Skip to main content
SpringerLink
Log in

Probing Endothelial Cell Mechanics through Connexin 43 Disruption

  • Published:
Experimental Mechanics Aims and scope Submit manuscript

Abstract

The endothelium has been established to generate intercellular stresses and suggested to transmit these intercellular stresses through cell-cell junctions, such as VE-Cadherin and ZO-1, for example. Although the previously mentioned molecules reflect the appreciable contributions both adherens junctions and tight junctions are believed to have in endothelial cell intercellular stresses, in doing so they also reveal the obscure relationship that exists between gap junctions and intercellular stresses. Therefore, to bring clarity to this relationship we disrupted expression of the endothelial gap junction connexin 43 (Cx43) by exposing confluent human umbilical vein endothelial cells (HUVECs) to a low (0.2 μg/mL) and high (2 μg/mL) concentration of 2,5-dihydroxychalcone (chalcone), a known Cx43 inhibitor. To evaluate the impact Cx43 disruption had on endothelial cell mechanics we utilized traction force microscopy and monolayer stress microscopy to measure cell-substrate tractions and cell-cell intercellular stresses, respectively. HUVEC monolayers exposed to a low concentration of chalcone produced average normal intercellular stresses that were on average 17% higher relative to control, while exposure to a high concentration of chalcone yielded average normal intercellular stresses that were on average 55% lower when compared to control HUVEC monolayers. HUVEC maximum shear intercellular stresses were observed to decrease by 16% (low chalcone concentration) and 66% (high chalcone concentration), while tractions exhibited an almost 2-fold decrease under high chalcone concentration. In addition, monolayer cell velocities were observed to decrease by 19% and 35% at low chalcone and high chalcone concentrations, respectively. Strain energies were also observed to decrease by 32% and 85% at low and high concentration of chalcone treatment, respectively, when compared to control. The findings we present here reveal for the first time the contribution Cx43 has to endothelial biomechanics.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5
Fig. 6
Fig. 7
Fig. 8
Fig. 9

Similar content being viewed by others

References

  1. Al-Soudi A, Kaaij MH, Tas SW (2017) Endothelial cells: from innocent bystanders to active participants in immune responses. Autoimmun Rev 16(9):951–962. https://doi.org/10.1016/j.autrev.2017.07.008

    Article  Google Scholar 

  2. Rajendran P, Rengarajan T, Thangavel J, Nishigaki Y, Sakthisekaran D, Sethi G, Nishigaki I (2013) The vascular endothelium and human diseases. Int J Biol Sci 9(10):1057–1069. https://doi.org/10.7150/ijbs.7502

    Article  Google Scholar 

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

    Article  Google Scholar 

  4. Widlansky ME, Gokce N, Keaney JF Jr, Vita JA (2003) The clinical implications of endothelial dysfunction. J Am Coll Cardiol 42(7):1149–1160

    Article  Google Scholar 

  5. Hadi HA, Carr CS, Al Suwaidi J (2005) Endothelial dysfunction: cardiovascular risk factors, therapy, and outcome. Vasc Health Risk Manag 1(3):183–198

    Google Scholar 

  6. Islam MM, Beverung S, Steward R Jr (2017) Bio-inspired microdevices that mimic the human vasculature. Micromachines 8(10). https://doi.org/10.3390/mi8100299

  7. Lu D, Kassab GS (2011) Role of shear stress and stretch in vascular mechanobiology. J R Soc Interface 8(63):1379–1385. https://doi.org/10.1098/rsif.2011.0177

    Article  Google Scholar 

  8. Warren KM, Islam MM, LeDuc PR, Steward R (2016) 2D and 3D Mechanobiology in human and nonhuman systems. ACS Appl Mater Interfaces 8(34):21869–21882. https://doi.org/10.1021/acsami.5b12064

    Article  Google Scholar 

  9. Steward R, Tambe D, Hardin CC, Krishnan R, Fredberg JJ (2015) Fluid shear, intercellular stress, and endothelial cell alignment. Am J Physiol Cell Physiol 308(8):C657–C664. https://doi.org/10.1152/ajpcell.00363.2014

    Article  Google Scholar 

  10. Steward RL Jr, Cheng CM, Wang DL, LeDuc PR (2010) Probing cell structure responses through a shear and stretching mechanical stimulation technique. Cell Biochem Biophys 56(2–3):115–124. https://doi.org/10.1007/s12013-009-9075-2

    Article  Google Scholar 

  11. Steward RL Jr, Cheng CM, Ye JD, Bellin RM, LeDuc PR (2011) Mechanical stretch and shear flow induced reorganization and recruitment of fibronectin in fibroblasts. Sci Rep 1:147. https://doi.org/10.1038/srep00147

    Article  Google Scholar 

  12. Steward RL Jr, Tan C, Cheng CM, LeDuc PR (2015) Cellular force signal integration through vector logic gates. J Biomech 48(4):613–620. https://doi.org/10.1016/j.jbiomech.2014.12.047

    Article  Google Scholar 

  13. Ashina K, Tsubosaka Y, Nakamura T, Omori K, Kobayashi K, Hori M, Ozaki H, Murata T (2015) Histamine induces vascular hyperpermeability by increasing blood flow and endothelial barrier disruption in vivo. PLoS One 10(7):e0132367. https://doi.org/10.1371/journal.pone.0132367

    Article  Google Scholar 

  14. Lum H, Malik AB (1996) Mechanisms of increased endothelial permeability. Can J Physiol Pharmacol 74(7):787–800

    Google Scholar 

  15. Rabiet MJ, Plantier JL, Rival Y, Genoux Y, Lampugnani MG, Dejana E (1996) Thrombin-induced increase in endothelial permeability is associated with changes in cell-to-cell junction organization. Arterioscler Thromb Vasc Biol 16(3):488–496

    Article  Google Scholar 

  16. van Hinsbergh VW, Koolwijk P (2008) Endothelial sprouting and angiogenesis: matrix metalloproteinases in the lead. Cardiovasc Res 78(2):203–212. https://doi.org/10.1093/cvr/cvm102

    Article  Google Scholar 

  17. Chiquet M (1999) Regulation of extracellular matrix gene expression by mechanical stress. Matrix Biol 18(5):417–426

    Article  Google Scholar 

  18. Hosseini Y, Agah M, Verbridge SS (2015) Endothelial cell sensing, restructuring, and invasion in collagen hydrogel structures. Integr Biol (Camb) 7(11):1432–1441. https://doi.org/10.1039/c5ib00207a

    Article  Google Scholar 

  19. Sieminski AL, Hebbel RP, Gooch KJ (2004) The relative magnitudes of endothelial force generation and matrix stiffness modulate capillary morphogenesis in vitro. Exp Cell Res 297(2):574–584. https://doi.org/10.1016/j.yexcr.2004.03.035

    Article  Google Scholar 

  20. Fournier MF, Sauser R, Ambrosi D, Meister JJ, Verkhovsky AB (2010) Force transmission in migrating cells. J Cell Biol 188(2):287–297. https://doi.org/10.1083/jcb.200906139

    Article  Google Scholar 

  21. Lange JR, Fabry B (2013) Cell and tissue mechanics in cell migration. Exp Cell Res 319(16):2418–2423. https://doi.org/10.1016/j.yexcr.2013.04.023

    Article  Google Scholar 

  22. Trepat X, Wasserman MR, Angelini TE, Millet E, Weitz DA, Butler JP, Fredberg JJ (2009) Physical forces during collective cell migration. Nat Phys 5:426. https://doi.org/10.1038/nphys1269 https://www.nature.com/articles/nphys1269#supplementary-information

    Article  Google Scholar 

  23. De Pascalis C, Etienne-Manneville S (2017) Single and collective cell migration: the mechanics of adhesions. Mol Biol Cell 28(14):1833–1846. https://doi.org/10.1091/mbc.E17-03-0134

    Article  Google Scholar 

  24. Gov N (2011) Cell mechanics: moving under peer pressure. Nat Mater 10(6):412–414. https://doi.org/10.1038/nmat3036

    Article  Google Scholar 

  25. Perrault CM, Brugues A, Bazellieres E, Ricco P, Lacroix D, Trepat X (2015) Traction forces of endothelial cells under slow shear flow. Biophys J 109(8):1533–1536. https://doi.org/10.1016/j.bpj.2015.08.036

    Article  Google Scholar 

  26. Tambe DT, Hardin CC, Angelini TE, Rajendran K, Park CY, Serra-Picamal X, Zhou EH, Zaman MH, Butler JP, Weitz DA, Fredberg JJ, Trepat X (2011) Collective cell guidance by cooperative intercellular forces. Nat Mater 10(6):469–475. https://doi.org/10.1038/nmat3025

    Article  Google Scholar 

  27. Hardin C, Rajendran K, Manomohan G, Tambe DT, Butler JP, Fredberg JJ, Martinelli R, Carman CV, Krishnan R (2013) Glassy dynamics, cell mechanics, and endothelial permeability. J Phys Chem B 117(42):12850–12856. https://doi.org/10.1021/jp4020965

    Article  Google Scholar 

  28. Tambe DT, Croutelle U, Trepat X, Park CY, Kim JH, Millet E, Butler JP, Fredberg JJ (2013) Monolayer stress microscopy: limitations, artifacts, and accuracy of recovered intercellular stresses. PLoS One 8(2):e55172. https://doi.org/10.1371/journal.pone.0055172

    Article  Google Scholar 

  29. Butler JP, Tolić-Nørrelykke IM, Fabry B, Fredberg JJ (2002) Traction fields, moments, and strain energy that cells exert on their surroundings. Am J Phys Cell Phys 282(3):C595–C605. https://doi.org/10.1152/ajpcell.00270.2001

    Article  Google Scholar 

  30. Cho Y, Young Park E, Ko E, Park J-S, Shin J (2016) Recent advances in biological uses of traction force microscopy. 17. https://doi.org/10.1007/s12541-016-0166-x

  31. Hardin CC, Chattoraj J, Manomohan G, Colombo J, Nguyen T, Tambe D, Fredberg JJ, Birukov K, Butler JP, Del Gado E, Krishnan R (2018) Long-range stress transmission guides endothelial gap formation. Biochem Biophys Res Commun 495(1):749–754. https://doi.org/10.1016/j.bbrc.2017.11.066

    Article  Google Scholar 

  32. Elineni KK, Gallant ND (2011) Regulation of cell adhesion strength by peripheral focal adhesion distribution. Biophys J 101(12):2903–2911. https://doi.org/10.1016/j.bpj.2011.11.013

    Article  Google Scholar 

  33. Mierke CT, Fischer T, Puder S, Kunschmann T, Soetje B, Ziegler WH (2017) Focal adhesion kinase activity is required for actomyosin contractility-based invasion of cells into dense 3D matrices. Sci Rep 7:42780. https://doi.org/10.1038/srep42780

    Article  Google Scholar 

  34. Bazzoni G, Dejana E (2004) Endothelial cell-to-cell junctions: molecular organization and role in vascular homeostasis. Physiol Rev 84(3):869–901. https://doi.org/10.1152/physrev.00035.2003

    Article  Google Scholar 

  35. Gulino-Debrac D (2013) Mechanotransduction at the basis of endothelial barrier function. Tissue Barriers 1(2):e24180. https://doi.org/10.4161/tisb.24180

    Article  Google Scholar 

  36. Tarbell JM (2010) Shear stress and the endothelial transport barrier. Cardiovasc Res 87(2):320–330. https://doi.org/10.1093/cvr/cvq146

    Article  Google Scholar 

  37. le Duc Q, Shi Q, Blonk I, Sonnenberg A, Wang N, Leckband D, de Rooij J (2010) Vinculin potentiates E-cadherin mechanosensing and is recruited to actin-anchored sites within adherens junctions in a myosin II–dependent manner. J Cell Biol 189(7):1107–1115

    Article  Google Scholar 

  38. DeMaio L, Chang YS, Gardner TW, Tarbell JM, Antonetti DA (2001) Shear stress regulates occludin content and phosphorylation. Am J Physiol Heart Circ Physiol 281(1):H105–H113. https://doi.org/10.1152/ajpheart.2001.281.1.H105

    Article  Google Scholar 

  39. Liu Z, Tan JL, Cohen DM, Yang MT, Sniadecki NJ, Ruiz SA, Nelson CM, Chen CS (2010) Mechanical tugging force regulates the size of cell-cell junctions. Proc Natl Acad Sci U S A 107(22):9944–9949. https://doi.org/10.1073/pnas.0914547107

    Article  Google Scholar 

  40. Ng MR, Besser A, Brugge JS, Danuser G (2014) Mapping the dynamics of force transduction at cell-cell junctions of epithelial clusters. Elife 3:e03282. https://doi.org/10.7554/eLife.03282

    Article  Google Scholar 

  41. Figueroa XF, Duling BR (2009) Gap junctions in the control of vascular function. Antioxid Redox Signal 11(2):251–266. https://doi.org/10.1089/ars.2008.2117

    Article  Google Scholar 

  42. Nielsen MS, Axelsen LN, Sorgen PL, Verma V, Delmar M, Holstein-Rathlou NH (2012) Gap junctions. Compr Physiol 2(3):1981–2035. https://doi.org/10.1002/cphy.c110051

    Article  Google Scholar 

  43. Sohl G, Willecke K (2004) Gap junctions and the connexin protein family. Cardiovasc Res 62(2):228–232. https://doi.org/10.1016/j.cardiores.2003.11.013

    Article  Google Scholar 

  44. Haefliger JA, Nicod P, Meda P (2004) Contribution of connexins to the function of the vascular wall. Cardiovasc Res 62(2):345–356. https://doi.org/10.1016/j.cardiores.2003.11.015

    Article  Google Scholar 

  45. Marquez-Rosado L, Solan JL, Dunn CA, Norris RP, Lampe PD (2012) Connexin43 phosphorylation in brain, cardiac, endothelial and epithelial tissues. Biochim Biophys Acta 1818(8):1985–1992. https://doi.org/10.1016/j.bbamem.2011.07.028

    Article  Google Scholar 

  46. de Wit C, Roos F, Bolz SS, Pohl U (2003) Lack of vascular connexin 40 is associated with hypertension and irregular arteriolar vasomotion. Physiol Genomics 13(2):169–177. https://doi.org/10.1152/physiolgenomics.00169.2002

    Article  Google Scholar 

  47. Simon AM, McWhorter AR (2002) Vascular abnormalities in mice lacking the endothelial gap junction proteins connexin37 and connexin40. Dev Biol 251(2):206–220

    Article  Google Scholar 

  48. Liao Y, Day KH, Damon DN, Duling BR (2001) Endothelial cell-specific knockout of connexin 43 causes hypotension and bradycardia in mice. Proc Natl Acad Sci U S A 98(17):9989–9994. https://doi.org/10.1073/pnas.171305298

    Article  Google Scholar 

  49. Walker DL, Vacha SJ, Kirby ML, Lo CW (2005) Connexin43 deficiency causes dysregulation of coronary vasculogenesis. Dev Biol 284(2):479–498. https://doi.org/10.1016/j.ydbio.2005.06.004

    Article  Google Scholar 

  50. Kwak BR, Pepper MS, Gros DB, Meda P (2001) Inhibition of endothelial wound repair by dominant negative connexin inhibitors. Mol Biol Cell 12(4):831–845

    Article  Google Scholar 

  51. Yuan D, Sun G, Zhang R, Luo C, Ge M, Luo G, Hei Z (2015) Connexin 43 expressed in endothelial cells modulates monocyteendothelial adhesion by regulating cell adhesion proteins. Mol Med Rep 12(5):7146–7152. https://doi.org/10.3892/mmr.2015.4273

    Article  Google Scholar 

  52. Lee Y-N, Yeh H-I, Tian T-Y, Lu W-W, Ko Y-S, Tsai C-H (2002) 2′,5′-Dihydroxychalcone down-regulates endothelial connexin43 gap junctions and affects MAP kinase activation. Toxicology 179(1):51–60. https://doi.org/10.1016/S0300-483X(02)00289-5

    Article  Google Scholar 

  53. Hsieh HK, Lee TH, Wang JP, Wang JJ, Lin CN (1998) Synthesis and anti-inflammatory effect of chalcones and related compounds. Pharm Res 15(1):39–46

    Article  Google Scholar 

  54. Lin CN, Lee TH, Hsu MF, Wang JP, Ko FN, Teng CM (1997) 2′,5′-Dihydroxychalcone as a potent chemical mediator and cyclooxygenase inhibitor. J Pharm Pharmacol 49(5):530–536

    Article  Google Scholar 

  55. Stroka KM, Aranda-Espinoza H (2011) Endothelial cell substrate stiffness influences neutrophil transmigration via myosin light chain kinase-dependent cell contraction. Blood 118(6):1632–1640. https://doi.org/10.1182/blood-2010-11-321125

    Article  Google Scholar 

  56. Pepper MS, Montesano R, el Aoumari A, Gros D, Orci L, Meda P (1992) Coupling and connexin 43 expression in microvascular and large vessel endothelial cells. Am J Phys Cell Phys 262(5):C1246–C1257. https://doi.org/10.1152/ajpcell.1992.262.5.C1246

    Article  Google Scholar 

  57. Nagasawa K, Chiba H, Fujita H, Kojima T, Saito T, Endo T, Sawada N (2006) Possible involvement of gap junctions in the barrier function of tight junctions of brain and lung endothelial cells. J Cell Physiol 208(1):123–132. https://doi.org/10.1002/jcp.20647

    Article  Google Scholar 

Download references

Acknowledgements

This work was supported by the University of Central Florida start-up funds and the National Heart, Lung, And Blood Institute of the National Institute of Health under award K25HL132098.

Author information

Authors and Affiliations

  1. Department of Mechanical and Aerospace Engineering, University of Central Florida, Orlando, FL, USA

    M. M. Islam & R. L. Steward Jr

  2. Burnett School of Biomedical Sciences, College of Medicine, University of Central Florida, Orlando, FL, USA

    R. L. Steward Jr

Authors
  1. M. M. Islam
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. R. L. Steward Jr
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to R. L. Steward Jr.

Electronic supplementary material

Supplementary figure 1

3D rendering of average normal intercellular stress (Pa) distribution of HUVEC monolayers. Figure labels are as follows—average normal intercellular stresses of control (a, b and c), 0.2 μg/mL chalcone treatment conditions (d, e and f) and 2 μg/mL chalcone treatment condition (g, h and i) are shown at before chalcone treatment (at 30 min, labels a, d and g), after chalcone treatment (at 2 h, labels b, e and h) and at the end of experiment (at 6 h, labels c, f and i). (PPTX 351 kb)

Supplementary figure 2

3D rendering of maximum shear intercellular stress (Pa) distribution of HUVEC monolayer. Figure labels are as follows—maximum shear intercellular stresses of control (a, b and c), 0.2 μg/mL chalcone treatment conditions (d, e and f) and 2 μg/mL chalcone treatment condition (g, h and i) are shown at before chalcone treatment (at 30 min, labels a, d and g), after chalcone treatment (at 2 h, labels b, e and h) and at the end of experiment (at 6 h, labels c, f and i). (PPTX 330 kb)

Supplementary figure 3

DAPI (blue), Tight junction (ZO-1, green) and Adherens junction (VE-Cadherin, red) staining of HUVEC monolayers at control (a, b and c, respectively) and at 2 μg/mL chalcone treatment conditions (d, e and f, respectively) after 6 h of experiment. Scale bar 200 × 200 μm. (PPTX 458 kb)

Supplementary figure 4

Cx40 (green) and Cx37 (red) staining of HUVEC monolayers at control (b and c, respectively) and 2 μg/mL chalcone treatment conditions (e and f, respectively) after 6 h of experiment. Scale bar 200 × 200 μm. (PPTX 533 kb)

Supplementary figure 5

F-actin staining of HUVEC monolayers at control (a) and 6 h of chalcone treatment at 0.2 μg/mL (b) and 2 μg/mL (c). Scale bar 200 × 200 μm. (PPTX 316 kb)

Supplementary figure 6

Measurement of polyacrylamide gel height using fluorescence microscopy. A 3D volume rendering of our polyacrylamide gel was reconstructed from a series of z-stack images. Gel height was found ~100 μm. (PPTX 345 kb)

Supplementary figure 7

Cell velocity measurements. Displacements (μm) were computed from consecutive phase contrast images and subsequently converted to velocities (μm/min). (PPTX 609 kb)

Supplementary figure 8

Maximum and Minimum principal stresses (σmax & σmin) distribution of HUVEC monolayers after an hour of chalcone treatment at control (figure a & d), 0.2 μg/mL chalcone treatment condition (figure b& e) and 2 μg/mL chalcone treatment condition (figure c & f), respectively. Bar 500 × 500 μm. (PPTX 542 kb)

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Islam, M.M., Steward, R.L. Probing Endothelial Cell Mechanics through Connexin 43 Disruption. Exp Mech 59, 327–336 (2019). https://doi.org/10.1007/s11340-018-00445-4

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s11340-018-00445-4

Keywords

Search

Navigation