Enzyme Autotaxin Research Articles

Imaging Pulmonary Fibrosis and Treatment Efficacy In Vivo with Autotaxin-Specific PET Ligand [18F]ATX-1905.

Idiopathic pulmonary fibrosis (IPF) is a fatal disease characterized by unpredictable progression and limited therapeutic options. Current diagnosis relies on high resolution computed tomography (HRCT), which may not adequately capture early signs of deterioration. The enzyme autotaxin (ATX) emerges as a prominently expressed extracellular secretory enzyme in the lungs of IPF patients. The objective of this study was to evaluate the effectiveness of 18F-labeled ATX-targeted tracer [18F]ATX-1905, in comparison with [18F]FDG, for early fibrosis diagnosis, disease evolution monitoring, and treatment efficacy assessment in bleomycin-induced pulmonary fibrosis (BPF) models. To assess treatment efficacy, mice were treated with two commonly used drugs for IPF, pirfenidone or nintedanib, from Day 9 to Day 23 postbleomycin administration. Lung tissue assessments encompassed inflammation severity via H&E staining, and Ashcroft scoring via Masson staining, alongside quantification of ATX expression through ELISA. Positron emission tomography (PET) imaging employing [18F]FDG and [18F]ATX-1905 tracked disease progression pre- and post-treatment. The extent of pulmonary fibrosis corresponded to changes in ATX expression levels in the BPF mouse model. Notably, [18F]ATX-1905 exhibited elevated uptake in BPF lungs during the progression of the disease, particularly evident at the early stage (Day 9). This uptake was inhibited by an ATX inhibitor, PF-8380, underscoring the specificity of the radiotracer. Conversely, [18F]FDG uptake, peaking at Day 15, decreased subsequently, likely reflective of diminished inflammation. A 2-week treatment regimen using either pirfenidone or nintedanib resulted in notable reductions of ATX expression levels and fibrosis degrees within lung tissues, based on ELISA and Masson staining, as evidenced by PET imaging with [18F]ATX-1905. [18F]FDG uptake also decreased following the treatment period. Additionally, PET/CT imaging extended to a nonhuman primate (NHP) BPF model. The uptake of [18F]ATX-1905 (SUVmax = 2.2) was significantly higher than that of [18F]FDG (SUVmax = 0.7) in fibrotic lung tissue. Using our novel ATX-specific radiotracer [18F]ATX-1905 and PET/CT imaging, we demonstrated excellent ability in early fibrosis detection, disease monitoring, and treatment assessment within lungs of the BPF mouse models. [18F]ATX-1905 displayed remarkable specificity for ATX expression and high sensitivity for ATX alterations, suggesting its potential for monitoring varying ATX expression in lungs of IPF patients.

Altered cortical synaptic lipid signaling leads to intermediate phenotypes of mental disorders

Excitation/inhibition (E/I) balance plays important roles in mental disorders. Bioactive phospholipids like lysophosphatidic acid (LPA) are synthesized by the enzyme autotaxin (ATX) at cortical synapses and modulate glutamatergic transmission, and eventually alter E/I balance of cortical networks. Here, we analyzed functional consequences of altered E/I balance in 25 human subjects induced by genetic disruption of the synaptic lipid signaling modifier PRG-1, which were compared to 25 age and sex matched control subjects. Furthermore, we tested therapeutic options targeting ATX in a related mouse line. Using EEG combined with TMS in an instructed fear paradigm, neuropsychological analysis and an fMRI based episodic memory task, we found intermediate phenotypes of mental disorders in human carriers of a loss-of-function single nucleotide polymorphism of PRG-1 (PRG-1R345T/WT). Prg-1R346T/WT animals phenocopied human carriers showing increased anxiety, a depressive phenotype and lower stress resilience. Network analysis revealed that coherence and phase-amplitude coupling were altered by PRG-1 deficiency in memory related circuits in humans and mice alike. Brain oscillation phenotypes were restored by inhibtion of ATX in Prg-1 deficient mice indicating an interventional potential for mental disorders.

Lysophosphatidic acid receptor 1 inhibition: a potential treatment target for pulmonary fibrosis.

Lysophosphatidic acid (LPA)-mediated activation of LPA receptor 1 (LPAR1) contributes to the pathophysiology of fibrotic diseases such as idiopathic pulmonary fibrosis (IPF) and systemic sclerosis (SSc). These diseases are associated with high morbidity and mortality despite current treatment options. The LPA-producing enzyme autotaxin (ATX) and LPAR1 activation contribute to inflammation and mechanisms underlying fibrosis in preclinical fibrotic models. Additionally, elevated levels of LPA have been detected in bronchoalveolar lavage fluid from patients with IPF and in serum from patients with SSc. Thus, ATX and LPAR1 have gained considerable interest as pharmaceutical targets to combat fibrotic disease and inhibitors of these targets have been investigated in clinical trials for IPF and SSc. The goals of this review are to summarise the current literature on ATX and LPAR1 signalling in pulmonary fibrosis and to help differentiate the novel inhibitors in development. The mechanisms of action of ATX and LPAR1 inhibitors are described and preclinical studies and clinical trials of these agents are outlined. Because of their contribution to numerous physiologic events underlying fibrotic disease, ATX and LPAR1 inhibition presents a promising therapeutic strategy for IPF, SSc and other fibrotic diseases that may fulfil unmet needs of the current standard of care.

An Autotaxin-Induced Ocular Hypertension Mouse Model Reflecting Physiological Aqueous Biomarker.

Animal models of ocular hypertension (OH) have been developed to understand the pathogenesis of glaucoma and facilitate drug discovery. However, many of these models are fraught with issues, including severe intraocular inflammation and technical challenges. Lysophosphatidic acid (LPA) is implicated in trabecular meshwork fibrosis and increased resistance of aqueous outflow, factors that contribute to high intraocular pressure (IOP) in human open-angle glaucoma. We aimed to elevate IOP by increasing expression of the LPA-producing enzyme autotaxin (ATX) in mouse eyes. Tamoxifen-inducible ATX transgenic mice were developed. Tamoxifen was administered to six- to eight-week-old mice via eye drops to achieve ATX overexpression in the eye. IOP and retinal thickness were measured over time, and retinal flat-mount were evaluated to count retinal ganglion cells (RGCs) loss after three months. Persistent elevation of ATX expression in mouse eyes was confirmed through immunohistochemistry and LysoPLD activity measurement. ATX Tg mice exhibited significantly increased IOP for nearly two months following tamoxifen treatment, with no anterior segment changes or inflammation. Immunohistochemical analysis revealed enhanced expression of extracellular matrix near the angle after two weeks and three months of ATX induction. This correlated with reduced outflow facility, indicating that sustained ATX overexpression induces angle fibrosis, elevating IOP. Although inner retinal layer thickness remained stable, peripheral retina showed a notable reduction in RGC cell count. These findings confirm the successful creation of an open-angle OH mouse model, in which ATX expression in the eye prompts fibrosis near the angle and maintains elevated IOP over extended periods.

Abstract A025: Autotaxin-lysolipid signaling suppresses a CCL11- eosinophil axis to promote pancreatic cancer progression

Abstract Among all cancer types, pancreatic ductal adenocarcinoma (PDAC) has one of the worst outcomes. In recent studies, metabolic and microenvironmental reprogramming in PDAC has been shown to facilitate tumor progression by promoting immune escape mechanisms, however these mechanisms are still poorly understood. Recently, our lab established that autotaxin (ATX) - lysophosphatidic acid (LPA) signaling in PDAC enhances tumorigenicity, but the immunomodulatory function of this signal pathway remains largely unexplored. We now show that ablation of the LPA-producing enzyme autotaxin (ATX) in PDAC cells can reduce tumor burden in mice and reprogram the tumor immune microenvironment by increasing tumor eosinophil infiltration. The functional role of eosinophils in the context of PDAC is a poorly understood area. The presence of eosinophils in pancreatic cancer has been reported in a few case reports: for example, prior studies identified neutrophils and eosinophils together as a group, expressing high levels of CD66b. We confirmed the presence of eosinophils in PDAC in mice as well as patients, and we found that rare patient samples with the highest intratumoral eosinophil abundance had the longest survival rates. Genetic or pharmacologic ATX inhibition increased intratumoral eosinophils, which promote tumor cell apoptosis locally and suppress tumor progression. When eosinophils were present, primary tumor growth was modestly reduced, but metastatic spread was greatly reduced. Mechanistically, ATX suppresses eosinophil accumulation via an autocrine feedback loop wherein ATX-LPA signaling negatively regulates activity of AP-1 transcription factor c-Jun, in turn suppressing expression of potent eosinophil chemoattractant CCL11 (Eotaxin-1). This is the first study that highlights the anti-tumor potential of eosinophils in pancreatic cancer. Citation Format: Sohinee Bhattacharyya, Chet Oon, Holly Sandborg, Terry Morgan, Mara H. Sherman. Autotaxin-lysolipid signaling suppresses a CCL11- eosinophil axis to promote pancreatic cancer progression [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Pancreatic Cancer; 2023 Sep 27-30; Boston, Massachusetts. Philadelphia (PA): AACR; Cancer Res 2024;84(2 Suppl):Abstract nr A025.

Autotaxin-lysolipid signaling suppresses a CCL11-eosinophil axis to promote pancreatic cancer progression.

Lipids and their modifying enzymes regulate diverse features of the tumor microenvironment and cancer progression. The secreted enzyme autotaxin (ATX) hydrolyzes extracellular lysophosphatidylcholine to generate the multifunctional lipid mediator lysophosphatidic acid (LPA) and supports the growth of several tumor types, including pancreatic ductal adenocarcinoma (PDAC). Here we show that ATX suppresses the accumulation of eosinophils in the PDAC microenvironment. Genetic or pharmacologic ATX inhibition increased the number of intratumor eosinophils, which promote tumor cell apoptosis locally and suppress tumor progression. Mechanistically, ATX suppresses eosinophil accumulation via an autocrine feedback loop, wherein ATX-LPA signaling negatively regulates the activity of the AP-1 transcription factor c-Jun, in turn suppressing the expression of the potent eosinophil chemoattractant CCL11 (eotaxin-1). Eosinophils were identified in human PDAC specimens, and rare individuals with high intratumor eosinophil abundance had the longest overall survival. Together with recent findings, this study reveals the context-dependent, immune-modulatory potential of ATX-LPA signaling in cancer.

Insights into autotaxin- and lysophosphatidate-mediated signaling in the pancreatic ductal adenocarcinoma tumor microenvironment: a survey of pathway gene expression.

Lysophosphatidate (LPA)-mediated signaling is a vital component of physiological wound healing, but the pathway is subverted to mediate chronic inflammatory signaling in many pathologies, including cancers. LPA, as an extracellular signaling molecule, is produced by the enzyme autotaxin (ATX, gene name ENPP2) and signals through six LPA receptors (LPARs). Its signaling is terminated by turnover via the ecto-activity of three lipid phosphate phosphatases (LPPs, gene names PLPP1-3). Many pharmacological developments against the LPA-signaling axis are underway, primarily against ATX. An ATX inhibitor against pancreatic ductal adenocarcinoma (PDAC), a very aggressive disease with limited systemic therapeutic options, is currently in clinical trials, and represents the first in-class drug against LPA signaling in cancers. In the present study, we surveyed the expression of ATX, LPARs, and LPPs in human PDACs and their clinical outcomes in two large independent cohorts, the Cancer Genome Atlas (TCGA) and GSE21501. Correlation among gene expressions, biological function and the cell composition of the tumor microenvironment were analysed using gene set enrichment analysis and cell cyber-sorting with xCell. ENPP2, LPAR1, LPAR4, LPAR5, LPAR6, PLPP1, and PLPP2 were significantly elevated in PDACs compared to normal pancreatic tissue, whereas LPAR2, LPAR3, and PLPP3 where downregulated (all P≤0.003). Only ENPP2 demonstrated survival differences, with overall survival favoring ENPP2-high patients (hazard ration 0.5-0.9). ENPP2 was also the only gene with enriched gene patterns for inflammatory and tissue repair gene sets. Epithelial (cancer) cells had increased LPAR2, LPAR5 and PLPP2 expression, and decreased ENPP2, LPAR1, PLPP1, and PLPP3 gene expression (all P<0.02). Tumor fibroblasts had increased ENPP2, LPAR2, LPAR4, PLPP1, and PLPP3 expression and decreased LPAR2, LPAR5, and PLPP2 expression in both cohorts (all P≤0.01). Immune cell populations were not well correlated to gene expression in PDACs, but across both cohorts, cytolytic scores were increased in high-expressing ENPP2, LPAR1, LPAR6, PLPP1, and PLPP3 tumors (P<0.01). Overall, in PDACs, ENPP2 may switch from an anti-to-pro tumor promoting gene with disease progression. LPAR2 and PLPP2 inhibition are also predicted to have potential therapeutic utility. Future multi-omics investigations are necessarily to validate which LPA signaling components are high-value candidates for pharmacological manipulation in PDAC treatment.

Autotaxin suppresses cytotoxic T cells via LPAR5 to promote anti-PD-1 resistance in non-small cell lung cancer.

Non-small cell lung cancers that harbor concurrent KRAS and TP53 (KP) mutations are immunologically warm tumors with partial responsiveness to anti-PD-(L)1 blockade; however, most patients observe little or no durable clinical benefit. To identify novel tumor-driven resistance mechanisms, we developed a panel of KP murine lung cancer models with intrinsic resistance to anti-PD-1 and queried differential gene expression between these tumors and anti-PD-1-sensitive tumors. We found that the enzyme autotaxin (ATX), and the metabolite it produces, lysophosphatidic acid (LPA), were significantly upregulated in resistant tumors and that ATX directly modulated antitumor immunity, with its expression negatively correlating with total and effector tumor-infiltrating CD8+ T cells. Pharmacological inhibition of ATX, or the downstream receptor LPAR5, in combination with anti-PD-1 was sufficient to restore the antitumor immune response and efficaciously control lung tumor growth in multiple KP tumor models. Additionally, ATX was significantly correlated with inflammatory gene signatures, including a CD8+ cytolytic score in multiple lung adenocarcinoma patient data sets, suggesting that an activated tumor-immune microenvironment upregulates ATX and thus provides an opportunity for cotargeting to prevent acquired resistance to anti-PD-1 treatment. These data reveal the ATX/LPA axis as an immunosuppressive pathway that diminishes the immune checkpoint blockade response in lung cancer.

Dose-Response Activity-Based DNA-Encoded Library Screening.

Dose-response, or "conforming" behavior, increases confidence in a screening hit's authenticity. Here, we demonstrate dose-response solid-phase DNA-encoded library (DEL) screening. Compound dose in microfluidic droplets is modulated via the UV intensity of photocleavage from DEL beads. A 55,296-member DEL was screened at different UV intensities against model enzyme drug targets factor Xa (FXa) and autotaxin (ATX). Both screens yielded photochemical dose-dependent hit rates (FXa hit rates of 0.08/0.05% at 100/30% UV exposure; ATX hit rates of 0.24/0.08% at 100/20% UV exposure). FXa hits contained structures reflective of FXa inhibitors and four hits inhibited FXa (IC50 = 4.2 ± 0.1, 7.4 ± 0.3, 9.0 ± 0.3, and 19 ± 2 μM.) The top ATX hits (two dihydrobenzamidazolones and a tetrahydroisoquinoline) were validated as inhibitors (IC50 = 7 ± 2, 13 ± 2, and 1 ± 0.3 μM). Photochemical dose-response DEL screening data prioritized hits for synthesis, the rate-limiting step in DEL lead identification.

Immunohistochemical evaluation of autotaxin and lubricin in mild osteoarthritic rat model performing moderate physical activity

Levels of the enzyme autotaxin (ATX) are elevated in synovial fluid and plasma of osteoarthritic patients, correlating positively with radiographic and symptomatic severity of the disease. Therefore, ATX is studied as potential marker for the progression of osteoarthritis (OA), whereas the chondrocyte-secreted glycoprotein Lubricin has chondroprotective properties. The aim of this study was to evaluate the expression of ATX and Lubricin in healthy and mild OA rat articular cartilage of femur, tibia and patella, and to analyse the effect of a protocol of moderate physical activity on their expressions. Mild OA resulted from anterior cruciate ligament transection and rats exercised on a treadmill for 12 weeks. Computerized staining intensity of immunostaining was used to evaluate ATX and Lubricin expressions. Higher expressions of ATX were found in femur and tibia of OA rats, suggesting that this molecule could participate in the progression of the disease, although not involved in the patella. In the femur, physical activity performed by OA rats was able to lower ATX expression, encouraging the evidence that joint movement is beneficial for the cartilage, although no significant differences in Lubricin expression were detected in femur, tibia and patella. This evidence might shade some light about the role of ATX, Lubricin and physical exercise in OA progression.

Lysophosphatidic Acid Signaling and microRNAs: New Roles in Various Cancers.

A wide range of microRNAs (miRNAs) are coded for in the human genome and contribute to the regulation of gene expression. MiRNAs are able to degrade mRNAs and/or prevent the RNA transcript from being translated through complementary binding of the miRNA seed region (nucleotide 2-8) to the 3’-untranslated regions of many mRNAs. Although miRNAs are involved in almost all processes of normal human cells, they are also involved in the abnormal functions of cancer cells. MiRNAs can play dual regulatory roles in cancer, acting either as tumor suppressors or as tumor promoters, depending on the target, tumor type, and stage. In the current review, we discuss the present status of miRNA modulation in the setting of lysophosphatidic acid (LPA) signaling. LPA is produced from lysophosphatidylcholine by the enzyme autotaxin and signals via a range of G protein-coupled receptors to affect cellular processes, which ultimately causes changes in cell morphology, survival, proliferation, differentiation, migration, and adhesion. Several studies have identified miRNAs that are over-expressed in response to stimulation by LPA, but their functional roles have not yet been fully clarified. Since RNA-based treatments hold tremendous promise in the area of personalized medicne, many efforts have been made to bring miRNAs into clinical trials, and this field is evolving at an increasing pace.

New role for enzyme autotaxin in stroke excitotoxicity.

New role for enzyme autotaxin in stroke excitotoxicity.

Abstract 362: Endothelial Increase In Autotaxin Regulates Cerebral Vascular Permeability During Ischemic Reperfusion

Introduction: Pathological progression of stroke is aided by ischemic reperfusion during the treatment procedure. Various molecular signals initiated during ischemic reperfusion exacerbates the disease leading to endothelial permeability. However, a detailed molecular therapeutic regimen with r-TPA and mechanical thrombectomy could limit the detrimental effects. Lysophosphatidic acid (LPA) is a bioactive phospholipid regulated in physiological and pathological conditions. LPA is produced by the enzyme autotaxin (ATX). ATX-LPA axis have been observed to deteriorate physiological status in various diseases. In cerebral ischemic-reperfusion, the ATX-LPA axis could regulate the endothelium disruption. Hypothesis: Increased ATX expression in endothelium disrupts the endothelial barrier by increasing LPA during ischemic-reperfusion. Methods: For the in-vitro study, oxygen and glucose deprivation and reperfusion (OGDR) were carried out in mouse brain microvascular endothelial cells (MBMEC).For invivo transient middle cerebral artery occlusion with 90 minutes of ischemia and 24 hours of reperfusion (I/R) was used as a stroke model in mice. Electric Cell Substrate Impedance System (ECIS) and Evan Blue was used for permeability study for invitro and invivo respectively. AR-2 probe fluorescence assay was used to measure ATX activity. Results: ATX expression was increased (P&lt;0.05) in MBMEC with OGDR suggesting ATX regulation in endothelial cells following ischemic-reperfusion. Permeability measured with ECIS in MBMEC showed that LPA treatment increased the permeability in a time and concentration-dependent manner. The protein levels of junctional proteins such as βcatenin, Claudin5, VEcadherin, and Zo1 weresignificantly (P&lt;0.05) decreased with LPA exposure. In invivo stroke model ATX enzymatic activity is prominently raised (P&lt;0.01) in I/R mice compared to sham mice. Increased ATX activity coheres with the Evans Blue permeability in the mice brain. Simultaneously LPA levels are elevated in I/R mice. LPA produced during the process can disrupt the blood-brain barrier, increasing cerebral permeability. Conclusion: Inhibition of the ATX-LPA axis prospects to be a better therapeutic avenue for cerebral ischemic reperfusion.

Cerebral Ischemic Reperfusion Increases Autotaxin Expression Elevating Brain Endothelial Permeability

IntroductionIschemic reperfusion during stroke treatment increases the pathological progression of stroke. Reperfusion with r‐TPA and mechanical thrombectomy applied to stroke patients increases the risk of intracerebral hemorrhage. In addition, various molecular signals initiated during ischemic reperfusion exacerbate the disease due to increased endothelial permeability. However, a detailed molecular therapeutic regimen with r‐TPA and mechanical thrombectomy could limit the detrimental effects and increase the survival rate. Lysophosphatidic acid (LPA) is a bioactive phospholipid regulated in physiological and pathological conditions. LPA is produced by the enzyme autotaxin (ATX). ATX and LPA have been observed to deteriorate physiological status in various diseases such as myocardial infarction, Alzheimer’s disease, liver fibrosis, neuropathic pain etc. In cerebral ischemic‐reperfusion, the ATX‐LPA axis could initiate the endothelium disruption.HypothesisIncreased ATX expression in endothelium disrupts the endothelial barrier by increasing LPA during ischemic‐reperfusion.MethodsTransient middle cerebral artery occlusion with 90 minutes of ischemia and 24 hours of reperfusion (I/R) was used as a stroke model in mice. LPA was analyzed with mass spectrometry and immunohistochemistry. AR‐2 probe fluorescence assay was used to measure ATX activity. Evans Blue Permeability in vivo was measured with an infrared imager. For the in‐vitro study, oxygen and glucose deprivation and reperfusion (OGDR) were carried out in mouse brain microvascular endothelial cells (MBMEC).ResultsATX enzymatic activity is prominently raised (P&lt;0.01) in I/R mice compared to sham mice. Increased ATX activity coheres with the Evans Blue permeability in the mice brain. Simultaneously LPA levels are elevated (P&lt;0.01) in I/R mice. LPA was increased in brain tissue along the vasculature, indicating an unfavorable role of ATX in maintaining endothelial integrity. LPA receptors expression was regulated following stroke with upregulation of LPAR1, LPAR2, LPAR5, and LPAR6 following I/R. Permeability measured in MBMEC showed that LPA treatment increased the permeability in a time and concentration‐dependent manner. ATX expression was increased (P&lt;0.05) in MBMEC with OGDR suggesting ATX regulation in endothelial cells following ischemic‐reperfusion. LPA produced during the process can disrupt the blood‐brain barrier, increasing cerebral permeability.ConclusionAfter a stroke, enhanced LPA production by ATX can disrupt the blood‐brain barrier, leading to brain edema and neural cell damage followed by irreversible brain injury. Inhibition of the ATX‐LPA axis prospects to be a better therapeutic avenue for cerebral ischemic reperfusion.

Inhibition of the enzyme autotaxin reduces cortical excitability and ameliorates the outcome in stroke.

Stroke penumbra injury caused by excess glutamate is an important factor in determining stroke outcome; however, several therapeutic approaches aiming to rescue the penumbra have failed, likely due to unspecific targeting and persistent excitotoxicity, which continued far beyond the primary stroke event. Synaptic lipid signaling can modulate glutamatergic transmission via presynaptic lysophosphatidic acid (LPA) 2 receptors modulated by the LPA-synthesizing molecule autotaxin (ATX) present in astrocytic perisynaptic processes. Here, we detected long-lasting increases in brain ATX concentrations after experimental stroke. In humans, cerebrospinal fluid ATX concentration was increased up to 14 days after stroke. Using astrocyte-specific deletion and pharmacological inhibition of ATX at different time points after experimental stroke, we showed that inhibition of LPA-related cortical excitability improved stroke outcome. In transgenic mice and in individuals expressing a single-nucleotide polymorphism that increased LPA-related glutamatergic transmission, we found dysregulated synaptic LPA signaling and subsequent negative stroke outcome. Moreover, ATX inhibition in the animal model ameliorated stroke outcome, suggesting that this approach might have translational potential for improving the outcome after stroke.

The ATX-LPA Axis Regulates Vascular Permeability during Cerebral Ischemic-Reperfusion.

Endothelial permeability is a major complication that must be addressed during stroke treatment. Study of the mechanisms underlying blood–brain barrier (BBB) disruption and management of the hypoxic stress-induced permeability of the endothelium following reperfusion are both urgently needed for stroke management. Lysophosphatidic acid (LPA), a bioactive lipid essential for basic cellular functions, causes unfavorable outcomes during stroke progression. LPA-producing enzyme autotaxin (ATX) is regulated in ischemic stroke. We used an electrical cell-substrate impedance sensor (ECIS) to measure endothelial permeability. Mitochondrial bioenergetics were obtained using a Seahorse analyzer. AR-2 probe fluorescence assay was used to measure ATX activity. LPA increased endothelial permeability and reduced junctional protein expression in mouse brain microvascular endothelial cells (MBMEC). LPA receptor inhibitors Ki16425 and AM095 attenuated the LPA-induced changes in the endothelial permeability and junctional proteins. LPA significantly diminished mitochondrial function in MBMEC. ATX was upregulated (p < 0.05) in brain microvascular endothelial cells under hypoxic reperfusion. ATX activity and permeability were attenuated with the use of an ATX inhibitor in a mouse stroke model. The upregulation of ATX with hypoxic reperfusion leads to LPA production in brain endothelial cells favoring permeability. Inhibition of the ATX–LPA–LPAR axis could be therapeutically targeted in stroke to achieve better outcomes.

Abstract TP245: Unfavorable Role Of Autotaxin Lysophosphatidic Acid Axis During Ischemic Reperfusion In Stroke.

Introduction: Stroke is a leading cause of death, also serious cause of long-term disability in the US. r-TPA and mechanical thrombectomy apply to 5-10% of patients, while others have the risk of intracerebral hemorrhage. Ischemic reperfusion following stroke exacerbates the condition and initiates various molecular signals. However, with a better therapeutic regimen, the detrimental effects can be limited. Lysophosphatidic acid (LPA) is a bioactive phospholipid regulated in physiological and pathological conditions. LPA is formed by the enzyme Autotaxin (ATX). ATX-LPA axis affects endothelial permeability in ischemic stroke. Hypothesis: ATX-LPA axis favors endothelial permeability in ischemic stroke. Methods: Transient middle cerebral artery occlusion with 90 minutes of ischemia and 24 hours of reperfusion (I/R) was used as a stroke model in mice. LPA was analyzed in plasma with mass spectrometry and in tissue with immunohistochemistry. AR-2 probe fluorescence assay was used to measure ATX activity. Evans Blue Permeability in vivo was measured with infrared imager. Mitochondrial bioenergetics was measured with an XF24 analyzer. Permeability was studied using Electric Cell Substrate Impedance System (ECIS). Results: ATX enzymatic activity is prominently raised in I/R mice compared to sham mice. Increased ATX activity coheres with the Evans Blue permeability in the mice brain. Simultaneously LPA levels are elevated (P&lt;0.01) in I/R mice. LPA was increased in brain tissue along the vasculature, indicating an unfavorable role of ATX in maintaining endothelial integrity. LPA receptors were regulated differentially following stroke. Permeability measured in MBMEC showed that LPA treatment increased the permeability in a time and concentration-dependent manner. LPA treatment also increased superoxide in MBMEC suggesting redox imbalance with LPA exposure. Mitochondrial superoxide elevated following I/R was limited with ATX inhibitors which also restricted the permeability compared to that of the I/R mice Conclusion: LPA produced by ATX plays a significant role in ischemic stroke, increasing BBB permeability and elevating mitochondrial dysfunction. Targeting the ATX-LPA axis could be a better therapeutic avenue for stroke management.

Structure-based discovery of (S)-2-amino-6-(4-fluorobenzyl)-5,6,11,11a-tetrahydro-1H-imidazo[1',5':1,6]pyrido[3,4-b]indole-1,3(2H)-dione as low nanomolar, orally bioavailable autotaxin inhibitor.

Inhibition of extracellular secreted enzyme autotaxin (ATX) represents an attractive strategy for the development of new therapeutics to treat various diseases and a few inhibitors entered in clinical trials. We herein describe structure-based design, synthesis, and biological investigations revealing a potent and orally bioavailable ATX inhibitor 1. During the molecular docking and scoring studies within the ATX enzyme (PDB-ID: 4ZGA), the S-enantiomer (Gscore=-13.168kcal/mol) of the bound ligand PAT-494 scored better than its R-enantiomer (Gscore=-9.562kcal/mol) which corroborated with the reported observation and analysis of the results suggested the scope of manipulation of the hydantoin substructure in PAT-494. Accordingly, the docking-based screening of a focused library of 10 compounds resulted in compound 1 as a better candidate for pharmacological studies. Compound 1 was synthesized from L-tryptophan and evaluated against ATX enzymatic activities with an IC50 of 7.6 and 24.6nM in biochemical and functional assays, respectively. Further, ADME-PK studies divulged compound 1 as non-cytotoxic (19.02% cell growth inhibition at 20μM in human embryonic kidney cells), metabolically stable against human liver microsomes (CLint =15.6μl/min/mg; T1/2 =113.2min) with solubility of 4.82μM and orally bioavailable, demonstrating its potential to be used for in vivo experiments.

Plasma Concentrations of Lysophosphatidic Acid and Autotaxin in Abstinent Patients with Alcohol Use Disorder and Comorbid Liver Disease.

Lysophosphatidic acid (LPA) is an endogenous lysophospholipid and a bioactive lipid that is synthesized by the enzyme autotaxin (ATX). The ATX–LPA axis has been associated with cognitive dysfunction and inflammatory diseases, mainly in a range of nonalcoholic liver diseases. Recently, preclinical and clinical evidence has suggested a role of LPA signaling in alcohol use disorder (AUD) and AUD-related cognitive function. However, the ATX–LPA axis has not been sufficiently investigated in alcoholic liver diseases. An exploratory study was conducted in 136 participants, 66 abstinent patients with AUD seeking treatment for alcohol (alcohol group), and 70 healthy control subjects (control group). The alcohol group was divided according to the presence of comorbid liver diseases (i.e., fatty liver/steatosis, alcoholic steatohepatitis, or cirrhosis). All participants were clinically evaluated, and plasma concentrations of total LPA and ATX were measured using enzyme-linked immunosorbent assays. Data were primarily analyzed using analysis of covariance (ANCOVA) while controlling for age, body mass index, and sex. Logistic regression models were created to assess the association of the ATX–LPA axis and AUD or liver disease. LPA and ATX were log10-transformed to fit the assumptions of parametric testing.The main results were as follows: total LPA and ATX concentrations were dysregulated in the alcohol group, and patients with AUD had significantly lower LPA (F(1,131) = 10.677, p = 0.001) and higher ATX (F(1,131) = 8.327, p = 0.005) concentrations than control subjects; patients with AUD and liver disease had significantly higher ATX concentrations (post hoc test, p < 0.05) than patients with AUD but not liver disease; significant correlations between AUD-related variables and concentrations of LPA and ATX were only found in the non-liver disease subgroup (the duration of alcohol abstinence with LPA and ATX (r = +0.33, p < 0.05); and the severity of AUD with ATX (rho = −0.33, p < 0.05)); and a logistic regression model with LPA, ATX, and AUD-related variables showed an excellent discriminative power (area under the curve (AUC) = 0.915, p < 0.001) for distinguishing patients with AUD and comorbid liver disease. In conclusion, our data show that the ATX–LPA axis is dysregulated in AUD and suggest this lipid signaling, in combination with relevant AUD-related variables, as a reliable biomarker of alcoholic liver diseases.

LPA and Autotaxin: Potential Drug Targets in Asthma?

Lysophosphatidic acid (LPA) is a versatile lysolipid, and activates a variety of signaling cascades in many cell types. Extracellular LPA is produced from lysophosphatidylcholine (LPC) by the enzyme autotaxin (ATX), and binds to a family of G-protein coupled receptors on its target cells. Research by many groups continues to support the idea that LPA, and the ATX-LPA axis, have important roles in asthma and allergic airway inflammation. In vitro studies have shown that LPA activates many cell types implicated in airway inflammation, including eosinophils, mast cells, dendritic cells, lymphocytes, airway epithelial cells, and airway smooth muscle cells. In animal models ATX and LPA receptor antagonists have been shown to attenuate allergic airway inflammation and hyperreactivity, cardinal features of asthma in humans. ATX and LPA antagonists are currently under active development to treat lung fibrosis, cancer, and other conditions. If compounds with acceptable safety profiles can be identified, then it seems likely that they will be useful in inflammatory lung diseases like asthma.