiogenesis through vascular endothelial growth factor (VEGF)-mediated apoptosis. Journal of Cellular Physiology, 219, 449-458. doi:10.1038/npre.2008.1758.1
[12] Thommen, R., Humar, R., Misevic, G., Pepper, M.S., Hahn, A.W., John, M. and Battegay, E.J. (1997) PDGFBB increases endothelial migration on cord movements during angiogenesis in vitro. Journal of Cellular Biochemistry, 64, 403-413. doi:10.1002/(SICI)1097-4644(19970301)64:3<403::AID-JCB7>3.0.CO;2-Z
[13] Lange, S., Heger, J., Euler, G., Wartenberg, M., Piper, H.M. and Sauer, H. (2009) Platelet-Derived growth factor BB stimulates vasculogenesis of embryonic stem cell-derived endothelial cells by calcium-mediated generation of reactive oxygen species. Cardiovascular Research, 81, 159-168. doi:10.1093/cvr/cvn258
[14] Hao, X., Silva, E.A., Mansson-Broberg E.A., Grinnemo, K-H., Siddiqui, A.J., Dellgren, G., Wardell E., Brodin, L.A., Mooney, D.J. and Sylvén, C. (2007) Angiogenic effects of sequential release of VEGF-A165 and PDGF-BB with alginate hydrogels after myocardial infarction. Cardiovascular Research, 75, 178-185. doi:10.1016/j.cardiores.2007.03.028
[15] Stavridis, M.P., Collins, B.J. and Storey, K.G. (2010) Retinoic acid orchestrates fibroblast growth factor signalling to drive embryonic stem cell differentiation. Development, 137, 881-890. doi:10.1242/dev.043117
[16] Elizalde, C., Campa, V.M., Caro, M., Schlangen, K., Aransay, A.M., Del Mar Vivanco, M. and Kypta, R.M. (2010) Distinct roles for wnt-4 and wnt-11 during retinoic acid-induced neuronal differentiation. Stem Cells, 29, 141-153. doi:10.1002/stem.562
[17] Seifert, T., Stoelting, S., Wagner, T. and Peters, S.O. (2008) Vasculogeneic maturation of E14 embryonic stem cells with evidence of early vascular endothelial growth factor independency. Differentiation, 76, 857-867. doi:10.1111/j.1432-0436.2008.00271.x
[18] Kawamura, H., Li, X., Harper, S.J., Bates, D.O. and Claesson-Welsh, L. (2008) Vascular endothelial growth factor (VEGF)-A165b is a weak in vitro agonist for VEGF receptor-2 due to lack of coreceptor binding and deficient regulation of kinase activity. Cancer Research, 68, 4683-4692. doi:10.1158/0008-5472.CAN-07-6577
[19] Cao, R., Bjorndahl, M.A., Religa, P., Clasper, S., Garvin, S., Galter D., Meister, B., Ikomi, F., Tritsaris, K., Dissing, S., Ohhashi, T., Jackson, D.G., and Cao, Y. (2004) PDGFBB induces intratumoral lymphangiogenesis and promotes lymphatic metastasis. Cancer Cell, 6, 333-345. doi:10.1016/j.ccr.2004.08.034
[20] Yamashita, J.K. (2007) Differentiation of arterial, venous, and lymphatic endothelial cells from vascular progenitors. Trends in Cardiovascular Medicine, 17, 59-63. doi:10.1016/j.tcm.2007.01.001
[21] You, L.R., Lin, F.J., Lee, C.T., DeMayo, F.J., Tsai, M.J. and Tsai, S.Y. (2005) Suppression of Notch signalling by the COUP-TFII transcription factor regulates vein identity. Nature, 435, 98-104.
[22] Herbert, S.P., Huisken, J., Kim, T.N., Feldman, M.E., Houseman, B.T., Wang, R.A., Shokat, K.M. and Stainier, D.Y. (2009) Arterial-Venous segregation by selective cell sprouting: An alternative mode of blood vessel formation. Science, 326, 294-298. doi:10.1126/science.1178577
[23] Yamamizu, K., Matsunaga, T., Uosaki, H., Fukushima, H., Katayama, S., Hiraoka-Kanie, M., Mitani, K. and Yamashita, J.K. (2010) Convergence of Notch and beta-catenin signaling induces arterial fate in vascular progenitors. Journal of Cell Biology, 189, 325-338.
[24] Seki, T., Hong, K.H. and Oh, S.P. (2006) Nonoverlapping expression patterns of ALK1 and ALK5 reveal distinct roles of each receptor in vascular development. Laboratory Investigation, 86, 116-129. doi:10.1038/labinvest.3700376
[25] Kokudo, T., Suzuki, Y., Yoshimatsu, Y., Yamazaki, T., Watabe, T. and Miyazono, K. (2008) Snail is required for TGFbeta-induced endothelial-mesenchymal transition of embryonic stem cell-derived endothelial cells. Journal of Cell Science, 121, 3317-3324. doi:10.1242/jcs.028282

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