Attenuation of Blood-Brain Barrier Breakdown and Hyperpermeability by Calpain Inhibition

Abstract

Blood-brain barrier (BBB) breakdown and the associated microvascular hyperpermeability followed by brain edema are hallmark features of several brain pathologies, including traumatic brain injuries (TBI). Recent studies indicate that pro-inflammatory cytokine interleukin-1β (IL-1β) that is up-regulated following traumatic injuries also promotes BBB dysfunction and hyperpermeability, but the underlying mechanisms are not clearly known. The objective of this study was to determine the role of calpains in mediating BBB dysfunction and hyperpermeability and to test the effect of calpain inhibition on the BBB following traumatic insults to the brain. In these studies, rat brain microvascular endothelial cell monolayers exposed to calpain inhibitors (calpain inhibitor III and calpastatin) or transfected with calpain-1 siRNA demonstrated attenuation of IL-1β-induced monolayer hyperpermeability. Calpain inhibition led to protection against IL-1β-induced loss of zonula occludens-1 (ZO-1) at the tight junctions and alterations in F-actin cytoskeletal assembly. IL-1β treatment had no effect on ZO-1 gene (tjp1) or protein expression. Calpain inhibition via calpain inhibitor III and calpastatin decreased IL-1β-induced calpain activity significantly (p < 0.05). IL-1β had no detectable effect on intracellular calcium mobilization or endothelial cell viability. Furthermore, calpain inhibition preserved BBB integrity/permeability in a mouse controlled cortical impact model of TBI when studied using Evans blue assay and intravital microscopy. These studies demonstrate that calpain-1 acts as a mediator of IL-1β-induced loss of BBB integrity and permeability by altering tight junction integrity, promoting the displacement of ZO-1, and disorganization of cytoskeletal assembly. IL-1β-mediated alterations in permeability are neither due to the changes in ZO-1 expression nor cell viability. Calpain inhibition has beneficial effects against TBI-induced BBB hyperpermeability.

Keywords: calpain; inflammation; inhibitor; interleukin 1 (IL-1); vascular biology.

FIGURE 1.

Calpain inhibitor III and calpastatin pretreatment attenuates IL-1β treatment-induced monolayer hyperpermeability and calpain activity. Calpain inhibitor III (A, n = 4; p < 0.05) and calpastatin (B, n = 4; p < 0.05) pretreatment attenuates IL-1β-induced monolayer hyperpermeability significantly. Knockdown of calpain-1 by siRNA attenuates IL-1β treatment-induced monolayer hyperpermeability (C, n = 4; p < 0.05). Monolayer permeability is expressed as a percentage control of FITC-dextran-10 kDa fluorescence intensity, plotted on y axis. Calpastatin and calpain inhibitor III pretreatment attenuates IL-1β treatment-induced calpain activity significantly (D, n = 4; p < 0.05). Calpain activity is expressed as RFU, plotted on the y axis. Data are expressed as mean ± % S.E. *a indicates significant increase compared with the control group; *b indicates significant decrease compared with the IL-1β treatment group.

FIGURE 2.

IL-1β treatment (at concentrations 1, 10, and 100 ng/ml) as well as various time points (1, 2, and 4 h) induces calpain activity significantly (A and B, n = 5; p < 0.05). Calpain activity is expressed as RFU, plotted on the y axis. Data are expressed as mean ± S.E. *a indicates significant increase compared with the control (0 h) group.

FIGURE 3.

Calpain inhibitor III treatment of various concentrations (0. 1, 1, 10, and 50 μm) decreases IL-1β-induced calpain activity and monolayer hyperpermeability significantly (A and B, n = 5; p < 0.05) in RBMEC. Calpain activity is expressed as RFU, plotted on the y axis. Monolayer permeability is expressed as FITC-dextran fluorescence intensity (% control). Data are expressed as mean ± S.E. *a indicates significant increase compared with the control (0 μm) group). *b indicates significant decrease compared with the 0 μm group.

FIGURE 4.

IL-1β treatment-induced ZO-1 junctional disruption and F-actin stress fiber formation is reduced by pretreatment with calpain inhibitor III (A–D). IL-1β treatment-induced ZO-1 junctional disruption and F-actin stress fiber formation are shown by white arrows in A and B, respectively. ZO-1 junctional integrity and F-actin stress fiber formation were assessed using immunofluorescence localization and rhodamine phalloidin techniques, respectively (n = 4 for each study). The changes in ZO-1 localization and the formation of F-actin stress fibers were further determined using ImageJ software and presented as arbitrary units (C and D, respectively; p < 0.05). Under normal physiological conditions actin filaments are randomly distributed throughout the cell, but agents that induce endothelial hyperpermeability induce them to reorganize themselves into stress fibers that are linear parallel bundles across the cell interior (following IL-1β treatment as pointed by the arrows in B) and also exhibit increased binding to rhodamine phalloidin (as shown in B and D).

FIGURE 5.

IL-1β treatment does not induce ZO-1 mRNA or protein expression. Fluorescence intensity from RT-PCR studies is expressed as relative expression of ZO-1 normalized to GAPDH, plotted on y axis (n = 3; A). Data are represented as mean ± S.E. ZO-1 protein expression did not change following IL-1β treatment (n = 4; B).

FIGURE 6.

IL-1β treatment does not induce cell death. IL-1β treatment did not alter cell viability up to 4 h. Treatment of hydrogen peroxide used as a positive control showed a significant decrease in cell viability compared with the untreated and IL-1β treatment groups (n = 5; p < 0.05). Data are expressed as mean ± % S.E. *a indicates significant decrease compared with the control group.

FIGURE 7.

IL-1β treatment does not induce intracellular calcium mobilization. A demonstrates the effect of IL-1β treatment on intracellular calcium mobilization (F) relative to basal Ca²⁺ (F₀) when measured 10 min after the application of DMSO (vehicle control), IL-1β (10 ng/ml), TG (2 μm), and Iono (1 μm) and displayed as mean ± S.E. (n = 4). B and C are the representative intracellular free calcium recordings ([Ca²⁺]_i, mean ± S.E.) showing cytosolic Ca²⁺ levels coupled to TG (2 μm, B) or IL-1β (100 ng/ml, C) stimulation. The top bars indicate the type of extracellular solutions applied to the RBMECs, and the vertical lines on the x axis indicate the time of solution exchange. D displays averaged peak values of [Ca²⁺]_i in response to TG (n = 40 cells) and IL-1β (n = 30 cells), respectively.

FIGURE 8.

Calpain inhibitor III treatment attenuates TBI-induced BBB hyperpermeability studied by Evans blue dye extravasation method. Vehicle control group subjected to TBI demonstrated a significant increase in Evans blue leakage compared with the sham injury group (A, p < 0.05). Calpain inhibitor III (10 μg/g body weight of the animal) pre- or post-treatment attenuated TBI-induced Evans blue leakage into the extravascular tissue space significantly (A, p < 0.05). Pictorial representation of the brain tissue from various groups is shown in B. Each group consisted of five animals. Sham injury group is used as the baseline for all comparisons. Data are expressed as nanograms/brain cortex ± S.E. *a indicates significant increase compared with the sham injury group; *b indicates significant decrease compared with the vehicle + TBI group.

FIGURE 9.

Intravital microscopy imaging of mouse brain demonstrates the protective effect of calpain inhibitor III against TBI-induced BBB hyperpermeability. Mice subjected to TBI demonstrated a significant increase in BBB permeability at 60 min as evidenced by enhanced FITC-dextran fluorescence compared with the sham injury group (A and B, n = 5 each group, p < 0.05). Calpain inhibitor III (10 μg/g body weight of the animal; n = 4) treatment 15 min following TBI attenuated TBI-induced BBB hyperpermeability as evidenced by the extravasation of FITC-dextran into the extravascular space compared with TBI + vehicle group (A and B, p < 0.05). Data are expressed as a change in relative fluorescence to the initial image (0 time point) in each group, and the sham injury group is used as the baseline for all comparisons. *a indicates significant increase compared with the sham injury group; *b indicates significant decrease compared with the vehicle + TBI group.