Inhibition of 2-AG hydrolysis differentially regulates blood brain barrier permeability after injury

Abstract

Background: Acute neurological insults caused by infection, systemic inflammation, ischemia, or traumatic injury are often associated with breakdown of the blood-brain barrier (BBB) followed by infiltration of peripheral immune cells, cytotoxic proteins, and water. BBB breakdown and extravasation of these peripheral components into the brain parenchyma result in inflammation, oxidative stress, edema, excitotoxicity, and neurodegeneration. These downstream consequences of BBB dysfunction can drive pathophysiological processes and play a substantial role in the morbidity and mortality of acute and chronic neurological insults, and contribute to long-term sequelae. Preserving or rescuing BBB integrity and homeostasis therefore represents a translational research area of high therapeutic potential.

Methods: Induction of general and localized BBB disruption in mice was carried out using systemic administration of LPS and focal photothrombotic ischemic insult, respectively, in the presence and absence of the monoacylglycerol lipase (MAGL) inhibitor, CPD-4645. The effects of CPD-4645 treatment were assessed by gene expression analysis performed on neurovascular-enriched brain fractions, cytokine and inflammatory mediator measurement, and functional assessment of BBB permeability. The mechanism of action of CPD-4645 was studied pharmacologically using inverse agonists/antagonists of the cannabinoid receptors CB1 and CB2.

Results: Here, we demonstrate that the neurovasculature exhibits a unique transcriptional signature following inflammatory insults, and pharmacological inhibition of MAGL using a newly characterized inhibitor rescues the transcriptional profile of brain vasculature and restores its functional homeostasis. This pronounced effect of MAGL inhibition on blood-brain barrier permeability is evident following both systemic inflammatory and localized ischemic insults. Mechanistically, the protective effects of the MAGL inhibitor are partially mediated by cannabinoid receptor signaling in the ischemic brain insult.

Conclusions: Our results support considering MAGL inhibitors as potential therapeutics for BBB dysfunction and cerebral edema associated with inflammatory brain insults.

Keywords: 2-arachidonoylglycerol; Blood-brain barrier; Monoacylglycerol lipase; Neuroinflammation; Neurovasculature.

Fig. 1

Transcriptomic profiling of neurovascular unit after LPS challenge. a Expression profile of cell type-specific makers in neurovasculature preparation. b Volcano plot of statistically significant upregulated (green) and downregulated (red) genes in the neurovascular unit after LPS challenge. c Gene ontology clusters for up- and downregulated genes identified using DAVID bioinformatics resource. Neurovasculature was isolated 4 h post last LPS dose. Data are means ± SEM, n = 4/5 mice per group

Fig. 2

Pharmacokinetic and pharmacodynamic profiling of CPD-4645 in naïve mouse brain. a Structure of the 2-AG hydrolysis inhibitor CPD-4645. Total CPD-4645 concentrations in brain and plasma (b) and bulk levels of brain 2-AG (c) and AA (d) at given time points following single 10 mg/kg subcutaneous dose of CPD-4645 in CD1 mice. Data are means ± SEM, n = 3/5 mice per group

Fig. 3

Pharmacodynamic and anti-inflammatory activity of CPD-4645 in LPS-challenged mouse brain. Inhibition of MAGL by CPD-4645 resulted in significant elevation of brain 2-AG (a) and concomitant reduction in brain AA (b). Levels of the proinflammatory cytokines, IL1β (c) and IL6 (d), were significantly modulated in brain tissue following LPS challenge and CPD-4645 treatment. Bar graphs were plotted with mean ± SEM and data analyzed using one-way analysis of variance (ANOVA) with Bonferroni post-hoc comparisons. n = 10 mice per group. Significance is shown as *p < 0.05; **p < 0.01; ****p < 0.0001

Fig. 4

CPD-4645 alters LPS-induced gene expression profiles. a Volcano plots showing expression changes of the LPS upregulated genes (left, green points) after treatment with CPD-4645 (right). b Bubble plot showing changes in expression of genes that are induced by LPS and related to cytokines and inflammation. The y-axis represents the log2-fold change due to LPS challenge while the x-axis represents the log2-fold change due to CPD-4645 treatment. Dotted line represents a return to basal expression level. Size of the bubbles represents the multiple comparison adjusted p values for vehicle versus CPD-4645 treatment. Neurovasculature was isolated 4 h post last LPS dose. n = 4/5 mice per group

Fig. 5

Genes related to BBB (dys) function and proteases are differentially expressed after LPS challenge and treatment with CPD-4645. a Changes in the expression of 40 genes related to BBB function and dysfunction. The y-axis represents the log2-fold change due to LPS challenge while the x-axis represents the log2-fold change due to CPD-4645 treatment. Dotted line represents a return to basal expression level. Size of the bubbles represents the multiple comparison adjusted p values for vehicle vs. CPD-4645 treatment. b Expression levels of selected BBB genes. c Expression levels of selected extracellular proteases. n = 4–5 mice/group. Significance is shown as adjusted p values *p ≤ 0.05, **p < 0.01, *** p < 0.001. Differential gene expression was analyzed using the limma package in R/Bioconductor (see Methods)

Fig. 6

Inhibition of 2-AG hydrolysis reduces LPS-induced BBB permeability. a, b Fibrinogen levels in b plasma and the a ratio of brain to plasma fibrinogen were assessed by ELISA. n = 5/7 mice per group. c, d Fluorescent immunostaining in the striatum for fibrinogen (red) and vascular marker (CD31; green) demonstrated leakage of fibrinogen into the brain with vehicle treatment, whereas vascular integrity was preserved when (e, f) MAGL was inhibited. g Extravascular fibrinogen was semi-quantitated in fluorescently labeled sections of the striatum. Bar graphs were plotted with mean ± SEM and data analyzed using one-way analysis of variance (ANOVA) with Tukey post-hoc comparisons. n = 5/7 mice per group. Significance is shown as *p < 0.05, **p < 0.01. Scale bar = 20 μm

Fig. 7

CPD-4645 rescues BBB integrity after ischemic challenge. The rose bengal photothrombosis model induces a focal ischemic injury that leads to BBB dysfunction. CPD-4645 administered 30 min post lesion (a, b) reduced the penumbra size (a) and BBB permeability (b) as assessed by extravasation of Cadavarin⁵⁵⁵ into the brain parenchyma. CPD-4645 administered 6 h post ischemic lesion (c, d) reduces penumbra size (c) and BBB permeability (d) as assessed by extravasation of 70 kDa FITC conjugated dextran in the brain parenchyma. Data were plotted with means ± SEM and data analyzed using unpaired t test (n = 5 mice/group). Significance is shown as *p ≤ 0.05 and **p < 0.01 compared to vehicle group

Fig. 8

CPD-4645 rescues BBB integrity via endocannabinoid dependent and independent mechanisms. Blockade of endocannabinoid signaling with rimonabant and AM630 (3 mg/kg) does not reverse the BBB protective effects of MAGL inhibition in the inflammation-driven LPS model (a), whereas blockade of endocannabinoid signaling partially reverses the protective effects of MAGL inhibition (administered 30 min post lesion) in the photothrombotic ischemia-driven model (b) as measured by extravasation of 70 kDa FITC-dextran into the brain parenchyma. Data were plotted with means ± SEM (n = 6–9 mice/group) and analyzed with one-way ANOVA with Bonferroni’s post-hoc test. Significance is shown as *p ≤ 0.05, **p ≤ 0.01