Quantitative Phenotyping of Cell–Cell Junctions to Evaluate ZO-1 Presentation in Brain Endothelial Cells
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.
KeywordsMatrix stiffness Mechanotransduction Mechanobiology Blood–brain barrier
The authors acknowledge Kyle Thomas at Yellow Basket, LLC (firstname.lastname@example.org) 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.
- 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
- 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.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
- 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.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
- 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
- 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
- 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