Autotaxin Activity Research Articles

Bronchopulmonary dysplasia demonstrates dysregulated autotaxin/lysophosphatidic acid signaling in a neonatal mouse model.

Bronchopulmonary dysplasia (BPD) is a chronic lung disease affecting premature infants who require oxygen supplementation and ventilator therapy to support their underdeveloped lungs. Autotaxin (ATX), an enzyme that generates the bioactive phospholipid lysophosphatidic acid (LPA), which acts via G-protein coupled receptors, has been implicated in numerous pulmonary diseases. In this study, we explored the pathophysiological role of the ATX/LPA signaling pathway in BPD. Neonatal mice were exposed to normoxia or hyperoxia (85%) for 14 days from birth while being treated with vehicle, ATX inhibitor or LPA receptor 1 (LPA1) inhibitor. In vitro studies utilized human lung fibroblast (HLF) cells exposed to room air, 85% oxygen, or LPA for varying time periods. Supernatants and cells were collected for assays and Western blotting. Animals exposed to hyperoxia showed elevated expression of ATX, ATX activity, and LPA1. Inhibiting ATX or LPA1 improved alveolarization, reduced inflammation, and mitigated extracellular matrix deposition and lysyl oxidase (LOX) expression. LPA1 inhibition leading to reduced LOX expression was associated with a reduction in phosphorylation of AKT. Hyperoxia increases the expression of ATX and LPA1 associated with increased LOXin the lungs. Targeting the ATX/LPA1 pathway could be a potential therapeutic approach to BPD. Exposure to hyperoxia increases the expression and activity of autotaxin (ATX), as well as expression of LPA receptor 1 (LPA1). Increased expression of ATX influences extra cellular matrix (ECM) remodeling. Inhibitors targeting the ATX/LPA pathway could offer a new therapeutic approach to bronchopulmonary dysplasia (BPD), potentially mitigating ECM deposition and improving lung development.

Safety and clinical efficacy of IOA-289, a novel autotaxin inhibitor, plus gemcitabine and nab-paclitaxel (GnP) in patients with previously untreated metastatic pancreatic ductal adenocarcinoma (mPDAC).

e15130 Background: Autotaxin (ATX) plays a critical role in inflammation and resistance mechanisms in a wide range of malignancies. IOA-289 is a novel inhibitor of ATX, with anti-fibrotic, immune-enhancing and anti-tumour activity. This first-in-human trial evaluated safety and tolerability, pharmacokinetic (PK)/pharmacodynamic (PD) profile of IOA-289 in combination with GnP. Methods: IOA-289 was investigated, in untreated mPDAC, in a dose escalation study evaluating the continuous twice daily dosing (BID). IOA-289 was initially administered for 7 days as a monotherapy (Cycle 0) before adding GnP at standard doses in 3 weekly schedule followed by 1 week pause (Cycle 1 onwards). Primary objective: Safety and tolerability. Secondary objectives: PK; PD (e.g., plasma LPA and serum CA19-9); radiographic responses (RECIST 1.1); PFS and OS. Results: Cohort 1 assessed the safety at 100 mg BID (n = 4), cohort 2 at 200 mg (n = 4), and cohort 3 at 400 mg (n = 5). Safety assessment shows no treatment-emergent adverse events led to study drug discontinuation, or a dose limiting toxicity. Most toxicities were consistent with the known toxicity profile of GnP. Median time on treatment for cohort 1 was 4.8 months, for cohort 2 and cohort 3 patients are still under treatment and clinical outcomes are not yet established but are projected to surpass the observation from cohort 1. Five patients are currently remain active on treatment No clinical responses were observed in cohort 1. In cohort 2, 2 out of 4 patients (50%) achieved a Partial Response (PR) at cycle 5 and 7 respectively, which was confirmed and durable. No mature data is available yet for later cohorts. Exposure to the compound resulted in reductions in multiple LPA species, with a protocol-defined inhibition of > 50% for over 24 hrs. Consistent reductions in levels of CA19-9 have been observed since cohort 2 (200 mg BID), with > 50% reduction already from cycle 2, with durable reductions lasting longer than 4 months. No mature data is available for later cohorts. Conclusions: IOA-289 is well tolerated at all dose levels in combination with standard GnP. Patients in the higher dose cohorts are experiencing robust CA19-9 reductions consistent with ORR. Occurrence of PRs occurred later than compared to historical controls and were durable, suggesting a differentiated mode of action. Clinical trial information: NCT05586516 .

Autotaxin inhibition attenuates the aortic valve calcification by suppressing inflammation-driven fibro-calcific remodeling of valvular interstitial cells

BackgroundPatients with fibro-calcific aortic valve disease (FCAVD) have lipid depositions in their aortic valve that engender a proinflammatory impetus toward fibrosis and calcification and ultimately valve leaflet stenosis. Although the lipoprotein(a)-autotaxin (ATX)-lysophosphatidic acid axis has been suggested as a potential therapeutic target to prevent the development of FCAVD, supportive evidence using ATX inhibitors is lacking. We here evaluated the therapeutic potency of an ATX inhibitor to attenuate valvular calcification in the FCAVD animal models.MethodsATX level and activity in healthy participants and patients with FCAVD were analyzed using a bioinformatics approach using the Gene Expression Omnibus datasets, enzyme-linked immunosorbent assay (ELISA), immunohistochemistry, and western blotting. To evaluate the efficacy of ATX inhibitor, interleukin-1 receptor antagonist-deficient (Il1rn-/-) mice and cholesterol-enriched diet-induced rabbits were used as the FCAVD models, and primary human valvular interstitial cells (VICs) from patients with calcification were employed.ResultsThe global gene expression profiles of the aortic valve tissue of patients with severe FCAVD demonstrated that ATX gene expression was significantly upregulated and correlated with lipid retention (r = 0.96) or fibro-calcific remodeling-related genes (r = 0.77) in comparison to age-matched non-FCAVD controls. Orally available ATX inhibitor, BBT-877, markedly ameliorated the osteogenic differentiation and further mineralization of primary human VICs in vitro. Additionally, ATX inhibition significantly attenuated fibrosis-related factors’ production, with a detectable reduction of osteogenesis-related factors, in human VICs. Mechanistically, ATX inhibitor prohibited fibrotic changes in human VICs via both canonical and non-canonical TGF-β signaling, and subsequent induction of CTGF, a key factor in tissue fibrosis. In the in vivo FCAVD model system, ATX inhibitor exposure markedly reduced calcific lesion formation in interleukin-1 receptor antagonist-deficient mice (Il1rn-/-, P = 0.0210). This inhibition ameliorated the rate of change in the aortic valve area (P = 0.0287) and mean pressure gradient (P = 0.0249) in the FCAVD rabbit model. Moreover, transaortic maximal velocity (Vmax) was diminished with ATX inhibitor administration (mean Vmax = 1.082) compared to vehicle control (mean Vmax = 1.508, P = 0.0221). Importantly, ATX inhibitor administration suppressed the effects of a high-cholesterol diet and vitamin D2-driven fibrosis, in association with a reduction in macrophage infiltration and calcific deposition, in the aortic valves of this rabbit model.ConclusionsATX inhibition attenuates the development of FCAVD while protecting against fibrosis and calcification in VICs, suggesting the potential of using ATX inhibitors to treat FCAVD.

Lipoprotein(a) As a Potential Predictive Factor for Earlier Aortic Valve Replacement in Patients with Bicuspid Aortic Valve.

Bicuspid aortic valve (BAV) affects 0.5-2% of the general population and constitutes the major cause of severe aortic valve stenosis (AVS) in individuals ≤70 years. The aim of the present study was to evaluate the parameters that may provide information about the risk of AVS developing in BAV patients, with particular emphasis on lipoprotein(a) (Lp(a)), which is a well-recognized risk factor for stenosis in the general population. We also analyzed the impact of autotaxin (ATX) and interleukin-6 (IL-6) as parameters potentially related to the pathomechanism of Lp(a) action. We found that high Lp(a) levels (>50 mg/dL) occurred significantly more frequently in patients with AVS than in patients without AVS, both in the group below and above 45 years of age (p = 0.036 and p = 0.033, respectively). Elevated Lp(a) levels were also strictly associated with the need for aortic valve replacement (AVR) at a younger age (p = 0.016). However, the Lp(a) concentration did not differ significantly between patients with and without AVS. Similarly, we observed no differences in ATX between the analyzed patient groups, and both ATX activity and concentration correlated significantly with Lp(a) level (R = 0.465, p < 0.001 and R = 0.599, p < 0.001, respectively). We revealed a significantly higher concentration of IL-6 in young patients with AVS. However, this observation was not confirmed in the group of patients over 45 years of age. We also did not observe a significant correlation between IL-6 and Lp(a) or between CRP and Lp(a) in any of the analyzed groups of BAV patients. Our results demonstrate that a high level of Lp(a), greater than 50 mg/dL, may be a significant predictive factor for earlier AVR. Lp(a)-related parameters, such as ATX and IL-6, may be valuable in providing information about the additional cardiovascular risks associated with developing AVS.

Autotaxin Inhibition with IOA-289 Decreases Breast Tumor Growth in Mice Whereas Knockout of Autotaxin in Adipocytes Does Not.

Breast cancer cells produce negligible quantities of autotaxin. Instead, previous work indicated that adipocytes in the inflamed adipose tissue adjacent to breast tumors are a major source of autotaxin secretion that drives breast tumor growth, metastasis, and the loss of efficacy for chemotherapy and radiotherapy. To test this hypothesis, we used mice with an adipocyte-specific knock out of autotaxin. The lack of autotaxin secretion from adipocytes failed to decrease the growth of orthotopic E0771 breast tumors in syngeneic C57BL/6 mice and the growth and lung metastasis of spontaneous breast tumors in MMTV-PyMT mice. However, the inhibition of autotaxin with IOA-289 decreased the growth of E0771 tumors, indicating that another source of autotaxin is responsible for tumor growth. Tumor-associated fibroblasts and leukocytes produce the majority of autotoxin transcripts in the E0771 breast tumors, and we hypothesize that they are the main sources of ATX that drive breast tumor growth. Autotaxin inhibition with IOA-289 increased the numbers of CD8α+-T-cells in the tumors. This was accompanied by decreases in the concentrations of CXCL10, CCL2, and CXCL9 in the plasma and LIF, TGFβ1, TGFβ2, and prolactin in the tumors. Bioinformatics analysis of human breast tumor databases showed that autotaxin (ENPP2) is expressed mainly in endothelial cells and fibroblasts. Autotaxin expression correlated significantly with increases in IL-6 cytokine receptor ligand interactions, signaling by LIF, TGFβ, and prolactin. This confirms the relevance of results from autotaxin inhibition in the mouse model. We propose that inhibiting autotaxin activity that is derived from cells presenting breast tumors such as fibroblasts, leukocytes, or endothelial cells changes the tumor micro-environment in such a way as to inhibit tumor growth.

Endothelial Specific Deletion of Autotaxin Improves Stroke Outcomes

Autotaxin (ATX) is an extracellular secretory enzyme (lysophospholipase D) that catalyzes the hydrolysis of lysophosphatidyl choline to lysophosphatidic acid (LPA). The ATX-LPA axis is a well-known pathological mediator of liver fibrosis, metastasis in cancer, pulmonary fibrosis, atherosclerosis, and neurodegenerative diseases. Also, it is believed that LPA may cause vascular permeability. In ischemic stroke, vascular permeability leading to hemorrhagic transformation is a major limitation for therapies and an obstacle to stroke management. So, in this study, we generated an endothelial-specific ATX deletion in mice (ERT2 ATX-/-) to observe stroke outcomes in a mouse stroke model to analyze the role of endothelial ATX. The AR2 probe and Evans Blue staining were used to perform the ATX activity and vascular permeability assays, respectively. Laser speckle imaging was used to observe the cerebral blood flow following stroke. In this study, we observed that stroke outcomes were alleviated with endothelial deletion of ATX. Permeability and infarct volume was reduced in ERT2 ATX-/- mice compared to ischemia-reperfusion (I/R) only mice. In addition, cerebral blood flow was retained in ERT2 ATX-/- compared to I/R mice. The outcomes in the stroke model are alleviated due to limited LPA concentration, reduced ATX concentration, and ATX activity in ERT2 ATX-/- mice. In this study, we observed that ATX produced by the endothelium could be a major factor responsible for increases in permeability and infarcts, and adverse effects on the cerebral blood flow during cerebral ischemic reperfusion. LPA produced specifically by endothelial cells in the vasculature can disrupt the BBB leading ultimately to neuronal loss and glial activation. With the deletion of ATX from the endothelium, the microvasculature might be more resilient due to reduced LPA. We found evidence that the functional outcomes of ischemic stroke were alleviated with ATX deletion. Therefore, endothelial ATX can be targeted to make better and more effective therapies for stroke management. his research was supported by Louisiana State University Health Sciences-Shreveport Intramural Grant 110101074A to SM; National Institutes of Health grants HL141998 and HL141998-01S1 to SM; grants AA025744, AA026708, and AA025744-02S1 to MP; grants HL122354 and HL145753 to MSB; P20GM12130 to Dr. Christopher Kevil. This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Endothelial Specific Deletion of Autotaxin Improves Stroke Outcomes.

Autotaxin (ATX) is an extracellular secretory enzyme (lysophospholipase D) that catalyzes the hydrolysis of lysophosphatidyl choline to lysophosphatidic acid (LPA). The ATX-LPA axis is a well-known pathological mediator of liver fibrosis, metastasis in cancer, pulmonary fibrosis, atherosclerosis, and neurodegenerative diseases. Additionally, it is believed that LPA may cause vascular permeability. In ischemic stroke, vascular permeability leading to hemorrhagic transformation is a major limitation for therapies and an obstacle to stroke management. Therefore, in this study, we generated an endothelial-specific ATX deletion in mice (ERT2 ATX-/-) to observe stroke outcomes in a mouse stroke model to analyze the role of endothelial ATX. The AR2 probe and Evans Blue staining were used to perform the ATX activity and vascular permeability assays, respectively. Laser speckle imaging was used to observe the cerebral blood flow following stroke. In this study, we observed that stroke outcomes were alleviated with the endothelial deletion of ATX. Permeability and infarct volume were reduced in ERT2 ATX-/- mice compared to ischemia-reperfusion (I/R)-only mice. In addition, the cerebral blood flow was retained in ERT2 ATX-/- compared to I/R mice. The outcomes in the stroke model are alleviated due to the limited LPA concentration, reduced ATX concentration, and ATX activity in ERT2 ATX-/- mice. This study suggests that endothelial-specific ATX leads to increased LPA in the brain vasculature following ischemic-reperfusion and ultimately disrupts vascular permeability, resulting in adverse stroke outcomes.

Serum autotaxin levels correlate with the severity of pruritus in intrahepatic cholestasis of pregnancy.

This study aimed to examine autotaxin (ATX) concentrations in the serum of pregnant women complicated with intrahepatic cholestasis of pregnancy (ICP) and compare them with individuals with uncomplicated healthy pregnancies. This prospective case-control study took place with 83 pregnant women. The study group included 43 pregnant women presenting with a singleton pregnancy diagnosed with ICP in their third trimester of pregnancy. The diagnostic power of the ATX variable was examined by receiver operating characteristic analysis, and the cut-off value calculated according to the Youden index was summarized with the related sensitivity and specificity points. The mean serum concentration of maternal ATX was significantly higher in the ICP cases (8.91 ± 2.69 pg/mL) compared to the pregnant women in the control group (3.59 ± 1.39 ng/mL, p < 0.001). According to the Youden index, a 5.80 ng/mL cut-off value of serum ATX concentrations can be used to diagnose ICP with 97.7% sensitivity and 97.5% specificity. A significant highly positive correlation was found between maternal serum ATX levels and maternal serum total bile acid levels (r=0.633 and p < 0.001) and itch intensity, which was objectified by the visual analog scale score (r=0.951 and p < 0.001). Maternal serum ATX levels were significantly increased in ICP patients as compared with healthy pregnant women. Also, serum ATX activity was highly correlated with the itch intensity. We consider that ATX might represent a robust, accurate, and reliable circulating biomarker to diagnose ICP.

Autotaxin Activity in Chronic Subdural Hematoma: A Prospective Clinical Study.

Autotaxin (ATX) is the ectoenzyme producing the bulk of lysophosphatidic acid (LPA) in circulation. ATX and LPA-mediated signaling (the ATX-LPA axis) play critical roles in the vascular and nervous system development. In adults, this axis contributes to diverse processes, including coagulation, inflammation, fibroproliferation and angiogenesis under physiological and/or pathophysiological conditions. Given evidence implicating several of these processes in chronic subdural hematoma (CSDH) pathogenesis and development, we assessed ATX activity in CSDH patients. Twenty-eight patients were recruited. Blood and hematoma fluid were collected. Enzymatic assays were used to establish serum and hematoma ATX activity. Enzyme-linked immunosorbent assays were used to establish hematoma beta trace (BT) levels, a cerebrospinal fluid (CSF) marker, in a hematoma. ATX activity was nearly three folds higher in hematoma compared to serum (P < 0.001). There was no significant correlation between BT levels and ATX activity in a hematoma. The present results show, for the first time, that ATX is catalytically active in the hematoma fluid of CSDH patients. Moreover, our findings of significantly elevated ATX activity in hematoma compared to serum, implicate the ATX-LPA axis in CSDH pathophysiology. The CSF origin of ATX could not be inferred with the present results. Additional research is warranted to establish the significance of the ATX-LPA axis in CSDH and its potential as a biomarker and/or therapeutic target.

The Covert Surge: Murine Bile Acid Levels Are Associated With Pruritus in Pediatric Autoimmune Sclerosing Cholangitis.

ObjectivesThe exact etiology of pruritus in chronic cholestasis is unknown. Pruritus intensity does not correlate with common biochemical indices and there is a lack of biomarkers guiding diagnosis and treatment. We explored profiles of bile acids (BA) and muricholic acids (MCA) as well as autotaxin (ATX) antigen levels as potential circulating biomarkers of pruritus in pediatric patients.MethodsIn 27 pediatric cholestatic patients [autoimmune sclerosing cholangitis (ASC) n = 20 (with pruritus n = 6, without pruritus n = 14); progressive familial intrahepatic cholestasis (PFIC) n = 7 (with pruritus n = 5, without pruritus n = 2)] and 23 age-matched controls pruritus was assessed by a visual analog scale of pruritus (PVAS). We obtained profiles of serum human BA including MCA using a mass-spectrometry assay and ATX antigen levels with a commercial ELISA.ResultsPFIC and ASC patients exhibited significantly higher BA-, and MCA levels, than healthy controls, but only PFIC patients showed elevated ATX antigen levels higher [median: 1,650 ng/ml, interquartile rang (IQR): 776.9–3,742] compared to controls (median: 315.9 ng/ml, IQR: 251.1–417.2; PFIC p = 0.0003). ASC patients with pruritus showed only a minor increase in total BA (tBA) levels (median: 76.5 μmol/L, IQR: 54.7–205), but strikingly higher T-conjugated BA (median: 16.4 μmol/L, IQR: 8.9–41.4) and total MCA (tMCA) (median: 1.15 μmol/L, IQR: 0.77–2.44) levels compared to ASC patients without pruritus (tBA median: 24.3 μmol/L, IQR: 16.2–80.8; p < 0.0408; T-conjugated BA median: 1.3 μmol/L, IQR: 0.8–4.9; p = 0.0023; tMCA median: 0.30 μmol/L, IQR: 0.13–0.64, p = 0.0033). BA/MCA profiles distinctly differed depending on presence/absence of pruritus. Different from PFIC patients, ATX antigen levels were not significantly elevated in ASC patients with (median: 665.8 ng/ml, IQR: 357.8–1,203) and without pruritus (median: 391.0 ng/ml, IQR: 283.2–485.6). In ASC patients, tBA, tMCA, and ATX antigen levels did not correlate with pruritus severity.ConclusionDespite the same underlying disease, pediatric ASC patients with pruritus exhibit significantly altered BA profiles and MCA levels compared to ASC patients without pruritus. ATX antigen levels seem to have little diagnostic or prognostic meaning in ASC patients. An increased ATX activity alone seems not to be causal for pruritus genesis in ASC patients.Clinical Trial Registration[www.drks.de], identifier [DRKS00026913].

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.

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.

MNK/eIF4E Ser209 Inhibitor eFT-508 Prevents and Therapeutically Treats Fibrosis in a Murine Model of Restrictive Chronic Lung Allograft Rejection

<h3>Purpose</h3> Lung transplant is limited by poor outcomes due to chronic lung allograft dysfunction, CLAD. A fulminant CLAD phenotype, restrictive allograft syndrome (RAS), with limited therapies underscores the need to understand underlying mechanisms. MNK1 kinase is a key translational regulator of cellular functions via eIF4E serine 209 phosphorylation. Here we investigate the role of MNK/eIF4E Ser209 in mesenchymal cell activation and the therapeutic effectiveness of eFT-508 in a murine model of RAS. <h3>Methods</h3> Human lung allograft derived MCs treated with eFT-508 (10uM x 24h) were immunoblotted for collagen I, alpha smooth muscle actin, CD44, autotaxin. A modified boyden chamber assay was utilized for migration. We recently reported a murine RAS model, B6D2F1/J into C57BL6/J. Mice were administered eFT-508 5mg/kg/daily/gavage in a preventive group days 7-28, or therapeutic group days 14-28. <h3>Results</h3> Human allograft derived MCs from CLAD patients demonstrated 4-fold increase in MNK1 (Thr197/202) and eIF4E Ser209 compared to non-fibrotic MCs. Upon eFT-508 treatment, we observed reduced eIF4E Ser209, collagen I, alpha smooth muscle actin and SPARC expression, and migration (n=7; p<0.05). CD44 and autotaxin protein expression was decreased upon eFT-508 treatment, along with secreted autotaxin activity (n=9; p=0.0005). <i>In vivo</i> preventive treatment with eFT-508 decreased airway fibrosis upon histological examination utilizing trichrome staining. Pleural thickness measurements were also reduced compared to vehicle treated controls. A significant reduction in hydroxyproline content, autotaxin secretion, and activity were also noted in eFT-508 treated transplant lung homogenates (n=7; p<0.001). Therapeutic treatment with eFT-508 reduced hydroxyproline content compared to vehicle treated allografts (n=4; p=0.0252). <h3>Conclusion</h3> MNK/eIF4E Ser209 inhibition reduced fibrotic protein expression and migration, demonstrating a role for this pathway in MC activation. <i>In vivo</i> MNK/eIF4E Ser209 inhibition reduced graft fibrosis by preventive and therapeutic treatment regimens in a murine model of RAS. This data highlights a novel therapeutic option for preventing development and progression of RAS.

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.

Investigation of novel ATX inhibitor metabolites by UHPLC-orbitrap-MS/MS and molecular docking studies

Lysophosphatidic acid (LPA) mediated signaling is relevant to fibrosis and the proliferation of pulmonary cells, such as the promotion of fibroblast accumulation and stimulation of the TGF-β signal. One of the essential mechanisms to reduce lysophosphatidic acid production is inhibiting autotaxin activity, which can block the hydrolysis of a lysophosphatidylcholine (LPC) into LPA, leading to the alleviation of the development of pulmonary fibrosis. Recently, 3,4-difluorobenzyl(1-ethyl-5-(4-((4-hydroxypiperidin-1-yl)-methyl)-thiazol-2-yl)-1H-indol-3-yl)carbamate (NAI59), a novel autotaxin inhibitor, showed outstanding anti-pulmonary fibrosis therapeutic effectiveness; however, the metabolic information of NAI59 has not been comprehensively expounded yet. Therefore, in this study, we aim to identify the metabolites of NAI59 in rats using ultra-high-performance liquid chromatography-high-resolution tandem mass spectrometry, and we analyze the potential anti-fibrosis of metabolites in plasma using molecular docking with the crystal structure autotaxin. As a result, a total of 24 metabolites, namely, 15 phase I metabolites and 9 phase II metabolites, were identified, all of which are the novel discovered metabolites. The metabolic pathways of NAI59 were primarily hydroxylation, oxidation, hydrogenation, glucuronic acid conjugation, sulfate conjugation, acetylation, and glucosylation. The molecular docking result showed that the identified metabolites in plasma had great affinities upon docking into the active site of autotaxin, indicating they may have potential anti-pulmonary fibrosis activity. The interaction plots revealed the importance of fluorophenyl and the hydroxyl groups on piperidine that contribute toward the formation of hydrogen bonds with autotaxin. Additionally, we found that the metabolite possessed a better docking result when the structure contained a glucuronic acid group. To the best of our knowledge, this study is the first research into the metabolic fate of NAI59 in rats, and we analyze the potential anti-fibrosis aspects of metabolites using molecular docking. This study provides a metabolic rationale for further preclinical research and imparts new insights for developing improved ATX inhibitors.

Structural and PK-guided identification of indole-based non-acidic autotaxin (ATX) inhibitors exhibiting high in vivo anti-fibrosis efficacy in rodent model

In recent decades, pharmacological targeting of the autotaxin (ATX)/lysophosphatidic acid (LPA) axis accounted for excellent disease management benefits. Herein, to extend the scope of structure-activity relationships (SARs), fifteen indole-based carbamate derivatives (1–15) were prepared to evaluate the ATX inhibitory potency. Among them, compound 4 bearing morpholine moiety was identified as the optimal ATX inhibitor (0.41 nM), superior to the positive control GLPG1690 (2.90 nM). To resolve the intractable issue of poor pharmacokinetic (PK) property, urea moiety was introduced as a surrogate of carbamate which furnished compounds 16–30. The dedicated modification identified the diethanolamine entity 30 with satisfactory water solubility and PK profiles with a minimum sacrifice of ATX inhibition (2.17 nM). The most promising candidate 30 was evaluated for anti-fibrosis effect in a bleomycin challenged mice lung fibrosis model. Upon treatment with 30, the in vivo ATX activity in both lung homogenate and broncheoalveolar fluid (BALF) sample was significantly down-regulated. Furthermore, the gene expression of pro-fibrotic cytokines transforming growth factor-β (TGF-β), interleukin- 6 (IL-6) and tumor necrosis factor-α (TNF-α) in lung tissue was reduced to normal level. Collectively, the promising biological effects may advocate potential application of 30 in fibrosis relevant diseases.

Targeting autotaxin impacts disease advance in the SOD1-G93A mouse model of amyotrophic lateral sclerosis.

A preclinical strategy to broaden the search of potentially effective treatments in amyotrophic lateral sclerosis (ALS) relies on identifying factors controlling motor neuron (MN) excitability. These partners might be part of still unknown pathogenic pathways and/or useful for the design of new interventions to affect disease progression. In this framework, the bioactive membrane‐derived phospholipid lysophosphatidic acid (LPA) affects MN excitability through LPA receptor 1 (LPA1). Furthermore, LPA1 knockdown is neuroprotective in transgenic ALS SOD1‐G93A mice. On this basis, we raised the hypothesis that the major LPA‐synthesizing ectoenzyme, autotaxin (ATX), regulates MN excitability and is a potential target to modulate disease development in ALS mice. We show here that PF‐8380, a specific ATX inhibitor, reduced intrinsic membrane excitability (IME) of hypoglossal MNs in brainstem slices, supporting that baseline ATX activity regulates MN IME. PF‐8380‐induced alterations were prevented by a small‐interfering RNA directed against mRNA for lpa1 . These outcomes support that impact of ATX‐originated lysophospholipids on MN IME engages, at least, the G‐protein‐coupled receptor LPA1. Interestingly, mRNA atx levels increased in the spinal cord of pre‐symptomatic (1–2 months old) SOD1‐G93A mice, thus preceding MN loss. The rise in transcripts levels also occurred in cultured spinal cord MNs from SOD1‐G93A embryos, suggesting that mRNA atx upregulation in MNs is an etiopathogenic event in the ALS cell model. Remarkably, chronic administration in the drinking water of the orally bioavailable ATX inhibitor PF‐8380 delayed MN loss, motor deterioration and prolonged life span in ALS mice. Treatment also led to a reduction in LPA1‐immunoreactive patches in transgenic animals mostly in MNs. These outcomes support that neuroprotective effects of interfering with ATX in SOD1‐G93A mice rely, at least in part, on LPA1 knockdown in MNs. Therefore, we propose ATX as a potential target and/or a biomarker in ALS and highlight ATX inhibitors as reasonable tools with therapeutic usefulness for this lethal pathology.

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

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

Reduced Cerebrospinal Fluid Levels of Lysophosphatidic Acid Docosahexaenoic Acid in Patients With Major Depressive Disorder and Schizophrenia.

BackgroundLysophosphatidic acid (LPA) is involved in numerous biological processes, including neurodevelopment, chronic inflammation, and immunologic response in the central nervous system. Autotaxin (ATX) is a secreted enzyme that produces LPA from lysophosphatidylcholine (LPC). Previous studies have demonstrated decreased protein levels of ATX in cerebrospinal fluid (CSF) of patients with major depressive disorder (MDD). Based on those studies, the current study investigated the levels of lysophospholipids species including LPA and related metabolic enzymes, in CSF of patients with MDD and schizophrenia (SCZ).MethodsThe levels of lysophospholipids species and related metabolic enzymes were measured with either liquid chromatography-tandem mass spectrometry or enzyme-linked immunosorbent assay. Japanese patients were diagnosed with DSM-IV-TR. CSF was obtained from age- and sex-matched healthy controls (n = 27) and patients with MDD (n = 26) and SCZ (n = 27).ResultsOf all lysophospholipids species, the levels of LPA 22:6 (LPA - docosahexaenoic acid) were significantly lower in patients with MDD and SCZ than in healthy controls. These levels were negatively correlated with several clinical symptomatic scores of MDD, but not those of SCZ. In addition, the levels of LPA 22:6 were significantly correlated with the levels of LPC 22:6 among all 3 groups. On the other hand, the levels of LPA 22:6 were not correlated with ATX activity in patients with MDD and SCZ.ConclusionThe lower levels of LPA 22:6 in patients with MDD and SCZ suggest an abnormality of LPA 22:6 metabolism. In addition, several depressive symptoms in patients with MDD were significantly associated with the lower levels of LPA 22:6, suggesting an involvement of LPA 22:6 in the pathophysiology of MDD.