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

  • Kelsey M. Gray
  • Dakota B. Katz
  • Erica G. Brown
  • Kimberly M. StrokaEmail author
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.

Keywords

Matrix stiffness Mechanotransduction Mechanobiology Blood–brain barrier 

Notes

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.

Supplementary material

10439_2019_2266_MOESM1_ESM.pdf (1.5 mb)
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)

References

  1. 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.CrossRefGoogle Scholar
  2. 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.CrossRefGoogle Scholar
  3. 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.Google Scholar
  4. 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.CrossRefGoogle Scholar
  5. 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.CrossRefGoogle Scholar
  6. 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.CrossRefGoogle Scholar
  7. 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.CrossRefGoogle Scholar
  8. 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.CrossRefGoogle Scholar
  9. 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.CrossRefGoogle Scholar
  10. 10.
    Cheney, R. E. Myosins in cell junctions. Bioarchitecture 2:158–170, 2012.CrossRefGoogle Scholar
  11. 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.CrossRefGoogle Scholar
  12. 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.CrossRefGoogle Scholar
  13. 13.
    Dorland, Y. L., and S. Huveneers. Cell–cell junctional mechanotransduction in endothelial remodeling. Cell. Mol. Life Sci. 74:279–292, 2017.CrossRefGoogle Scholar
  14. 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.CrossRefGoogle Scholar
  15. 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. 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.CrossRefGoogle Scholar
  17. 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.CrossRefGoogle Scholar
  18. 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.CrossRefGoogle Scholar
  19. 19.
    Grammas, P., J. Martinez, and B. Miller. Cerebral microvascular endothelium and the pathogenesis of neurodegenerative diseases. Expert Rev. Mol. Med. 13:e19, 2011.CrossRefGoogle Scholar
  20. 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.CrossRefGoogle Scholar
  21. 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.CrossRefGoogle Scholar
  22. 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.CrossRefGoogle Scholar
  23. 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.CrossRefGoogle Scholar
  24. 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.CrossRefGoogle Scholar
  25. 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.CrossRefGoogle Scholar
  26. 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.CrossRefGoogle Scholar
  27. 27.
    Kohn, J. C., M. C. Lampi, and C. A. Reinhart-King. Age-related vascular stiffening: causes and consequences. Front. Genet. 6:112, 2015.CrossRefGoogle Scholar
  28. 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.CrossRefGoogle Scholar
  29. 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.CrossRefGoogle Scholar
  30. 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.CrossRefGoogle Scholar
  31. 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.CrossRefGoogle Scholar
  32. 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.CrossRefGoogle Scholar
  33. 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.CrossRefGoogle Scholar
  34. 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.CrossRefGoogle Scholar
  35. 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.CrossRefGoogle Scholar
  36. 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.CrossRefGoogle Scholar
  37. 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.CrossRefGoogle Scholar
  38. 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.CrossRefGoogle Scholar
  39. 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.CrossRefGoogle Scholar
  40. 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.CrossRefGoogle Scholar
  41. 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.CrossRefGoogle Scholar
  42. 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.CrossRefGoogle Scholar
  43. 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.CrossRefGoogle Scholar
  44. 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.CrossRefGoogle Scholar
  45. 45.
    Stroka, K. M., and H. Aranda-espinoza. Neutrophils display biphasic relationship between migration and substrate stiffness. Cell Motil. Cytoskeleton 66:328–341, 2009.CrossRefGoogle Scholar
  46. 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.CrossRefGoogle Scholar
  47. 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.CrossRefGoogle Scholar
  48. 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.CrossRefGoogle Scholar
  49. 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.CrossRefGoogle Scholar
  50. 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.CrossRefGoogle Scholar
  51. 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. 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.CrossRefGoogle Scholar
  53. 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.CrossRefGoogle Scholar
  54. 54.
    Zieman, S. J., V. Melenovsky, and D. A. Kass. Mechanisms, pathophysiology, and therapy of arterial stiffness. Aterioscler. Thromb. Vasc. Biol. 25:932–943, 2005CrossRefGoogle Scholar

Copyright information

© Biomedical Engineering Society 2019

Authors and Affiliations

  1. 1.Fischell Department of BioengineeringUniversity of MarylandCollege ParkUSA
  2. 2.Biophysics ProgramUniversity of MarylandCollege ParkUSA
  3. 3.Center for Stem Cell Biology and Regenerative MedicineUniversity of MarylandBaltimoreUSA
  4. 4.Marlene and Stewart Greenebaum Comprehensive Cancer CenterUniversity of MarylandBaltimoreUSA

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