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

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.

Keywords: blood-brain barrier (BBB); drug delivery; matrix metalloproteinases (MMPs); neurodegenerative diseases; tissue Inhibitors of metalloproteinases (TIMPs).

Fig. 1

A schematic of the blood-brain barrier model. The simplified in vitro blood brain barrier (BBB) model uses rat brain endothelial cell (RBMEC). RBMECs were grown on the Transwell^® inserts to form a confluent monolayer. The permeability was measured after adding FITC-conjugated dextran (MW: 10 kDa) to the apical chamber while samples were taken from the basolateral chamber at 30- or 15-min time intervals for 2 h.

Fig. 2

Confirming the RBMEC growth by TEER and microscopy. (a) The TEER value was measured immediately after transferring the cells (n = 17) into 24-well Transwells (time = 0) and after 24 h. (b) The bright field microscopy images (Fisherbrand^™ Entry Level Research Grade Inverted Microscope, Fisher Scientific, USA, at 10X magnification) of RBMEC on Transwells, are shown after 24 h incubation at 37°C incubator when the TEER value exceeded 150 Ω × cm². The top image is blank (no cells, collagen/fibronectin-coated Transwell only) and the bottom is the RBMECs grown after 24 h incubation.

Fig. 3

The permeability level of RBMEC and 3T3 cells was compared over time. (a) Comparison between blank (no cells, collagen/fibronectin-coated Transwell only), RBMEC, and 3T3 cells. Time points were taken every 30 min. (b) Permeability value for 3T3 cells vs. RBMEC, (c) The RBMEC TEER value exceeded 150 Ω × cm² but the TEER value of 3T3 cells was less than 10 Ω × cm². Experiment was performed in duplicate with the 12-well Transwell (V_c,t = 1500 μl × S_b,t / S_a).

Fig. 4

The permeability of RBMEC and 3T3 cells after treating with different concentration of MMP-3 cd was compared in time dependence manner. (a) Time point value (left) and Pe value bar graphs (right) for permeability of 3T3 cells before and after MMP-3 cd (M-3) treatment, (b) Time point value (left) and Pe value bar graphs (right) for permeability RBMEC before and after MMP-3 cd (M-3) treatment. Experiment was performed with 12-well Transwells (V_c,t = 1500 μl × S_b,t / S_a) and repeated at least three times. The error bars represent standard deviation.

Fig. 5

Effect of MMP-3 cd and MMP-9 cd and their complex with TIMP-1 on permeability of RBMEC. (a) MMP-3 cd disrupts the tight junctions of RBMECs in a dose-dependent manner based on 5 replicate experiments from 3 different groups of RBMEC leading to a nearly 18-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-3 cd, permeability was slightly over two and half of baseline (p-value: **** < 0.0001). Experiment was performed in 24-well Transwells (V_c,t = 600 μl × S_b,t / S_a). (b) MMP-9 cd disrupts the tight junctions of RBMEC in a dose-dependent manner based on an average of 5 different wells from 3 different groups of RBMEC leading to a nearly 2.5-fold increase over baseline permeability at 1.5 μM of MMP-9 cd. In the presence of 1.5 μM of both TIMP-1 and MMP-9 cd, permeability was slightly over one and half of baseline (p-value: **** < 0.0001). (M-3: MMP-3 cd, M-9: MMP-9 cd, T-1: TIMP-1). Experiment was performed in 24-well Transwells (V_c,t = 600 μl × S_b,t / S_a).

Fig. 6

TEER of MMP-3 cd and MMP-9 cd alone or with TIMP-1 on RBMEC. (a) The TEER value of RBMEC after treating with MMP-3 cd alone or with MMP-3 cd in complex with TIMP-1, and (b) with MMP-9 cd alone or in complex with TIMP-1 was compared at 60-min intervals for 2 h. (M-3: MMP-3 cd, M-9: MMP-9 cd, T1: TIMP-1). Each experiment repeated three times, and the error bar shows the standard deviation.

Fig. 7

Confirming the effect of MMP-3 cd alone or with TIMP-1 on RBMEC by ICC. (a) RBMEC on 35 glass base dishes without treatment, after 1 h treatment with 1.5 μM MMP-3 cd and after 1 h treatment with preincubated 1.5 μM MMP-3 cd and 1.5 μM TIMP-1 by inverted bright field microscope at 10X magnification (b) ICC image of RBMEC, as primary antibody 1/50 Zo-1 used and as secondary antibody AlexaFluor 488 (rabbit) used by confocal fluorescent microscope at 60X magnification (M-3: MMP-3 cd, T-1: TIMP-1). The arrows highlight some of the observations for disruption (white, MMP-3 treated), or protection (yellow, TIMP-1 in complex with MMP-3 treated).