Abstract
Microvascular endothelial cells from human neonatal foreskin were grown in vitro until a three-dimensional network of capillary-like structures was formed. All stages of the angiogenic cascade could be observed in this in vitro model, including the formation of an internal lumen. The microscopy focused on morphology, formation of an internal lumen, role of the extracellular matrix, polarity of the cells, and the time-course of the angiogenic cascade. Bright-field microscopy revealed cells arranged circularly side by side and the internal lumen of capillary-like structures was verified by electron microscopy. Immunolabeling revealed a peritubular localization of collagen IV. Reporter gene expression after the formation of capillary-like structures was marginally higher than control expression, but clearly lower than the expression of cells at the stage of proliferation. Highest transfection efficiencies were obtained using vectors with the CMV promoter and the long fragment of the Ets-1 promoter. This is a first study of transfection efficiencies mapped for stages of in vitro angiogenesis. We describe here the morphological features of a long-term in vitro model of angiogenesis of human microvascular endothelial cells that could be used for transfection studies, without the provision of an extracellular matrix substrate. The cells self-create their own extracellular matrix to proliferate and form a three-dimensional network of capillary-like structures with an internal lumen.
References
Auerbach, R., Akhtar, N., Lewis, R.L., and Shinners, B.L. (2000). Angiogenesis assays: problems and pitfalls. Cancer Metast. Rev.19, 167–172.10.1023/A:1026574416001Search in Google Scholar
Bevilacqua, M.P., Stengelin, S., Gimbrone, M.A. Jr., and Seed, B. (1989). Endothelial leukocyte molecule 1: an inducible receptor for neutrophils related to complement regulatory proteins and lectins. Science243, 1160–1165.10.1126/science.2466335Search in Google Scholar
Bischoff, J., Brasel, C., Kräling, B.M., and Vranovska, K. (1997). E-selectin is upregulated in proliferating endothelial cells in vitro. Microcirculation4, 279–287.10.3109/10739689709146791Search in Google Scholar
Bishop, E.T., Bell, G.T., Bloor, S., Broom, I.J., Hendry, N.F.K., and Wheatley, D.N. (1999). An in vitro model of angiogenesis: basic features. Angiogenesis3, 335–344.10.1023/A:1026546219962Search in Google Scholar
Bolon, I., Gouyer, V., Devouassoux, M., Vandenbunder, B., Wernert, N., Moro, D., Brambilla, C., and Brambilla, E. (1995). Expression of c-ets-1, collagenase 1, and urokinase-type plasminogen activator genes in lung carcinomas. Am. J. Pathol.147, 1298–1310.Search in Google Scholar
Bonanno, E., Iurlaro, M., Madri, J.A., and Nicosia, R.F. (2000) Type IV collagen modulates angiogenesis and neovessel survival in the rat aorta model. In Vitro Cell Dev. Biol. Anim.36, 336–340.10.1290/1071-2690(2000)036<0336:TICMAA>2.0.CO;2Search in Google Scholar
Chalupowicz, D.G., Chowdhury, Z.A., Bach, T.L., Barsigian, C., and Martinez, J. (1995). Fibrin II induces endothelial cell capillary tube formation. J. Cell Biol.130, 207–215.10.1083/jcb.130.1.207Search in Google Scholar
Erbe, D.V., Barry, A., Presta, L.G., Norton, C.R., Ramos, R.J., Burns, D.K., Rumberger, J.M., Narasinga Rao, B.N., Foxall, C., Brandley, B.K., and Lasky, K.A. (1992). Identification of an E-selectin region critical for carbohydrate recognition and cell adhesion. J. Cell Biol.119, 215–227.10.1083/jcb.119.1.215Search in Google Scholar
Feder, J., Marasa, J.C., and Olander, J.V. (1983). The formation of capillary-like tubes by calf aortic endothelial cells grown in vitro. J. Cell. Physiol.116, 1–6.10.1002/jcp.1041160102Search in Google Scholar
Folkman, J., and Haudenschild, C. (1980). Angiogenesis by capillary endothelial cells in culture. Trans. Ophthalmol. Soc. UK100, 346-353.Search in Google Scholar
Haralabopoulos, G.C., Grandt, D.S., Kleinman, H.K., Lelkes, P.I., Papaioannou, S.P., and Maragoudakis, M.E. (1994). Inhibitors of basement membrane collagen synthesis prevent endothelial cell alignment in Matrigel in vitro and angiogenesis in vivo. Lab. Invest.71, 575–582.Search in Google Scholar
Iljin, K., Dube, A., Kontusaari, S., Korhonen, J., Lahtinen, I., Oettgen, P., and Alitalo, K. (1999). Role of ets factors in the activity and endothelial cell specificity of the mouse Tie gene promoter. FASEB J.13, 377–386.10.1096/fasebj.13.2.377Search in Google Scholar
Ingber, D.E., and Folkman, J. (1989). Mechanochemical switching between growth and differentiation during fibroblast growth factor-stimulated angiogenesis in vitro: role of extracellular matrix. J. Cell Biol.109, 317–330.10.1083/jcb.109.1.317Search in Google Scholar
Kappel, A., Rönicke, V., Damert, A., Flamme, I., Risau, W., and Breier, G. (1999). Identification of vascular endothelial growth factor (VEGF) receptor-2 (Flk-1) promoter/enhancer sequences sufficient for angioblast and endothelial cell-specific transcription in transgenic mice. Blood93, 4284–4292.10.1182/blood.V93.12.4284Search in Google Scholar
Kaufman, D.S., Lewis, R.L., Hanson, E.T., Auerbach, R., Plendl, J., and Thomson, J.A. (2004). Functional endothelial cells isolated from rhesus monkey embryonic stem cells. Blood103, 1325–1333.10.1182/blood-2003-03-0799Search in Google Scholar
Kola, I., Brookes, S., Green, A.R., Garber, R., Tymms, M., Papas, T.S., and Seth, A. (1993). The Ets1 transcription factor is widely expressed during murine embryo development and is associated with mesodermal cells involved in morphogenetic processes such as organ formation. Proc. Natl. Acad. Sci. USA90, 7588–7592.10.1073/pnas.90.16.7588Search in Google Scholar
Koolwijk, P., Peters E., van der Vecht, B., Hornig, C., Weich, H.A., Alitalo, K., Hicklin, D.J., Wu, Y., Witte, L., and van Hinsbergh, V.W. (2001). Involvement of VEGFR-2 (kdr/flk-1) but not VEGFR-1 (flt-1) in VEGF-A- and VEGF-C-induced tube formation by human microvascular endothelial cells in fibrin matrices in vitro. Angiogenesis4, 53–60.10.1023/A:1016637700638Search in Google Scholar
Kräling, B.M., Razon, M.J., Boon, L.M., Zurakowski, D., Seachord, C., Darveau, R.P., Mulliken, J.B., Corless, C.L., and Bischoff, J. (1996). E-Selectin is present in proliferating endothelial cells in human hemangiomas. Am. J. Pathol.148, 1181–1191.Search in Google Scholar
Lawley, T.J., and Kubota, Y. (1989). Induction of morphologic differentiation of endothelial cells in culture. J. Invest. Dermatol.93, 59S–61S.Search in Google Scholar
Madri, J.A., and Williams, S.K. (1983). Capillary endothelial cell cultures: phenotypic modulation by matrix components. J. Cell Biol.97, 153–165.10.1083/jcb.97.1.153Search in Google Scholar
Montesano, R., and Orci, L. (1985). Tumor-promoting phorbol esters induce angiogenesis in vitro. Cell42, 469–477.10.1016/0092-8674(85)90104-7Search in Google Scholar
Montesano, R., Orci, L., and Vassalli, P. (1983). In vitro rapid organization of endothelial cells into capillary-like networks is promoted by collagen matrices. J. Cell Biol.97, 1648–1652.10.1083/jcb.97.5.1648Search in Google Scholar PubMed PubMed Central
Montesano, R., Vassalli, J.D., Baird, A., Guillemin, R., and Orci, L. (1986). Basic fibroblast growth factor induces angiogenesis in vitro. Proc. Natl. Acad. Sci. USA83, 7297–7301.10.1073/pnas.83.19.7297Search in Google Scholar PubMed PubMed Central
Nehls, V., and Drenckhahn, D. (1995). A microcarrier-based cocultivation system for the investigation of factors and cells involved in angiogenesis in three-dimensional fibrin matrices in vitro. Histochem. Cell Biol.104, 459–466.10.1007/BF01464336Search in Google Scholar PubMed
Pelletier, L., Regnard, J., Fellmann, D., and Charbord, P. (2000). An in vitro model for the study of human bone marrow angiogenesis: role of hematopoietic cytokines. Lab. Invest.80, 501–511.10.1038/labinvest.3780056Search in Google Scholar PubMed
Peters, K., Troyer, D., Kummer, S., Kirkpatrick, C.J., and Rauterberg, J. (2002). Apoptosis causes lumen formation during angiogenesis in vitro. Microvasc. Res.64, 334–338.10.1006/mvre.2002.2438Search in Google Scholar
Plendl, J., Neumüller, C., Vollmar, A., Auerbach, R., and Sinowatz, F. (1996). Isolation and characterization of endothelial cells from different organs of fetal pigs. Anat. Embryol.194, 445–456.10.1007/BF00185992Search in Google Scholar
Plendl, J., Snyman, C., Naidoo, S., Sawant, S., Mahaber, R., and Bhoola, K. (2000). Expression of tissue kallikrein and kinin receptors in angiogenic microvascular endothelial cells. Biol. Chem.381, 1103–1115.10.1515/BC.2000.135Search in Google Scholar
Plendl, J., Gilligan, B.J., Wang, S.-J., Lewis, R., Shiners, B., Vandenbroeck, K., and Auerbach, R. (2002). Primitive endothelial cell lines from the porcine embryonic yolk sac. In Vitro Cell. Dev. Biol. Anim.38, 334–342.10.1290/1071-2690(2002)038<0334:PECLFT>2.0.CO;2Search in Google Scholar
Pröls, F., Loser, B., and Marx, M. (1998). Differential expression of osteopontin, PC4, and CEC5, a novel mRNA species, during in vitro angiogenesis. Exp. Cell. Res.239, 1–10.Search in Google Scholar
Richardson, K.C., Jarett, L., and Finke, E.H. (1960). Embedding in epoxy resins for ultrathin sectioning in electron microscopy. Stain. Techn.35, 313.10.3109/10520296009114754Search in Google Scholar
Rönicke, V., Risau, W., and Breier, G. (1996). Characterization of the endothelium-specific murine vascular endothelial growth factor receptor-2 (Flk-1) promoter. Circ. Res.79, 277–285.10.1161/01.RES.79.2.277Search in Google Scholar
Sakuda, H., Nakashima, Y., Kuriyama, S., and Sueishi, K. (1992). Media conditioned by smooth muscle cells cultured in a variety of hypoxic environments stimulates in vitro angiogenesis. A relationship to transforming growth factor β1. Am. J. Pathol.141, 1507–1516.Search in Google Scholar
Schlaeger, T.M., Bartunkova, S., Lawitts, J.A., Teichmann, G., Risau, W., Deutsch, U., and Sato, T.N. (1997). Uniform vascular-endothelial-cell-specific gene expression in both embryonic and adult transgenic mice. Proc. Natl. Acad. Sci. USA94, 3058–3063.10.1073/pnas.94.7.3058Search in Google Scholar
Schor, A.M., Schor, S.L., and Allen, T.D. (1983). Effects of culture conditions on the proliferation, morphology and migration of bovine aortic endothelial cells. J. Cell Sci.62, 267-285.10.1242/jcs.62.1.267Search in Google Scholar
Sun, X.T., Ding, Y.T., Yan, X.G., Wu, L.Y., Li, Q., Cheng, N., Qiu, Y.D., and Zhang, M.Y. (2004). Angiogenic synergistic effect of basic fibroblast growth factor and vascular endothelial growth factor in an in vitro quantitative microcarrier-based three-dimensional fibrin angiogenesis system. World J. Gastroenterol.10, 2524–2528.10.3748/wjg.v10.i17.2524Search in Google Scholar
Thurston, G., McLean, J.W., Rizen, M., Baluk, P., Haskell, A., Murphy, T.J., Hanahan, D., and McDonald, D.M. (1998). Cationic liposomes target angiogenic endothelial cells in tumors and chronic inflammation in mice. J. Clin. Invest.101, 1401–1413.10.1172/JCI965Search in Google Scholar
Tilling, T., Engelbertz, C., Decker, S., Korte, D., Hüwel, S., and Galla, H.J. (2002). Expression and adhesive properties of basement membrane proteins in cerebral capillary endothelial cell cultures. Cell Tissue Res.310, 19–29.10.1007/s00441-002-0604-1Search in Google Scholar PubMed
Ueda A., Koga M., Ikeda M., Kudo S., and Tanishita, K. (2004). Effect of shear stress on microvessel network formation of endothelial cells with in vitro three-dimensional model. Am. J. Physiol. Heart Circ. Physiol.287, H994–H1002.10.1152/ajpheart.00400.2003Search in Google Scholar PubMed
Vailhé, B., Ronot, X., Tracqui, P., Usson, Y., and Tranqui, L. (1997). In vitro angiogenesis is modulated by the mechanical properties of fibrin gels and is related to α(v)β3 integrin localization. In Vitro Cell. Dev. Biol. Anim.33, 763–773.10.1007/s11626-997-0155-6Search in Google Scholar PubMed
Vailhé, B., Vittet, D., and Feige, J.J. (2001). In vitro models of vasculogenesis and angiogenesis. Lab. Invest.81, 439–452.10.1038/labinvest.3780252Search in Google Scholar PubMed
Velázquez, O.C., Snyder, R., Liu, Z.J., Fairman, R.M., and Herlyn, M. (2002). Fibroblast-dependent differentiation of human microvascular endothelial cells into capillary-like, three dimensional networks. FASEB J.16, 1316–1318.10.1096/fj.01-1011fjeSearch in Google Scholar PubMed
Wernert, N., Raes, M.B., Lassalle, P., Dehouck, M.P., Goselin, B., Vandenbunder, B., and Stehelin, D. (1992). C-ets1 proto-oncogene is a transcription factor expressed in endothelial cells during tumor vascularization and other forms of angiogenesis in humans. Am. J. Pathol.140, 119–127.Search in Google Scholar
Wilting, J., Christ, B., Yuan, L., and Eichmann, A. (2003). Cellular and molecular mechanisms of embryonic haemangiogenesis and lymphangiogenesis. Naturwissenschaften90, 433–448.10.1007/s00114-003-0455-ySearch in Google Scholar PubMed
©2005 by Walter de Gruyter Berlin New York