Increase in Blood-Brain Barrier (BBB) Permeability Is Regulated by MMP3 via the ERK Signaling Pathway

Abstract

Background: The blood-brain barrier (BBB) regulates the exchange of molecules between the brain and peripheral blood and is composed primarily of microvascular endothelial cells (BMVECs), which form the lining of cerebral blood vessels and are linked via tight junctions (TJs). The BBB is regulated by components of the extracellular matrix (ECM), and matrix metalloproteinase 3 (MMP3) remodels the ECM's basal lamina, which forms part of the BBB. Oxidative stress is implicated in activation of MMPs and impaired BBB. Thus, we investigated whether MMP3 modulates BBB permeability.

Methods: Experiments included in vivo assessments of isoflurane anesthesia and dye extravasation from brain in wild-type (WT) and MMP3-deficient (MMP3-KO) mice, as well as in vitro assessments of the integrity of monolayers of WT and MMP3-KO BMVECs and the expression of junction proteins.

Results: Compared to WT mice, measurements of isoflurane usage and anesthesia induction time were higher in MMP3-KO mice and lower in WT that had been treated with MMP3 (WT+MMP3), while anesthesia emergence times were shorter in MMP3-KO mice and longer in WT+MMP3 mice than in WT. Extravasation of systemically administered dyes was also lower in MMP3-KO mouse brains and higher in WT+MMP3 mouse brains, than in the brains of WT mice. The results from both TEER and Transwell assays indicated that MMP3 deficiency (or inhibition) increased, while MMP3 upregulation reduced barrier integrity in either BMVEC or the coculture. MMP3 deficiency also increased the abundance of TJs and VE-cadherin proteins in BMVECs, and the protein abundance declined when MMP3 activity was upregulated in BMVECs, but not when the cells were treated with an inhibitor of extracellular signal related-kinase (ERK).

Conclusion: MMP3 increases BBB permeability following the administration of isoflurane by upregulating the ERK signaling pathway, which subsequently reduces TJ and VE-cadherin proteins in BMVECs.

Figure 1

Experimental design. Experiments were conducted both in vivo and in vitro, as indicated. In vivo experiments were conducted in wild-type mice (WT), MMP3-KO mice (KO) mice, and WT mice administered MMP3 (WT + MMP3) and included assessments of susceptibility to isoflurane anesthesia and BBB permeability to intravenously administered dyes (Evans blue, sodium-FITC). In vitro assessments included measurements of barrier integrity via TEER using ECIS system in monolayers of BMVECs and in cocultures of BMVECs and astrocytes via Transwell assay. The abundance of junctional proteins was also evaluated via Western blot.

Figure 2

MMP3 is predominantly expressed in BMVECs. (a) MMP3 mRNA abundance was evaluated via q-PCR in BMVECs and astrocytes isolated from the brains of WT (BMVEC-WT and Astrocyte-WT) and MMP3-KO (BMVEC-KO and Astrocyte-KO) mice; results were normalized to measurements in BMVEC-WT. ^∗∗∗∗p < 0.0001, ^####p < 0.0001. (b) MMP3 protein abundance was evaluated via ELISA in culture medium from the indicated cell populations. ^∗∗∗∗p < 0.0001, ^####p < 0.0001. (c) MMP3 mRNA abundance was evaluated in microvascular endothelial cells (MVECs) isolated from the brains, hearts, lungs, kidneys, and spleens of WT mice results were normalized to measurements in brain MVECs. ^∗∗∗∗p < 0.0001.

Figure 3

MMP3 enhances the anesthetic effect of isoflurane. (a) The genotypes of WT (WT+/+), heterozygous MMP3-KO (MMP3–/+), and homozygous MMP3-KO (MMP3–/–) mice were confirmed via PCR; the WT and KO alleles were identified with 517-bp and 670-bp fragments, respectively. (b) MMP3 protein abundance was evaluated via ELISA in the serum of WT and homozygous MMP3-KO (KO) mice. ^∗∗∗∗p < 0.0001. (c–e) WT mice, MMP3-KO mice, and WT mice treated with MMP3 were anesthetized with inhaled isoflurane, and the (c) time to the onset of anesthesia, (d) time to emergence from anesthesia, and (e) total volume of isoflurane was recorded. ^∗∗∗∗p < 0.0001.

Figure 4

MMP3 increases BBB permeability. (a, b) Evans blue dye was administered via tail-vein intracardial injection to WT mice, MMP3-KO mice (KO), and WT mice that had been treated with MMP3. Two hours later, saline was injected to clear the dye from the vasculature, and the brains were harvested. Extravasation of the dye was evaluated (a) qualitatively in brain images and (b) quantitatively via spectrophotometric measurements of absorbance (632-nm wavelength). Dye quantities were calculated by comparing absorbance measurements to a standard curve. ^∗∗∗∗p < 0.0001, ^∗∗∗p < 0.001. (c) Sodium-FITC was administered via tail-vein injection to mice, and dye extravasation was quantified 2 hours later via fluorescence intensity. Dye quantities were calculated by comparing fluorescence measurements to a standard curve. ^∗∗∗p < 0.001.

Figure 5

MMP3 reduces the integrity of BMVEC monolayers. (a, c) TEER measurements were recorded in monolayers of WT and MMP3-KO (KO) BMVECs during treatment with the indicated combinations of phosphate-buffered saline (PBS), isoflurane, MMP3, and/or NNGH; treatment was administered one hour after TEER was initiated. (b, d) Endothelial barrier integrity was evaluated by calculating the mean TEER. ^∗∗∗∗p < 0.0001, ^####p < 0.0001. (e, f) WT or MMP3-KO (KO) BMVECs were grown with primary mouse astrocytes in Transwell chambers and treated with PBS, LPS, LPS, and NNGH, or MMP3, as indicated; then, the chambers were suspended in the wells of a 6-well plate, (e) red (40KD) or (f) blue (3KD) FITC-dextran was added to the chamber, and permeability was evaluated 24 hours later by measuring the intensity of dextran fluorescence in the plate wells. ^∗∗∗∗p < 0.0001, ^∗∗∗p < 0.001, ^∗∗p < 0.010, ^####p < 0.0001. Quantified data are summarized for 5 independent experiments.

Figure 6

MMP3 regulates the abundance of junction proteins in BMVECs. (a–f) The abundance of MMP3, ZO-1, Occludin, VE-cadherin, and Claudin 5 were (a) evaluated via Western blot in WT BMVECs, WT BMVECs that had been treated with MMP3 (WT+MMP3), and MMP3-KO BMVECs (KO). Cofilin abundance was also evaluated to confirm equal loading, and then (b) MMP3, (c) ZO-1, (d) Occludin, (e) VE-cadherin, and (f) Claudin 5 measurements were quantified via normalization to Cofilin. ^∗∗∗∗p < 0.0001, ∗∗∗p < 0.001, ^∗∗p < 0.010. (g) WT, WT+MMP3, and MMP3-KO BMVECs were incubated with ZO-1 primary antibodies and fluorescent secondary antibodies (bar = 20 μm), nuclei were counterstained with DAPI, and then (h) ZO-1 abundance was quantified via measurements of fluorescence intensity. ^∗∗∗∗p < 0.0001. Quantified data are summarized for 3 experiments.

Figure 7

MMP3 regulation of BMVEC junction proteins is mediated by ERK. WT and MMP3-KO (KO) BMVECs were treated with LPS, saline, and/or an ERK inhibitor as indicated; then (a) MMP3, (b) phosphorylated ERK (p-ERK), (c) ZO-1, (d) Occludin, (e) VE-cadherin, and (f) Claudin 5 abundance were evaluated via Western blot and normalized to Cofilin abundance (^∗p < 0.001 vs. WT+PBS in a; ^∗p < 0.010 vs. WT+PBS, ^#p < 0.010 for all other panels (Please verify)). Quantified data are summarized for 3 experiments.

Figure 8

Schematic model for MMP3 regulation of BBB permeability. MMP3 is produced by BMVECs in response to external factors and phosphorylates ERK, which subsequently disrupts tight junctions between adjacent ECs, thereby increasing BBB permeability. BBB integrity can be preserved via ERK inhibition or by blocking MMP3 activity with NNGH.