Skip to main content

Quantitative Phenotyping of Cell–Cell Junctions to Evaluate ZO-1 Presentation in Brain Endothelial Cells

Annals of Biomedical Engineering volume 47, pages 1675–1687 (2019)Cite this article

Abstract

The selective permeability of the blood–brain barrier (BBB) is controlled by tight junction-expressing brain endothelial cells. The integrity of these junctional proteins, which anchor to actin via zonula occludens (e.g., ZO-1), plays a vital role in barrier function. While disrupted junctions are linked with several neurodegenerative diseases, the mechanisms underlying disruption are not fully understood. This is largely due to the lack of appropriate models and efficient techniques to quantify edge-localized protein. Here, we developed a novel junction analyzer program (JAnaP) to semi-automate the quantification of junctional protein presentation. Because significant evidence suggests a link between myosin-II mediated contractility and endothelial barrier properties, we used the JAnaP to investigate how biochemical and physical cues associated with altered contractility influence ZO-1 presentation in brain endothelial cells. Treatment with contractility-decreasing agents increased continuous ZO-1 presentation; however, this increase was greatest on soft gels of brain-relevant stiffness, suggesting improved barrier maturation. This effect was reversed by biochemically inhibiting protein phosphatases to increase cell contractility on soft substrates. These results promote the use of brain-mimetic substrate stiffness in BBB model design and motivates the use of this novel JAnaP to provide insight into the role of junctional protein presentation in BBB physiology and pathologies.

This is a preview of subscription content, access via your institution.

Access options

Buy single article

Instant access to the full article PDF.

USD 39.95

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

Figure 1
Figure 2
Figure 3
Figure 4
Figure 5

References

  1. Abbott, N. J., and A. Friedman. Overview and introduction: the blood-brain barrier in health and disease. Epilepsia 53(Suppl 6):1–6, 2012.

    Article  PubMed  PubMed Central  Google Scholar 

  2. Abdullahi, W., D. Tripathi, and P. T. Ronaldson. Blood-brain barrier dysfunction in ischemic stroke: targeting tight junctions and transporters for vascular protection. Am. J. Physiol. Physiol. 315:C343–C356, 2018.

    Article  CAS  Google Scholar 

  3. Adamson, R. H., B. Liu, G. N. Fry, L. L. Rubin, and F. E. Curry. Microvascular permeability and number of tight junctions are modulated by cAMP. Am. Physiol. Soc. 274:H1885–H1894, 1998.

    CAS  Google Scholar 

  4. Andrews, A. M., E. M. Lutton, S. F. Merkel, R. Razmpour, and S. H. Ramirez. Mechanical injury induces brain endothelial-derived microvesicle release: implications for cerebral vascular injury during traumatic brain injury. Front. Cell. Neurosci. 10:43, 2016.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  5. Beese, M., K. Wyss, M. Haubitz, and T. Kirsch. Effect of cAMP derivates on assembly and maintenance of tight junctions in human umbilical vein endothelial cells. BMC Cell Biol. 11:68, 2010.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  6. Birukova, A. A., X. Tian, I. Cokic, Y. Beckham, M. L. Gardel, and K. G. Birukov. Endothelial barrier disruption and recovery is controlled by substrate stiffness. Microvasc. Res. 87:50–57, 2013.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  7. Boutouyrie, P., A. I. Tropeano, R. Asmar, I. Gautier, A. Benetos, P. Lacolley, and S. Laurent. Aortic stiffness is an independent predictor of primary coronary events in hypertensive patients: a longitudinal study. Hypertens. (Dallas, Tex. 1979) 39:10–15, 2002.

    Article  CAS  Google Scholar 

  8. Byfield, F. J., R. K. Reen, T. P. Shentu, I. Levitan, and K. J. Gooch. Endothelial actin and cell stiffness is modulated by substrate stiffness in 2D and 3D. J. Biomech. 42:1114–1119, 2009.

    Article  PubMed  PubMed Central  Google Scholar 

  9. Chen, Y., F. Shen, J. Liu, and G.-Y. Yang. Arterial stiffness and stroke: de-stiffening strategy, a therapeutic target for stroke. BMJ 2:65–72, 2017.

    Article  Google Scholar 

  10. Cheney, R. E. Myosins in cell junctions. Bioarchitecture 2:158–170, 2012.

    Article  PubMed  PubMed Central  Google Scholar 

  11. Cho, Y.-E., D.-S. Ahn, K. G. Morgan, and Y.-H. Lee. Enhanced contractility and myosin phosphorylation induced by Ca 21-independent MLCK activity in hypertensive rats. Cardiovasc. Res. 91:162–170, 2011.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  12. Dejana, E., F. Orsenigo, and M. G. Lampugnani. The role of adherens junctions and VE-cadherin in the control of vascular permeability. J. Cell Sci. 121:2115, 2008.

    Article  CAS  PubMed  Google Scholar 

  13. Dorland, Y. L., and S. Huveneers. Cell–cell junctional mechanotransduction in endothelial remodeling. Cell. Mol. Life Sci. 74:279–292, 2017.

    Article  CAS  PubMed  Google Scholar 

  14. Eigenmann, D. E., G. Xue, K. S. Kim, A. V. Moses, M. Hamburger, and M. Oufir. Comparative study of four immortalized human brain capillary endothelial cell lines, hCMEC/D3, hBMEC, TY10, and BB19, and optimization of culture conditions, for an in vitro blood-brain barrier model for drug permeability studies. Fluids Barriers CNS 10:33, 2013.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  15. Escribano, J., M. B. Chen, E. Moeendarbary, X. Cao, V. Shenoy, J. Manuel Garcia-Aznar, R. D. Kamm, and F. Spill. Balance of mechanical forces drives endothelial gap formation and may facilitate cancer and immune-cell extravasation. 2018. https://doi.org/10.1101/375931

  16. Essler, M., J. M. Staddon, P. C. Weber, and M. Aepfelbacher. Cyclic AMP blocks bacterial lipopolysaccharide-induced myosin light chain phosphorylation in endothelial cells through inhibition of rho/rho kinase signaling. J. Immunol. 164:6543–6549, 2000.

    Article  CAS  PubMed  Google Scholar 

  17. Fanning, A. S., B. J. Jameson, L. A. Jesaitis, and J. M. Anderson. The tight junction protein ZO-1 establishes a link between the transmembrane protein occludin and the actin cytoskeleton. J. Biol. Chem. 273:29745–29753, 1998.

    Article  CAS  PubMed  Google Scholar 

  18. Goutal, S., M. Gerstenmayer, S. Auvity, F. Caillé, S. Mériaux, I. Buvat, B. Larrat, and N. Tournier. Physical blood–brain barrier disruption induced by focused ultrasound does not overcome the transporter-mediated efflux of erlotinib. J. Control. Release 292:210–220, 2018.

    Article  CAS  PubMed  Google Scholar 

  19. Grammas, P., J. Martinez, and B. Miller. Cerebral microvascular endothelium and the pathogenesis of neurodegenerative diseases. Expert Rev. Mol. Med. 13:e19, 2011.

    Article  CAS  PubMed  Google Scholar 

  20. Hamilla, S. M., K. M. Stroka, and H. Aranda-Espinoza. VE-cadherin-independent cancer cell incorporation into the vascular endothelium precedes transmigration. PLoS ONE 9:e109748, 2014.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  21. Hemphill, M. A., S. Dauth, C. J. Yu, B. E. Dabiri, and K. K. Parker. Traumatic brain injury and the neuronal microenvironment: a potential role for neuropathological mechanotransduction. Neuron 85:1177–1192, 2015.

    Article  CAS  PubMed  Google Scholar 

  22. Hsu, J., D. Serrano, T. Bhowmick, K. Kumar, Y. Shen, Y. C. Kuo, C. Garnacho, and S. Muro. Enhanced endothelial delivery and biochemical effects of α-galactosidase by ICAM-1-targeted nanocarriers for Fabry disease. J. Control. Release 149:323–331, 2011.

    Article  CAS  PubMed  Google Scholar 

  23. Huveneers, S., J. Oldenburg, E. Spanjaard, G. van der Krogt, I. Grigoriev, A. Akhmanova, H. Rehmann, and J. de Rooij. Vinculin associates with endothelial VE-cadherin junctions to control force-dependent remodeling. J. Cell Biol. 196:641–652, 2012.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  24. Kakei, Y., M. Akashi, T. Shigeta, T. Hasegawa, and T. Komori. Alteration of cell–cell junctions in cultured human lymphatic endothelial cells with inflammatory cytokine stimulation. Lymphat. Res. Biol. 12:136–143, 2014.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  25. Katt, M. E., R. M. Linville, L. N. Mayo, Z. S. Xu, and P. C. Searson. Functional brain-specific microvessels from iPSC-derived human brain microvascular endothelial cells: the role of matrix composition on monolayer formation. Fluids Barriers CNS 15:7, 2018.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  26. Klein, E. A., L. Yin, D. Kothapalli, P. Castagnino, F. J. Byfield, T. Xu, I. Levental, E. Hawthorne, P. A. Janmey, and R. K. Assoian. Cell-cycle control by physiological matrix elasticity and in vivo tissue stiffening. Curr. Biol. 19:1511–1518, 2009.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  27. Kohn, J. C., M. C. Lampi, and C. A. Reinhart-King. Age-related vascular stiffening: causes and consequences. Front. Genet. 6:112, 2015.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  28. Kothapalli, D., S.-L. Liu, Y. H. Bae, J. Monslow, T. Xu, E. A. Hawthorne, F. J. Byfield, P. Castagnino, S. Rao, D. J. Rader, E. Puré, M. C. Phillips, S. Lund-Katz, P. A. Janmey, and R. K. Assoian. Cardiovascular protection by ApoE and ApoE-HDL linked to suppression of ECM gene expression and arterial stiffening. Cell Rep. 2:1259–1271, 2012.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  29. Krishnan, R., D. D. Klumpers, C. Y. Park, K. Rajendran, X. Trepat, J. van Bezu, V. W. M. M. van Hinsbergh, C. V. Carman, J. D. Brain, J. J. Fredberg, J. P. Butler, and G. P. van Nieuw Amerongen. Substrate stiffening promotes endothelial monolayer disruption through enhanced physical forces. Am. J. Physiol. Cell Physiol. 300:146–154, 2011.

    Article  CAS  Google Scholar 

  30. Li, A. Q., L. Zhao, T. F. Zhou, M. Q. Zhang, and X. M. Qin. Exendin-4 promotes endothelial barrier enhancement via PKA-and Epac1-dependent Rac1 activation. Am. J. Physiol. Physiol. 308:C164–C175, 2015.

    Article  CAS  Google Scholar 

  31. Li, B., W.-D. Zhao, Z.-M. Tan, W.-G. Fang, L. Zhu, and Y.-H. Chen. Involvement of Rho/ROCK signalling in small cell lung cancer migration through human brain microvascular endothelial cells. FEBS Lett. 580:4252–4260, 2006.

    Article  CAS  PubMed  Google Scholar 

  32. Li, C.-H., M.-K. Shyu, C. Jhan, Y.-W. Cheng, C.-H. Tsai, C.-W. Liu, C.-C. Lee, R.-M. Chen, and J.-J. Kang. Gold nanoparticles increase endothelial paracellular permeability by altering components of endothelial tight junctions, and increase blood-brain barrier permeability in mice. Toxicol. Sci. 148:192–203, 2015.

    Article  CAS  PubMed  Google Scholar 

  33. Mckee, C. T., J. A. Last, P. Russell, and C. J. Murphy. Indentation vs. tensile measurements of young’s modulus for soft biological tissues. Tissue Eng. Part B 17:155–164, 2011.

    Article  Google Scholar 

  34. McRae, M. P., L. M. LaFratta, B. M. Nguyen, J. J. Paris, K. F. Hauser, and D. E. Conway. Characterization of cell-cell junction changes associated with the formation of a strong endothelial barrier. Tissue Barriers 6:1–9, 2018.

    Article  CAS  Google Scholar 

  35. Mesiwala, A. H., L. Farrell, H. J. Wenzel, D. L. Silbergeld, L. A. Crum, H. R. Winn, and P. D. Mourad. High-intensity focused ultrasound selectively disrupts the blood-brain barrier in vivo. Ultrasound Med. Biol. 28:389–400, 2002.

    Article  PubMed  Google Scholar 

  36. Mierke, C. T., N. Bretz, and P. Altevogt. Contractile forces contribute to increased glycosylphosphatidylinositol-anchored receptor CD24-facilitated cancer cell invasion. J. Biol. Chem. 286:34858–34871, 2011.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  37. Newell-Litwa, K. A., R. Horwitz, and M. L. Lamers. Non-muscle myosin II in disease: mechanisms and therapeutic opportunities. Dis. Model. Mech. 8:1495–1515, 2015.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  38. Nieuw Amerongen, V. G. P., C. M. L. Beckers, I. D. Achekar, S. Zeeman, R. J. P. Musters, and V. W. M. Van Hinsbergh. Involvement of Rho kinase in endothelial barrier maintenance. Arterioscler. Thromb. Vasc. Biol. 27:2332–2339, 2007.

    Article  CAS  PubMed  Google Scholar 

  39. Nitz, T., T. Eisenblatter, K. Psathaki, and H.-J. Gaï. Serum-derived factors weaken the barrier properties of cultured porcine brain capillary endothelial cells in vitro. Brain Res. 981:30–40, 2003.

    Article  CAS  PubMed  Google Scholar 

  40. Onken, M. D., O. L. Mooren, S. Mukherjee, S. T. Shahan, J. Li, and J. A. Cooper. Endothelial monolayers and transendothelial migration depend on mechanical properties of the substrate. Cytoskeleton 71:695–706, 2014.

    Article  CAS  PubMed  Google Scholar 

  41. Peloquin, J., J. Huynh, R. M. Williams, and C. A. Reinhart-King. Indentation measurements of the subendothelial matrix in bovine carotid arteries. J. Biomech. 44:815–821, 2011.

    Article  PubMed  Google Scholar 

  42. Raman, P. S., C. D. Paul, K. M. Stroka, and K. Konstantopoulos. Probing cell traction forces in confined microenvironments. Lab Chip 13:4599–4607, 2013.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  43. Semyachkina-Glushkovskaya, O., J. Kurths, E. Borisova, S. Sokolovski, V. Mantareva, I. Angelov, A. Shirokov, N. Navolokin, N. Shushunova, A. Khorovodov, M. Ulanova, M. Sagatova, I. Agranivich, O. Sindeeva, A. Gekalyuk, A. Bodrova, and E. Rafailov. Photodynamic opening of blood-brain barrier. Biomed. Opt. Express 8:5040–5048, 2017.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  44. Sheikov, N., N. McDannold, N. Vykhodtseva, F. Jolesz, and K. Hynynen. Cellular mechanisms of the blood-brain barrier opening induced by ultrasound in presence of microbubbles. Ultrasound Med. Biol. 30:979–989, 2004.

    Article  PubMed  Google Scholar 

  45. Stroka, K. M., and H. Aranda-espinoza. Neutrophils display biphasic relationship between migration and substrate stiffness. Cell Motil. Cytoskeleton 66:328–341, 2009.

    Article  CAS  PubMed  Google Scholar 

  46. Stroka, K. M., and H. Aranda-Espinoza. Endothelial cell substrate stiffness influences neutrophil transmigration via myosin light chain kinase-dependent cell contraction. Blood 118:1632–1640, 2011.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  47. Stroka, K. M., I. Levitan, and H. Aranda-Espinoza. OxLDL and substrate stiffness promote neutrophil transmigration by enhanced endothelial cell contractility and ICAM-1. J. Biomech. 45:1828–1834, 2012.

    Article  PubMed  PubMed Central  Google Scholar 

  48. Tornavaca, O., M. Chia, N. Dufton, L. O. Almagro, D. E. Conway, A. M. Randi, M. A. Schwartz, K. Matter, and M. S. Balda. ZO-1 controls endothelial adherens junctions, cell–cell tension, angiogenesis, and barrier formation. J. Cell Biol. 208:821–838, 2015.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  49. Wallez, Y., and P. Huber. Endothelial adherens and tight junctions in vascular homeostasis, inflammation and angiogenesis. Biochim. Biophys. Acta Biomembr. 1778:794–809, 2008.

    Article  CAS  Google Scholar 

  50. Wang, Y. L., and R. J. Pelham. Preparation of a flexible, porous polyacrylamide substrate for mechanical studies of cultured cells. Methods Enzymol. 298:489–496, 1998.

    Article  CAS  PubMed  Google Scholar 

  51. Wilhelm, I., C. Fazakas, and I. A. Krizbai. In vitro models of the blood-brain barrier. Acta Neurobiol. Exp. 71:113–128, 2011.

    Google Scholar 

  52. Winger, R. C., J. E. Koblinski, T. Kanda, R. M. Ransohoff, and W. A. Muller. Rapid remodeling of tight junctions during paracellular diapedesis in a human model of the blood-brain barrier. J. Immunol. 193:2427–2437, 2014.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  53. Wong, K. H. K., J. G. Truslow, and J. Tien. The role of cyclic AMP in normalizing the function of engineered human blood microvessels in microfluidic collagen gels. Biomaterials 31:4706–4714, 2010.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  54. Zieman, S. J., V. Melenovsky, and D. A. Kass. Mechanisms, pathophysiology, and therapy of arterial stiffness. Aterioscler. Thromb. Vasc. Biol. 25:932–943, 2005

    Article  CAS  Google Scholar 

Download references

Acknowledgments

The authors acknowledge Kyle Thomas at Yellow Basket, LLC (kyle@yellowbasket.io) for software development support. The authors also acknowledge funding from the Burroughs Wellcome Career Award at the Scientific Interface (to KMS), the Fischell Fellowship in Biomedical Engineering and the Dr. Mabel S. Spencer Award for Excellence in Graduate Achievement (to KMG), and the University of Maryland.

Author information

Authors and Affiliations

  1. Fischell Department of Bioengineering, University of Maryland, College Park, MD, 20742, USA

    Kelsey M. Gray, Dakota B. Katz, Erica G. Brown & Kimberly M. Stroka

  2. Biophysics Program, University of Maryland, College Park, MD, 20742, USA

    Kimberly M. Stroka

  3. Center for Stem Cell Biology and Regenerative Medicine, University of Maryland, Baltimore, MD, 21201, USA

    Kimberly M. Stroka

  4. Marlene and Stewart Greenebaum Comprehensive Cancer Center, University of Maryland, Baltimore, MD, 21201, USA

    Kimberly M. Stroka

Authors
  1. Kelsey M. Gray
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Dakota B. Katz
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Erica G. Brown
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Kimberly M. Stroka
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Kimberly M. Stroka.

Additional information

Associate Editor Dan Elson oversaw the review of this article.

Publisher's Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Electronic supplementary material

Below is the link to the electronic supplementary material.

10439_2019_2266_MOESM1_ESM.pdf

Supplementary material 1 See supplementary material for the determination of JAnaP thresholds (Figs. S1–S3) and program validation (Table S.I.), full statistical analysis (Table S.II-S.V), additional shape factors for Figs. 3–5 (Figs. S4, S6, and S5), correlation between junction and monolayer coverage (Fig. S7), and for the AFM method and results (Method S1 and Fig. S5) (PDF 1562 kb)

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

Gray, K.M., Katz, D.B., Brown, E.G. et al. Quantitative Phenotyping of Cell–Cell Junctions to Evaluate ZO-1 Presentation in Brain Endothelial Cells. Ann Biomed Eng 47, 1675–1687 (2019). https://doi.org/10.1007/s10439-019-02266-5

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s10439-019-02266-5

Keywords

  • Matrix stiffness
  • Mechanotransduction
  • Mechanobiology
  • Blood–brain barrier