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Influence of Substrate Stiffness on Barrier Function in an iPSC-Derived In Vitro Blood-Brain Barrier Model

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Abstract

Introduction

Vascular endothelial cells respond to a variety of biophysical cues such as shear stress and substrate stiffness. In peripheral vasculature, extracellular matrix (ECM) stiffening alters barrier function, leading to increased vascular permeability in atherosclerosis and pulmonary edema. The effect of ECM stiffness on blood-brain barrier (BBB) endothelial cells, however, has not been explored. To investigate this topic, we incorporated hydrogel substrates into an in vitro model of the human BBB.

Methods

Induced pluripotent stem cells were differentiated to brain microvascular endothelial-like (BMEC-like) cells and cultured on hydrogel substrates of varying stiffness. Cellular changes were measured by imaging, functional assays such as transendothelial electrical resistance (TEER) and p-glycoprotein efflux activity, and bulk transcriptome readouts.

Results

The magnitude and longevity of TEER in iPSC-derived BMEC-like cells is enhanced on compliant substrates. Quantitative imaging shows that BMEC-like cells form fewer intracellular actin stress fibers on substrates of intermediate stiffness (20 kPa relative to 1 and 150 kPa). Chemical induction of actin polymerization leads to a rapid decline in TEER, agreeing with imaging readouts. P-glycoprotein activity is unaffected by substrate stiffness. Modest differences in RNA expression corresponding to specific signaling pathways were observed as a function of substrate stiffness.

Conclusions

iPSC-derived BMEC-like cells exhibit differences in passive but not active barrier function in response to substrate stiffness. These findings may provide insight into BBB dysfunction during neurodegeneration, as well as aid in the optimization of more complex three-dimensional neurovascular models utilizing compliant hydrogels.

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References

  1. Birukova, A. A., F. T. Arce, N. Moldobaeva, S. M. Dudek, J. G. N. Garcia, R. Lal, and K. G. Birukov. Endothelial permeability is controlled by spatially defined cytoskeletal mechanics: AFM force mapping of pulmonary endothelial monolayer. Nanomed. Nanotechnol. Biol. Med. 5:30, 2009.

    Article  Google Scholar 

  2. Bogatcheva, N. V., and A. D. Verin. The role of cytoskeleton in the regulation of vascular endothelial barrier function. Microvasc. Res. 76:202–207, 2008.

    Article  Google Scholar 

  3. Bubb, M. R., A. M. Senderowicz, E. A. Sausville, K. L. Duncan, and E. D. Korn. Jasplakinolide, a cytotoxic natural product, induces actin polymerization and competitively inhibits the binding of phalloidin to F-actin. J. Biol. Chem. 269:14869–14871, 1994.

    Article  Google Scholar 

  4. Califano, J. P., and C. A. Reinhart-King. A balance of substrate mechanics and matrix chemistry regulates endothelial cell network assembly. Cell. Mol. Bioeng. 1:122–132, 2008.

    Article  Google Scholar 

  5. Carmeliet, P., and R. K. Jain. Molecular mechanisms and clinical applications of angiogenesis. Nature. 473:298–307, 2011.

    Article  Google Scholar 

  6. Cecelja, M., and P. Chowienczyk. Role of arterial stiffness in cardiovascular disease. JRSM Cardiovasc. Dis. 1:1–10, 2012.

    Article  Google Scholar 

  7. Faley, S. L., E. H. Neal, J. X. Wang, A. M. Bosworth, C. M. Weber, K. M. Balotin, E. S. Lippmann, and L. M. Bellan. iPSC-derived brain endothelium exhibits stable, long-term barrier function in perfused hydrogel scaffolds. Stem Cell Rep. 12:474–487, 2019.

    Article  Google Scholar 

  8. Gray, K. M., and K. M. Stroka. Vascular endothelial cell mechanosensing: New insights gained from biomimetic microfluidic models. Semin. Cell Dev. Biol. 71:106–117, 2017.

    Article  Google Scholar 

  9. Greene, W., and S.-J. Gao. Actin dynamics regulate multiple endosomal steps during Kaposi’s sarcoma-associated herpesvirus entry and trafficking in endothelial cells. PLOS Pathog. 5:e1000512, 2009.

    Article  Google Scholar 

  10. Hawkins, B. T., and T. P. Davis. The blood-brain barrier/neurovascular unit in health and disease. Pharmacol. Rev. 57:173–185, 2005.

    Article  Google Scholar 

  11. Hollmann, E. K., A. K. Bailey, A. V. Potharazu, M. D. Neely, A. B. Bowman, and E. S. Lippmann. Accelerated differentiation of human induced pluripotent stem cells to blood–brain barrier endothelial cells. Fluids Barriers CNS. 14:9, 2017.

    Article  Google Scholar 

  12. 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  Google Scholar 

  13. Huynh, J., et al. Age-related intimal stiffening enhances endothelial permeability and leukocyte transmigration. Sci. Transl. Med. 3:112ra122, 2011.

    Article  Google Scholar 

  14. Johnson, B. D., K. J. Mather, and J. P. Wallace. Mechanotransduction of shear in the endothelium: Basic studies and clinical implications. Vasc. Med. 16:365–377, 2011.

    Article  Google Scholar 

  15. Kalaria, R. N., and A. B. Pax. Increased collagen content of cerebral microvessels in Alzheimer’s disease. Brain Res. 705:349–352, 1995.

    Article  Google Scholar 

  16. Kimbrough, I. F., S. Robel, E. D. Roberson, and H. Sontheimer. Vascular amyloidosis impairs the gliovascular unit in a mouse model of Alzheimer’s disease. Brain. 138:3716–3733, 2015.

    Article  Google Scholar 

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

    Article  Google Scholar 

  18. Krishnan, R., D. D. Klumpers, C. Y. Park, K. Rajendran, X. Trepat, J. van Bezu, V. W. 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:C146–C154, 2010.

    Article  Google Scholar 

  19. Kroon, D.-J. Hessian based Frangi Vesselness filter (https://www.mathworks.com/matlabcentral/fileexchange/24409-hessian-based-frangi-vesselness-filter). MATLAB Central File Exchange, [Retrieved 2020] 2009.

  20. Kumar, K. K., et al. Cellular manganese content is developmentally regulated in human dopaminergic neurons. Sci. Rep. 4:6801, 2014.

    Article  Google Scholar 

  21. Lippmann, E. S., S. M. Azarin, J. E. Kay, R. A. Nessler, H. K. Wilson, A. Al-Ahmad, S. P. Palecek, and E. V. Shusta. Derivation of blood-brain barrier endothelial cells from human pluripotent stem cells. Nat. Biotechnol. 30:783–791, 2012.

    Article  Google Scholar 

  22. Lippmann, E. S., A. Al-Ahmad, S. M. Azarin, S. P. Palecek, and E. V. Shusta. A retinoic acid-enhanced, multicellular human blood-brain barrier model derived from stem cell sources. Sci. Rep. 4:4160, 2014.

    Article  Google Scholar 

  23. Mammoto, A., T. Mammoto, M. Kanapathipillai, C. W. Yung, E. Jiang, A. Jiang, K. Lofgren, E. P. S. Gee, and D. E. Ingber. Control of lung vascular permeability and endotoxin-induced pulmonary oedema by changes in extracellular matrix mechanics. Nat. Commun. 4:1759, 2013.

    Article  Google Scholar 

  24. Mancardi, G. L., F. Perdelli, C. Rivano, A. Leonardi, and O. Bugiani. Thickening of the basement membrane of cortical capillaries in Alzheimer’s disease. Acta Neuropathol. (Berl.). 49:79–83, 1980.

    Article  Google Scholar 

  25. McKee, C. T., J. A. Last, P. Russell, and C. J. Murphy. Indentation versus tensile measurements of Young’s modulus for soft biological tissues. Tissue Eng. B. 17:155–164, 2011.

    Article  Google Scholar 

  26. Montagne, A., S. R. Barnes, M. D. Sweeney, M. R. Halliday, A. P. Sagare, Z. Zhao, A. W. Toga, R. E. Jacobs, C. Y. Liu, L. Amezcua, M. G. Harrington, H. C. Chui, M. Law, and B. V. Zlokovic. Blood-brain barrier breakdown in the aging human hippocampus. Neuron. 85:296–302, 2015.

    Article  Google Scholar 

  27. Neal, E. H., N. A. Marinelli, Y. Shi, P. M. McClatchey, K. M. Balotin, D. R. Gullett, K. A. Hagerla, A. B. Bowman, K. C. Ess, J. P. Wikswo, and E. S. Lippmann. A simplified, fully defined differentiation scheme for producing blood-brain barrier endothelial cells from human iPSCs. Stem Cell Rep. 12:1380–1388, 2019.

    Article  Google Scholar 

  28. Neal, E. H., K. A. Katdare, Y. Shi, N. A. Marinelli, K. A. Hagerla, and E. S. Lippmann. Influence of basal media composition on barrier fidelity within human pluripotent stem cell-derived blood-brain barrier models. bioRxiv, 2021.03.01.433282, 2021.

  29. Poole, K. M., D. R. McCormack, C. A. Patil, C. L. Duvall, and M. C. Skala. Quantifying the vascular response to ischemia with speckle variance optical coherence tomography. Biomed. Opt. Express. 5:4118–4130, 2014.

    Article  Google Scholar 

  30. Sabbagh, M. F., and J. Nathans. A genome-wide view of the de-differentiation of central nervous system endothelial cells in culture. eLife. 9:e51276, 2020.

    Article  Google Scholar 

  31. Stebbins, M. J., E. S. Lippmann, M. G. Faubion, R. Daneman, S. P. Palecek, and E. V. Shusta. Activation of RARα, RARγ, or RXRα increases barrier tightness in human induced pluripotent stem cell-derived brain endothelial cells. Biotechnol. J. 13:1700093, 2018.

    Article  Google Scholar 

  32. Sweeney, M. D., Z. Zhao, A. Montagne, A. R. Nelson, and B. V. Zlokovic. Blood-brain barrier: from physiology to disease and back. Physiol. Rev. 99:21–78, 2018.

    Article  Google Scholar 

  33. Tarbell, J. M. Shear stress and the endothelial transport barrier. Cardiovasc. Res. 87:320–330, 2010.

    Article  Google Scholar 

  34. Van Itallie, C. M., A. S. Fanning, A. Bridges, and J. M. Anderson. ZO-1 stabilizes the tight junction solute barrier through coupling to the perijunctional cytoskeleton. Mol. Biol. Cell. 20:3930–3940, 2009.

    Article  Google Scholar 

  35. van Nieuw Amerongen, 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  Google Scholar 

  36. VanderBurgh, J.A., and C.A. Reinhart-King. Chapter Ten - The Role of Age-Related Intimal Remodeling and Stiffening in Atherosclerosis. In: Advances in Pharmacology, edited by R.A. Khalil. Academic Press, 2018, pp. 365–391.

  37. Wang, J., S. Vasaikar, Z. Shi, M. Greer, and B. Zhang. WebGestalt 2017: a more comprehensive, powerful, flexible and interactive gene set enrichment analysis toolkit. Nucleic Acids Res. 45:W130–W137, 2017.

    Article  Google Scholar 

  38. Waschke, J., F. E. Curry, R. H. Adamson, and D. Drenckhahn. Regulation of actin dynamics is critical for endothelial barrier functions. Am. J. Physiol.-Heart Circ. Physiol. 288:H1296–H1305, 2005.

    Article  Google Scholar 

  39. Watanabe, K., M. Ueno, D. Kamiya, A. Nishiyama, M. Matsumura, T. Wataya, J. B. Takahashi, S. Nishikawa, S. Nishikawa, K. Muguruma, and Y. Sasai. A ROCK inhibitor permits survival of dissociated human embryonic stem cells. Nat. Biotechnol. 25:681–686, 2007.

    Article  Google Scholar 

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Acknowledgments

Funding for this work was provided by a Ben Barres Early Career Acceleration Award from the Chan Zuckerberg Initiative (Grant 2018-191850 to ESL), the BrightFocus Foundation (Grant A20170945 to ESL), National Institutes of Health grants R21 NS106510 (to ESL), R01 NS110665 (to ESL), R61 NS112445 (to ESL), and K01 EB030039 (to KPO), and National Science Foundation grant 1846860 (to ESL). BJO was supported by the Vanderbilt Interdisciplinary Training Program in Alzheimer’s Disease (T32 AG058524). Support for RNA sequencing was provided by the Vanderbilt VANTAGE core facility, which is supported in part by a Clinical and Translational Science Award (5UL1 RR024975), the Vanderbilt Ingram Cancer Center (P30 CA68485), the Vanderbilt Vision Center (P30 EY08126), a CTSA award from the National Center for Advancing Translational Sciences (UL1 TR002243), and the National Center for Research Resources (G20 RR030956). CTSA award UL1 TR002243 also provided pilot funding for this project. The authors would like to thank Dr. Jean-Philippe Cartailler for helpful discussions on RNA sequencing experiments, Dr. Cynthia Reinhart-King and Wenjun Wang for helpful discussions and training on polyacrylamide hydrogel synthesis, and Dr. Anthony Hmelo and John Thornton for guidance with AFM measurements.

Data Accessibility

RNA sequencing data have been uploaded to ArrayExpress under the accession number E-MTAB-10336.

Conflict of interest

Allison Bosworth, Hyosung Kim, Kristin O’Grady, Isabella Richter, Lynn Lee, Brian O’Grady, and Ethan Lippmann declare no conflicts of interest.

Ethical Approval

No approvals were required for this study.

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Authors and Affiliations

  1. Department of Biomedical Engineering, Vanderbilt University, Nashville, TN, USA

    Allison M. Bosworth, Isabella Richter & Ethan S. Lippmann

  2. Department of Chemical and Biomolecular Engineering, Vanderbilt University, Nashville, TN, USA

    Hyosung Kim, Lynn Lee, Brian J. O’Grady & Ethan S. Lippmann

  3. Department of Radiology and Radiological Sciences, Vanderbilt University Medical Center, Nashville, TN, USA

    Kristin P. O’Grady

  4. Vanderbilt University Institute of Imaging Science, Vanderbilt University Medical Center, Nashville, TN, USA

    Kristin P. O’Grady

  5. Interdisciplinary Materials Science Program, Vanderbilt University, Nashville, TN, USA

    Brian J. O’Grady & Ethan S. Lippmann

  6. Department of Neurology, Vanderbilt University Medical Center, Nashville, TN, USA

    Ethan S. Lippmann

  7. Vanderbilt Brain Institute, Vanderbilt University, Nashville, TN, USA

    Ethan S. Lippmann

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  1. Allison M. Bosworth
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Correspondence to Ethan S. Lippmann.

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Bosworth, A.M., Kim, H., O’Grady, K.P. et al. Influence of Substrate Stiffness on Barrier Function in an iPSC-Derived In Vitro Blood-Brain Barrier Model. Cel. Mol. Bioeng. 15, 31–42 (2022). https://doi.org/10.1007/s12195-021-00706-8

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