Abstract
Several approaches have been described for improving transfection efficiencies of endothelial cells but the general observations have indicated that yields of transfected endothelial cells are low, irrespective of the techniques used. Here we describe a transfection procedure performed by means of electroporation, with efficiencies up to 85%, by optimizing several parameters such as the electroporation buffer, number of cells, voltage, capacitance and pulse length. The protocol was applied to three endothelial cell types (HUVECs, HUAECs and HMEC-1) commonly used in ‘in vitro’ angiogenic assays. We did not observe functional impairment between transfected and non-transfected cells in their adhesion to different components of the extracellular matrix, migration, or the development of capillary-like structures. Our experiments show that this electroporation procedure does not alter the physiology of endothelial cells and can be applied to functional studies, as exemplified by the successful transfection of the isoform 1 of calcipressin 1 (CALP1L).
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Abbreviations
- bFGF:
-
basic fibroblast growth factor
- EBM:
-
endothelial cell basal medium
- EBM-2:
-
endothelial cell basal medium-2
- GFP:
-
green fluorescent protein
- HMEC-1:
-
human microvascular endothelial cells
- HUAECs:
-
human umbilical arterial endothelial cells
- HUVECs:
-
human umbilical vein endothelial cells
References
J Folkman (1992) ArticleTitleThe role of angiogenesis in tumor growth. Semin Cancer Biol 3 65–71
J Folkman (1995) ArticleTitleAngiogenesis in cancer, vascular, rheumatoid and other disease. Nat Med 1 27–31
Carmeliet P, Jain RK. Angiogenesis in cancer and other diseases. Nature 407, 249–57.
Kumar CC. Integrin alpha v beta 3 as a therapeutic target for blocking tumor-induced angiogenesis. Curr Drug Targets 4: 123–31.
EW Ades FJ Candal RA Swerlick (1992) ArticleTitleHMEC-1: Establishment of an immortalized human microvascular endothelial cell line. J Invest Dermatol 99 683–90
R Fontijn C Hop HJ Brinkman et al. (1995) ArticleTitleMaintenance of vascular endothelial cell-specific properties after immortalization with an amphotrophic replication-deficient retrovirus containing human papilloma virus 16 E6/E7 DNA. Exp Cell Res 216 199–207
R Sipehia G Martucci (1995) ArticleTitleHigh-efficiency transformation of human endothelial cells by Apo E-mediated transfection with plasmid DNA. Biochem Biophys Res Commun 214 206–211
FC Tanner DP Carr GJ Nabel EG Nabel (1997) ArticleTitleTransfection of human endothelial cells. Cardiovasc Res 35 522–8
M Teifel LT Heine S Milbredt P Friedl (1997) ArticleTitleOptimization of transfection of human endothelial cells. Endothelium 5 21–35
AC Nathwani KM Gale KD Pemberton et al. (1994) ArticleTitleEfficient gene transfer into human umbilical vein endothelial cells allows functional analysis of the human tissue factor gene promoter. Br J Haematol 88 122–8
RA Kotnis MM Thompson SL Eady et al. (1995) ArticleTitleOptimisation of gene transfer into vascular endothelial cells using electroporation. Eur J Vasc Endovasc Surg 9 71–9
T Ear P Giguere A Fleury et al. (2001) ArticleTitleHigh efficiency transient transfection of genes in human umbilical vein endothelial cells by electroporation. J Immunol Meth 257 41–9
AF Merrick LD Shewring GJ Sawyer et al. (1996) ArticleTitleComparison of adenovirus gene transfer to vascular endothelial cells in cell culture, organ culture, and in vivo. Transplantation 62 1085–9
CJ Wrighton R Hofer-Warbinek T Moll et al. (1996) ArticleTitleInhibition of endothelial cell activation by adenovirus-mediated expression of I kappa B alpha, an inhibitor of the transcription factor NF-kappa B. J Exp Med 183 1013–22
IH Maxwell C Kaletta K Naujoks F Maxwell (2003) ArticleTitleTargeting diphtheria toxin A-chain transcription to activated endothelial cells using an E-selectin promoter. Angiogenesis 6 31–8
PJ Keely JK Westwick IP Whitehead et al. (1997) ArticleTitleCdc42 and Rac1 induce integrin-mediated cell motility and invasiveness through PI(3)K. Nature 390 632–6
JF Marshall DC Rutherford AC McCartney et al. (1995) ArticleTitleAlpha v beta 1 is a receptor for vitronectin and fibrinogen, and acts with alpha 5 beta 1 to mediate spreading on fibronectin. J Cell Sci 108 IssueIDPt 3 1227–38
T Ishida RK Kundu E Yang et al. (2003) ArticleTitleTargeted disruption of endothelial cell-selective adhesion molecule inhibits angiogenic processes in vitro and in vivo. J Biol Chem 278 34598–604
L Genesca A Aubareda JJ Fuentes et al. (2003) ArticleTitlePhosphorylation of calcipressin 1 increases its ability to inhibit calcineurin and decreases calcipressin half-life. Biochem J 374 567–75
K Tei N Kawakami-Kimura O Taguchi et al. (2002) ArticleTitleRoles of cell adhesion molecules in tumor angiogenesis induced by cotransplantation of cancer and endothelial cells to nude rats. Cancer Res 62 6289–96
S Soeda Y Shibata H Shimeno (1997) ArticleTitleInhibitory effect of oversulfated fucoidan on tube formation by human vascular endothelial cells. Biol Pharm Bull 20 1131–5
H Kurzen S Schmitt H Naher T Mohler (2003) ArticleTitleInhibition of angiogenesis by non-toxic doses of temozolomide. Anticancer Drugs 14 515–22
D Ivanov M Philippova V Tkachuk et al. (2004) ArticleTitleCell adhesion molecule T-cadherin regulates vascular cell adhesion, phenotype and motility. Exp Cell Res 293 207–18
D Hanahan J Folkman (1996) ArticleTitlePatterns and emerging mechanisms of the angiogenic switch during tumorigenesis. Cell 86 353–64
J Folkman (1971) ArticleTitleTumor angiogenesis: Therapeutic implications. N Engl J Med 285 1182–6
MU Naik D Vuppalanchi UP Naik (2003) ArticleTitleEssential role of junctional adhesion molecule-1 in basic fibroblast growth factor-induced endothelial cell migration. Arterioscler Thromb Vasc Biol 23 2165–71
JG Rozic C Chakraborty PK Lala (2001) ArticleTitleCyclooxygenase inhibitors retard murine mammary tumor progression by reducing tumor cell migration, invasiveness and angiogenesis Int J Cancer. 93 497–506
Z Lang M Guerrera R Li S Ladisch (2001) ArticleTitleGanglioside GD1a enhances VEGF-induced endothelial cell proliferation and migration. Biochem Biophys Res Commun 282 1031–7
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Hernández, J.L., Coll, T. & Ciudad, C.J. A highly efficient electroporation method for the transfection of endothelial cells. Angiogenesis 7, 235–241 (2004). https://doi.org/10.1007/s10456-004-4180-8
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DOI: https://doi.org/10.1007/s10456-004-4180-8