Lysophosphatidic Acid Treatment Research Articles

Treatment with lysophosphatidic acid improves glomerulonephritis through the suppression of macrophage activation in a murine model of systemic lupus erythematosus.

Several therapeutic agents have been developed and used for the clinical treatment of systemic lupus erythematosus (SLE). In cases where SLE is accompanied by severe organ failures, such as neuropsychiatric lupus erythematosus (NPSLE) and acute onset of lupus nephritis, the use of potent immunosuppressive drugs, such as cyclophosphamide, is necessary. However, potent immunosuppressive drugs are known to increase infection risks. Thus, the development of therapeutic agents with novel mechanisms is urgently required. Previously, we reported that treatment with lysophosphatidic acid (LPA) prevents depression-like behaviours by suppressing microglial activation in MRL/lpr mice. In this study, we examined whether the treatment with LPA improves glomerulonephritis by affecting systemic immunity in MRL/lpr mice. Eighteen-week-old MRL/lpr mice were treated with a vehicle or LPA for 3 weeks. After treatment, the glomerular inflammation and damage parameters were compared between the 2 groups. Moreover, we examined the effects of LPA on immune cells by flow cytometry using isolated splenocytes. LPA treatment in MRL/lpr mice significantly reduced the daily urinary albumin content and suppressed the CD68-positive cells and Periodic acid-Schiff (PAS)-positive areas in the glomeruli. The treatment also suppressed plasma anti-dsDNA antibodies and inflammatory cytokines in MRL/lpr mice. Although LPA did not significantly affect the total number of splenocytes, the treatment significantly reduced CD11b+Ly6G-Ly6C- cells (mature macrophages), as well as CD11b+Ly6G-Ly6C-CD68+ cells (activated mature macrophages). These results suggest that LPA may improve glomerulonephritis by suppressing macrophage activation in MRL/lpr mice.

Lysophosphatidic acid down-regulates human RIPK4 mRNA in keratinocyte- derived cell lines.

The tight control of proliferating keratinocytes is vital to the successful function of the skin. Differentiation of dividing cells is necessary to form a skin barrier. The same dividing cells are necessary to heal wounds and when malignant form tumors. RIPK4, a serine-threonine kinase, plays critical roles in these processes. Its loss of function was associated with pathological keratinocyte proliferation and development of squamous cell carcinoma (SCC) in humans and mice. The current study extends previous findings in the importance of RIPK4 in keratinocyte proliferation. A serum-derived phospholipid, lysophosphatidic acid (LPA), was identified as an important biologic inhibitor of RIPK4. LPA functions by inhibiting the transcription of RIPK4 mRNA. LPA treatment led to increased keratinocyte proliferation, and this was compromised in cells with reduced RIPK4 expression. The current study may help to explain the mechanism by which RIPK4 was downregulated during SCC progression and provide insights on RIPK4 functions. It may also allow for targeting of RIPK4 through a natural component of serum.

Lysophosphatidic acid exerts protective effects on HEI-OC1 cells against cytotoxicity of cisplatin by decreasing apoptosis, excessive autophagy, and accumulation of ROS

Lysophosphatidic acid (LPA) is an active phospholipid signaling molecule that binds to six specific G protein-coupled receptors (LPA1-6) on the cell surface and exerts a variety of biological functions, including cell migration and proliferation, morphological changes, and anti-apoptosis. The earliest study from our group demonstrated that LPA treatment could restore cochlear F-actin depolymerization induced by noise exposure, reduce hair cell death, and thus protect hearing. However, whether LPA could protect against cisplatin-induced ototoxicity and which receptors play the major role remain unclear. To this end, we integrated the HEI-OC1 mouse cochlear hair cell line and zebrafish model, and found that cisplatin exposure induced a large amount of reactive oxygen species accumulation in HEI-OC1 cells, accompanied by mitochondrial damage, leading to apoptosis and autophagy. LPA treatment significantly attenuated autophagy and apoptosis in HEI-OC1 cells after cisplatin exposure. Further investigation revealed that all LPA receptors except LPA3 were expressed in HEI-OC1 cells, and the mRNA expression level of LPA1 receptor was significantly higher than that of other receptors. When LPA1 receptor was silenced, the protective effect of LPA was reduced and the proportion of apoptosis cells was increased, indicating that LPA-LPA1 plays an important role in protecting HEI-OC1 cells from cisplatin-induced apoptosis. In addition, the behavioral trajectory and in vivo fluorescence imaging results showed that cisplatin exposure caused zebrafish to move more actively, and the movement speed and distance were higher than those of the control and LPA groups, while LPA treatment reduced the movement behavior. Cisplatin caused hair cell death and loss in zebrafish lateral line, and LPA treatment significantly protected against hair cell death and loss. LPA has a protective effect on hair cells in vitro and in vivo against the cytotoxicity of cisplatin, and its mechanism may be related to reducing apoptosis, excessive autophagy and ROS accumulation.

Abstract 13046: Lysophosphatidic Acid Activates Spinal TRPV1 Signaling and Contributes to Myocardial Ischemia/Reperfusion Injury

Introduction: Lysophosphatidic acid (LPA) has been shown to directly activate transient receptor potential vanilloid 1 (TRPV1) on primary nociceptors via the binding site of K710 to produce acute pain. However, it is unclear whether LPA participates in myocardial ischemia/reperfusion (I/R) injury by activating TRPV1 receptors. Hypothesis: We hypothesize that LPA contributes to myocardial I/R injury by acting on TRPV1 receptors in the spinal cord, and that blocking the interaction between LPA and TRPV1 could exert cardioprotective effects. Methods: Wild type male C57BL/6 mice underwent myocardial I/R injury, and LPA levels in cerebrospinal fluid (CSF) were measured. LPA synthesis inhibitor HA-130 or exogenous LPA were intrathecally administrated during myocardial ischemia. Additionally, CRISPR/Cas-9 edited TRPV1K710N knock-in mice and a peptide V1-Cal targeting the K710 region were utilized to block the interaction of LPA with TRPV1. Results: The levels of LPA in CSF of the I/R group were found to be 1.5-fold higher than those of the sham group (Fig.1B, p=0.0001). Intrathecal administration of HA130 reduced infarct size (Fig.1C, p=0.0021) and CSF LPA levels (Fig.1D, p=0.0018). The CSF LPA levels were positively correlated with infarct size (Fig.1E, p<0.0001). Intrathecal administration of LPA caused a 1.4-fold rise in infarct size relative to I/R group in wild type mice (Fig.1F, p<0.0001), but not in TRPV1K710N mice (Fig.1F, p>0.9999). LPA treatment enhanced the activation of spinal TRPV1 receptors following I/R injury in wild type mice, but not in TRPV1K710N mice (Fig.1 G-I). Further, the V1-Cal peptide reduced myocardial I/R injury, as well as the protein levels of TRPV1 and pTRPV1 in spinal cord (Fig.1 J-M). Conclusions: Our results suggest that LPA activates spinal TRPV1 signaling and contributes to myocardial I/R injury. Blockade of the interaction between LPA and TRPV1 in spinal cord may be a promising therapeutic approach for myocardial ischemic injury.

Simulated microgravity attenuates skin wound healing by inhibiting dermal fibroblast migration via F-actin/YAP signaling pathway.

Skin and its cell components continuously subject to extrinsic and intrinsic mechanical forces and are mechanical sensitive. Disturbed mechanical homeostasis may lead to changes in skin functions. Gravity is the integral mechanical force on the earth, however, how gravity contributes to the maintenance of skin function and how microgravity in space affects the wound healing are poorly understood. Here, using microgravity analogs, we show that simulated microgravity (SMG) inhibits the healing of cutaneous wound and the accumulation of dermal fibroblasts in the wound bed. In vitro, SMG inhibits the migration of human foreskin fibroblast cells (HFF-1), and decreases the F-actin polymerization and YAP (yes-associated protein) activity. The SMG-inhibited migration can be recovered by activating YAP or F-actin polymerization using lysophosphatidic acid (LPA) or jasplakinolide (Jasp), suggesting the involvement of F-actin/YAP signaling pathway in this process. In SMG rats, LPA treatment improves the cutaneous healing with increased dermal fibroblasts in the wound bed. Together, our results demonstrate that SMG attenuates the cutaneous wound healing by inhibiting dermal fibroblast migration, and propose the crucial role of F-actin/YAP mechano-transduction in the maintenance of skin homeostasis under normal gravity, and YAP as a possible therapeutic target for the skin care of astronauts in space.

Treatment with lysophosphatidic acid prevents microglial activation and depression-like behaviours in a murine model of neuropsychiatric systemic lupus erythematosus.

Neuropsychiatric systemic lupus erythematosus (NPSLE) is an incurable disease characterised by neuropsychiatric symptoms, particularly depression. Novel therapeutic options for NPSLE are urgently needed. Several previous reports have suggested that both microglial activation and impaired neurogenesis may be involved in the progression of depression. In contrast, the administration of lysophosphatidic acid (LPA) ameliorates depression and anxiety. Therefore, in the present study, we determined whether treatment with LPA affects microglial activation, impaired neurogenesis, and abnormal behaviour in MRL/lpr mice. In both tail suspension test and forced swim test, the MRL/lpr mice exhibited a significant increase in total immobility time compared with MRL/+ mice. Treatment with LPA significantly suppressed the prolonged immobility time in MRL/lpr mice. In contrast, pretreatment with ki16425 (a specific antagonist of LPA receptor 1 and 3) significantly reversed the effects of LPA. Furthermore, MRL/lpr mice exhibited impairments in spatial working memory and visual cognitive memory, which were suppressed by LPA treatment. The expression levels of TMEM119, CD68, GFAP, and caspase-3 in the hippocampus and prefrontal cortex of MRL/lpr mice were significantly higher than those in MRL/+ mice. Treatment with LPA inhibited these increases in MRL/lpr mice. Pretreatment with ki16425 reversed LPA-mediated inhibition of microglial activation. The quantity of sodium fluorescein that leaked into the brain tissues in MRL/lpr mice were significantly higher than that in MRL/+ mice. Treatment with LPA tended to decrease the sodium fluorescein leakage. These findings suggest that treatment with LPA may regulate microglial activation, which is important in the pathogenesis of NPSLE, as well as blood-brain-barrier weakening and abnormal behaviour.

Alteration of Cellular Energy Metabolism through LPAR2-Axin2 Axis in Gastric Cancer.

Lysophosphatidic acid (LPA), a multifunctional endogenous phospholipid, plays a vital role in cellular homeostasis and the malignant behavior of cancer cells through G-protein-coupled receptors. However, the role of LPA in β-catenin-mediated gastric cancer is unknown. Here, we have noted the high expression of LPAR2 in human gastric cancer tissues, and that LPA treatment significantly increased the proliferation, migration, and invasion of human gastric cancer cells. Results from our biochemical experiments showed that an LPA exposure increased the expression of β-catenin and its nuclear localization, increased the phosphorylation of glycogen synthase kinase 3β (GSK-3β), decreased the expression of Axin2, and increased the expression of the target genes of the β-catenin signaling pathway. The LPA2 receptor (LPAR2) antagonist significantly reduced the LPA-induced nuclear localization of β-catenin, the primary signaling event. The knockdown of LPAR2 in the gastric cancer cell lines robustly reduced the LPA-induced β-catenin activity. An LPA exposure increased the ATP production by both oxidative phosphorylation and glycolysis, and this effect was abrogated with the addition of an LPAR2 antagonist and XAV393, which stabilizes the Axin and inhibits the β-catenin signaling pathway. Based on our findings, the possibility that LPA contributes to gastric cancer initiation and progression through the β-catenin signaling pathway as well as by the dysregulation of the energy metabolism via the LPAR2 receptor and Axin2, respectively, provides a novel insight into the mechanism of and possible therapeutic targets of gastric cancer.

Lysophosphatidic Acid Improves Human Sperm Motility by Enhancing Glycolysis and Activating L-Type Calcium Channels.

Until now, the molecular mechanisms underlining sperm motility defect causing male infertility are still poorly understood. Safe and effective compounds or drugs that can improve sperm motility are also very limited. Lysophosphatidic acid (LPA) is a naturally occurring phospholipid and a bioactive intermediate with multiple biological activities. It has been detected in various body fluids such as serum, plasma, saliva, tears, blister fluids, hen egg white, and ascites from patients with ovarian cancer. LPA is also abundant in seminal plasma and follicular fluid. It enhances follicle stimulation, improves oocyte fertilization, and promotes early embryonic development and embryo implantation. However, the physiological role of LPA in the male reproductive system remains unknown. Here, our study showed that LPA significantly improved the motility parameters of human sperm hyperactivation in a dose-dependent manner. The LPA-induced elevation of sperm motility is dependent on bovine serum albumin (BSA) but independent of the classical BSA-induced sAC/cAMP/PKA signaling pathway. The enhancement of sperm motility by LPA could not be blocked by CCCP, a respiratory inhibitor suppressing mitochondrial ATP production. Moreover, LPA improved the activity of triosephosphate isomerase in glycolysis. Meanwhile, LPA treatment significantly increased ATP and phosphoenolpyruvate levels and decreased ADP content during sperm glycolysis. Notably, none of known or identified LPA receptors was detected in human sperm. Further investigations showed that LPA promoted sperm motility through L-type calcium channels. In summary, this study revealed the involvement of LPA in the regulation for human sperm motility by enhancing glycolysis and activating L-type calcium channels. The current findings may shed new light on the understanding of causes of asthenozoospermia, and indicate that LPA could be used as a novel therapeutic agent to improve sperm function and fertilizing capacity.

Abstract 5810: LPAR2 depletion suppresses gastric cancer via alteration of mitochondrial energy metabolism

Abstract Introduction: Gastric cancer is the second most common cause of cancer-related death worldwide. Altered metabolism is considered a primary hallmark of tumorigenesis, as it can regulate important processes associated with proliferation, migration, and invasion. Lysophosphatidic acid (LPA), a multifunctional endogenous phospholipid, plays a vital role in cellular homeostasis and malignant behavior of the cancer cells through G-protein coupled receptors. Although several signaling pathways have been reported as a critical mediator of gastric cancer, LPA-LPAR2 axis-mediated alteration of energy metabolism in gastric cancer progression has not been established yet. Therefore, our study focused on the molecular mechanism of LPA-LPAR2 axis-mediated alteration of mitochondrial bioenergetics during gastric cancer progression. Method: TCGA database analysis was carried out to check the mRNA levels of the LPA receptors. Expression of LPA receptors was investigated in human gastric cancer samples by immunoblotting, RT-PCR, and immunohistochemical analysis. We elucidated the functional effects of LPA on gastric cancer by performing ECIS proliferation, migration assay, scratch assay, and transwell invasion assay in gastric cancer cells. To confirm the involvement of the specific LPA receptor on gastric cancer progression, we treated the cells with LPA1-3 receptor blocker (Ki16425) and LPA2 receptor-specific antagonist. Following LPA receptor blockade, we analyzed the glycolytic and mitochondrial functions by measuring extracellular acidification rate (ECAR) and oxygen consumption rate (OCR) using a Seahorse XF24 analyzer. Result: Our TCGA dataset analysis, LPA ELISA, and the Western blotting result showed the presence of LPA receptors in human gastric cancer tissues, specifically LPAR2 was robustly increased (P<0.001) in human gastric cancer tissue samples, and LPA level was significantly higher (P<0.001), suggesting a possible role of LPA in gastric cancer. LPA treatment increased proliferation, migration, and invasion activity in AGS and NCI-N87 gastric cancer cell lines. In contrast, LPA receptor antagonist Ki16425 abrogated the LPA-induced effect. Our seahorse analysis results showed that LPA treatment increased the OCR (P<0.001) and the ECAR (P<0.001), whereas LPAR2 specific receptor antagonists abrogated this effect (P<0.001). Conclusion: Together, our results suggested that the LPA-LPAR2 axis mediates gastric cancer initiation and progression by increasing energy metabolism via oxidative phosphorylation and glycolysis. Thus, targeting the LPAR2 receptor may give us a novel therapeutic approach to treat gastric cancer. Citation Format: Hosne Ara, Susmita Bhattarai, Sudha Sharma, Utsab Subedi, Xiuping Yu, Md. Shenuarin Bhuiyan, Sumitra Miriyala, Manikandan Panchatcharam. LPAR2 depletion suppresses gastric cancer via alteration of mitochondrial energy metabolism [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5810.

Lysophosphatidic acid stimulates pericyte migration via LPA receptor 1

Lysophosphatidic acid (LPA) is a bioactive compound known to regulate various vascular functions. However, despite the fact that many vascular functions are regulated by peri-vascular cells such as pericytes, the effect of LPA on brain pericytes has not been fully evaluated. Thus, we designed this study to evaluate the effects of LPA on brain pericytes. These experiments revealed that while LPA receptors (LPARs) are expressed in cultured pericytes from mouse brains, LPA treatment does not influence the proliferation of these cells but does have a profound impact on their migration, which is regulated via the expression of LPAR1. LPAR1 expression was also detected in human pericyte culture and LPA treatment of these cells also induced migration. Taken together these findings imply that LPA-LPAR1 signaling is one of the key mechanisms modulating pericyte migration, which may help to control vascular function during development and repair processes.

Functional MYO5B Loss Alters Lipid Metabolic Pathways in Intestinal Progenitor Cells

Background and Aims: Differentiation of intestinal epithelial cells involves cell division inhibition, cell lineage choice, and brush border elaboration. Myosin Vb (MYO5B) is a motor protein that is critical for cell polarization and membrane protein trafficking in intestinal epithelial cells and its deficiency causes both hyperproliferation and microvillus defects. Recently, we reported that a bioactive phospholipid, lysophosphatidic acid (LPA), can promote cell differentiation and nutrient absorption in tamoxifen‐induced MYO5B knockout mice in vivo and in enteroids. Our RNA‐sequencing data with KEGG pathway and GO enrichment analysis indicated that MYO5B loss disrupts stem cell characteristics, cell lineage differentiation, and energy metabolic pathways. We hypothesize that the alteration of metabolic pathways in progenitor cells causes the disruption of stem cell function.Methods: Adult Villin‐CreERT2;Myo5bflox/flox or littermate control (Myo5bflox/flox) mice received a single dose of tamoxifen and the intestinal tissues were analyzed 4 days after the tamoxifen injection. Fresh frozen sections of jejunum were analyzed by imaging mass spectrometry (IMS). Immunostaining for key metabolic enzymes were utilized to support the RNA‐seq and IMS observations.Results: Using RNA‐sequencing, MYO5B deficient epithelial cells showed significant decreases in transcripts for enzymes that mediate fatty acid ß‐oxidation compared to control mouse jejunum. Instead, Acsl3, Acat2, and Hmgcs2 were significantly upregulated, proteins that are important for lipogenesis, cholesterol ester synthesis, and ketogenesis, respectively. Consistent with these results, IMS demonstrated that MYO5B deficient tissues accumulated long‐chain fatty acids, phosphatidylinositol, cholesterol derivatives, and nucleotide products in the mucosa (Figure 1). Immunostaining signals of HMGCS2 in control mouse jejunum is limited to the crypt stem cells, whereas MYO5B‐deficient intestine showed expanded HMGCS2 expression in the proliferative cell zone. Interestingly, systemic LPA treatment in MYO5B‐deficient mice further increased the expression of HMGCS2 in both proliferating and differentiated epithelial cells (Figure 2). These observations suggest that MYO5B loss induces a starvation‐like phenotype in proliferative epithelial cells and that LPA treatment increased ketone body production as alternative energy fuels, resulting in the promotion of epithelial cell differentiation.Conclusion: MYO5B loss impairs fatty acid oxidation in the intestinal progenitor cells, which likely leads to cell differentiation deficits. Alteration of metabolic pathways might be a therapeutic target for epithelial disfunction seen in diarrheal diseases.

Roles for CCN proteins in Lysophosphatidic Acid and Sphingosine 1‐Phosphate Signaling in Prostate Cancer Cells

IntroductionCysteine rich angiogenic factor (CCN1/Cyr61) and connective tissue growth factor (CCN2/CTGF) are members of the CCN family of matricellular proteins. These secreted proteins interact with extracellular matrix proteins and integrins to modulate cell signaling. CCN proteins have been implicated in survival and other mitogenic responses in various cancers. CCN1 and CCN2 are the two early inducible genes in this family. Lysophosphatidic acid (LPA) and sphingosine‐1‐phosphate (S1P) are lipid growth factors known to contribute to tumor progression in prostate cancer. In previous work, our group showed that LPA stimulates proliferation and migration in prostate cancer cells via the G protein‐coupled receptor LPAR1. Free fatty acid receptor 4 (FFAR4) agonists, inhibit LPA‐induced responses in prostate cancer cells. We also demonstrated that CCN1 expression is induced by LPA in human prostate cancer cells. However, the roles of CCN1 and CCN2 in cancer cell signaling have not been fully elucidated.HypothesisWe hypothesize that CCN proteins contribute to LPA‐ and S1P‐induced adhesion and mitogenic signaling in prostate cancer cells.Methods and ResultsPC‐3 human prostate cancer cells were used in the current study. For all experiments, cells were serum starved for 24 hours and then treated with and without 10 µM LPA or S1P. Immunoblotting showed that CCN1 and CCN2 were induced by LPA after 2‐5 hours in PC‐3 cells. The response was inhibited by the LPA receptor antagonist Ki1625. TUG‐891, an FFAR4 agonist, inhibited LPA‐induced CCN1 expression in PC‐3 cells. S1P also induced CCN1 expression in PC‐3 cells. In related experiments, whole cell extracts, and extracellular matrix were subjected to immunoblotting for CCN1, and immunofluorescence microscopy was used to localize the induced CCN1. CCN1 protein levels in extracellular matrix were increased 2‐5 hours after LPA treatment, as compared to untreated controls, and CCN1 was detected outside the cells by confocal microscopy. In cell adhesion assays, LPA‐treated PC‐3 cells exhibited an increase in adhesion to fibronectin‐coated 96‐well plates that was detected by 2 hours. Erk MAPK activation in response to LPA was biphasic, with the later phase occurring after CCN1/2 induction.ConclusionThe results of this study suggest that CCN proteins may play roles in LPA‐ and S1P‐induced signaling in prostate cancer cells and are thus potential therapeutic targets.

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<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<0.05) decreased with LPA exposure. In invivo stroke model ATX enzymatic activity is prominently raised (P<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<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<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<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.

LPAR2 receptor stimulates progression of gastric cancer through β‐catenin signaling pathway

IntroductionLysophosphatidic acid (LPA) is a simple lipid that has been implicated in cellular homeostasis and pathology of different cancer by binding with G‐protein coupled receptors (LPAR1‐6). The aberrant Wnt signaling pathway has been reported to facilitate a crucial role in tumorigenesis and therapy responses. Although dysregulated β‐catenin signaling pathway has been established as a critical mediator of gastric cancer, whether LPA and LPAR2 play a role in the β ‐catenin signaling pathway during gastric cancer progression remains elusive.ObjectiveOur present study explored an unknown role of LPA and LPAR2 in LPA‐induced activation of β‐catenin and its downstream signaling mechanism in β‐catenin mediated gastric cancer.MethodWe analyzed the mRNA levels of different LPA receptors in human gastric cancer by ULCAN database using TCGA dataset. LPA ELISA, Western blotting, and immunohistochemical analysis were done in the human normal stomach and cancerous stomach to measure the LPA level and check the expression of different LPA receptors. To investigate the functional role of LPAR2 in LPA‐mediated gastric cancer progression, we performed ECIS proliferation, migration assay, scratch assay, and transwell invasion assay in LPA or LPA with LPAR2 antagonist treated cells. To elucidate the molecular mechanism of LPA‐mediated gastric cancer progression, we performed Western blotting, Real‐time PCR, Luciferase assay, Immunocytochemistry, and nuclear fractionation in gastric cancer cells treated with LPA or LPA with LPAR2 antagonist.ResultWe confirmed a robust increase in LPA level and significantly increased expression of LPAR2 receptor in human gastric cancer tissue samples (P<0.001) by LPA ELISA, western blotting analysis, and immunohistochemistry, suggesting a possible role of LPA in gastric cancer through LPAR2 receptor. In cultured AGS and NCI‐N87 gastric cancer cell line, LPA treatment increased proliferation, migration, and invasion activity, whereas LPAR2 receptor antagonist abrogated the LPA induced effect. Further, when the cells were treated with LPA, it increased the β‐catenin activity, nuclear localization of β ‐catenin, and the expression of the downstream target genes of the β‐catenin signaling pathway. In addition, we confirmed LPA‐induced activation of the β‐catenin signaling pathway through the LPAR2 receptor by using LPAR2 antagonist and by knockdown of LPAR2 where LPAR2 antagonist treatment or LPAR2 knockdown rescued LPA induced activation of the β ‐catenin signaling pathway in both cell lines.Conclusionwe established a novel LPA‐LPAR2‐β‐catenin signaling pathway in the progression of gastric cancer. All our results support that LPAR2 is a potential marker and target for developing novel therapeutic strategies in the treatment of gastric cancer.

Lysophosphatidic Acid Promotes the Expansion of Cancer Stem Cells via TRPC3 Channels in Triple-Negative Breast Cancer.

Triple-negative breast cancer (TNBC) is a highly aggressive cancer for which targeted therapeutic agents are limited. Growing evidence suggests that TNBC originates from breast cancer stem cells (BCSCs), and elucidation of the molecular mechanisms controlling BCSC proliferation will be crucial for new drug development. We have previously reported that the lysosphingolipid sphingosine-1-phosphate mediates the CSC phenotype, which can be identified as the ALDH-positive cell population in several types of human cancer cell lines. In this study, we have investigated additional lipid receptors upregulated in BCSCs. We found that lysophosphatidic acid (LPA) receptor 3 was highly expressed in ALDH-positive TNBC cells. The LPAR3 antagonist inhibited the increase in ALDH-positive cells after LPA treatment. Mechanistically, the LPA-induced increase in ALDH-positive cells was dependent on intracellular calcium ion (Ca2+), and the increase in Ca2+ was suppressed by a selective inhibitor of transient receptor potential cation channel subfamily C member 3 (TRPC3). Moreover, IL-8 production was involved in the LPA response via the activation of the Ca2+-dependent transcriptional factor nuclear factor of activated T cells. Taken together, our findings provide new insights into the lipid-mediated regulation of BCSCs via the LPA-TRPC3 signaling axis and suggest several potential therapeutic targets for TNBC.

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<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.

Cell differentiation is disrupted by MYO5B loss through Wnt/Notch imbalance.

Functional loss of myosin Vb (MYO5B) induces a variety of deficits in intestinal epithelial cell function and causes a congenital diarrheal disorder, microvillus inclusion disease (MVID). The impact of MYO5B loss on differentiated cell lineage choice has not been investigated. We quantified the populations of differentiated epithelial cells in tamoxifen-induced, epithelial cell–specific MYO5B-knockout (VilCreERT2 Myo5bfl/fl) mice utilizing digital image analysis. Consistent with our RNA-sequencing data, MYO5B loss induced a reduction in tuft cells in vivo and in organoid cultures. Paneth cells were significantly increased by MYO5B deficiency along with expansion of the progenitor cell zone. We further investigated the effect of lysophosphatidic acid (LPA) signaling on epithelial cell differentiation. Intraperitoneal LPA significantly increased tuft cell populations in both control and MYO5B-knockout mice. Transcripts for Wnt ligands were significantly downregulated by MYO5B loss in intestinal epithelial cells, whereas Notch signaling molecules were unchanged. Additionally, treatment with the Notch inhibitor dibenzazepine (DBZ) restored the populations of secretory cells, suggesting that the Notch pathway is maintained in MYO5B-deficient intestine. MYO5B loss likely impairs progenitor cell differentiation in the small intestine in vivo and in vitro, partially mediated by Wnt/Notch imbalance. Notch inhibition and/or LPA treatment may represent an effective therapeutic approach for treatment of MVID.

Abstract 1987: Lysophosphatidic acid's unknown role in the initiation and progression of gastric cancer through LPAR2 receptor and beta-catenin signaling pathway by altering the energy metabolism

Abstract Introduction: Lysophosphatidic acid (LPA), a multifunctional endogenous phospholipid, plays a vital role in cellular homeostasis and malignant behavior of the cancer cells through G-protein coupled receptors. Although dysregulated beta-catenin signaling pathway has been reported as a critical mediator of gastric cancer, LPA's role in beta-catenin mediated gastric cancer is unknown. Hence, our study aims to determine LPA's functional role and its downstream signaling mechanism in beta-catenin mediated gastric cancer. Method: We used commercially available tissue lysates from the human normal and cancerous stomach to check the LPA level and the expression of LPA receptors by ELISA, western blotting, and Immunohistochemical analysis. To examine LPA's functional effect in gastric cancer, we performed ECIS proliferation, migration assay, scratch assay, and transwell invasion assay in LPA treated gastric cancer cells. Next, to elucidate LPA's molecular mechanism mediated gastric cancer progression, we performed Western blotting, and Real-time PCR of beta-catenin and its downstream target genes in LPA treated cells. To investigate the LPA-induced beta-catenin activity, Luciferase assay, Immunocytochemistry, and nuclear fractionation were done in LPA-treated cells. TCGA database analysis was carried out to check the mRNA level of beta-catenin and its downstream target genes and LPA receptors. Furthermore, superoxide production and Seahorse analysis were performed in the LPA treated cells to determine the oxidative stress and mitochondrial energy metabolism. Result: LPA ELISA and Western blotting of LPA receptors in human gastric cancer tissues showed that the LPA level is significantly higher (P<0.001), and LPAR2 is robustly increased (P<0.001)in human gastric cancer tissue samples suggesting a possible role of LPA in gastric cancer. In cultured gastric cancer cell line, LPA treatment increased proliferation, migration, and invasion activity, whereas LPA receptor antagonist abrogated the LPA induced effect. Similarly, LPA treatment leads to activate the beta-catenin and increased the expression of the downstream target genes of the beta-catenin signaling pathway. LPA-induced activation of beta-catenin signaling pathway was abrogated with the addition of LPA receptor antagonist and knockdown of LPAR2. Besides, LPA treatment increased oxidative stress and increased glucose uptake and ATP production (P<0.001) by both oxidative phosphorylation and glycolysis, whereas LPA antagonist abrogated this effect. Conclusion: Together, all of our results suggested that LPA mediates gastric cancer initiation and progression through the beta-catenin signaling pathway via the LPAR2 receptor by altering energy metabolism. Our findings will provide a novel insight into gastric cancer treatment. Citation Format: Hosne Ara, Susmita Bhattarai, Sudha Sharma, Utsab Subedi, MD.Shenuarin Bhuiyan, Xiuping Yu, Sumitra Miriyala, Manikandan Panchatcharam. Lysophosphatidic acid's unknown role in the initiation and progression of gastric cancer through LPAR2 receptor and beta-catenin signaling pathway by altering the energy metabolism [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1987.

Primed to die: an investigation of the genetic mechanisms underlying noise-induced hearing loss and cochlear damage in homozygous Foxo3-knockout mice

The prevalence of noise-induced hearing loss (NIHL) continues to increase, with limited therapies available for individuals with cochlear damage. We have previously established that the transcription factor FOXO3 is necessary to preserve outer hair cells (OHCs) and hearing thresholds up to two weeks following mild noise exposure in mice. The mechanisms by which FOXO3 preserves cochlear cells and function are unknown. In this study, we analyzed the immediate effects of mild noise exposure on wild-type, Foxo3 heterozygous (Foxo3+/−), and Foxo3 knock-out (Foxo3−/−) mice to better understand FOXO3’s role(s) in the mammalian cochlea. We used confocal and multiphoton microscopy to examine well-characterized components of noise-induced damage including calcium regulators, oxidative stress, necrosis, and caspase-dependent and caspase-independent apoptosis. Lower immunoreactivity of the calcium buffer Oncomodulin in Foxo3−/− OHCs correlated with cell loss beginning 4 h post-noise exposure. Using immunohistochemistry, we identified parthanatos as the cell death pathway for OHCs. Oxidative stress response pathways were not significantly altered in FOXO3’s absence. We used RNA sequencing to identify and RT-qPCR to confirm differentially expressed genes. We further investigated a gene downregulated in the unexposed Foxo3−/− mice that may contribute to OHC noise susceptibility. Glycerophosphodiester phosphodiesterase domain containing 3 (GDPD3), a possible endogenous source of lysophosphatidic acid (LPA), has not previously been described in the cochlea. As LPA reduces OHC loss after severe noise exposure, we treated noise-exposed Foxo3−/− mice with exogenous LPA. LPA treatment delayed immediate damage to OHCs but was insufficient to ultimately prevent their death or prevent hearing loss. These results suggest that FOXO3 acts prior to acoustic insult to maintain cochlear resilience, possibly through sustaining endogenous LPA levels.