Arteriosclerosis, Thrombosis, and Vascular Biology. 1997;17:1599-1604
© 1997 American Heart Association, Inc.
Articles |
A General Strategy for Isolation of Endothelial Cells From Murine Tissues
Characterization of Two Endothelial Cell Lines From the Murine Lung and Subcutaneous Sponge Implants
From the Istituto Ricerche Farmacologiche Mario Negri, Milan, Italy, and Departamento de Quimica Biologica, Universidad de Buenos Aires, Buenos Aires, Argentina (R.W.De C.).
Correspondence to Dr Annunciata Vecchi, Istituto Ricerche Farmacologiche Mario Negri, via Eritrea 62, 20157 Milano, Italy. E-mail vecchi{at}irfmn.mnegri.it.
Abstract A rapid, reproducible method for the isolation of
murine endothelial cells (ECs) has been developed.
Murine ECs were highly enriched by collagenase digestion of
mechanically minced lung and subcutaneous sponge implants followed by
specific selection with rat anti-mouse CD31 (ie, PECAM-1) monoclonal
antibody–coated magnetic beads (Dynabeads). Pure EC populations were
isolated from primary cultures by a second cycle of immunomagnetic
selection. The cells from the lung were then cloned by a
limiting-dilution method to exclude the possibility of
nonendothelial cell contamination. Of the 300 cells
plated, 29 clones (
10%) were obtained. The clones were positive for
CD31 as measured by flow cytometry, and one clone from the lungs (1G11)
and the cells from sponge implants (designated as SIECs) were then
subjected to subsequent culture in vitro for 40 and 30 passages (up to
5 months), respectively. Characterization was performed on cells
between passage 3 and 10. Both cell types formed contact-inhibited
monolayers on gelatin and capillary-like "tubes" on Matrigel.
However, 1G11 cells exhibited a "cobblestone" morphology, whereas
SIECs had a fibroblast-like appearance at confluence. By flow
cytometry and enzyme-linked immunosorbent assay, these cells
constitutively expressed CD31, VE-cadherin (cadherin-5), CD34, ICAM-1,
VCAM-1, and P-selectin. After stimulation with 30 ng/mL of tumor
necrosis factor-
, the cells became positive for E-selectin (at 4
hours poststimulation) and the expression of ICAM-1, VCAM-1, and
P-selectin was upregulated (after 24 hours of stimulation). The
presence of VE-cadherin in 1G11 cells and SIECs was confirmed by
fluorescence microscopy and Northern blot analysis. The
phenotype and morphology of both cell types were stable during
5 months of culture, and there was no evidence of overgrowth by
contaminating cells. Taken together, the approach outlined herein may
provide a general strategy for the isolation and culture of ECs from a
variety of murine tissues. The general strategy outlined here is
simple, effective, and flexible, allowing the inclusion of further
positive or negative selection steps.
Key Words: endothelial cell lines • mouse • CD31 • CD34 • VE-cadherin
This article has been cited by other articles:
 |
|
 |
|
  M. D. Michaud, G. A. Robitaille, J.-P. Gratton, and D. E. Richard Sphingosine-1-Phosphate: A Novel Nonhypoxic Activator of Hypoxia-Inducible Factor-1 in Vascular Cells Arterioscler. Thromb. Vasc. Biol., June 1, 2009; 29(6): 902 - 908. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. L. Fehrenbach, G. Cao, J. T. Williams, J. M. Finklestein, and H. M. DeLisser Isolation of murine lung endothelial cells Am J Physiol Lung Cell Mol Physiol, June 1, 2009; 296(6): L1096 - L1103. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 L. Maddaluno, S. E. Verbrugge, C. Martinoli, G. Matteoli, A. Chiavelli, Y. Zeng, E. D. Williams, M. Rescigno, and U. Cavallaro The adhesion molecule L1 regulates transendothelial migration and trafficking of dendritic cells J. Exp. Med., March 16, 2009; 206(3): 623 - 635. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 D. Mariappan, J. Winkler, S. Chen, H. Schulz, J. Hescheler, and A. Sachinidis Transcriptional profiling of CD31(+) cells isolated from murine embryonic stem cells. Genes Cells, February 1, 2009; 14(2): 243 - 260. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
X. Ye, J. Ding, X. Zhou, G. Chen, and S. F. Liu Divergent roles of endothelial NF-{kappa}B in multiple organ injury and bacterial clearance in mouse models of sepsis J. Exp. Med., June 9, 2008; 205(6): 1303 - 1315. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 D. Belotti, C. Calcagno, A. Garofalo, D. Caronia, E. Riccardi, R. Giavazzi, and G. Taraboletti Vascular Endothelial Growth Factor Stimulates Organ-Specific Host Matrix Metalloproteinase-9 Expression and Ovarian Cancer Invasion Mol. Cancer Res., April 1, 2008; 6(4): 525 - 534. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 H. Stabile, S. Mitola, E. Moroni, M. Belleri, S. Nicoli, D. Coltrini, F. Peri, A. Pessi, L. Orsatti, F. Talamo, et al. Bone morphogenic protein antagonist Drm/gremlin is a novel proangiogenic factor Blood, March 1, 2007; 109(5): 1834 - 1840. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 B. Loeffler, J. Heeren, M. Blaeser, H. Radner, D. Kayser, B. Aydin, and M. Merkel Lipoprotein lipase-facilitated uptake of LDL is mediated by the LDL receptor J. Lipid Res., February 1, 2007; 48(2): 288 - 298. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 A. H. Licht, O. T. Pein, L. Florin, B. Hartenstein, H. Reuter, B. Arnold, P. Lichter, P. Angel, and M. Schorpp-Kistner JunB is required for endothelial cell morphogenesis by regulating core-binding factor {beta} J. Cell Biol., December 18, 2006; 175(6): 981 - 991. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 G. Bix, R. Castello, M. Burrows, J. J. Zoeller, M. Weech, R. A. Iozzo, C. Cardi, M. L. Thakur, C. A. Barker, K. Camphausen, et al. Endorepellin In Vivo: Targeting the Tumor Vasculature and Retarding Cancer Growth and Metabolism. J Natl Cancer Inst, November 15, 2006; 98(22): 1634 - 1646. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
C. Chiodoni, M. Iezzi, C. Guiducci, S. Sangaletti, I. Alessandrini, C. Ratti, F. Tiboni, P. Musiani, D. N. Granger, and M. P. Colombo Triggering CD40 on endothelial cells contributes to tumor growth J. Exp. Med., October 30, 2006; 203(11): 2441 - 2450. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 T. Matsui, M. Kanai-Azuma, K. Hara, S. Matoba, R. Hiramatsu, H. Kawakami, M. Kurohmaru, P. Koopman, and Y. Kanai Redundant roles of Sox17 and Sox18 in postnatal angiogenesis in mice J. Cell Sci., September 1, 2006; 119(17): 3513 - 3526. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 A. J. Bagnall, N. F. Kelland, F. Gulliver-Sloan, A. P. Davenport, G. A. Gray, M. Yanagisawa, D. J. Webb, and Y. V. Kotelevtsev Deletion of Endothelial Cell Endothelin B Receptors Does Not Affect Blood Pressure or Sensitivity to Salt Hypertension, August 1, 2006; 48(2): 286 - 293. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 B. Teng, H. R. Ansari, P. J. Oldenburg, J. Schnermann, and S. J. Mustafa Isolation and characterization of coronary endothelial and smooth muscle cells from A1 adenosine receptor-knockout mice Am J Physiol Heart Circ Physiol, April 1, 2006; 290(4): H1713 - H1720. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 S. Mitola, M. Belleri, C. Urbinati, D. Coltrini, B. Sparatore, M. Pedrazzi, E. Melloni, and M. Presta Cutting Edge: Extracellular High Mobility Group Box-1 Protein Is a Proangiogenic Cytokine J. Immunol., January 1, 2006; 176(1): 12 - 15. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 T. Milovanova, S. Chatterjee, Y. Manevich, I. Kotelnikova, K. DeBolt, M. Madesh, J. S. Moore, and A. B. Fisher Lung endothelial cell proliferation with decreased shear stress is mediated by reactive oxygen species Am J Physiol Cell Physiol, January 1, 2006; 290(1): C66 - C76. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 T. Zhang, H.-C. Sun, Y. Xu, K.-Z. Zhang, L. Wang, L.-X. Qin, W.-Z. Wu, Y.-K. Liu, S.-L. Ye, and Z.-Y. Tang Overexpression of Platelet-Derived Growth Factor Receptor {alpha} in Endothelial Cells of Hepatocellular Carcinoma Associated with High Metastatic Potential Clin. Cancer Res., December 15, 2005; 11(24): 8557 - 8563. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
Q. Zhang, I. Matsuzaki, S. Chatterjee, and A. B. Fisher Activation of endothelial NADPH oxidase during normoxic lung ischemia is KATP channel dependent Am J Physiol Lung Cell Mol Physiol, December 1, 2005; 289(6): L954 - L961. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. Coma, F. X. Guix, I. Uribesalgo, G. Espuna, M. Sole, D. Andreu, and F. J. Munoz Lack of oestrogen protection in amyloid-mediated endothelial damage due to protein nitrotyrosination Brain, July 1, 2005; 128(7): 1613 - 1621. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
L. Rossig, C. Urbich, T. Bruhl, E. Dernbach, C. Heeschen, E. Chavakis, K.-i. Sasaki, D. Aicher, F. Diehl, F. Seeger, et al. Histone deacetylase activity is essential for the expression of HoxA9 and for endothelial commitment of progenitor cells J. Exp. Med., June 6, 2005; 201(11): 1825 - 1835. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 A. Vidal, S. Zacharoulis, W. Guo, D. Shaffer, F. Giancotti, A. H. Bramley, C. de la Hoz, K. K. Jensen, D. Kato, D. D. MacDonald, et al. p130Rb2 and p27kip1 cooperate to control mobilization of angiogenic progenitors from the bone marrow PNAS, May 10, 2005; 102(19): 6890 - 6895. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
C. J. Favre, M. Mancuso, K. Maas, J. W. McLean, P. Baluk, and D. M. McDonald Expression of genes involved in vascular development and angiogenesis in endothelial cells of adult lung Am J Physiol Heart Circ Physiol, November 1, 2003; 285(5): H1917 - H1938. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
H. Kataoka, J. R. Hamilton, D. D. McKemy, E. Camerer, Y.-W. Zheng, A. Cheng, C. Griffin, and S. R. Coughlin Protease-activated receptors 1 and 4 mediate thrombin signaling in endothelial cells Blood, November 1, 2003; 102(9): 3224 - 3231. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 P. Dell'Era, R. Ronca, L. Coco, S. Nicoli, M. Metra, and M. Presta Fibroblast Growth Factor Receptor-1 Is Essential for In Vitro Cardiomyocyte Development Circ. Res., September 5, 2003; 93(5): 414 - 420. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
D. Cai, M. Xaymardan, J. M. Holm, J. Zheng, J. R. Kizer, and J. M. Edelberg Age-associated impairment in TNF-{alpha} cardioprotection from myocardial infarction Am J Physiol Heart Circ Physiol, July 11, 2003; 285(2): H463 - H469. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 Y.-C. Lim, G. Garcia-Cardena, J. R. Allport, M. Zervoglos, A. J. Connolly, M. A. Gimbrone Jr., and F. W. Luscinskas Heterogeneity of Endothelial Cells from Different Organ Sites in T-Cell Subset Recruitment Am. J. Pathol., May 1, 2003; 162(5): 1591 - 1601. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 K. ILJIN, T. V. PETROVA, T. VEIKKOLA, V. KUMAR, M. POUTANEN, and K. ALITALO A fluorescent Tie1 reporter allows monitoring of vascular development and endothelial cell isolation from transgenic mouse embryos FASEB J, November 1, 2002; 16(13): 1764 - 1774. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. Corada, L. Zanetta, F. Orsenigo, F. Breviario, M. G. Lampugnani, S. Bernasconi, F. Liao, D. J. Hicklin, P. Bohlen, and E. Dejana A monoclonal antibody to vascular endothelial-cadherin inhibits tumor angiogenesis without side effects on endothelial permeability Blood, July 18, 2002; 100(3): 905 - 911. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 R. C. Elble, G. Ji, K. Nehrke, J. DeBiasio, P. D. Kingsley, M. I. Kotlikoff, and B. U. Pauli Molecular and Functional Characterization of a Murine Calcium-activated Chloride Channel Expressed in Smooth Muscle J. Biol. Chem., May 17, 2002; 277(21): 18586 - 18591. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
E. Camerer, H. Kataoka, M. Kahn, K. Lease, and S. R. Coughlin Genetic Evidence That Protease-activated Receptors Mediate Factor Xa Signaling in Endothelial Cells J. Biol. Chem., May 3, 2002; 277(18): 16081 - 16087. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J. R. Allport, Y.-C. Lim, J. M. Shipley, R. M. Senior, S. D. Shapiro, N. Matsuyoshi, D. Vestweber, and F. W. Luscinskas Neutrophils from MMP-9- or neutrophil elastase-deficient mice show no defect in transendothelial migration under flow in vitro J. Leukoc. Biol., May 1, 2002; 71(5): 821 - 828. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 F. Liao, J. F. Doody, J. Overholser, B. Finnerty, R. Bassi, Y. Wu, E. Dejana, P. Kussie, P. Bohlen, and D. J. Hicklin Selective Targeting of Angiogenic Tumor Vasculature by Vascular Endothelial-cadherin Antibody Inhibits Tumor Growth without Affecting Vascular Permeability Cancer Res., May 1, 2002; 62(9): 2567 - 2575. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
E. A. Lidington, R. M. Rao, F. M. Marelli-Berg, P. S. Jat, D. O. Haskard, and J. C. Mason Conditional immortalization of growth factor-responsive cardiac endothelial cells from H-2Kb-tsA58 mice Am J Physiol Cell Physiol, January 1, 2002; 282(1): C67 - C74. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 F. Vinals and J. Pouyssegur Transforming Growth Factor {beta}1 (TGF-{beta}1) Promotes Endothelial Cell Survival during In Vitro Angiogenesis via an Autocrine Mechanism Implicating TGF-{alpha} Signaling Mol. Cell. Biol., November 1, 2001; 21(21): 7218 - 7230. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 L. J. Hutley, A. C. Herington, W. Shurety, C. Cheung, D. A. Vesey, D. P. Cameron, and J. B. Prins Human adipose tissue endothelial cells promote preadipocyte proliferation Am J Physiol Endocrinol Metab, November 1, 2001; 281(5): E1037 - E1044. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
G. Condorelli, U. Borello, L. De Angelis, M. Latronico, D. Sirabella, M. Coletta, R. Galli, G. Balconi, A. Follenzi, G. Frati, et al. Cardiomyocytes induce endothelial cells to trans-differentiate into cardiac muscle: Implications for myocardium regeneration PNAS, September 4, 2001; (2001) 191217898. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M.-A. Impagnatiello, S. Weitzer, G. Gannon, A. Compagni, M. Cotten, and G. Christofori Mammalian Sprouty-1 and -2 Are Membrane-anchored Phosphoprotein Inhibitors of Growth Factor Signaling in Endothelial Cells J. Cell Biol., March 5, 2001; 152(5): 1087 - 1098. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
F. Liao, Y. Li, W. O’Connor, L. Zanetta, R. Bassi, A. Santiago, J. Overholser, A. Hooper, P. Mignatti, E. Dejana, et al. Monoclonal Antibody to Vascular Endothelial-cadherin Is a Potent Inhibitor of Angiogenesis, Tumor Growth, and Metastasis Cancer Res., December 1, 2000; 60(24): 6805 - 6810. [Abstract] [Full Text]
|
|
|
|
|
|
A. Compagni, P. Wilgenbus, M.-A. Impagnatiello, M. Cotten, and G. Christofori Fibroblast Growth Factors Are Required for Efficient Tumor Angiogenesis Cancer Res., December 1, 2000; 60(24): 7163 - 7169. [Abstract] [Full Text]
|
|
|
|
|
|
F. M. Marelli-Berg, D. Scott, I. Bartok, E. Peek, J. Dyson, and R. I. Lechler Activated Murine Endothelial Cells Have Reduced Immunogenicity for CD8+ T Cells: A Mechanism of Immunoregulation? J. Immunol., October 15, 2000; 165(8): 4182 - 4189. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
Y. Zhang, E. C. Griffith, J. Sage, T. Jacks, and J. O. Liu Cell cycle inhibition by the anti-angiogenic agent TNP-470 is mediated by p53 and p21WAF1/CIP1 PNAS, June 6, 2000; 97(12): 6427 - 6432. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
G. Balconi, R. Spagnuolo, and E. Dejana Development of Endothelial Cell Lines From Embryonic Stem Cells : A Tool for Studying Genetically Manipulated Endothelial Cells In Vitro Arterioscler. Thromb. Vasc. Biol., June 1, 2000; 20(6): 1443 - 1451. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
D. E. Richard, E. Berra, E. Gothie, D. Roux, and J. Pouyssegur p42/p44 Mitogen-activated Protein Kinases Phosphorylate Hypoxia-inducible Factor 1alpha (HIF-1alpha ) and Enhance the Transcriptional Activity of HIF-1 J. Biol. Chem., November 12, 1999; 274(46): 32631 - 32637. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
F. Vinals, J. C. Chambard, and J. Pouyssegur p70 S6 Kinase-mediated Protein Synthesis Is a Critical Step for Vascular Endothelial Cell Proliferation J. Biol. Chem., September 17, 1999; 274(38): 26776 - 26782. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. Corada, M. Mariotti, G. Thurston, K. Smith, R. Kunkel, M. Brockhaus, M. G. Lampugnani, I. Martin-Padura, A. Stoppacciaro, L. Ruco, et al. Vascular endothelial-cadherin is an important determinant of microvascular integrity in vivo PNAS, August 17, 1999; 96(17): 9815 - 9820. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
F. Vinals and J. Pouyssegur Confluence of Vascular Endothelial Cells Induces Cell Cycle Exit by Inhibiting p42/p44 Mitogen-Activated Protein Kinase Activity Mol. Cell. Biol., April 1, 1999; 19(4): 2763 - 2772. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
I. Martin-Padura, S. Lostaglio, M. Schneemann, L. Williams, M. Romano, P. Fruscella, C. Panzeri, A. Stoppacciaro, L. Ruco, A. Villa, et al. Junctional Adhesion Molecule, a Novel Member of the Immunoglobulin Superfamily That Distributes at Intercellular Junctions and Modulates Monocyte Transmigration J. Cell Biol., July 13, 1998; 142(1): 117 - 127. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. Presta, M. Belleri, A. Vecchi, J. Hesselgesser, A. Mantovani, and R. Horuk Noncompetitive, Chemokine-mediated Inhibition of Basic Fibroblast Growth Factor-induced Endothelial Cell Proliferation J. Biol. Chem., April 3, 1998; 273(14): 7911 - 7919. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
G. Condorelli, U. Borello, L. De Angelis, M. Latronico, D. Sirabella, M. Coletta, R. Galli, G. Balconi, A. Follenzi, G. Frati, et al. From the Cover: Cardiomyocytes induce endothelial cells to trans-differentiate into cardiac muscle: Implications for myocardium regeneration PNAS, September 11, 2001; 98(19): 10733 - 10738. [Abstract] [Full Text] [PDF]
|
|