Impedance analysis of GPCR-mediated changes in endothelial barrier function: overview and fundamental considerations for stable and reproducible measurements

Abstract

The past 20 years has seen significant growth in using impedance-based assays to understand the molecular underpinning of endothelial and epithelial barrier function in response to physiological agonists and pharmacological and toxicological compounds. Most studies on barrier function use G protein-coupled receptor (GPCR) agonists which couple to fast and transient changes in barrier properties. The power of impedance-based techniques such as electric cell-substrate impedance sensing (ECIS) resides in its ability to detect minute changes in cell layer integrity label-free and in real-time ranging from seconds to days. We provide a comprehensive overview of the biophysical principles, applications, and recent developments in impedance-based methodologies. Despite extensive application of impedance analysis in endothelial barrier research, little attention has been paid to data analysis and critical experimental variables, which are both essential for signal stability and reproducibility. We describe the rationale behind common ECIS data presentation and interpretation and illustrate practical guidelines to improve signal intensity by adapting technical parameters such as electrode layout, monitoring frequency, or parameter (resistance versus impedance magnitude). Moreover, we discuss the impact of experimental parameters, including cell source, liquid handling, and agonist preparation on signal intensity and kinetics. Our discussions are supported by experimental data obtained from human microvascular endothelial cells challenged with three GPCR agonists, thrombin, histamine, and sphingosine-1-phosphate.

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Abbreviations

AC:

Alternating current

BBB:

Blood brain barrier

DC:

Direct current

ECIS:

Electric cell-substrate impedance sensing

FITC:

Fluorescein-isothiocyanate

GPCR:

G protein-coupled receptor

HDMEC:

Human dermal microvascular endothelial cells

His:

Histamine

HUVEC:

Human umbilical vein endothelial cells

ITO:

Indium tin oxide

RVSMC:

Rat vascular smooth muscle cells

S1P:

Sphingosine-1-phosphate

TEER:

Transendothelial (/epithelial) electrical resistance

Thr:

Thrombin

8W1E:

8 Wells

1 Electrode per well:

(1 Working electrode/1 counter electrode)

8W10E:

8 Wells

10 Electrodes per well:

(Working electrode consisting of 10 small electrodes/1 counter electrode)

8W10E+:

8 Wells

40 Electrodes per well:

(40 Electrodes distributed on interdigitated finger pattern)

References

  1. 1.

    Adam AP, Sharenko AL, Pumiglia K, Vincent PA (2010) Src-induced tyrosine phosphorylation of VE-cadherin is not sufficient to decrease barrier function of endothelial monolayers. J Biol Chem 285(10):7045–7055. doi:10.1074/jbc.M109.079277

    PubMed Central  CAS  PubMed  Article  Google Scholar 

  2. 2.

    Aman J, van Bezu J, Damanafshan A, Huveneers S, Eringa EC, Vogel SM, Groeneveld AB, Vonk Noordegraaf A, van Hinsbergh VW, van Nieuw Amerongen GP (2012) Effective treatment of edema and endothelial barrier dysfunction with imatinib. Circulation 126(23):2728–2738. doi:10.1161/CIRCULATIONAHA.112.134304

    CAS  PubMed  Article  Google Scholar 

  3. 3.

    Anderson JM, Van Itallie CM (2009) Physiology and function of the tight junction. Cold Spring Harbor Perspect Biol 1(2):a002584. doi:10.1101/cshperspect.a002584

    Article  Google Scholar 

  4. 4.

    Artus C, Glacial F, Ganeshamoorthy K, Ziegler N, Godet M, Guilbert T, Liebner S, Couraud PO (2014) The Wnt/planar cell polarity signaling pathway contributes to the integrity of tight junctions in brain endothelial cells. J Cerebral Blood Flow Metabol Off J Int Soc Cerebral Blood Flow Metabol 34(3):433–440. doi:10.1038/jcbfm.2013.213

    CAS  Article  Google Scholar 

  5. 5.

    Atienza JM, Yu N, Wang X, Xu X, Abassi Y (2006) Label-free and real-time cell-based kinase assay for screening selective and potent receptor tyrosine kinase inhibitors using microelectronic sensor array. J Biomol Screen 11(6):634–643. doi:10.1177/1087057106289334

    CAS  PubMed  Article  Google Scholar 

  6. 6.

    Atienza JM, Zhu J, Wang X, Xu X, Abassi Y (2005) Dynamic monitoring of cell adhesion and spreading on microelectronic sensor arrays. J Biomol Screen 10(8):795–805. doi:10.1177/1087057105279635

    CAS  PubMed  Article  Google Scholar 

  7. 7.

    Bagnaninchi PO, Drummond N (2011) Real-time label-free monitoring of adipose-derived stem cell differentiation with electric cell-substrate impedance sensing. Proc Natl Acad Sci U S A 108(16):6462–6467. doi:10.1073/pnas.1018260108

    PubMed Central  CAS  PubMed  Article  Google Scholar 

  8. 8.

    Barsoukov E, Macdonald JR (2005) Impedance spectroscopy: theory, experiment, and applications. Wiley, Hoboken

    Google Scholar 

  9. 9.

    Bazzoni G (2006) Endothelial tight junctions: permeable barriers of the vessel wall. Thromb Haemost 95(1):36–42

    CAS  PubMed  Google Scholar 

  10. 10.

    Becker PM, Verin AD, Booth MA, Liu F, Birukova A, Garcia JG (2001) Differential regulation of diverse physiological responses to VEGF in pulmonary endothelial cells. Am J Physiol Lung Cell Molec Physiol 281(6):L1500–L1511

    CAS  Google Scholar 

  11. 11.

    Becker PM, Waltenberger J, Yachechko R, Mirzapoiazova T, Sham JS, Lee CG, Elias JA, Verin AD (2005) Neuropilin-1 regulates vascular endothelial growth factor-mediated endothelial permeability. Circ Res 96(12):1257–1265. doi:10.1161/01.RES.0000171756.13554.49

    CAS  PubMed  Article  Google Scholar 

  12. 12.

    Benson K, Cramer S, Galla HJ (2013) Impedance-based cell monitoring: barrier properties and beyond. Fluids Barriers CNS 10(1):5. doi:10.1186/2045-8118-10-5

    PubMed Central  PubMed  Article  Google Scholar 

  13. 13.

    Bernas MJ, Cardoso FL, Daley SK, Weinand ME, Campos AR, Ferreira AJ, Hoying JB, Witte MH, Brites D, Persidsky Y, Ramirez SH, Brito MA (2010) Establishment of primary cultures of human brain microvascular endothelial cells to provide an in vitro cellular model of the blood–brain barrier. Nat Protoc 5(7):1265–1272. doi:10.1038/nprot.2010.76

    PubMed Central  CAS  PubMed  Article  Google Scholar 

  14. 14.

    Birukova AA, Cokic I, Moldobaeva N, Birukov KG (2009) Paxillin is involved in the differential regulation of endothelial barrier by HGF and VEGF. Am J Respir Cell Mol Biol 40(1):99–107. doi:10.1165/rcmb.2008-0099OC

    PubMed Central  CAS  PubMed  Article  Google Scholar 

  15. 15.

    Bogatcheva NV, Zemskova MA, Kovalenkov Y, Poirier C, Verin AD (2009) Molecular mechanisms mediating protective effect of cAMP on lipopolysaccharide (LPS)-induced human lung microvascular endothelial cells (HLMVEC) hyperpermeability. J Cell Physiol 221(3):750–759. doi:10.1002/jcp.21913

    PubMed Central  CAS  PubMed  Article  Google Scholar 

  16. 16.

    Bogatcheva NV, Zemskova MA, Poirier C, Mirzapoiazova T, Kolosova I, Bresnick AR, Verin AD (2011) The suppression of myosin light chain (MLC) phosphorylation during the response to lipopolysaccharide (LPS): beneficial or detrimental to endothelial barrier? J Cell Physiol 226(12):3132–3146. doi:10.1002/jcp.22669

    PubMed Central  CAS  PubMed  Article  Google Scholar 

  17. 17.

    Cecchelli R, Aday S, Sevin E, Almeida C, Culot M, Dehouck L, Coisne C, Engelhardt B, Dehouck MP, Ferreira L (2014) A stable and reproducible human blood–brain barrier model derived from hematopoietic stem cells. PLoS One 9(6):e99733. doi:10.1371/journal.pone.0099733

    PubMed Central  PubMed  Article  Google Scholar 

  18. 18.

    Chatterjee A, Snead C, Yetik-Anacak G, Antonova G, Zeng J, Catravas JD (2008) Heat shock protein 90 inhibitors attenuate LPS-induced endothelial hyperpermeability. Am J Physiol Lung Cell Molec Physiol 294(4):L755–L763. doi:10.1152/ajplung.00350.2007

    CAS  Article  Google Scholar 

  19. 19.

    Chen XL, Nam JO, Jean C, Lawson C, Walsh CT, Goka E, Lim ST, Tomar A, Tancioni I, Uryu S, Guan JL, Acevedo LM, Weis SM, Cheresh DA, Schlaepfer DD (2012) VEGF-induced vascular permeability is mediated by FAK. Dev Cell 22(1):146–157. doi:10.1016/j.devcel.2011.11.002

    PubMed Central  CAS  PubMed  Article  Google Scholar 

  20. 20.

    Cohen-Kashi Malina K, Cooper I, Teichberg VI (2009) Closing the gap between the in-vivo and in-vitro blood–brain barrier tightness. Brain Res 1284:12–21. doi:10.1016/j.brainres.2009.05.072

    CAS  PubMed  Article  Google Scholar 

  21. 21.

    Cooper JA, Del Vecchio PJ, Minnear FL, Burhop KE, Selig WM, Garcia JG, Malik AB (1987) Measurement of albumin permeability across endothelial monolayers in vitro. J Appl Physiol 62(3):1076–1083

    CAS  PubMed  Google Scholar 

  22. 22.

    Daniels BP, Cruz-Orengo L, Pasieka TJ, Couraud PO, Romero IA, Weksler B, Cooper JA, Doering TL, Klein RS (2013) Immortalized human cerebral microvascular endothelial cells maintain the properties of primary cells in an in vitro model of immune migration across the blood brain barrier. J Neurosci Methods 212(1):173–179. doi:10.1016/j.jneumeth.2012.10.001

    PubMed Central  CAS  PubMed  Article  Google Scholar 

  23. 23.

    Davies PF, Remuzzi A, Gordon EJ, Dewey CF Jr, Gimbrone MA Jr (1986) Turbulent fluid shear stress induces vascular endothelial cell turnover in vitro. Proc Natl Acad Sci U S A 83(7):2114–2117

    PubMed Central  CAS  PubMed  Article  Google Scholar 

  24. 24.

    Davis GE, Senger DR (2005) Endothelial extracellular matrix: biosynthesis, remodeling, and functions during vascular morphogenesis and neovessel stabilization. Circ Res 97(11):1093–1107. doi:10.1161/01.RES.0000191547.64391.e3

    CAS  PubMed  Article  Google Scholar 

  25. 25.

    De Blasio BF, Laane M, Walmann T, Giaever I (2004) Combining optical and electrical impedance techniques for quantitative measurement of confluence in MDCK-I cell cultures. BioTechniques 36 (4):650–654, 656, 658 passim

  26. 26.

    de Vries HE, Blom-Roosemalen MC, de Boer AG, van Berkel TJ, Breimer DD, Kuiper J (1996) Effect of endotoxin on permeability of bovine cerebral endothelial cell layers in vitro. J Pharmacol Experiment Therapeutics 277(3):1418–1423

    Google Scholar 

  27. 27.

    DeMaio L, Tarbell JM, Scaduto RC Jr, Gardner TW, Antonetti DA (2004) A transmural pressure gradient induces mechanical and biological adaptive responses in endothelial cells. Am J Physiol Heart Circulat Physiol 286(2):H731–H741. doi:10.1152/ajpheart.00427.2003

    CAS  Article  Google Scholar 

  28. 28.

    DePaola N, Phelps JE, Florez L, Keese CR, Minnear FL, Giaever I, Vincent P (2001) Electrical impedance of cultured endothelium under fluid flow. Ann Biomed Eng 29(8):648–656

    CAS  PubMed  Article  Google Scholar 

  29. 29.

    Dewi BE, Takasaki T, Kurane I (2004) In vitro assessment of human endothelial cell permeability: effects of inflammatory cytokines and dengue virus infection. J Virol Methods 121(2):171–180. doi:10.1016/j.jviromet.2004.06.013

    CAS  PubMed  Article  Google Scholar 

  30. 30.

    Dubrovskyi O, Birukova AA, Birukov KG (2013) Measurement of local permeability at subcellular level in cell models of agonist- and ventilator-induced lung injury. Lab Investig J Technical Methods Pathol 93(2):254–263. doi:10.1038/labinvest.2012.159

    CAS  Article  Google Scholar 

  31. 31.

    Ehret R, Baumann W, Brischwein M, Schwinde A, Stegbauer K, Wolf B (1997) Monitoring of cellular behaviour by impedance measurements on interdigitated electrode structures. Biosensors Bioelectro 12(1):29–41

    CAS  Article  Google Scholar 

  32. 32.

    Ehret R, Baumann W, Brischwein M, Schwinde A, Wolf B (1998) On-line control of cellular adhesion with impedance measurements using interdigitated electrode structures. Med Biol Eng Comput 36(3):365–370

    CAS  PubMed  Article  Google Scholar 

  33. 33.

    Estrada R, Zeng Q, Lu H, Sarojini H, Lee JF, Mathis SP, Sanchez T, Wang E, Kontos CD, Lin CY, Hla T, Haribabu B, Lee MJ (2008) Up-regulating sphingosine 1-phosphate receptor-2 signaling impairs chemotactic, wound-healing, and morphogenetic responses in senescent endothelial cells. J Biol Chem 283(44):30363–30375. doi:10.1074/jbc.M804392200

    PubMed Central  CAS  PubMed  Article  Google Scholar 

  34. 34.

    Faurobert E, Rome C, Lisowska J, Manet-Dupe S, Boulday G, Malbouyres M, Balland M, Bouin AP, Keramidas M, Bouvard D, Coll JL, Ruggiero F, Tournier-Lasserve E, Albiges-Rizo C (2013) CCM1-ICAP-1 complex controls beta1 integrin-dependent endothelial contractility and fibronectin remodeling. J Cell Biol 202(3):545–561. doi:10.1083/jcb.201303044

    PubMed Central  CAS  PubMed  Article  Google Scholar 

  35. 35.

    Franke RP, Grafe M, Schnittler H, Seiffge D, Mittermayer C, Drenckhahn D (1984) Induction of human vascular endothelial stress fibres by fluid shear stress. Nature 307(5952):648–649

    CAS  PubMed  Article  Google Scholar 

  36. 36.

    Garcia JG, Liu F, Verin AD, Birukova A, Dechert MA, Gerthoffer WT, Bamberg JR, English D (2001) Sphingosine 1-phosphate promotes endothelial cell barrier integrity by Edg-dependent cytoskeletal rearrangement. J Clin Invest 108(5):689–701. doi:10.1172/JCI12450

    PubMed Central  CAS  PubMed  Article  Google Scholar 

  37. 37.

    Garcia JG, Siflinger-Birnboim A, Bizios R, Del Vecchio PJ, Fenton JW 2nd, Malik AB (1986) Thrombin-induced increase in albumin permeability across the endothelium. J Cell Physiol 128(1):96–104. doi:10.1002/jcp.1041280115

    CAS  PubMed  Article  Google Scholar 

  38. 38.

    Giaever I, Keese CR (1984) Monitoring fibroblast behavior in tissue culture with an applied electric field. Proc Natl Acad Sci U S A 81(12):3761–3764

    PubMed Central  CAS  PubMed  Article  Google Scholar 

  39. 39.

    Giaever I, Keese CR (1991) Micromotion of mammalian cells measured electrically. Proc Natl Acad Sci U S A 88(17):7896–7900

    PubMed Central  CAS  PubMed  Article  Google Scholar 

  40. 40.

    Heijink IH, van Oosterhout A, Kapus A (2010) Epidermal growth factor receptor signalling contributes to house dust mite-induced epithelial barrier dysfunction. Euro Respirat J 36(5):1016–1026. doi:10.1183/09031936.00125809

    CAS  Article  Google Scholar 

  41. 41.

    Herron CR, Lowery AM, Hollister PR, Reynolds AB, Vincent PA (2011) p120 regulates endothelial permeability independently of its NH2 terminus and Rho binding. Am J Physiol Heart Circulat Physiol 300(1):H36–H48. doi:10.1152/ajpheart.00812.2010

    CAS  Article  Google Scholar 

  42. 42.

    Hofmann U, Michaelis S, Winckler T, Wegener J, Feller KH (2013) A whole-cell biosensor as in vitro alternative to skin irritation tests. Biosens Bioelectron 39(1):156–162. doi:10.1016/j.bios.2012.07.075

    CAS  PubMed  Article  Google Scholar 

  43. 43.

    Hoheisel D, Nitz T, Franke H, Wegener J, Hakvoort A, Tilling T, Galla HJ (1998) Hydrocortisone reinforces the blood–brain barrier in a serum-free culture system. Biochem Biophys Res Commun 224(1):312–316

    Article  Google Scholar 

  44. 44.

    Janshoff A, Wegener J, Sieber M, Galla HJ (1996) Double-mode impedance analysis of epithelial cell monolayers cultured on shear wave resonators. Eur Biophys J 25:93–103

    CAS  PubMed  Article  Google Scholar 

  45. 45.

    Karczewski J, Troost FJ, Konings I, Dekker J, Kleerebezem M, Brummer RJ, Wells JM (2010) Regulation of human epithelial tight junction proteins by Lactobacillus plantarum in vivo and protective effects on the epithelial barrier. Am J Physiol Gastrointestinal Liver Physiol 298(6):G851–G859. doi:10.1152/ajpgi.00327.2009

    CAS  Article  Google Scholar 

  46. 46.

    Keese CR, Bhawe K, Wegener J, Giaever I (2002) Real-time impedance assay to follow the invasive activities of metastatic cells in culture. BioTechniques 33 (4):842–844, 846, 848–850

  47. 47.

    Keese CR, Wegener J, Walker SR, Giaever I (2004) Electrical wound-healing assay for cells in vitro. Proc Natl Acad Sci U S A 101(6):1554–1559. doi:10.1073/pnas.0307588100

    PubMed Central  CAS  PubMed  Article  Google Scholar 

  48. 48.

    Kim YV, Di Cello F, Hillaire CS, Kim KS (2004) Differential Ca2+ signaling by thrombin and protease-activated receptor-1-activating peptide in human brain microvascular endothelial cells. Am J Physiol Cell Physiol 286(1):C31–C42. doi:10.1152/ajpcell.00157.2003

    CAS  PubMed  Article  Google Scholar 

  49. 49.

    Kobayashi K, Tsubosaka Y, Hori M, Narumiya S, Ozaki H, Murata T (2013) Prostaglandin D2-DP signaling promotes endothelial barrier function via the cAMP/PKA/Tiam1/Rac1 pathway. Arterioscler Thromb Vasc Biol 33(3):565–571. doi:10.1161/ATVBAHA.112.300993

    CAS  PubMed  Article  Google Scholar 

  50. 50.

    Kobayashi N, Nishi T, Hirata T, Kihara A, Sano T, Igarashi Y, Yamaguchi A (2006) Sphingosine 1-phosphate is released from the cytosol of rat platelets in a carrier-mediated manner. J Lipid Res 47(3):614–621. doi:10.1194/jlr. M500468-JLR200

    CAS  PubMed  Article  Google Scholar 

  51. 51.

    Konya V, Ullen A, Kampitsch N, Theiler A, Philipose S, Parzmair GP, Marsche G, Peskar BA, Schuligoi R, Sattler W, Heinemann A (2013) Endothelial E-type prostanoid 4 receptors promote barrier function and inhibit neutrophil trafficking. The Journal of allergy and clinical immunology 131 (2):532–540 e531-532. doi:10.1016/j.jaci.2012.05.008

  52. 52.

    Kuo YC, Su CH, Liu CY, Chen TH, Chen CP, Wang HS (2009) Transforming growth factor-beta induces CD44 cleavage that promotes migration of MDA-MB-435 s cells through the up-regulation of membrane type 1-matrix metalloproteinase. Int J Cancer J Int du Cancer 124(11):2568–2576. doi:10.1002/ijc.24263

    CAS  Article  Google Scholar 

  53. 53.

    Lamontagne D, Pohl U, Busse R (1992) Mechanical deformation of vessel wall and shear stress determine the basal release of endothelium-derived relaxing factor in the intact rabbit coronary vascular bed. Circ Res 70(1):123–130

    CAS  PubMed  Article  Google Scholar 

  54. 54.

    Lien CF, Mohanta SK, Frontczak-Baniewicz M, Swinny JD, Zablocka B, Gorecki DC (2012) Absence of glial alpha-dystrobrevin causes abnormalities of the blood–brain barrier and progressive brain edema. J Biol Chem 287(49):41374–41385. doi:10.1074/jbc.M112.400044

    PubMed Central  CAS  PubMed  Article  Google Scholar 

  55. 55.

    Liu Q, Yu J, Xiao L, Tang JC, Zhang Y, Wang P, Yang M (2009) Impedance studies of bio-behavior and chemosensitivity of cancer cells by micro-electrode arrays. Biosens Bioelectron 24(5):1305–1310. doi:10.1016/j.bios.2008.07.044

    CAS  PubMed  Article  Google Scholar 

  56. 56.

    Lo CM, Keese CR, Giaever I (1993) Monitoring motion of confluent cells in tissue culture. Exp Cell Res 204(1):102–109. doi:10.1006/excr.1993.1014

    CAS  PubMed  Article  Google Scholar 

  57. 57.

    Lo CM, Keese CR, Giaever I (1994) pH changes in pulsed CO2 incubators cause periodic changes in cell morphology. Exp Cell Res 213(2):391–397. doi:10.1006/excr.1994.1214

    CAS  PubMed  Article  Google Scholar 

  58. 58.

    Lo CM, Keese CR, Giaever I (1999) Cell-substrate contact: another factor may influence transepithelial electrical resistance of cell layers cultured on permeable filters. Exp Cell Res 250(2):576–580. doi:10.1006/excr.1999.4538

    CAS  PubMed  Article  Google Scholar 

  59. 59.

    Lorenowicz MJ, Fernandez-Borja M, Kooistra MR, Bos JL, Hordijk PL (2008) PKA and Epac1 regulate endothelial integrity and migration through parallel and independent pathways. Eur J Cell Biol 87(10):779–792. doi:10.1016/j.ejcb.2008.05.004

    CAS  PubMed  Article  Google Scholar 

  60. 60.

    Lovelady DC, Friedman J, Patel S, Rabson DA, Lo CM (2009) Detecting effects of low levels of cytochalasin B in 3 T3 fibroblast cultures by analysis of electrical noise obtained from cellular micromotion. Biosens Bioelectron 24(7):2250–2254. doi:10.1016/j.bios.2008.09.033

    PubMed Central  CAS  PubMed  Article  Google Scholar 

  61. 61.

    Luissint AC, Federici C, Guillonneau F, Chretien F, Camoin L, Glacial F, Ganeshamoorthy K, Couraud PO (2012) Guanine nucleotide-binding protein Galphai2: a new partner of claudin-5 that regulates tight junction integrity in human brain endothelial cells. J Cerebral Blood Flow Metabol Off J Int Soc Cerebral Blood Flow Metabol 32(5):860–873. doi:10.1038/jcbfm.2011.202

    CAS  Article  Google Scholar 

  62. 62.

    Lvovich VF (2012) Impedance spectroscopy: applications to electrochemical and dielectricphenomena. Wiley, Hoboken

    Google Scholar 

  63. 63.

    Lynch JJ, Ferro TJ, Blumenstock FA, Brockenauer AM, Malik AB (1990) Increased endothelial albumin permeability mediated by protein kinase C activation. J Clin Invest 85(6):1991–1998. doi:10.1172/JCI114663

    PubMed Central  CAS  PubMed  Article  Google Scholar 

  64. 64.

    McAdams ET, Lackermeier A, McLaughlin JA, Macken D, Jossinet J (1995) The linear and nonlinear electrical properties of the electrode-electrolyte interface. Biosens Bioelectron 10:76–74

    Google Scholar 

  65. 65.

    McLaughlin JN, Shen L, Holinstat M, Brooks JD, Dibenedetto E, Hamm HE (2005) Functional selectivity of G protein signaling by agonist peptides and thrombin for the protease-activated receptor-1. J Biol Chem 280(26):25048–25059. doi:10.1074/jbc.M414090200

    CAS  PubMed  Article  Google Scholar 

  66. 66.

    Mehta D, Malik AB (2006) Signaling mechanisms regulating endothelial permeability. Physiol Rev 86(1):279–367. doi:10.1152/physrev.00012.2005

    CAS  PubMed  Article  Google Scholar 

  67. 67.

    Michaelis S, Rommel CE, Endell J, Goring P, Wehrsporn R, Steinem C, Janshoff A, Galla HJ, Wegener J (2013) Macroporous silicon chips for laterally resolved, multi-parametric analysis of epithelial barrier function. Lab Chip 12:2329–2336

    Article  CAS  Google Scholar 

  68. 68.

    Michaelis S, Wegener J, Robelek R (2013) Label-free monitoring of cell-based assays: combining impedance analysis with SPR for multiparametric cell profiling. Biosens Bioelectron 49:63–70

    CAS  PubMed  Article  Google Scholar 

  69. 69.

    Michel CC (1996) Transport of macromolecules through microvascular walls. Cardiovasc Res 32(4):644–653

    CAS  PubMed  Article  Google Scholar 

  70. 70.

    Monaghan-Benson E, Wittchen ES (2011) In vitro analyses of endothelial cell permeability. Methods Mol Biol 763:281–290. doi:10.1007/978-1-61779-191-8_19

    CAS  PubMed  Article  Google Scholar 

  71. 71.

    Moore E, Rawley O, Wood T, Galvin P (2009) Monitoring of cell growth in vitro using biochips packaged with indium tin oxide sensors. Sensors Actators B-Chem 139:187–193

    CAS  Article  Google Scholar 

  72. 72.

    Moy AB, Blackwell K, Kamath A (2002) Differential effects of histamine and thrombin on endothelial barrier function through actin-myosin tension. Am J Physiol Heart Circulat Physiol 282(1):H21–H29

    CAS  Google Scholar 

  73. 73.

    Murata N, Sato K, Kon J, Tomura H, Yanagita M, Kuwabara A, Ui M, Okajima F (2000) Interaction of sphingosine 1-phosphate with plasma components, including lipoproteins, regulates the lipid receptor-mediated actions. Biochem J 352(Pt 3):809–815

    PubMed Central  CAS  PubMed  Article  Google Scholar 

  74. 74.

    Murata T, Aritake K, Tsubosaka Y, Maruyama T, Nakagawa T, Hori M, Hirai H, Nakamura M, Narumiya S, Urade Y, Ozaki H (2013) Anti-inflammatory role of PGD2 in acute lung inflammation and therapeutic application of its signal enhancement. Proc Natl Acad Sci U S A 110(13):5205–5210. doi:10.1073/pnas.1218091110

    PubMed Central  CAS  PubMed  Article  Google Scholar 

  75. 75.

    Mycielska ME, Djamgoz MB (2004) Cellular mechanisms of direct-current electric field effects: galvanotaxis and metastatic disease. J Cell Sci 117(Pt 9):1631–1639. doi:10.1242/jcs.01125

    CAS  PubMed  Article  Google Scholar 

  76. 76.

    Nakagawa S, Deli MA, Kawaguchi H, Shimizudani T, Shimono T, Kittel A, Tanaka K, Niwa M (2009) A new blood–brain barrier model using primary rat brain endothelial cells, pericytes and astrocytes. Neurochem Int 54(3–4):253–263. doi:10.1016/j.neuint.2008.12.002

    CAS  PubMed  Article  Google Scholar 

  77. 77.

    Panke O, Weigel W, Schmidt S, Steude A, Robitzki AA (2011) A cell-based impedance assay for monitoring transient receptor potential (TRP) ion channel activity. Biosens Bioelectron 26(5):2376–2382. doi:10.1016/j.bios.2010.10.015

    PubMed  Article  CAS  Google Scholar 

  78. 78.

    Paolinelli R, Corada M, Ferrarini L, Devraj K, Artus C, Czupalla CJ, Rudini N, Maddaluno L, Papa E, Engelhardt B, Couraud PO, Liebner S, Dejana E (2013) Wnt activation of immortalized brain endothelial cells as a tool for generating a standardized model of the blood brain barrier in vitro. PLoS One 8(8):e70233. doi:10.1371/journal.pone.0070233

    PubMed Central  CAS  PubMed  Article  Google Scholar 

  79. 79.

    Parker JC, Stevens T, Randall J, Weber DS, King JA (2006) Hydraulic conductance of pulmonary microvascular and macrovascular endothelial cell monolayers. Am J Physiol Lung Cell Molec Physiol 291(1):L30–L37. doi:10.1152/ajplung.00317.2005

    CAS  Article  Google Scholar 

  80. 80.

    Rahman ARA, Lo CM, Bhansali S (2006) A micro-electrode array biosensor for impedance spectroscopy of human umbilical vein endothelial cells. Sens Actuators B-Chem 118:115–120

    CAS  Article  Google Scholar 

  81. 81.

    Ramirez-Icaza G, Mohammed KA, Nasreen N, Van Horn RD, Hardwick JA, Sanders KL, Tian J, Ramirez-Icaza C, Johnson MT, Antony VB (2004) Th2 cytokines IL-4 and IL-13 downregulate paxillin expression in bronchial airway epithelial cells. J Clin Immunol 24(4):426–434. doi:10.1023/B:JOCI.0000029111.27168.c6

    CAS  PubMed  Article  Google Scholar 

  82. 82.

    Rehder D, Iden S, Nasdala I, Wegener J, Brickwedde MK, Vestweber D, Ebnet K (2006) Junctional adhesion molecule-a participates in the formation of apico-basal polarity through different domains. Exp Cell Res 312(17):3389–3403. doi:10.1016/j.yexcr.2006.07.004

    CAS  PubMed  Article  Google Scholar 

  83. 83.

    Riethmuller C, Jungmann P, Wegener J, Oberleithner H (2006) Bradykinin shifts endothelial fluid passage from para- to transcellular routes. Pflugers Arch - Eur J Physiol 453(2):157–165. doi:10.1007/s00424-006-0121-2

    CAS  Article  Google Scholar 

  84. 84.

    Rosen H, Stevens RC, Hanson M, Roberts E, Oldstone MB (2013) Sphingosine-1-phosphate and its receptors: structure, signaling, and influence. Annu Rev Biochem 82:637–662. doi:10.1146/annurev-biochem-062411-130916

    CAS  PubMed  Article  Google Scholar 

  85. 85.

    Schiller KR, Maniak PJ, O’Grady SM (2010) Cystic fibrosis transmembrane conductance regulator is involved in airway epithelial wound repair. Am J Physiol Cell Physiol 299(5):C912–C921. doi:10.1152/ajpcell.00215.2010

    PubMed Central  CAS  PubMed  Article  Google Scholar 

  86. 86.

    Schlegel N, Waschke J (2009) Impaired cAMP and Rac 1 signaling contribute to TNF-alpha-induced endothelial barrier breakdown in microvascular endothelium. Microcirculation 16(6):521–533. doi:10.1080/10739680902967427

    CAS  PubMed  Article  Google Scholar 

  87. 87.

    Schneider D, Tarantola M, Janshoff A (2011) Dynamics of TGF-beta induced epithelial-to-mesenchymal transition monitored by electric cell-substrate impedance sensing. Biochim Biophys Acta 1813(12):2099–2107. doi:10.1016/j.bbamcr.2011.07.016

    CAS  PubMed  Article  Google Scholar 

  88. 88.

    Schnoor M, Lai FP, Zarbock A, Klaver R, Polaschegg C, Schulte D, Weich HA, Oelkers JM, Rottner K, Vestweber D (2011) Cortactin deficiency is associated with reduced neutrophil recruitment but increased vascular permeability in vivo. J Experiment Med 208(8):1721–1735. doi:10.1084/jem.20101920

    CAS  Article  Google Scholar 

  89. 89.

    Seebach J, Dieterich P, Luo F, Schillers H, Vestweber D, Oberleithner H, Galla HJ, Schnittler HJ (2000) Endothelial barrier function under laminar fluid shear stress. Lab Investig J Tech Methods Pathol 80(12):1819–1831

    CAS  Article  Google Scholar 

  90. 90.

    Shinde AV, Motiani RK, Zhang X, Abdullaev IF, Adam AP, Gonzalez-Cobos JC, Zhang W, Matrougui K, Vincent PA, Trebak M (2013) STIM1 controls endothelial barrier function independently of Orai1 and Ca2+ entry. Sci Signal 6(267):ra18. doi:10.1126/scisignal.2003425

    PubMed Central  PubMed  Article  CAS  Google Scholar 

  91. 91.

    Shivanna M, Rajashekhar G, Srinivas SP (2010) Barrier dysfunction of the corneal endothelium in response to TNF-alpha: role of p38 MAP kinase. Invest Ophthalmol Vis Sci 51(3):1575–1582. doi:10.1167/iovs. 09-4343

    PubMed Central  PubMed  Article  Google Scholar 

  92. 92.

    Solly K, Wang X, Xu X, Strulovici B, Zheng W (2004) Application of real-time cell electronic sensing (RT-CES) technology to cell-based assays. Assay Drug Dev Technol 2(4):363–372. doi:10.1089/1540658041850544

    CAS  PubMed  Article  Google Scholar 

  93. 93.

    Spindler V, Peter D, Harms GS, Asan E, Waschke J (2011) Ultrastructural analysis reveals cAMP-dependent enhancement of microvascular endothelial barrier functions via Rac1-mediated reorganization of intercellular junctions. Am J Pathol 178(5):2424–2436. doi:10.1016/j.ajpath.2011.01.014

    PubMed Central  CAS  PubMed  Article  Google Scholar 

  94. 94.

    Stamatovic SM, Keep RF, Andjelkovic AV (2008) Brain endothelial cell-cell junctions: how to “open” the blood brain barrier. Curr Neuropharmacol 6(3):179–192. doi:10.2174/157015908785777210

    PubMed Central  CAS  PubMed  Article  Google Scholar 

  95. 95.

    Stolwijk JA, Hartmann C, Balani P, Albermann S, Keese CR, Giaever I, Wegener J (2011) Impedance analysis of adherent cells after in situ electroporation: non-invasive monitoring during intracellular manipulations. Biosens Bioelectron 26:4720–4727

    CAS  PubMed  Article  Google Scholar 

  96. 96.

    Sun M, Fu H, Cheng H, Cao Q, Zhao Y, Mou X, Zhang X, Liu X, Ke Y (2012) A dynamic real-time method for monitoring epithelial barrier function in vitro. Anal Biochem 425(2):96–103. doi:10.1016/j.ab.2012.03.010

    CAS  PubMed  Article  Google Scholar 

  97. 97.

    Szulcek R, Beckers CM, Hodzic J, de Wit J, Chen Z, Grob T, Musters RJ, Minshall RD, van Hinsbergh VW, van Nieuw Amerongen GP (2013) Localized RhoA GTPase activity regulates dynamics of endothelial monolayer integrity. Cardiovasc Res 99(3):471–482. doi:10.1093/cvr/cvt075

    PubMed Central  CAS  PubMed  Article  Google Scholar 

  98. 98.

    Szulcek R, Bogaard HJ, van Nieuw Amerongen GP (2014) Electric cell-substrate impedance sensing for the quantification of endothelial proliferation, barrier function, and motility. Journal of visualized experiments : JoVE (85). doi:10.3791/51300

  99. 99.

    Tang VW, Goodenough DA (2003) Paracellular ion channel at the tight junction. Biophys J 84(3):1660–1673. doi:10.1016/S0006-3495(03)74975-3

    PubMed Central  CAS  PubMed  Article  Google Scholar 

  100. 100.

    Tarantola M, Marel AK, Sunnick E, Adam H, Wegener J, Janshoff A (2010) Dynamics of human cancer cell lines monitored by electrical and acoustic fluctuation analysis. Integ Biol Quant Biosci Nano Macro 2(2–3):139–150. doi:10.1039/b920815a

    CAS  Google Scholar 

  101. 101.

    ten Klooster JP, Jaffer ZM, Chernoff J, Hordijk PL (2006) Targeting and activation of Rac1 are mediated by the exchange factor beta-Pix. J Cell Biol 172(5):759–769. doi:10.1083/jcb.200509096

    PubMed Central  PubMed  Article  CAS  Google Scholar 

  102. 102.

    Tiruppathi C, Malik AB, Del Vecchio PJ, Keese CR, Giaever I (1992) Electrical method for detection of endothelial cell shape change in real time: assessment of endothelial barrier function. Proc Natl Acad Sci U S A 89(17):7919–7923

    PubMed Central  CAS  PubMed  Article  Google Scholar 

  103. 103.

    Turner MR (1992) Flows of liquid and electrical current through monolayers of cultured bovine arterial endothelium. J Physiol 449:1–20

    PubMed Central  CAS  PubMed  Article  Google Scholar 

  104. 104.

    Utoguchi N, Ikeda K, Saeki K, Oka N, Mizuguchi H, Kubo K, Nakagawa S, Nayumi T (1995) Ascorbic acid stimulates barrier function of cultured endothelial cell monolayer. J Cell Physiol 163(2):393–399

    CAS  PubMed  Article  Google Scholar 

  105. 105.

    Van Itallie CM, Anderson JM (2004) The role of claudins in determining paracellular charge selectivity. Proc Am Thorac Soc 1(1):38–41. doi:10.1513/pats.2306013

    PubMed  Article  CAS  Google Scholar 

  106. 106.

    von Wedel-Parlow M, Schrot S, Lemmen J, Treeratanapiboon L, Wegener J, Galla HJ (2011) Neutrophils cross the BBB primarily on transcellular pathways: an in vitro study. Brain Res 1367:62–76. doi:10.1016/j.brainres.2010.09.076

    Article  CAS  Google Scholar 

  107. 107.

    von Wedel-Parlow M, Wolte P, Galla HJ (2009) Regulation of major efflux transporters under inflammatory conditions at the blood–brain barrier in vitro. J Neurochem 111(1):111–118. doi:10.1111/j.1471-4159.2009.06305.x

    Article  CAS  Google Scholar 

  108. 108.

    Wallez Y, Huber P (2008) Endothelial adherens and tight junctions in vascular homeostasis, inflammation and angiogenesis. Biochim Biophys Acta 1778(3):794–809. doi:10.1016/j.bbamem.2007.09.003

    CAS  PubMed  Article  Google Scholar 

  109. 109.

    Wang Y, Alexander JS (2011) Analysis of endothelial barrier function in vitro. Methods Mol Biol 763:253–264. doi:10.1007/978-1-61779-191-8_17

    CAS  PubMed  Article  Google Scholar 

  110. 110.

    Wang Z, Ginnan R, Abdullaev IF, Trebak M, Vincent PA, Singer HA (2010) Calcium/Calmodulin-dependent protein kinase II delta 6 (CaMKIIdelta6) and RhoA involvement in thrombin-induced endothelial barrier dysfunction. J Biol Chem 285(28):21303–21312. doi:10.1074/jbc.M110.120790

    PubMed Central  CAS  PubMed  Article  Google Scholar 

  111. 111.

    Wegener J, Abrams D, Willenbrink W, Galla HJ, Janshoff A (2004) Automated multi-well device to measure transepithelial electrical resistances under physiological conditions. BioTechniques 37 (4):590, 592–594, 596–597

  112. 112.

    Wegener J, Hakvoort A, Galla HJ (2000) Barrier function of porcine choroid plexus epithelial cells is modulated by cAMP-dependent pathways in vitro. Brain Res 853(1):115–124

    CAS  PubMed  Article  Google Scholar 

  113. 113.

    Wegener J, Keese CR, Giaever I (2000) Electric cell-substrate impedance sensing (ECIS) as a noninvasive means to monitor the kinetics of cell spreading to artificial surfaces. Exp Cell Res 259(1):158–166. doi:10.1006/excr.2000.4919

    CAS  PubMed  Article  Google Scholar 

  114. 114.

    Wegener J, Seebach J (2014) Experimental tools to monitor the dynamics of endothelial barrier function: a survey of in vitro approaches. Cell Tissue Res 355(3):485–514. doi:10.1007/s00441-014-1810-3

    CAS  PubMed  Article  Google Scholar 

  115. 115.

    Weidenfeller C, Schrot S, Zozulya A, Galla HJ (2005) Murine brain capillary endothelial cells exhibit improved barrier properties under the influence of hydrocortisone. Brain Res 1053(1–2):162–174. doi:10.1016/j.brainres.2005.06.049

    CAS  PubMed  Article  Google Scholar 

  116. 116.

    Wilhelm I, Fazakas C, Krizbai IA (2011) In vitro models of the blood–brain barrier. Acta Neurobiol Exp 71(1):113–128

    Google Scholar 

  117. 117.

    Wilkerson BA, Grass GD, Wing SB, Argraves WS, Argraves KM (2012) Sphingosine 1-phosphate (S1P) carrier-dependent regulation of endothelial barrier: high density lipoprotein (HDL)-S1P prolongs endothelial barrier enhancement as compared with albumin-S1P via effects on levels, trafficking, and signaling of S1P1. J Biol Chem 287(53):44645–44653. doi:10.1074/jbc.M112.423426

    PubMed Central  CAS  PubMed  Article  Google Scholar 

  118. 118.

    Will C, Fromm M, Muller D (2008) Claudin tight junction proteins: novel aspects in paracellular transport. Periton Dial Int J Int Soc Periton Dial 28(6):577–584

    CAS  Google Scholar 

  119. 119.

    Wolf P, Rothermel A, Beck-Sickinger AG, Robitzki AA (2008) Microelectrode chip based real time monitoring of vital MCF-7 mamma carcinoma cells by impedance spectroscopy. Biosens Bioelectron 24(2):253–259. doi:10.1016/j.bios.2008.03.040

    CAS  PubMed  Article  Google Scholar 

  120. 120.

    Xiao C, Lachance B, Sunahara G, Luong JH (2002) Assessment of cytotoxicity using electric cell-substrate impedance sensing: concentration and time response function approach. Anal Chem 74(22):5748–5753

    CAS  PubMed  Article  Google Scholar 

  121. 121.

    Xiao C, Luong JH (2003) On-line monitoring of cell growth and cytotoxicity using electric cell-substrate impedance sensing (ECIS). Biotechnol Prog 19(3):1000–1005. doi:10.1021/bp025733x

    CAS  PubMed  Article  Google Scholar 

  122. 122.

    Xu M, Waters CL, Hu C, Wysolmerski RB, Vincent PA, Minnear FL (2007) Sphingosine 1-phosphate rapidly increases endothelial barrier function independently of VE-cadherin but requires cell spreading and Rho kinase. Am J Physiol Cell Physiol 293(4):C1309–C1318. doi:10.1152/ajpcell.00014.2007

    CAS  PubMed  Article  Google Scholar 

  123. 123.

    Yin F, Watsky MA (2005) LPA and S1P increase corneal epithelial and endothelial cell transcellular resistance. Invest Ophthalmol Vis Sci 46(6):1927–1933. doi:10.1167/iovs. 04-1256

    CAS  PubMed  Article  Google Scholar 

  124. 124.

    Yoshida Y, Okano M, Wang S, Kobayashi M, Kawasumi M, Hagiwara H, Mitsumata M (1995) Hemodynamic-force-induced difference of interendothelial junctional complexes. Ann N Y Acad Sci 748:104–120, discussion 120–101

    CAS  PubMed  Article  Google Scholar 

  125. 125.

    Yuan SY, Rigor RR (2010) Methods for measuring permeability. In: Yuan SY, Rigor RR (eds) Regulation of endothelial barrier function. Morgan & Claypool Life Sciences; 2010., San Rafael (CA),

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Acknowledgments

This work was partially supported by a postdoctoral fellowship from Applied Biophysics Inc. to J.S. and by grants R01HL097111 and R01 HL123364 from the NIH and grant 14GRNT18880008 from the American Heart Association to M.T. and partially by grant R01HL095566 from the NIH to K.M.

Conflict of interest

Judith Stolwijk is partially supported by a postdoctoral fellowship from Applied Biophysics Inc.

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Correspondence to Mohamed Trebak.

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Stolwijk, J.A., Matrougui, K., Renken, C.W. et al. Impedance analysis of GPCR-mediated changes in endothelial barrier function: overview and fundamental considerations for stable and reproducible measurements. Pflugers Arch - Eur J Physiol 467, 2193–2218 (2015). https://doi.org/10.1007/s00424-014-1674-0

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Keywords

  • ECIS
  • Endothelial cells
  • Barrier function
  • Impedance
  • Resistance
  • TEER