Disrupted Blood-Brain Barrier and Mitochondrial Impairment by Autotaxin-Lysophosphatidic Acid Axis in Postischemic Stroke

Abstract

Background The loss of endothelial integrity increases the risk of intracerebral hemorrhage during ischemic stroke. Adjunct therapeutic targets for reperfusion in ischemic stroke are in need to prevent blood-brain barrier disruption. Recently, we have shown that endothelial permeability is mediated by lysophosphatidic acid (LPA), but the role of autotaxin, which produces LPA, remains unclear in stroke. We investigate whether autotaxin/LPA axis regulates blood-brain barrier integrity after cerebral ischemia. Methods and Results Ischemic stroke was induced in mice by middle cerebral artery occlusion for 90 minutes, followed by 24-hour reperfusion. The therapeutic efficacy of autotaxin/LPA receptor blockade was evaluated using triphenyl tetrazolium chloride staining, Evans blue permeability, infrared imaging, mass spectrometry, and XF24 analyzer to evaluate blood-brain barrier integrity, autotaxin activity, and mitochondrial bioenergetics. In our mouse model of ischemic stroke, the mRNA levels of autotaxin were elevated 1.7-fold following the cerebral ischemia and reperfusion (I/R) group compared with the sham. The enzymatic activity of autotaxin was augmented by 4-fold in the I/R group compared with the sham. Plasma and brain tissues in I/R group showed elevated LPA levels. The I/R group also demonstrated mitochondrial dysfunction, as evidenced by decreased (P<0.01) basal oxygen consumption rate, mitochondrial ATP production, and spare respiratory capacity. Treatment with autotaxin inhibitors (HA130 or PF8380) or autotaxin/LPA receptor inhibitor (BrP-LPA) rescued endothelial permeability and mitochondrial dysfunction in I/R group. Conclusions Autotaxin-LPA signaling blockade attenuates blood-brain barrier disruption and mitochondrial function following I/R, suggesting targeting this axis could be a new therapeutic approach toward treating ischemic stroke.

Keywords: autotaxin; blood‐brain barrier; ischemic stroke; lysophosphatidic acid; permeability; superoxide radicals.

Figure 1. Elevated autotaxin (ATX) activity following ischemia and reperfusion (I/R).

A, Triphenyl tetrazolium chloride staining of mouse brain slices and respective infarct volume (percentage of the hemisphere) measured in sham and I/R mice. B, Mouse brain cryosections stained for microglia (Iba1) in sham and I/R mice ipsilateral cortex. Bar=200 µm. C, ATX mRNA expression was measured in ipsilateral cortex brain tissue lysate from sham and I/R mice. D, Enzymatic activity test for ATX measured with AR‐2 fluorescence, quantified as relative fluorescence unit (RFU) in sham and I/R mouse brain slices. E, Lysophosphatidylcholine subspecies in plasma from sham and I/R mice, measured with liquid chromatography–mass spectrometry, quantified relative to 18:1 LPC. All values are mean±SD compared with the sham group using Mann‐Whitney U test. DAPI indicates 4′,6‐diamidino‐2‐phenylindole. NI, near‐infrared.

Figure 2. Lysophosphatidic acid (LPA) escalation following ischemia and reperfusion (I/R).

A, LPA subspecies in plasma from sham and I/R mice, measured with high‐performance liquid chromatography–tandem mass spectrometry (liquid chromatography–mass spectrometry), quantified relative to 18:1 LPA. B, LPA ELISA immunoassay performed in ipsilateral brain tissue lysate and quantified as ng/mg protein in sham and I/R. C, Mouse brain cryosections stained for LPA (green fluorescence) and 4′,6‐diamidino‐2‐phenylindole (DAPI) (blue fluorescence) in sham and I/R mice; the arrows show magnified images of the respective regions in the brain Bar=1 mm for the whole brain, and bar=500 µm for magnified images. D, Immunofluorescence in mouse brain cryosections performed for LPA and cluster of differentiation 31 (CD31) in sham and I/R on ipsilateral and contralateral cortex. Bar=200 µm. E, LPA receptor mRNA expression was measured in brain tissue lysate from sham and I/R mice. All values are mean±SD compared with the sham group using Mann‐Whitney U test. L indicates left; LPAR, LPA receptor; OCR, oxygen consumption rate; and R, right.

Figure 3. Autotaxin (ATX) inhibitors in middle cerebral artery occlusion decrease ATX activity and lysophosphatidic acid (LPA).

A, Enzymatic activity test for ATX, measured with AR‐2 fluorescence, quantified as relative fluorescence unit (RFU), in sham, ischemia and reperfusion (I/R), HA130, and PF8380 mouse brain slices. B, Lysophosphatidylcholine (LPC) subspecies in plasma from sham, I/R, HA130, and PF8380 mice measured with high‐performance liquid chromatography–tandem mass spectrometry (liquid chromatography–mass spectrometry [LC‐MS]) quantified relative to 18:1 LPC. C, LPA subspecies in plasma from sham, I/R, HA130, and PF8380 mice measured with LC‐MS, quantified relative to 18:1 LPA. D and E, Graph represents oxygen consumption rate (OCR) (pmol/min per µg protein) measurements in isolated mitochondria from sham, I/R, HA130, PF8380, or BrP‐LPA mouse brain tissue at baseline and after sequential addition of oligomycin, carbonyl cyanide p‐trifluoro‐methoxyphenyl hydrazone (FCCP), and antimycin A+rotenone. F, Spare respiratory capacity, ATP turnover, and maximum respiration values analyzed in sham, I/R, HA130, PF8380, or BrP‐LPA mice. All values are mean±SD. *P<0.05, **P<0.01 (1‐way ANOVA, followed by the Tukey, post hoc test, was performed).

Figure 4. Biochemical assays improved with the use of inhibitors.

A, Triphenyl tetrazolium chloride staining of mouse brain slices and respective infarct volume (percentage of the hemisphere) measured in sham, ischemia and reperfusion (I/R), HA130, PF8380, or BrP‐LPA mice (n=5 in each group). B, Superoxide measured using high‐performance liquid chromatography in brain tissue lysate after staining with a dihydroethidium fluorescence probe in sham, I/R, HA130, PF8380, or BrP‐LPA mice. C through E, Superoxide dismutase (SOD) activity, catalase activity, and glutathione peroxidase activity quantified in brain tissue lysate from sham, I/R, HA130, PF8380, or BrP‐LPA mice. F, Glutathione levels were measured in sham, I/R, HA130, PF8380, or BrP‐LPA mice. All values are mean±SD (1‐way ANOVA, followed by the Tukey, post hoc test, was performed).

Figure 5. Improved endothelial permeability with the use of inhibitors.

A, Evans blue fluorescence measured and quantified with relative fluorescence unit (RFU) in whole brains of sham, ischemia and reperfusion (I/R), HA130, PF8380, or BrP‐LPA mice. B, Immunofluorescence staining for claudin‐5 in ipsilateral cortex of sham, I/R, HA130, PF8380, or BrP‐LPA mice. C, Immunofluorescence staining for zonula occludens‐1 in ipsilateral cortex of sham, I/R, HA130, PF8380, or BrP‐LPA mice. Bar=200 µm. All values are mean±SD (1‐way ANOVA, followed by the Tukey, post hoc test, was performed). CD31 indicates cluster of differentiation 31; and DAPI, 4′,6‐diamidino‐2‐phenylindole.