Inhibition Of Lysophosphatidic Acid Receptor Research Articles

Inhibition of lysophosphatidic acid receptor 2 attenuates neonatal chronic lung disease in mice by preserving vascular and alveolar development

AimBronchopulmonary dysplasia (BPD) is a common morbidity in extremely premature infants. Previous studies demonstrated the important role of lysophosphatidic acid (LPA) in inflammation in BPD. However, the role of LPA and its receptors in hyperoxia-induced vascular malformations in BPD remains to be elucidated. Methods and ResultsElevated plasma LPA levels were observed in mice with BPD compared to controls (792 vs. 607 ng/mL, p < 0.05). Inhibition of LPA signaling protected against hyperoxia-induced lung injury in neonatal mice, demonstrated by a 2.8-fold increase in pulmonary vascular density and a 14% reduction in alveolar enlargement. In vitro studies showed that LPA suppressed tube formation in human umbilical vein endothelial cells (HUVECs) by approximately 50%. LPA receptor 2 (LPA2) was identified as a functional LPA receptor in primary endothelial cells from the lungs of hyperoxic mice and in HUVECs under hyperoxic conditions. The LPA2 antagonist H2L5186303 enhanced the tube formation ability of HUVECs exposed to LPA, both under normoxia (4-fold) and hyperoxia (5-fold). Moreover, H2L5186303 significantly protected against hyperoxia-induced vascular malformation (2-fold) and improved alveolarization in neonatal mice (12% decrease in mean linear intercept, MLI). Early growth response 1 (EGR1) was characterized as a downstream target of LPA2, silencing EGR1 restored tube formation in HUVECs exposed to LPA and hyperoxia. ConclusionsOur in vitro and in vivo findings demonstrate that the inhibition of LPA/LPA2 signaling mitigates hyperoxia-induced pulmonary vascular malformations, suggesting the LPA/LPA2-dependent signaling pathway has therapeutic potential for extremely premature infants with BPD.

SiRNA Inhibition of Lysophosphatidic Acid Receptor 1 (LPAR1) Attenuates Kidney Fibrosis and Improves Kidney Function in a Rat Model of Hypertensive CKD

siRNA Inhibition of Lysophosphatidic Acid Receptor 1 (LPAR1) Attenuates Kidney Fibrosis and Improves Kidney Function in a Rat Model of Hypertensive CKD

Inhibition of lysophosphatidic acid receptor 1 relieves PMN recruitment in CNS via LPA1/TSP1/CXCR2 pathway and alleviates disruption on blood-brain barrier following intracerebral haemorrhage in mice

BackgroudThe frequencies of morbidity and impairment associated with spontaneous intracerebral haemorrhage (ICH) are comparatively high. Blood-brain barrier (BBB) integrity was compromised due to subsequent brain injury induced by ICH, which is crucial for a poor prognosis. Polymorphonuclear leukocyte (PMN) strongly modulate the disruption of BBB in the central nervous system (CNS). The lysophosphatidic acid receptor 1 (LPA1) mediated thrombospondin-1 (TSP1) regulation in astrocytes, which induce macrophage inflammatory protein 2(MIP2) secretion. MIP2 enhance PMN recruitment through CXC chemokine type 2 (CXCR2) activation. The purpose of this study was to investigate whether the LPA1-mediated inhibition of PMN recruitment and BBB protection after ICH is regulated by TSP1 and CXCR2 networks.MethodsICH induction was performed in CD1 mice using collagenase administration. AM966, a targeted LPA1 antagonist, was orally administered 1 and 12 h following ICH. further identify possible LPA1-mediated BBB protection mechanisms, we intracerebroventricularly (ICV) administered a CXCR2 ligand MIP2, as well as TSP1 CRISPR activation (ACT) with AM966. Consequently, we performed neurobehavioral, brain water content (BWC), Evans blue staining (EBS), immunofluorescence (IF), and western blot (WB) analyses.ResultsAfter ICH, astrocytes showed signs of LPA1, which peaked after 24 h, while PMN\\ displayed evidence of CXCR2. The AM966-mediated LPA1 suppression relieved PMN recruitment, diminished brain oedema, demonstrated extravasation (as evidenced by EBS), protected BBB integrity, and enhanced neurologic activity following ICH. AM966 treatment strongly reduced TSP1, CXCR2, Occludin, and Claudin-5 expressions and PMN recruitment following ICH, and their expressions were restored by MIP2 and TSP1 CRISPR (ACT).ConclusionsThis study shows that LAP1 suppression reduced PMN recruitment after ICH in mice via TSP1/CXCR2 signalling, which minimized BBB disruption and improved the CNS’s neurobehavioral functioning. Hence, LPA1 is a strong candidate for therapy to reduce PMN recruitment and offer protection of BBB integrity after ICH.

Inhibition of lysophosphatidic acid receptor 6 upregulated by the choline-deficient l-amino acid-defined diet prevents hepatocarcinogenesis in mice.

Hepatocellular carcinoma (HCC) is one of the most worrying tumors worldwide today, and its epidemiology is on the rise. Traditional pharmacological approaches have shown unfavorable results and exhibited many side effects. Hence, there is a need for new efficacious molecules with fewer side effects and improvements on traditional approaches. We previously showed that lysophosphatidic acid (LPA) supports hepatocarcinogenesis, and its effects are mainly mediated by LPA receptor 6 (LPAR6). We also reported that 9-xanthylacetic acid (XAA) acts as an antagonist of LPAR6 to inhibit the growth of HCC. Here, we report that LPAR6 is involved in the choline-deficient l-amino acid-defined(CDAA) diet-induced hepatocarcinogenesis in mice. Our data demonstrate that CDAA diet-induced metabolic imbalance stimulates LPAR6 expression in mice and that XAA counteracts diet-induced effects on hepatic lipid accumulation, fibrosis, inflammation, and HCC development. These conclusions are corroborated by results on LPAR6 gain and loss-of-function in HCC cells.

Inhibition of lysophosphatidic acid receptor 1\u20133 deteriorates experimental autoimmune encephalomyelitis by inducing oxidative stress

BackgroundLysophosphatidic acid receptors (LPARs) are G-protein-coupled receptors involved in many physiological functions in the central nervous system. However, the role of the LPARs in multiple sclerosis (MS) has not been clearly defined yet.MethodsHere, we investigated the roles of LPARs in myelin oligodendrocyte glycoprotein peptides-induced experimental autoimmune encephalomyelitis (EAE), an animal model of MS.ResultsPre-inhibition with LPAR1–3 antagonist Ki16425 deteriorated motor disability of EAElow. Specifically, LPAR1–3 antagonist (intraperitoneal) deteriorated symptoms of EAElow associated with increased demyelination, chemokine expression, cellular infiltration, and immune cell activation (microglia and macrophage) in spinal cords of mice compared to the sham group. This LPAR1–3 antagonist also increased the infiltration of CD4+/IFN-γ+ (Th1) and CD4+/IL-17+ (Th17) cells into spinal cords of EAElow mice along with upregulated mRNA expression of IFN-γ and IL-17 and impaired blood–brain barrier (BBB) in the spinal cord. The underlying mechanism for negative effects of LPAR1–3 antagonist was associated with the overproduction of reactive oxygen species (ROS)-generating nicotinamide adenine dinucleotide phosphate (NADPH) oxidases (NOX) 2 and NOX3. Interestingly, LPAR1/2 agonist 1-oleoyl-LPA (LPA 18:1) (intraperitoneal) ameliorated symptoms of EAEhigh and improved representative pathological features of spinal cords of EAEhigh mice.ConclusionsOur findings strongly suggest that some agents that can stimulate LPARs might have potential therapeutic implications for autoimmune demyelinating diseases such as MS.

Inhibition of lysophosphatidic acid receptor 1 attenuates neuroinflammation via PGE2/EP2/NOX2 signalling and improves the outcome of intracerebral haemorrhage in mice

Lysophosphatidic acid receptor 1 (LPA1) plays a critical role in proinflammatory processes in the central nervous system by modulating microglia activation. The aim of this study was to explore the anti-inflammatory effects and neurological function improvement of LPA1 inhibition after intracerebral haemorrhage (ICH) in mice and to determine whether prostaglandin E2 (PGE2), E-type prostaglandin receptor 2 (EP2), and NADPH oxidase 2 (NOX2) signalling are involved in LPA1-mediated neuroinflammation. ICH was induced in CD1 mice by autologous whole blood injection. AM966, a selective LPA1 antagonist, was administered by oral gavage 1 h and 12 h after ICH. The LPA1 endogenous ligand, LPA was administered to verify the effect of LPA1 activation. To elucidate potential inflammatory mechanisms of LPA1, the selective EP2 activator butaprost was administered by intracerebroventricular injection with either AM966 or LPA1 CRISPR knockout (KO). Water content of the brain, neurobehavior, immunofluorescence staining, and western blot were performed. After ICH, EP2 was expressed in microglia whereas LPA1 was expressed in microglia, neurons, and astrocytes, which peaked after 24 h. AM966 inhibition of LPA1 improved neurologic function, reduced brain oedema, and suppressed perihematomal inflammatory cells after ICH. LPA administration aggravated neurological deficits after ICH. AM966 treatment and LPA1 CRISPR KO both decreased the expressions of PGE2, EP2, NOX2, NF-κB, TNF-α, IL-6, and IL-1β expressions after ICH, which was reversed by butaprost. This study demonstrated that inhibition of LPA1 attenuated neuroinflammation caused by ICH via PGE2/EP2/NOX2 signalling pathway in mice, which consequently improved neurobehavioral functions and alleviated brain oedema. LPA1 may be a promising therapeutic target to attenuate ICH-induced secondary brain injury.

P3479Sequential stimulation and inhibition of lysophosphatidic acid receptor 4 are critical for cardiac differentiation and repair

Abstract Background The clinical application of cell therapy to repair the damaged heart needs to understand the precise differentiation process of stem cells and the characteristics of cardiac progenitor cells. Purpose We examined the cardiac-specific markers that expressed on the cell surface and determined their functional significance during cardiac differentiation. Methods and results We screened cell-surface expressing proteins on cardiac progenitor cells at differentiation day 3 compared to undifferentiated pluripotent stem cells (PSCs). Among candidates, we identified lysophosphatidic acid receptor 4 (LPAR4) that is a G protein-coupled receptor. During in vitro differentiation of mouse PSCs toward cardiac cells, LPAR4 expression peaked for 3–5 days and then and declined immediately. Also in vivo, LPAR4 was specifically expressed in the early stage of heart development in embryos and disappeared completely in adults, suggesting that stimulatory signal of LPAR4 at an early stage should be shut off for further progression of differentiation. We next have identified the LPAR4 downstream signaling molecule, p38MAPK, by comparing PSCs and LPAR4 knockdown PSCs. In both mouse and human PSCs, ODP (LPAR4 specific agonist) followed by p38MAPK blocker (SB203580) treatment significantly increased cardiac differentiation efficiency. Furthermore, we investigated whether LPAR4 is the maker for adult cardiac progenitor cells. We found that LPAR4-positive cells were rarely present in normal adult mouse hearts, but LPAR4-positive cells were increased when the heart was damaged. LPAR4-positive cells from adult hearts differentiated into cardiomyocytes. After myocardial infarction (MI), the sequential stimulation and inhibition of LPAR4 with ODP and p38MAPK blocker resulted in the reduction of infarct size and improvement of left ventricular dysfunction. Conclusion We demonstrated that LPAR4 is a cardiac progenitor-specific marker and its functional significance during cardiac differentiation and regeneration. Our findings provide a new insight in cell-free cardiac repair by the modulation of progenitor-specific downstream signaling. Acknowledgement/Funding Grants from “Strategic Center of Cell and Bio Therapy” (grant number: HI17C2085) and “Korea Research-Driven Hospital” (HI14C1277)

Inhibition of lysophosphatidic acid receptor ameliorates Sjögren's syndrome in NOD mice.

Lysophosphatidic acid (LPA), a bioactive lysophospholipid, is involved in the pathogenesis of chronic inflammatory and autoimmune diseases. In this study, we investigated the role of LPA/LPA receptor (LPAR) signaling in the pathogenesis of Sjögren's syndrome (SS). We found that autotaxin, an LPA producing enzyme, and LPAR1 and LPAR3 mRNA, and IL-17 mRNA were highly expressed in the exocrine glands of 20-week-old nonobese diabetic (NOD) mice, which show SS symptoms at this age, as compared with non-symptomatic 8-week-old NOD mice. In an adoptive transfer model using NOD lymphocytes, treatment with Ki16425, an LPAR1/3 antagonist, restored tear and saliva secretion and decreased symptoms of SS compared with the vehicle-treated group. IL-17 levels in serum and lacrimal glands were also significantly reduced by Ki16425 in recipient mice. In addition, Ki16425 treatment of 20-week-old NOD mice, which spontaneously developed SS, restored saliva volume. Treatment of NOD splenocytes with LPA induced the expression of IL-17 in a dose-dependent manner, and Ki16425 inhibited this increase. LPA stimulated the activation of ROCK2 and p38 MAPK; and inhibition of ROCK2 or p38 MAPK suppressed LPA-induced IL-17 expression. Our data suggest that LPAR signaling stimulates SS development by induction of IL-17 production via ROCK and p38 MAPK pathways. Thus, LPAR inhibition could be a possible therapeutic strategy for SS.

Deficiency or inhibition of lysophosphatidic acid receptor 1 protects against hyperoxia-induced lung injury in neonatal rats.

Blocking of lysophosphatidic acid (LPA) receptor (LPAR) 1 may be a novel therapeutic option for bronchopulmonary dysplasia (BPD) by preventing the LPAR1-mediated adverse effects of its ligand (LPA), consisting of lung inflammation, pulmonary arterial hypertension (PAH) and fibrosis. In Wistar rats with experimental BPD, induced by continuous exposure to 100% oxygen for 10 days, we determined the beneficial effects of LPAR1 deficiency in neonatal rats with a missense mutation in cytoplasmic helix 8 of LPAR1 and of LPAR1 and -3 blocking with Ki16425. Parameters investigated included survival, lung and heart histopathology, fibrin and collagen deposition, vascular leakage and differential mRNA expression in the lungs of key genes involved in LPA signalling and BPD pathogenesis. LPAR1-mutant rats were protected against experimental BPD and mortality with reduced alveolar septal thickness, lung inflammation (reduced influx of macrophages and neutrophils, and CINC1 expression) and collagen III deposition. However, LPAR1-mutant rats were not protected against alveolar enlargement, increased medial wall thickness of small arterioles, fibrin deposition and vascular alveolar leakage. Treatment of experimental BPD with Ki16425 confirmed the data observed in LPAR1-mutant rats, but did not reduce the pulmonary influx of neutrophils, CINC1 expression and mortality in rats with experimental BPD. In addition, Ki16425 treatment protected against PAH and right ventricular hypertrophy. LPAR1 deficiency attenuates pulmonary injury by reducing pulmonary inflammation and fibrosis, thereby reducing mortality, but does not affect alveolar and vascular development and, unlike Ki16425 treatment, does not prevent PAH in neonatal rats with experimental BPD.

Mechanosensitive PPAP2B Regulates Endothelial Responses to Atherorelevant Hemodynamic Forces.

PhosPhatidic Acid Phosphatase type 2B (PPAP2B), an integral membrane protein known as lipid phosphate phosphatase (LPP3) that inactivates lysophosphatidic acid, was implicated in coronary artery disease (CAD) by genome-wide association studies. However, it is unclear whether genome-wide association studies-identified coronary artery disease genes, including PPAP2B, participate in mechanotransduction mechanisms by which vascular endothelia respond to local atherorelevant hemodynamics that contribute to the regional nature of atherosclerosis. To establish the critical role of PPAP2B in endothelial responses to hemodynamics. Reduced PPAP2B was detected in vivo in mouse and swine aortic arch (AA) endothelia exposed to chronic disturbed flow, and in mouse carotid artery endothelia subjected to surgically induced acute disturbed flow. In humans, PPAP2B was reduced in the downstream part of carotid plaques where low shear stress prevails. In culture, reduced PPAP2B was measured in human aortic endothelial cells under atherosusceptible waveform mimicking flow in human carotid sinus. Flow-sensitive microRNA-92a and transcription factor KLF2 were identified as upstream inhibitor and activator of endothelial PPAP2B, respectively. PPAP2B suppression abrogated atheroprotection of unidirectional flow; inhibition of lysophosphatidic acid receptor 1 restored the flow-dependent, anti-inflammatory phenotype in PPAP2B-deficient cells. PPAP2B inhibition resulted in myosin light-chain phosphorylation and intercellular gaps, which were abolished by lysophosphatidic acid receptor 1/2 inhibition. Expression quantitative trait locus mapping demonstrated PPAP2B coronary artery disease risk allele is not linked to PPAP2B expression in various human tissues but significantly associated with reduced PPAP2B in human aortic endothelial cells. Atherorelevant flows dynamically modulate endothelial PPAP2B expression through miR-92a and KLF2. Mechanosensitive PPAP2B plays a critical role in promoting anti-inflammatory phenotype and maintaining vascular integrity of endothelial monolayer under atheroprotective flow.

Abstract 351: Mechano-sensitive Ppap2b Regulates Endothelial Responses to Athero-relevant Hemodynamic Forces

Rationale: PhosPhatidic-Acid-Phosphatase-type-2B (PPAP2B), an integral membrane protein that inactivates lysophosphatidic acid, was implicated in coronary artery disease (CAD) by genome-wide association studies (GWAS). However, it is unclear whether GWAS-identified CAD genes including PPAP2B participate in mechanotransduction mechanisms by which vascular endothelia respond to local athero-relevant blood flows that contribute to the regional nature of atherosclerosis. Approach and Results: Reduced endothelial PPAP2B was detected in vivo in swine aortic arch exposed to chronic disturbed flow and in mouse carotid artery subjected to surgically-induced acute disturbed flow. In culture, elevated PPAP2B was measured in human aortic endothelial cells (HAEC) under athero-protective flow mimicking hemodynamics of human distal carotid artery when compared with athero-susceptible waveform representing flow in carotid sinus. Disturbed flow-induced miR-92a was identified as a direct posttranscriptional inhibitor of mechano-sensitive PPAP2B. PPAP2B suppression abrogated athero-protection of unidirectional flow, shown by elevation of inflammatory genes. Inhibition of lysophosphatidic acid receptor 1 restored the flow-dependent, anti-inflammatory phenotype in PPAP2B-deficient cells. Moreover, PPAP2B inhibition resulted in myosin-light-chain phosphorylation and intercellular gaps, which were abolished by inhibition of lysophosphatidic acid receptors 1 and 2. Expression-quantitative-trait-locus-mapping demonstrated PPAP2B CAD risk allele is not linked to PPAP2B expression in various human tissues but significantly associated with reduced PPAP2B in HAEC. Conclusions: Athero-relevant flows dynamically modulate endothelial PPAP2B expression through miR-92a regulation. Mechano-sensitive PPAP2B plays an indispensable role in mediating cell alignment, promoting anti-inflammatory phenotype and maintaining vascular integrity of endothelial monolayer under athero-protective flow.

Abstract 846: Inhibition of lysophosphatidic acid receptor 1 radiosensitizes lung cancer cells

Abstract Despite advances in diagnostic and therapeutic modalities, lung cancer remains the leading cause of cancer-related mortality in the United States. In most cases, lung cancer presents as an unresectable mass requiring a combination of chemotherapy and radiotherapy (CRT) for treatment. A barrier to efficacy of this treatment is radioresistance. Radiation induced activation of pro-survival pathways are currently thought to play an important role in radioresistance. The enzyme cytosolic phospholipase A2 (cPLA2) has been identified as a key component in radiation induced pro-suvival pathways. Ionizing radiation activates cPLA2, which then cleaves phosphatidylcholine (PC) to yield lysophosphatidylcholine (LPC). LPC is then converted to lysophosphatidic acid (LPA) by autotaxin (ATX). LPA binds to distinct G-protein coupled receptors (GPCRs) on the plasma membrane; LPA1, LPA2 and LPA3, which are encoded by the endothelial differentiation gene family. These receptors are involved in the regulation of various aspects of cancer, including proliferation, migration and metastasis. Previously, we have shown that inhibition of cPLA2, as well as inhibition of ATX, was sufficient to enhance the sensitivity of cancer cells to radiation. However, the question remains as to whether inhibition of LPA receptor activity would have a similar effect. Thus, we studied the role of LPA receptors as mediators of radioresponse in human A549 and murine Lewis Lung Carcinoma (LLC) cell models. Quantitative polymerase chain reaction (QPCR) analysis of lung cancer cells showed that LPA1 was highly expressed in LLC and A549, and that both cell lines had nearly undetectable levels of LPA2 and LPA3 mRNA. Hence, we used Ki16425 and VPC12249, specific inhibitors of LPA1 and LPA3, to study the effect of LPA1 inhibition on clonogenic survival, cellular proliferation and invasion after irradiation. We found that pre-treatment of LLC and A549 cells with 10µM Ki16425 or 10µM VPC12249 significantly attenuated cell proliferation and viability after irradiation. Pre-treatment of LLC cells with 10µM Ki16425 or 10µM VPC12249 also reduced clonogenic survival. Transwell cell-invasion assays showed that the combination of LPA1 inhibitors with irradiation significantly reduced LPA-induced cell invasion in A549 and LLC cells. To investigate a potential mechanism for these effects, we also determined the activation of AKT and ERK signaling pathways. Inhibition of LPA1 resulted in reduced radiation-induced phosphorylation of both AKT and ERK. Overall, our results indicate that LPA1 is a mediator of pro-survival signaling in the adaptive response to radiation, and could serve as a viable target for enhancing the efficacy of radiation therapy of lung cancer. Note: This abstract was not presented at the meeting. Citation Format: David Y.A. Dadey, Rowan M. Karvas, Rama Kotipatruni, Jerry Jaboin, Dennis Hallahan, Dinesh Thotala. Inhibition of lysophosphatidic acid receptor 1 radiosensitizes lung cancer cells. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 846. doi:10.1158/1538-7445.AM2014-846