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TIMP-1 Protects Tight Junctions of Brain Endothelial Cells From MMP-Mediated Degradation

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Abstract

Objective

The blood–brain barrier (BBB) plays a critical role in central nervous system homeostasis, and the integrity of BBB is disrupted in many neurodegenerative diseases. Matrix metalloproteinases (MMPs) degrade the tight junctions (TJs) of endothelial cells and basement membrane components essential to BBB integrity, which leads to increased BBB permeability and allows inflammatory cells and neurotoxic substances to enter the brain. Tissue inhibitors of metalloproteinases (TIMPs), endogenous inhibitors of MMPs, regulate MMP activity, thereby maintaining BBB integrity.

Methods

The disruptive impacts of MMP-3 and MMP-9 on BBB and protective effect of TIMP-1 were investigated in a simplified in vitro model of the BBB, which was generated using rat brain microvascular endothelial cells (RBMEC). The main features of BBB formation, including permeability and the trans-endothelial electrical resistance (TEER), were monitored over time after the addition of MMP-3 and MMP-9 and their complexes with TIMP-1 inhibitor.

Results

Our results indicated that MMP-3 and MMP-9 caused a dose-dependent disruption of the BBB, with 1.5 µM MMPs resulting in an over threefold increase in permeability, while TIMP-1 inhibition protected the integrity of the BBB model and recovered TEER and permeability of RBMECs. The disruption and recovery of tight junction proteins of RBMECs after MMP and TIMP treatment were also detected using fluorescent microscopy.

Conclusion

MMP-9 and MMP-3 disrupt the BBB by degrading tight junctions in endothelial cells, and TIMP-1 could inhibit the disruptive effect of MMP-3 and MMP-9 by showing potential as therapeutic protein against MMP-related diseases where BBB disruption plays a role.

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Data Availability

The authors declare that the data supporting the findings of this study are available within the paper and its Supplementary Information files. Should any raw data files be needed in another format they are available from the corresponding author upon reasonable request.

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Acknowledgements

The authors acknowledge the UNR genomic center. The authors would also like to thank Dr. Robert Rendon for his help with confocal fluorescence microscopy and Dr. Eric Shusta for his scientific consultation on fluorescence microscopy in vitro BBB model.

Funding

M. R.-S. had funding support from NIH R03AG070511 and NIH R21HD109743.

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Authors and Affiliations

  1. Department of Chemical and Materials Engineering, University of Nevada, 1664 N. Virginia St, Reno, NV, 89557, USA

    Hannaneh Ahmadighadykolaei, Janet A. Lambert & Maryam Raeeszadeh-Sarmazdeh

  2. Department of Pharmacology, University of Nevada, Reno School of Medicine, Reno, NV, USA

    Janet A. Lambert

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M. R.-S., conception; H. A. and J. A. L., performing the experiments; H. A, J. A. L., M. R.-S., data analysis, writing and editing the manuscript drafts. M. R.-S. funding and supervision.

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Correspondence to Maryam Raeeszadeh-Sarmazdeh.

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ESM 1

Supplemental Fig S1. Effect of MMP-3 and TIMP-1 alone or with each other’s and their buffer on permeability of RBMEC. MMP-3 disrupts the TJs of RBMEC leading to a nearly 25-fold increase over baseline permeability at 1.5 µM of MMP-3-cd. In the presence of 1.5 µM of both TIMP-1 and MMP-3cd, permeability is slightly over twice baseline. The 1.5 µM of MMP-3 and 1.5 µM of both TIMP-1 and MMP-3cd shown highest effect on RBMEC so we tried these amounts for rest of experiments. No significant effect of buffers or TIMP-1 alone on RBMEC permeability was observed. Experiment was performed in 24-well Transwells (Vc,t = 600 µl × Sb,t / Sa) (PNG 288 kb)

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Ahmadighadykolaei, H., Lambert, J.A. & Raeeszadeh-Sarmazdeh, M. TIMP-1 Protects Tight Junctions of Brain Endothelial Cells From MMP-Mediated Degradation. Pharm Res 40, 2121–2131 (2023). https://doi.org/10.1007/s11095-023-03593-y

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