Biomolecules & Therapeutics 2024; 32(3): 319-328  https://doi.org/10.4062/biomolther.2024.052
Lysophosphatidic Acid Receptor 1 Plays a Pathogenic Role in Permanent Brain Ischemic Stroke by Modulating Neuroinflammatory Responses
Supriya Tiwari, Nikita Basnet and Ji Woong Choi*
Laboratory of Neuropharmacology, College of Pharmacy and Gachon Institute of Pharmaceutical Sciences, Gachon University, Incheon 21936, Republic of Korea
*E-mail: pharmchoi@gachon.ac.kr
Tel: +82-32-820-4955, Fax: +82-32-820-4829
The first two authors contributed equally to this work.
Received: March 25, 2024; Revised: April 8, 2024; Accepted: April 9, 2024; Published online: April 17, 2024.
© The Korean Society of Applied Pharmacology. All rights reserved.

This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/4.0/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.
Abstract
Lysophosphatidic acid receptor 1 (LPA1) plays a critical role in brain injury following a transient brain ischemic stroke. However, its role in permanent brain ischemic stroke remains unknown. To address this, we investigated whether LPA1 could contribute to brain injury of mice challenged by permanent middle cerebral artery occlusion (pMCAO). A selective LPA1 antagonist (AM152) was used as a pharmacological tool for this investigation. When AM152 was given to pMCAO-challenged mice one hour after occlusion, pMCAO-induced brain damage such as brain infarction, functional neurological deficits, apoptosis, and blood-brain barrier disruption was significantly attenuated. Histological analyses demonstrated that AM152 administration attenuated microglial activation and proliferation in injured brain after pMCAO challenge. AM152 administration also attenuated abnormal neuroinflammatory responses by decreasing expression levels of pro-inflammatory cytokines while increasing expression levels of anti-inflammatory cytokines in the injured brain. As underlying effector pathways, NF-κB, MAPKs (ERK1/2, p38, and JNKs), and PI3K/Akt were found to be involved in LPA1-dependent pathogenesis. Collectively, these results demonstrate that LPA1 can contribute to brain injury by permanent ischemic stroke, along with relevant pathogenic events in an injured brain.
Keywords: Lysophosphatidic acid receptor 1 (LPA1), AM152, Permanent brain ischemic stroke, Permanent middle cerebral artery occlusion (pMCAO), Microglia, Pro-inflammatory cytokines
INTRODUCTION

Lysophosphatidic acid (LPA) is well known as a main bioactive lysophospholipid along with sphingosine 1-phosphate (S1P). LPA is present in a variety of cells and influences cellular functions by binding to its specific six G protein-coupled receptors (LPA1 to LPA6) and activating diverse effector pathways (Yanagida and Shimizu, 2023). Receptor-mediated LPA signaling is known to contribute to the pathogenesis of many diseases, boosting research on drug development by targeting LPA receptors (Stoddard and Chun, 2015). Among LPA receptors, LPA1 has become a promising target for drug development to treat pulmonary fibrosis and psoriasis (Stoddard and Chun, 2015) due to clinical trials (ClinicalTrials.gov ID for pulmonary fibrosis, NCT01766817; ClinicalTrials.gov ID for psoriasis, NCT02763969) on its specific antagonist, AM152 (Palmer et al., 2018). In addition to these diseases, LPA1 has been identified as a critical pathogenic factor for other diseases such as systemic sclerosis, cancer, neuropathic pain, spinal cord injury, brain trauma, neuropsychiatric disorders, developmental disorders, and ischemic stroke through studies employing genetic or pharmacological tools (Choi and Chun, 2013; Gaire and Choi, 2021; Xiao et al., 2021). These findings strongly indicate that targeting LPA1 is a promising therapeutic strategy for drug development to treat diverse diseases.

Intriguingly, in brain ischemic stroke that occurs by a blockage or rupture of blood vessels and causes death or severe disability, LPA1 has been found to play a pathogenic role. Either LPA1 antagonist administration or its genetic deletion can attenuate brain injury such as brain infarction, neurological dysfunction, neuronal cell death, and pain (Halder et al., 2013; Gaire et al., 2019b, 2020). However, all these findings have been demonstrated solely in studies using a mouse model of transient brain ischemic stroke (a transient middle cerebral artery occlusion (tMCAO)-challenged mouse model), in which ischemia/reperfusion brain injury occurs. Indeed, there is no evidence for the role of LPA1 in permanent brain ischemic stroke, the other type of ischemic stroke. However, whether a therapeutic target is important for permanent brain ischemic stroke should be considered since 40-50% of stroke patients can undergo permanent ischemic stroke without reperfusion (Ma et al., 2020). In this respect, whether LPA1 could also play a pathogenic role in permanent brain ischemic stroke is worthy of investigation.

In the current study, we determined the pathogenic role of LPA1 in permanent brain ischemic stroke using a mouse model that was subjected to permanent middle cerebral artery occlusion (pMCAO; a challenge of MCAO without reperfusion). For this purpose, we used AM152, a well-known selective antagonist of LPA1, which was given to mice one hour after occlusion. The role of LPA1 in brain injury after pMCAO challenge was determined through analyses of brain infarction, functional neurological deficits, cell death, and blood-brain barrier (BBB) disruption. We also determined whether the pathogenic role of LPA1 could be associated with microglial activation, proliferation, and upregulation of pro-inflammatory cytokines in injured brains. Furthermore, we determined which effector pathways of LPA1 could be associated with brain injury.

MATERIALS AND METHODS

Animals

Male ICR mice (32 ± 2 g, six weeks old) were obtained from Orient Bio Co., Ltd. (Seongnam, Korea) and acclimatized to controlled conditions (diurnal lights of 12 h/12 h light/dark, humidity of 60 ± 10%, and temperature of 22 ± 2°C). All animal experiments were performed in compliance with guidelines from the Gachon Institutional Animal Care and Use Committee (GIACUC) (approved animal protocol number: GIACUC-R2021012).

pMCAO challenge

Mice were subjected to pMCAO challenge using an intraluminal suture occlusion method (Crupi et al., 2018). Briefly, mice were anesthetized with isoflurane (3% for induction and 1.5% for maintenance in an air mixture of 70% N2O with 30% O2). Under a stereo-dissecting microscope, a ventral neck incision of 1 cm was made, fat and fascia were removed, the right common carotid artery (CCA) was separated from the vagus nerve, and the external carotid artery (ECA) was ligated. MCAO was induced by inserting a 5-0 monofilament coated with silicone (9-mm-long with a tip with a diameter of 0.21-0.22 mm) from the point of internal CCA bifurcation to middle cerebral artery (MCA). Sham-operated mice received the same surgical procedure without MCAO. During the operation of pMCAO, the body temperature of each mouse was maintained at 37°C.

AM152 administration

After pMCAO challenge, mice were randomly assigned to a vehicle (1% DMSO in 10% Tween-80) or an AM152-administered group. AM152 was kindly provided by Dr. Dong Yun Shin (Gachon University, Incheon, Korea). To determine whether AM152 could exert neuroprotective effects against brain damage caused by pMCAO challenge, AM152 was administered to mice at different dosages (1.5, 5, and 15 mg/kg, p.o.) at one hour after occlusion.

Determination of functional neurological deficit score

Modified neurological severity score (mNSS) was used to assess functional neurological deficits. The neurological deficit score consists of the score for motor, sensory, balance, and reflex functions on an 18-point scale (0 for normal and 18 for maximum deficits) as previously described (Sapkota and Choi, 2022).

Determination of brain infarction volume

Brain infarction volume was measured one day after pMCAO challenge as previously described (Sapkota and Choi, 2022). Briefly, brains were removed from mice that were sacrificed with CO2 exposure, sliced, and made into coronal sections with a thickness of 2 mm using a brain matrix. Sections were then stained with 2% TTC (2,3,5-triphenyltetrazolium chloride) solution for 20 min at 37°C. Stained sections were photographed and infarction volume was analysed using ImageJ software (National Institute of Mental Health, Bethesda, MD, USA).

Histological analysis

Brain sampling and tissue preparation: Brain samples for histological analyses were obtained at one day or three days after pMCAO challenge. Mice were anesthetized with isoflurane, perfused with ice-cold phosphate buffered saline (PBS), and fixed with 4% paraformaldehyde (PFA). Harvested brains were further fixed in 4% PFA overnight, incubated in 30% sucrose solution for cryoprotection, and embedded in Tissue-Tek® Optimal Cutting Temperature compound. Brains were frozen with dry ice and sectioned into 20 µm sections using a cryostat (RD-2230, Round fin, Liaoning, China).

Fluoro-Jade B (FJB) staining: Cell death was evaluated by staining with FJB (Zhang et al., 2023). Cryostat sections were washed with distilled water and rehydrated with alcohol series (100% ethanol for 3 min, 70% ethanol for 1 min, and 30% ethanol for 1 min) and water. Rehydrated sections were oxidized in 0.06% potassium permanganate for 15 min, washed with water, and stained with 0.001% FJB in 0.1% acetic acid solution for 30 min at room temperature (R/T). Stained sections were rinsed with water, dried on a slide warmer, cleaned with xylene, and mounted with Entellan media (Merck, Darmstadt, Germany).

Claudin-5/CD31 double immunofluorescence: Claudin-5/CD31 double immunofluorescence was carried out to determine BBB disruption (Gaire et al., 2018a). Brain sections were washed, post-fixed with 4% PFA, incubated in 50 mM ammonium chloride for quenching autofluorescence, exposed to 1% H2O2 in 1% NaOH for antigen retrieval, and blocked with 1% FBS in PBS containing 0.3% Triton X-100. Sections were labeled with rat anti-CD31 (1:300, Dianova, Hamburg, Germany) and rabbit anti-claudin-5 (1:500, Santa Cruz Biotechnology, Dallas, TX, USA) antibodies overnight at 4°C. Sections were further labeled with AF488- and Cy3-conjugated secondary antibodies (1:1,000, Jackson ImmunoResearch, West Grove, PA, USA) for 2 h at R/T and mounted with VECTASHIELD® mounting media (Vector Laboratories, Burlingame, CA, USA).

Iba1 immunohistochemistry: Iba1 immunohistochemistry was carried out to evaluate microglial activation (Sapkota and Choi, 2022). Briefly, brain sections were oxidized with 1% H2O2, blocked with 1% FBS, and labeled with a rabbit anti-Iba1 antibody (1:500, Wako Pure Chemicals, Osaka, Japan) overnight at 4°C. Sections were then labeled with a biotinylated secondary antibody (1:200, Santa Cruz Biotechnology), incubated with ABC reagent (1:100, Vector Laboratories), and stained with a 3-3-diaminobenzidine substrate (Dako, Santa Clara, CA, USA) solution to visualize Iba1-immunopositive signals. Stained sections were dehydrated with ascending grades of alcohol, cleared with xylene, and mounted with Entellan media.

5-Bromo-2’-deoxyuridine (BrdU) incorporation and BrdU/Iba1 double immunofluorescence: Microglial proliferation was determined by BrdU/Iba1 double immunofluorescence (Gaire et al., 2019a). Briefly, BrdU (50 mg/kg, i.p., Sigma-Aldrich, St. Louis, MO, USA) was injected to mice every 12 h for 2 days after pMCAO challenge. At the end of the experiment, cryostat brain sections were obtained. For BrdU/Iba1 double immunofluorescence, sections were post-fixed with 4% PFA, exposed to 2N HCl for DNA denaturation, neutralized with 0.1 M borate buffer, blocked with 1% FBS, and labeled with rabbit anti-Iba1 and rat anti-BrdU (1:200, Abcam, Cambridge, UK) antibodies overnight at 4°C. Sections were then labeled with AF488- and Cy3-conjugated secondary antibodies for 2 h at R/T and mounted with VECTASHIELD®.

NF-κB/Iba1 double immunofluorescence: NF-κB/Iba1 double immunofluorescence was carried out to determine NF-κB expression in activated microglia as previously described (Gaire et al., 2019a). Brain sections were washed, post-fixed with 4% PFA, incubated in 50 mM ammonium chloride for quenching fluorescence, exposed to 1% H2O2 in 1% NaOH for antigen retrieval, and blocked with 1% FBS containing 0.3% Triton X-100 in PBS. Sections were labeled with mouse anti-NF-κB (1:200, Santa Cruz Biotechnology) and rabbit anti-Iba1 (1:500) antibodies overnight at 4°C. Section was further labeled with AF488- and Cy3-conjugated secondary antibodies for 2 h at R/T and mounted with VECTASHIELD®.

Image preparation and quantification: Images of brain sections were acquired using a bright-field and fluorescence microscope (BX53, Olympus, Tokyo, Japan) equipped with a DP72 camera (Olympus) or a laser scanning confocal microscope (Eclipse A1 Plus, Nikon, Tokyo, Japan). Four different images of each brain region were used to quantify the number of immunopositive cells.

Quantitative real-time PCR (qRT-PCR) analysis

Total RNA was extracted from the ipsilateral brain hemisphere using RNAiso Plus (Takara, Kusatsu, Japan). Total RNA (1 µg) was then used to synthesize cDNA with All-in-One First-Strand cDNA Synthesis SuperMix (TransGen Biotech, Haidian, China). qRT-PCR was carried out using a StepOnePlusTM qRT-PCR system (Applied Biosystems, Foster City, CA, USA) with Power SYBR Green PCR master mix (Life Technologies, Carlsbad, CA, USA) and corresponding primers (Table 1). The mRNA expression levels of target genes were quantified using the 2−ΔΔCT method and normalized to β-actin.

Table 1 Primer sets used for qRT-PCR analysis

GeneForwardReverse
TNF-α5'-AGGGTCTGGGCCATAGAACT-3'5'-CCACCACGCTCTTCTGTCTAC-3'
IL-1β5'-TGTGAAATGCCACCTTTTGA-3'5'-TTGTTGATGTGCTGCTGTGA-3'
IL-65'-GAGGATACCACTCCCAACAGACC-3'5'-AAGTGCATCATCGTTGTTCATACA-3'
TGF-β15ˈ-CAACCCAGGTCCTTCCTAAA-3ˈ5ˈ-GGAGAGCCCTGGATACCAAC-3ˈ
IL-45ˈ- TCTGTGGTGTTCTTCGTTGCT-3ˈ5ˈ-GTCATCCTGCTCTTCTTTCTCG-3ˈ
IL-105ˈ-TGGCCTTGTAGACACCTTGG-3ˈ5ˈ-AGCTGAAGACCCTCAGGATG-3ˈ
β-actin5'-AGCCTTCCTTCTTGGGTATG-3'5'-CTTCTGCATCCTGTCAGCAA-3'


Western blot analysis

Ipsilateral brain hemispheres were homogenized using a tissue lysis buffer (Ottimolyse II, JUBIOTECH, Daejeon, Korea) supplemented with a protease inhibitor cocktail (Complete™ mini, Roche, Mannheim, Germany). Proteins in samples were separated on 10% SDS-PAGE, transferred to PVDF membranes, and blocked with 5% skim milk. These membranes were incubated with primary antibodies against cleaved caspase-3, Bcl-2, phospho-ERK1/2, ERK1/2, phospho-JNK, JNK, phospho-p38, p38, phospho-Akt, Akt (1:1,000, Cell signaling Technology, Danvers, MA, USA), and β-actin (1:5,000, Sigma-Aldrich) overnight at 4°C, followed by incubation with respective secondary antibodies (1:10,000, Jackson ImmunoResearch) for 2 h at R/T. Protein bands were visualized with an enhanced chemiluminescence solution (Dongin Biotech Co., Seoul, Korea). Expression levels of target proteins were measured with ImageJ software .

Statistical analysis

Data are presented as mean ± SEM. Statistical differences between two groups were analysed by Student’s t-test and differences among multiple groups were analysed by one-way ANOVA followed by Newman-Keuls test. Statistical significance was set at p<0.05.

RESULTS

LPA1 contributes to brain damage after pMCAO challenge

To determine whether LPA1 could mediate brain damage after pMCAO challenge, its selective antagonist, AM152, was administered at different doses (1.5, 5, and 15 mg/kg) at one hour after occlusion. Neuroprotective effects of AM152 were determined at 1 day after pMCAO challenge by analysing brain infarction volume and neurological deficit score. In the vehicle-administered pMCAO group, severe brain infarction was developed in both cerebral cortex and striatum, which was significantly attenuated by AM152 administration (Fig. 1A). Similarly, AM152 administration significantly improved neurological functions of pMCAO-challenged mice compared with vehicle control administration (Fig. 1B). Neuroprotective effects of AM152 were more prominent at a dose of 5 mg/kg. This dosage was used for further experiments.

Figure 1. LPA1 antagonist attenuates brain damage after pMCAO challenge. Mice were challenged with pMCAO. AM152 at different doses (1.5, 5, and 15 mg/kg; p.o.) was then administered at one hour after occlusion. (A, B) Effects of AM152 on infarction volume (A) and functional neurological deficits (B) were determined at 1 day after pMCAO challenge. (A) Representative images of TTC-stained brain slices and quantification of brain infarction volume are shown. (B) Neurological score indicates functional neurological deficits. n=10 mice per group. *p<0.05 and ***p<0.001 versus vehicle-administered pMCAO group. (C) Effects of AM152 on cell death were determined by FJB staining at 1 day after pMCAO challenge. Representative images of FJB-stained sections are shown. Scale bars, 200 μm (top panel) and 50 μm (middle and bottom panel). (D) Effects of AM152 on apoptosis were determined by Western blot analysis on expression levels of Bcl-2, cleaved caspase-3, and β-actin proteins at 1 day after pMCAO challenge. Representative Western blots and quantification are shown. n=3 mice per group. *p<0.05 and **p<0.01 versus sham. #p<0.05 versus vehicle-administered pMCAO group. (E) Effects of AM152 on BBB disruption were determined by claudin-5/CD31 double immunofluorescence. Representative images of claudin-5/CD31-double immunopositive cells in the ischemic core region and quantification are shown. Scale bar, 50 µm. n=4 mice per group. ***p<0.001 versus sham. ##p<0.01 versus vehicle-administered pMCAO group.

Next, we determined whether LPA1 could be involved in cell death after pMCAO challenge by FJB staining at 1 day after pMCAO challenge (Fig. 1C). In the vehicle-administered pMCAO group, green fluorescence was observed in many cells of damaged cortex and striatum, indicating that cell death occurred in these areas (Fig. 1C). However, such cell death was markedly attenuated by AM152 administration (Fig. 1C). We further determined whether LPA1 could be involved in apoptosis by Western blot analysis for anti-apoptotic protein, Bcl-2, and apoptotic protein, cleaved caspase-3, at 1 day after pMCAO challenge. Both Bcl-2 downregulation and cleaved caspase 3 upregulation in injured brains after pMCAO challenge were significantly attenuated by AM152 administration (Fig. 1D).

To address whether LPA1 could be involved in BBB disruption after pMCAO challenge, expression levels of endothelial claudin-5 in ischemic core regions were determined at 1 day after pMCAO challenge by claudin-5/CD31 double immunofluorescence. Endothelial claudin-5 expression was significantly downregulated after pMCAO challenge, as evidenced by a reduced number of claudin-5/CD31-double immunopositive cells (Fig. 1E). However, such number was significantly increased after AM152 administration (Fig. 1E).

LPA1 contributes to microglial activation and proliferation in injured brains after pMCAO challenge

To determine whether LPA1-mediated brain damage is associated with microglial activation, a key pathogenic event of brain ischemic stroke (Yenari et al., 2010; Long et al., 2024), after pMCAO challenge, we carried out Iba1 immunohistochemistry to analyse key features of microglial activation, such as an increase in the number of Iba1-immunopositive cells and morphological changes of immunopositive cells from ramified microglia to amoeboid microglia (Kim et al., 2021). The number of Iba1-immunopositive cells was analysed in different regions (peri-ischemic and ischemic core regions) and at different time points (1 and 3 days after pMCAO challenge). Morphological changes were analysed in the ischemic core region at 3 days after pMCAO challenge. The number of Iba1-immunopositive cells was significantly increased in both regions (Fig. 2A, 2B) at both time points (Fig. 2A, 1 day; Fig. 2B, 3 days) in the vehicle-administered pMCAO group. This increase in the number of Iba1-immunopositive cells was significantly attenuated by AM152 administration (Fig. 2A, 2B). In addition, a massive morphological change of immunopositive cells from ramified to amoeboid was observed in the vehicle-administered pMCAO group (Fig. 2B), which was also significantly attenuated by AM152 administration (Fig. 2B).

Figure 2. LPA1 antagonist attenuates microglial activation and proliferation after pMCAO challenge. Mice were challenged with pMCAO. AM152 (5 mg/kg, p.o.) was then administered at one hour after occlusion. (A, B) Effects of AM152 on microglial activation were determined by Iba1 immunohistochemistry at 1 day (A) and 3 days (B) after pMCAO challenge. Representative images of Iba1-immunopositive cells in the peri-ischemic (‘P’) and ischemic core (‘C’) regions and quantification are shown. Dashed lines separate these two regions. Scale bars, 200 μm (top panels) and 50 μm (middle and bottom panels). The ratio of amoeboid to ramified Iba1-immunopositive cells in the ischemic core region (B) is shown. n=4 mice per group. **p<0.01 and ***p<0.001 versus sham. ##p<0.01 and ###p<0.001 versus vehicle-administered pMCAO group. (C) Effects of AM152 on microglial proliferation were determined by BrdU/Iba1 double immunofluorescence at 3 days after pMCAO challenge. Representative images of BrdU/Iba1-double immunopositive cells in the penumbra regions of injured brains and quantification are shown. Scale bar, 50 μm. n=4 mice per group. ***p<0.001 versus sham. ###p<0.001 versus vehicle-administered pMCAO group.

Figure 3. LPA1 antagonist suppresses mRNA expression levels of pro-inflammatory cytokines but enhances mRNA expression levels of anti-inflammatory cytokines after pMCAO challenge. Mice were challenged with pMCAO. AM152 (5 mg/kg, p.o.) was then administered at one hour after occlusion. Effects of AM152 on mRNA expression levels of pro-inflammatory cytokines such as TNF-α (A), IL-1β (B), and IL-6 (C) and anti-inflammatory cytokines such as TGF-β1 (D), IL-4 (E), and IL-10 (F) were determined by qRT-PCR analysis at 1 day after pMCAO challenge. n=3 mice per group. **p<0.01 and ***p<0.001 versus sham. #p<0.05, ##p<0.01, and ###p<0.001 versus vehicle-administered pMCAO group.

Proliferation of activated microglia in the penumbra region is another feature of microglial activation in brain ischemic stroke (Li et al., 2013; Deng et al., 2024). To address whether LPA1 could be involved in microglial proliferation after pMCAO challenge, BrdU incorporation into activated microglia in the penumbra regions was determined at 3 days after pMCAO challenge by BrdU/Iba1 double immunofluorescence. The number of BrdU/Iba1-double immunopositive cells was significantly increased in injured brains after pMCAO challenge (Fig. 2C), which was significantly attenuated by AM152 administration (Fig. 2C).

LPA1 regulates expression levels of pro- and anti-inflammatory cytokines in injured brains after pMCAO challenge

Activated microglia can modulate production of pro-inflammatory cytokines, contributing to neurodegeneration in brain ischemic stroke (Zhang et al., 2021). To address whether LPA1 could regulates this production in injured brains after pMCAO challenge, expression levels of pro-inflammatory cytokines were determined at 1 day after challenge by qRT-PCR analysis. Upregulation of TNF-α, IL-1β, and IL-6 mRNA expression levels in injured brains after pMCAO was significantly attenuated by AM152 administration (Fig. 3A-3C). We also determined whether LPA1 could affect expression levels of anti-inflammatory responses in injured brains by analysing mRNA expression levels of TGF-β1, IL-4, and IL-10 at 1 day after pMCAO challenge. Expression levels of these cytokines were significantly increased in the AM152-administered pMCAO group (Fig. 3D-3F).

NF-κB, MAPKs, and PI3K/Akt are involved in LPA1-dependent brain injury after pMCAO challenge

NF-κB is an important signaling molecule to mediate the upregulation of pro-inflammatory cytokines and brain injury in neuroinflammatory diseases, including brain ischemic stroke (Lawrence, 2009; Li et al., 2022a). Therefore, we determined NF-κB expression in injured brains at 1 day after pMCAO challenge. We also determined its expression in activated microglia since they are responsible for the production of pro-inflammatory cytokines via NF-κB (Zaghloul et al., 2020). Through NF-κB/Iba1 double immunofluorescence analysis, we found that NF-κB was upregulated in injured brains, particularly in activated microglia, after pMCAO challenge, as evidenced by the increase in the number of NF-κB/Iba1-double immunopositive cells (Fig. 4A). This upregulation was significantly attenuated by AM152 administration (Fig. 4A).

Figure 4. LPA1 antagonist attenuates NF-κB upregulation and MAPK activation but enhances PI3K/Akt activation after pMCAO challenge. Mice were challenged with pMCAO. AM152 (5 mg/kg, p.o.) was then administered at one hour after occlusion. (A) Effects of AM152 on NF-κB expression in activated microglia were determined by NF-κB(p65)/Iba1 double immunofluorescence at 1 day after pMCAO challenge. Representative images of NF-κB(p65) /Iba1-double immunopositive cells in ischemic core regions and quantification are shown. Scale bar, 50 μm. n=4 mice per group. ***p<0.001 versus sham. ###p<0.001 versus vehicle-administered pMCAO group. (B) Effects of AM152 on phosphorylation of MAPKs (ERK1/2, p38, and JNK) and PI3K/Akt were determined by Western blot analysis at 1 day after pMCAO challenge. Representative Western blots and quantification are shown. n=3 mice per group. *p<0.05, **p<0.01, and ***p<0.001 versus sham. #p<0.05, ##p<0.01 and ###p<0.001 versus vehicle-administered pMCAO group.

LPA1 can influence activation of MAPKs (ERK1/2, p38, and JNK) and PI3K/Akt as its effector pathways (Choi and Chun, 2013). These effector pathways are involved in LPA1-dependent pathogenesis of transient ischemic stroke (Gaire et al., 2019b). Moreover, activation of MAPKs can contribute to pro-inflammatory responses (Huang et al., 2023), whereas activation of PI3K/Akt can contribute to anti-inflammatory responses (Vergadi et al., 2017; He et al., 2022). Therefore, we determined whether these effector pathways of LPA1 could be involved in LPA1-dependent pathogenesis after pMCAO challenge by Western blot analysis. Phosphorylation levels of ERK1/2, p38, and JNK were increased in the vehicle-administered pMCAO group (Fig. 4B), indicating their activation in injured brains after pMCAO challenge. However, these phosphorylation levels were significantly attenuated by AM152 administration (Fig. 4B). In case of PI3K/Akt, Akt phosphorylation was significantly reduced in the vehicle-administered pMCAO group, whereas such reduction was attenuated by AM152 administration (Fig. 4B).

DISCUSSION

Considering that a specific antagonist of LPA1 (AM152) has been under clinical trials for pulmonary fibrosis (ClinicalTrials.gov ID: NCT01766817) and psoriasis (ClinicalTrials.gov ID: NCT02763969) (Stoddard and Chun, 2015), LPA1 can be a potential therapeutic target of drug development to treat other diseases in addition to pulmonary fibrosis and psoriasis. In fact, since the existence of LPA1 described in the 1990s (Hecht et al., 1996), lots of studies have revealed that this receptor is a key pathogenic factor of diverse diseases in addition to pulmonary fibrosis and psoriasis (Stoddard and Chun, 2015; Yanagida and Shimizu, 2023). Brain ischemic stroke is one of those diseases. Through previous studies by our group (Gaire et al., 2019b, 2020; Lee et al., 2020) and others (Halder et al., 2013), LPA1 has been identified to contribute to ischemia/reperfusion brain injury using a mouse model of transient brain ischemic stroke. The current study demonstrated that LPA1 also contributed to brain injury in permanent brain ischemic stroke using a mouse model of pMCAO. The current study also demonstrated LPA1-relevant pathogenic neuroinflammatory events, such as microglial activation, microglial proliferation, and regulation of inflammatory cytokines. As underlying molecular mechanisms of LPA1-dependent pathogenesis, NF-κB, MAPKs, and PI3K/Akt were identified. Therefore, the current study identified LPA1 as a pathogenic factor even for permanent brain ischemic stroke, along with associated pathogenic events and underlying molecular mechanisms (Fig. 5).

Figure 5. Schematic diagram showing a pathogenic role of LPA1 in permanent brain ischemic stroke. LPA1 contributed to brain injury in permanent brain ischemic stroke, which was revealed using a mouse model of pMCAO and an LPA1-specific antagonist. The pathogenic role of LPA1 was associated with neuroinflammatory responses, such as microglial activation, microglial proliferation, and upregulation of pro-inflammatory cytokines. As underlying molecular mechanisms, NF-κB, MAPKs, and PI3K/Akt were involved in the LPA1-dependent pathogenesis.

In a transient brain ischemic stroke, LPA1 has been found to play a critical role in brain damage during both acute phase (within 3 days) and chronic phase (Halder et al., 2013; Gaire et al., 2019b; Gaire et al., 2020). A genetic deletion (Halder et al., 2013) or knockdown (Gaire et al., 2019b) of LPA1 can attenuate tMCAO-induced ischemia/reperfusion brain injuries, supporting the pathogenic role of LPA1. This role was further affirmed using a specific LPA1 antagonist, AM095 (Gaire et al., 2019b) or AM152 (Gaire et al., 2020), in a mouse model of tMCAO. The current study revealed that LPA1 had a similar role in a permanent brain ischemic stroke as suppressing LPA1 activity by AM152 administration significantly attenuated brain infarction, functional neurological deficits, apoptosis, and BBB disruption in mice challenged with pMCAO. The current study also evaluated neuroprotective effects of AM152 because pMCAO challenge-induced brain injuries were significantly attenuated by delayed administration of AM152 (administered at one hour after pMCAO challenge), strongly indicating a potential of LPA1 antagonist as a therapeutic molecule for permanent brain ischemic stroke. Considering findings from previous studies and current studies using mouse models of transient (Halder et al., 2013; Gaire et al., 2019b, 2020) and permanent (the current study) brain ischemic stroke, targeting LPA1 would be a tempting strategy for drug development to treat brain ischemic stroke.

The best-studied core pathogenesis of brain ischemic stroke is microglial activation, leading to increased neuroinflammatory responses (Zhang et al., 2021). LPA1 is known to mediate not only physiological functions, but also pathological functions of microglia, resulting in tissue injuries (Gaire and Choi, 2021). The latter was clearly demonstrated in spinal cord injury (Santos-Nogueira et al., 2015), sepsis-induced brain injury (Kwon et al., 2018), and transient brain ischemic stroke (Gaire et al., 2019b, 2020). In particular, in injured brains caused by a transient ischemic stroke challenge (tMCAO challenge), it has been demonstrated that LPA1 contributes to microglial activation, such as an increase in the number of activated microglia, proliferation of microglia, and the number of amoeboid microglia (a shape of neurotoxic microglia) (Gaire et al., 2019b, 2020). This role of LPA1 was recapitulated in brain injuries after a permanent ischemic stroke challenge (pMCAO challenge) in the current study. These previous and current studies indicate that LPA1-driven brain injuries are associated with microglial activation in both types of ischemic stroke.

Activated microglia can produce pro- and anti-inflammatory cytokines and then contribute to tissue injuries and tissue protection, respectively (Guo et al., 2022; Long et al., 2024). In particular, previous in vitro and in vivo findings have demonstrated that LPA1 can regulate the production of pro-inflammatory cytokines (Kwon et al., 2018; Gaire et al., 2019b). Upregulation of TNF-α, IL-1β, and IL-6 at mRNA levels in lipopolysaccharide (LPS)-stimulated primary microglia was attenuated when LPA1 activity was suppressed by its antagonist (AM095, another well-known LPA1 antagonist) or a transient transfection of its specific siRNA (Gaire et al., 2019b). It has also been demonstrated that AM095 can attenuate TNF-α production from LPS-stimulated BV2 microglia overexpressing LPA1 (Kwon et al., 2018). In line with these in vitro findings, mRNA upregulation of TNF-α, IL-1β, and IL-6 in injured brains after tMCAO (Gaire et al., 2019b) and that of TNF-α in septic brains after LPS injection (Kwon et al., 2018) are attenuated by AM095 administration. The current study revealed that LPA1 played a similar role in pro-inflammatory responses in injured brains after pMCAO challenge as evidenced by reduced mRNA expression levels of pro-inflammatory cytokines (TNF-α, IL-1β, and IL-6) in injured brains after pMCAO challenge upon AM152 administration. The current study also revealed that LPA1 was involved in anti-inflammatory responses in injured brains after pMCAO challenge because AM152 administration enhanced mRNA levels of anti-inflammatory cytokines (TGF-β1, IL-4, and IL-10). Taken together, results from the current study indicate that LPA1 can regulate both pro- and anti-inflammatory responses in permanent brain ischemic stroke.

Regulatory roles of LPA1 in inflammatory responses could be interpreted as another role in regulating microglial polarization. In the latter aspect, activated microglia can be differentiated into pro-inflammatory M1- or anti-inflammatory M2-polarized cells to cause toxicity or enhance tissue repair (Guo et al., 2022; Long et al., 2024). In fact, pro-inflammatory cytokines such as TNF-α, IL-1β, and IL-6 are featured markers of M1 polarization and anti-inflammatory cytokines such as TGF-β1, IL-4, and IL-10 are markers of M2 polarization (Guo et al., 2022; Long et al., 2024). It has also been assumed that amoeboid microglia can be polarized into M1 cells in brain ischemic stroke (Jiang et al., 2020). In the current study, AM152 administration reduced mRNA expression levels of pro-inflammatory cytokines and enhanced those of anti-inflammatory cytokines in pMCAO-challenged brains. It also reduced the number of amoeboid microglia in these injured brains. Therefore, LPA1 might influence both M1 and M2 polarization in permanent brain ischemic stroke. This notion was further supported by findings from the current study to analyse LPA1-dependent effector pathways such as NF-κB, MAPKs, and PI3K/Akt. In general, activation of MAPKs and NF-κB is associated with M1 polarization (Lawrence, 2009; Li et al., 2022b). In contrast, activation of PI3K/Akt is associated with M2 polarization (Vergadi et al., 2017). In the current study, pMCAO challenge-induced upregulation of NF-κB and activation of MAPKs were attenuated by AM152 administration, while pMCAO-induced suppression of PI3K/Akt activity was reversed by AM152 administration. The regulatory role of LPA1 in M1 and M2 polarization was previously suggested in a transient brain ischemic stroke, in which suppressing LPA1 activity by AM095 attenuated upregulation of NF-κB, activation of MAPKs, and upregulation of pro-inflammatory cytokines but enhanced activation of PI3K/Akt in injured brains after tMCAO (Gaire et al., 2019b). These previous and present findings indicate that LPA1 can influence M1 and M2 polarization in both transient and permanent brain ischemic stroke.

In summary, the current study demonstrates that LPA1 contributes to brain injury in permanent brain ischemic stroke, along with underlying pathogenesis and molecular mechanisms. Considering previous findings on the pathogenic role of LPA1 in transient brain ischemic stroke, LPA1 can play as a pathogenic factor for both permanent brain ischemic stroke (the current study) and transient brain ischemic stroke (Halder et al., 2013; Gaire et al., 2019b, 2020; Lee et al., 2020). Since FTY720, the first drug targeting lysophospholipid receptors (Brinkmann et al., 2010), has been under clinical trials for brain ischemic stroke (Fu et al., 2014a, 2014b; Zhu et al., 2015), several LPA and S1P receptors such as LPA1 (Halder et al., 2013; Gaire et al., 2019b, 2020), LPA5 (Sapkota et al., 2020a, 2020b), S1P1 (Gaire et al., 2018a, 2019a), S1P2 (Sapkota et al., 2019), and S1P3 (Gaire et al., 2018b) have been identified as pathogenic factors for this disease through studies utilizing either pharmacological antagonists or genetic deletion. However, such roles of these receptors have been demonstrated in transient brain ischemic stroke, but not in permanent stroke even with a similar number of patients. The role of LPA1 in permanent stroke was finally revealed by the current study. However, roles of other LPA and S1P receptors in this type of stroke remain unclear. Therefore, it would be of interest to verify roles of other LPA and S1P receptors in permanent brain ischemic stroke in further studies.

ACKNOWLEDGMENTS

This work was supported by the National Research Foundation (NRF) of Korea (NRF-2021R1A2C1005520) and the Gachon University Research Fund of 2021 (GCU-202103430001) to JWC.

CONFLICT OF INTEREST

The authors have no conflicts of interest to disclose.

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