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.

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1. Introduction

The blood-brain barrier (BBB) regulates the exchange of molecules between the brain and peripheral blood and is composed primarily of endothelial cells (ECs), as well as pericytes and the endfeet of astrocytes [1]. Brain microvascular endothelial cells (BMVECs) form the lining of cerebral blood vessels and are linked to each other via tight junctions (TJs), and the structural stability of TJs is maintained, in part, by adherens junctions (AJs) [2, 3]. Numerous studies have shown that increases in BBB permeability are associated with the degradation of TJs and AJs in ECs [4–7], which can disrupt brain function [8, 9] by augmenting the passage of macrophages, leukocytes, endotoxins, bacteria, and drugs from the peripheral circulation into the brain [10–12]. Thus, the regulation of BBB permeability is crucial both for protecting the brain from harmful components of the peripheral circulation and for the treatment of central nervous system (CNS) disorders. The BBB is regulated primarily via interactions between its cellular components and the extracellular matrix (ECM); thus, matrix metalloproteinases (MMPs), which are the principal drivers of ECM degradation and remodeling, appear to have a key role in breaking down the BBB. [13]. Furthermore, activation of the extracellular signal-related kinase (ERK) pathway has been shown to induce BBB hyperpermeability by changing the composition of TJs [14–16], but TJ proteins in sheep pleura remained intact after exposure to MMP2 or MMP9 [17, 18], which suggests that these two MMPs regulate BBB permeability via an alternative mechanism. MMP3, another member of the MMP family, is a zinc-dependent protease that is activated through autocleavage, and activated MMP3 remodels the basal lamina of the ECM, which forms part of the BBB [19]. However, whether MMP3 regulates BBB integrity, and if so, whether its role is mediated by ERK signaling and/or changes in TJ stability, remains unclear.

For most molecules, passive permeability of the BBB is low, but inhaled anesthetics such as isoflurane are highly fat soluble and, consequently, can traverse the BBB and enter brain tissue [20]. Nevertheless, factors that increase BBB permeability augment the passage of isoflurane into the CNS and enhance anesthesia [9]. Thus, the experiments presented in this report evaluated the potential role of MMP3 in BBB permeability by combining in vivo assessments of isoflurane anesthesia and dye extravasation in wild-type (WT) and MMP3-deficient mice with in vitro assessments of the integrity of BMVEC cell layers and the expression of junction proteins. Our results indicate that MMP3 could be therapeutically targeted to manipulate BBB permeability and treat neurological disease.

2. Materials and Methods

2.1. Animals

All animal procedures were carried out in accordance with the guidelines provided by the Institutional Animal Care and Use Committee of the University of Chicago (Chicago, Illinois). The C57BL/6-MMP3 deficient (MMP3−/−) mice were a GEM Collection Model TTM-610 and were generated as described before [21]. The line was backcrossed for 12 generations with C57BL/6 mice, which were provided by the Taconic Biosciences company. A PCR-based analysis was employed to genotype mice. Mice were housed in a room at 22-24°C on a 12-hour light/dark cycle and received drinking water ad libitum. We used 8-week-old male mice in this study.

Genotyping: We isolated genomic DNA from mouse tail clips using the Puregene DNA isolation kit from Gentra Systems according to the manufacturer’s instruction. About 10 ng of the genomic DNA was used for PCR.

2.2. Experimental Design

The experimental design of the current study is described below and illustrated in Figure 1. All experiments were conducted in a blinded fashion to investigate the influence of MMP3 on the BBB integrity, we assigned all the mice to three different groups under isoflurane, including WT (MMP3+/+), KO (MMP3-/-), and WT+MMP3 (the mice were administered a recombinant human MMP3 via tail intravenous injection (iv), which was purchased from Abcam). Each experimental group contained 8 mice. In vivo, the BBB permeability was evaluated by measuring Evans blue and sodium-FITC-dextrans extravasation [22]. In vitro study, the BBB permeability was detected in an in vitro model of BBB. To evaluate the role of MMP3 on BBB junction proteins, we used the MMP3 recombinant human protein to stimulate brain microvascular endothelial cells (BMVECs). Afterward, we detected the protein level of MMP3, ZO-1, occludin, VE-cadherin, and claudin-5 by Western blot and fluorescence staining. To increase the MMP3 levels, lipopolysaccharide (LPS) in a dose of 100 μg/mL as a stimulus to increase the MMP3 levels [23, 24], and then, the ERK inhibitor FR1080204 [25] (purchased from Sigma-Aldrich) was administered to investigate the mechanism behind the effects of MMP3 on the BBB.

[figure omitted; refer to PDF]

In vivo assessments of MMP activity are technically challenging, which may partially explain why the results from previous investigations of MMPs in endothelial barrier function have been limited and somewhat inconsistent. For example, both MMP2 and MMP9 downregulated ZO-1 expression and enhanced BBB permeability in response to focal cerebral ischemia-reperfusion injury [39], but MMP9 deficiency did not reduce disruption of the BBB during viral encephalomyelitis [40]. The role of MMP3 in BBB maintenance is even less well-studied, but our observation that MMP3 increases BBB permeability by reducing the expression of several junction proteins, including ZO-1, claudin-5, and occludin, is consistent with a previous report that MMP3 disrupts the blood-spinal cord barrier [41]. Furthermore, ERK signaling appears to regulate TJ protein expression in the proximal epididymis of mice [42], and the results reported here indicate that MMP3-induced declines in TJ protein levels are strongly dependent on ERK activation: levels of ERK phosphorylation correlated positively with MMP3 activity and negatively with TJ protein levels, and ERK inhibition increased TJ protein levels in WT BMVEC monolayers, but not in monolayers of MMP3-KO BMVECs. The mechanism that links ERK phosphorylation to the disruption of BBB junctional proteins is unknown but could involve the ubiquitin ligase complex, which targets phosphorylated proteins for degradation [43]; these topics will be addressed in future studies.

MMPs are essential for normal brain function, but elevated levels of MMP expression are believed to contribute to pathological CNS conditions by damaging the neurovascular unit and disrupting the BBB, perhaps via the degradation of TJ and basement-membrane proteins [13, 44]. Oxidative stress is also implicated in activation of MMPs and impaired BBB [45]. Several studies have suggested that MMP2 and MMP9 may contribute to BBB breakdown [13, 17]—for example, MMP9 appears to mediate the loss of TJ stability observed in mice with 1,2-dichloroethane-induced brain edema [46]—and our observation that MMP3 mRNA levels were much higher in BMVECs than in MVECs from other organs (e.g., the heart, lungs, spleen, and kidney) suggests that it may have a unique and essential role in BMVECs, particularly since many of the ECM proteins that form the basal lamina of cerebral blood vessels can be proteolysed by MMP3 [19]. Isoflurane has also been linked to BBB disruption [47, 48], but may protect against acute lung injury by maintaining endothelial barrier integrity [11], so the effect of isoflurane on BBB permeability remains somewhat unclear.

One of the significant findings of this study is that MMP3 was associated with elevated anesthetic sensitivity and BBB opening. Because the BBB is the primary regulator of exchange between the peripheral circulation and the brain, and is the key surface through which systemically administered drugs access the CNS [8], the complexity of the BBB provides many unique opportunities for drug delivery. Thus, our results suggest that MMP3 may be a useful adjunctive treatment for enhancing the efficacy of other neurotherapeutics [49], which could reduce the occurrence and/or severity of side effects (as well as cost) by enabling patients to be treated with lower doses of the primary treatment [35, 50]. However, increases in BBB permeability could also enable blood-borne immune cells to enter the brain and provoke a neuroinflammatory response [51], as illustrated by growing evidence that BBB disruption is associated with brain inflammatory conditions such as Alzheimer’s disease and multiple sclerosis [52]. Oxidative stress is also implicated in MMP activation and blood-brain barrier injury [53]. However, despite enormous efforts, CNS drug discovery still relies on improving the BBB, and the success of CNS therapeutic development depends on techniques for modulating BBB permeability and the kinetics of drug distribution to ensure optimal CNS penetration [35].

Unlike other MMPs, MMP3 is predominantly expressed in ECs, and our results indicate that it is more highly expressed in the brain than in other organs; nevertheless, our in vivo experiments were conducted with global MMP3-KO mice, so our observations in these animals could have been influenced by the loss of MMP3 expression in non-ECs, as well as BMVECs. Furthermore, since direct MMP3 administration did not increase MMP3 levels in BMVECs, LPS was used to upregulate MMP3 expression in our in vitro studies and, consequently, we cannot exclude a potentially MMP3-independent role for LPS in TJ stability. Future studies will address these limitations by conducting experiments in EC-specific MMP3-KO mice, by investigating the use of other MMP3 inhibitors, and by determining how LPS regulates MMP3 expression and BBB integrity.

5. Conclusions

In summary, the results presented here are the first to reveal the function of MMP3 in the BBB and suggest that it has an essential role in the brain microvasculature that differs from its function in other vessels. We have shown that MMP3 increases BBB permeability by upregulating the ERK signaling pathway, which subsequently reduces TJ and AJ protein abundance in BMVECs. Oxidative stress often leads to impairment of BBB. Since the BBB is the primary regulator of exchange between the peripheral blood and the brain, our observations likely have important implications for treating neuroinflammatory conditions and other CNS disorders involving the endothelial MMP3 pathway.

Authors’ Contributions

Qin and Rongxue conceived the study design and wrote the manuscript. Qin performed experiments with assistance from Mei, Cristian, Lifeng, Albert, and Nikola. Qin analyzed the data. Qiong, John, and JK reviewed and contributed to the writing of the manuscript. Rongxue supervised the research. All the authors read and approved the final version of the manuscript.

Funding

Rongxue Wu is supported by 1R01HL140114-01A1. James K. Liao is supported by HL136962.

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