Discovering novel plasma biomarkers for ischemic stroke: Lipidomic and metabolomic analyses in an aged mouse model.

Ischemic stroke remains a leading cause of mortality and long-term disability worldwide, necessitating efforts to identify biomarkers for diagnosis, prognosis, and treatment monitoring. The present study aimed to identify novel plasma biomarkers of neurodegeneration and inflammation in a mouse model of stroke induced by distal middle cerebral artery occlusion. Using targeted lipidomic and global untargeted metabolomic profiling of plasma collected from aged male mice 24 h after stroke and weekly thereafter for 7 weeks, we discovered distinct acute and chronic signatures. In the acute phase, we observed elevations in myelin-associated lipids, including sphingomyelin (SM) and hexosylceramide (HCER) lipid species, indicating brain lipid catabolism. In the chronic phase, we identified 12-hydroxyeicosatetraenoic acid (12-HETE) as a putative biomarker of prolonged inflammation, consistent with our previous observation of a biphasic pro-inflammatory response to ischemia in the mouse brain. These results provide insight into the metabolic alterations detectable in the plasma after stroke and highlight the potential of myelin degradation products and arachidonic acid derivatives as biomarkers of neurodegeneration and inflammation, respectively. These discoveries lay the groundwork for further validation in human studies and may improve stroke management strategies.

Biomarkers for a less invasive strategy to predict children with eosinophilic esophagitis.

BACKGROUND: Noninvasive biomarkers for diagnosing and monitoring eosinophilic esophagitis (EoE) are currently lacking. This study evaluates 20 biomarkers in serum and saliva, aiming to assess their diagnostic potential in pediatric EoE patients and healthy individuals. METHODS: Blood and saliva from children undergoing upper endoscopy were analyzed for biomarkers, including absolute eosinophil count (AEC), eosinophil-derived neurotoxin (EDN), total and specific IgG4-antibodies (sIgG4), specific IgE-antibodies (sIgE) and 15-hydroxyeicosatetraenoic acid (15(S)-HETE). Some patients participated twice, forming a longitudinal cohort. The ability to use the biomarkers to predict the EoE diagnosis was evaluated. RESULTS: Analysis from 105 children divided into active EoE, remission, and healthy, revealed elevated levels of serum biomarkers (AEC, EDN, 15(S)-HETE, sIgG4, and sIgE) in active EoE compared to healthy individuals. A combination of biomarkers (AEC, EDN, sIgE to egg white and wheat) and symptoms showed an AUC of 0.92 in distinguishing between the three groups. We further showed that optimal cutoff values for these biomarkers could discriminate between active EoE and healthy with a sensitivity of 88% and a specificity of 100% in distinguishing EoE (active and in remission) from healthy. Longitudinally, levels of EDN, sIgG4 to Bos d 4, Bos d 5, Bos d 8, gliadin, and birch, and sIgE to milk decreased in patients progressing from active EoE to remission (p <.05). CONCLUSIONS: This study identified novel biomarkers associated with EoE and proposes a panel, together with symptoms, for effective discrimination between active EoE, EoE in remission, and healthy individuals. The findings may contribute to a less invasive diagnostic method and may be a potential surveillance tool for pediatric EoE patients.

Dickkopf-1 promotes vascular smooth muscle cell foam cell formation and atherosclerosis development through CYP4A11/SREBP2/ABCA1.

Vascular smooth muscle cells (VSMCs) are considered to be a crucial source of foam cells in atherosclerosis due to their low expression level of cholesterol exporter ATP-binding cassette transporter A1 (ABCA1) intrinsically. While the definite regulatory mechanisms are complicated and have not yet been fully elucidated, we previously reported that Dickkopf-1 (DKK1) mediates endothelial cell (EC) dysfunction, thereby aggravating atherosclerosis. However, the role of smooth muscle cell (SMC) DKK1 in atherosclerosis and foam cell formation remains unknown. In this study, we established SMC-specific DKK1-knockout (DKK1SMKO ) mice by crossbreeding DKK1flox/flox mice with TAGLN-Cre mice. Then, DKK1SMKO mice were crossed with APOE-/- mice to generate DKK1SMKO /APOE-/- mice, which exhibited milder atherosclerotic burden and fewer SMC foam cells. In vitro loss- and gain-of-function studies of DKK1 in primary human aortic smooth muscle cells (HASMCs) have proven that DKK1 prevented oxidized lipid-induced ABCA1 upregulation and cholesterol efflux and promoted SMC foam cell formation. Mechanistically, RNA-sequencing (RNA-seq) analysis of HASMCs as well as chromatin immunoprecipitation (ChIP) experiments showed that DKK1 mediates the binding of transcription factor CCAAT/enhancer-binding protein delta (C/EBPδ) to the promoter of cytochrome P450 epoxygenase 4A11 (CYP4A11) to regulate its expression. In addition, CYP4A11 as well as its metabolite 20-HETE-promoted activation of transcription factor sterol regulatory element-binding protein 2 (SREBP2) mediated the DKK1 regulation of ABCA1 in SMC. Furthermore, HET0016, the antagonist of CYP4A11, has also shown an alleviating effect on atherosclerosis. In conclusion, our results demonstrate that DKK1 promotes SMC foam cell formation during atherosclerosis via a reduction in CYP4A11-20-HETE/SREBP2-mediated ABCA1 expression.

12-HETE activates Müller glial cells: The potential role of GPR31 and miR-29.

Diabetic retinopathy (DR) is a neurovascular complication of diabetes, driven by an intricate network of cellular and molecular mechanisms. This study sought to explore the mechanisms by investigating the role of 12-hydroxyeicosatetraenoic acid (12-HETE), its receptor GPR31, and microRNA (miR-29) in the context of DR, specifically focusing on their impact on Müller glial cells. We found that 12-HETE activates Müller cells (MCs), elevates glutamate production, and induces inflammatory and oxidative responses, all of which are instrumental in DR progression. The expression of GPR31, the receptor for 12-HETE, was prominently found in the retina, especially in MCs and retinal ganglion cells, and was upregulated in diabetes. Interestingly, miR29 showed potential as a protective agent, mitigating the harmful effects of 12-HETE by attenuating inflammation and oxidative stress, and restoring the expression of pigment epithelium-derived factor (PEDF). Our results underline the central role of 12-HETE in DR progression through activation of a neurovascular toxic pathway in MCs and illuminate the protective capabilities of miR-29, highlighting both as promising therapeutic targets for the management of DR.

Lower serum 15-HETE level predicts nasal ILC2 accumulation during COX-1 inhibition in AERD.

BACKGROUND: Aspirin-exacerbated respiratory disease (AERD) is associated with high levels of cysteinyl leukotrienes, prostaglandin D2, and low levels of prostaglandin E2. Further, 15-hydroxyeicosatetraenoic acid (15-HETE) levels may have predictive value in therapeutic outcomes of aspirin desensitization. Accumulation of nasal group 2 innate lymphoid cells (ILC2s) has been demonstrated during COX-1 inhibition in AERD, although the relationships between tissue ILC2 accumulation, reaction symptom severity, and novel lipid biomarkers are unknown. OBJECTIVE: We sought to determine whether novel lipid mediators are predictive of nasal ILC2 accumulation and symptom scores during COX-1 inhibitor challenge in patients with AERD. METHODS: Blood and nasal scraping samples from patients with AERD were collected at baseline and COX-1 inhibitor reaction and then processed for flow cytometry for nasal ILC2s and serum for lipidomic analysis. RESULTS: Eight patients with AERD who were undergoing aspirin desensitization were recruited. Of the 161 eicosanoids tested, 42 serum mediators were detected. Baseline levels of 15-HETE were negatively correlated with the change in numbers of airway ILC2s (r = -0.6667; P = .0428). Docosahexaenoic acid epoxygenase metabolite 19,20-dihydroxy-4Z,7Z,10Z,13Z,16Z-docosapentaenoic acid (19,20-diHDPA) was positively correlated with both changes in airway ILC2s (r = 0.7143; P = .0305) and clinical symptom scores (r = 0.5000; P = .0081). CONCLUSION: Low levels of baseline 15-HETE predicted a greater accumulation of airway ILC2s in patients with AERD who were receiving COX-1 inhibition. Further, increases in the cytochrome P pathway metabolite 19,20-dihydroxy-4Z,7Z,10Z,13Z,16Z-docosapentaenoic acid (19,20-diHDPA) were associated with increased symptoms and nasal ILC2 accumulation. Future studies to assess how these mediators might control ILC2s may improve the understanding of AERD pathogenesis.

Pseudomonas aeruginosa cytochrome P450 CYP168A1 is a fatty acid hydroxylase that metabolizes arachidonic acid to the vasodilator 19-HETE.

Pseudomonas aeruginosa is a Gram-negative opportunistic human pathogen that is highly prevalent in individuals with cystic fibrosis (CF). A major problem in treating CF patients infected with P. aeruginosa is the development of antibiotic resistance. Therefore, the identification of novel P. aeruginosa antibiotic drug targets is of the utmost urgency. The genome of P. aeruginosa contains four putative cytochrome P450 enzymes (CYPs) of unknown function that have never before been characterized. Analogous to some of the CYPs from Mycobacterium tuberculosis, these P. aeruginosa CYPs may be important for growth and colonization of CF patients' lungs. In this study, we cloned, expressed, and characterized CYP168A1 from P. aeruginosa and identified it as a subterminal fatty acid hydroxylase. Spectral binding data and computational modeling of substrates and inhibitors suggest that CYP168A1 has a large, expansive active site and preferentially binds long chain fatty acids and large hydrophobic inhibitors. Furthermore, metabolic experiments confirm that the enzyme is capable of hydroxylating arachidonic acid, an important inflammatory signaling molecule present in abundance in the CF lung, to 19-hydroxyeicosatetraenoic acid (19-HETE; Km = 41 µM, Vmax = 220 pmol/min/nmol P450), a potent vasodilator, which may play a role in the pathogen's ability to colonize the lung. Additionally, we found that the in vitro metabolism of arachidonic acid is subject to substrate inhibition and is also inhibited by the presence of the antifungal agent ketoconazole. This study identifies a new metabolic pathway in this important human pathogen that may be of utility in treating P. aeruginosa infections.

Evidence that ABC transporter-mediated autocrine export of an eicosanoid signaling molecule enhances germ cell chemotaxis in the colonial tunicate Botryllus schlosseri.

The colonial ascidian Botryllus schlosseri regenerates the germline during repeated cycles of asexual reproduction. Germline stem cells (GSCs) circulate in the blood and migrate to new germline niches as they develop and this homing process is directed by a Sphigosine-1-Phosphate (S1P) gradient. Here, we find that inhibition of ABC transporter activity reduces migration of GSCs towards low concentrations of S1P in vitro In addition, inhibiting phospholipase A2 (PLA2) or lipoxygenase (Lox) blocks chemotaxis towards low concentrations of S1P. These effects can be rescued by addition of the 12-Lox product 12-S-HETE. Blocking ABC transporter, PLA2 or 12-Lox activity also inhibits homing of germ cells in vivo Using a live-imaging chemotaxis assay in a 3D matrix, we show that a shallow gradient of 12-S-HETE enhances chemotaxis towards low concentrations of S1P and stimulates motility. A potential homolog of the human receptor for 12-S-HETE, gpr31, is expressed on GSCs and differentiating vasa+ germ cells. These results suggest that 12-S-HETE might be an autocrine signaling molecule exported by ABC transporters that enhances chemotaxis in GSCs migrating towards low concentrations of S1P.

16R-HETE and 16S-HETE alter human cytochrome P450 1B1 enzyme activity probably through an allosteric mechanism.

Cytochrome P450 1B1 (CYP1B1) has been widely associated with the development of cardiac pathologies due to its ability to produce cardiotoxic metabolites like midchain hydroxyeicosatetraenoic acids (HETEs) from arachidonic acid (AA) through an allylic oxidation reaction. 16-HETE is a subterminal HETE that is also produced by CYP-mediated AA metabolism. 19-HETE is another subterminal HETE that was found to inhibit CYP1B1 activity, lower midchain HETEs, and have cardioprotective effects. However, the effect of 16-HETE enantiomers on CYP1B1 has not yet been investigated. We hypothesized that 16(R/S)-HETE could alter the activity of CYP1B1 and other CYP enzymes. Therefore, this study was carried out to investigate the modulatory effect of 16-HETE enantiomers on CYP1B1 enzyme activity, and to examine the mechanisms by which they exert these modulatory effects. To investigate whether these effects are specific to CYP1B1, we also investigated 16-HETE modulatory effects on CYP1A2. Our results showed that 16-HETE enantiomers significantly increased CYP1B1 activity in RL-14 cells, recombinant human CYP1B1, and human liver microsomes, as seen by the significant increase in 7-ethoxyresorufin deethylation rate. On the contrary, 16-HETE enantiomers significantly inhibited CYP1A2 catalytic activity mediated by the recombinant human CYP1A2 and human liver microsomes. 16R-HETE showed stronger effects than 16S-HETE. The sigmoidal binding mode of the enzyme kinetics data demonstrated that CYP1B1 activation and CYP1A2 inhibition occurred through allosteric regulation. In conclusion, our study provides the first evidence that 16R-HETE and 16S-HETE increase CYP1B1 catalytic activity through an allosteric mechanism.

The frequency of cytochrome 4F2 rs2108622 genetic variant and its effects on the lipid profile and complications of type II diabetes among a sample of patients in Jordan: A pilot study.

BACKGROUND: Cytochrome 4F2 (CYP4F2) is a major arachidonic acid-metabolizing enzyme which produces 20-Hydroxyeicosatetraenoic acid (20-HETE). It is found that 20-HETE is involved in the pathophysiology of many diseases, including diabetes mellitus. The genetic variants of CYP4F2 can affect its enzymatic activity as well as the 20-HETE production. AIMS: Our aim with this paper was to find out the genotype frequency of CYP4F2 rs2108622 C>T, the major functional variant in the CYP4F2 gene, among a sample of type II diabetes (TIIDM) and its effects on diabetes complications and lipid profile. METHODS: The CYP4F2 rs2108622 variant was genotyped among 90 healthy volunteers and 90 TIIDM patients that attending the University of Jordan Hospital, using the DNA Sanger sequencing method. The data of lipid profile and diabetes complications were obtained from the electronic records available in the hospital. RESULTS: We found that the frequency of CYP4F2 rs2108622C>T variant is significantly (P = 0.02) lower among TIIDM patients in comparison to healthy subjects using both co-dominant and dominant genotyping models. In addition, the CYP4F2 rs2108622 T/T genotype was significantly (P = 0.02) more frequent among TIIDM patients with retinopathy complications (OR=4.36, CI: 1.32-14.37). Lastly, the CYP4F2 rs2108622C>T variant was not associated (P > 0.05) with the glycaemic and lipid profile of patients. CONCLUSIONS: It can be concluded from this study that the frequency of CYP4F2 rs2108622 T/T genotype is lower among TIIDM, but this genotype is associated with an increased risk of retinopathy complications in patients of Jordanian origin. Further studies with a larger sample size are needed to validate the findings of this study.

17-(R/S)-hydroxyeicosatetraenoic acid (HETE) induces cardiac hypertrophy through the CYP1B1 in enantioselective manners.

Cardiac cellular hypertrophy is the increase in the size of individual cardiac cells. Cytochrome P450 1B1 (CYP1B1) is an extrahepatic inducible enzyme that is associated with toxicity, including cardiotoxicity. We previously reported that 19-hydroxyeicosatetraenoic acid (19-HETE) inhibited CYP1B1 and prevented cardiac hypertrophy in enantioselective manner. Therefore, our aim is to investigate the effect of 17-HETE enantiomers on cardiac hypertrophy and CYP1B1. Human adult cardiomyocyte (AC16) cells were treated with 17-HETE enantiomers (20 µM); cellular hypertrophy was evaluated by cell surface area and cardiac hypertrophy markers. In addition, CYP1B1 gene, protein and activity were assessed. Human recombinant CYP1B1 and heart microsomes of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD)-treated rats were incubated with 17-HETE enantiomers (10-80 nM). Our results demonstrated that 17-HETE induced cellular hypertrophy, which is manifested by increase in cell surface area and cardiac hypertrophy markers. 17-HETE enantiomers allosterically activated CYP1B1 and selectively upregulated CYP1B1 gene and protein expression in AC16 cells at uM range. In addition, CYP1B1 was allosterically activated by 17-HETE enantiomers at nM range in recombinant CYP1B1 and heart microsomes. In conclusion, 17-HETE acts as an autocrine mediator, leading to the cardiac hypertrophy through induction of CYP1B1 activity in the heart.

Isolation of human TRPA1 channel from transfected HEK293 cells and identification of alkylation sites after sulfur mustard exposure.

Transient receptor potential (TRP) channels are important in the sensing of pain and other stimuli. They may be triggered by electrophilic agonists after covalent modification of certain cysteine residues. Sulfur mustard (SM) is a banned chemical warfare agent and its reactivity is also based on an electrophilic intermediate. The activation of human TRP ankyrin 1 (hTRPA1) channels by SM has already been documented, however, the mechanism of action is not known in detail. The aim of this work was to purify hTRPA1 channel from overexpressing HEK293 cells for identification of SM-induced alkylation sites. To confirm hTRPA1 isolation, Western blot analysis was performed showing a characteristic double band at 125 kDa. Immunomagnetic separation was carried out using either an anti-His-tag or an anti-hTRPA1 antibody to isolate hTRPA1 from lysates of transfected HEK293 cells. The identity of the channel was confirmed by micro liquid chromatography-electrospray ionization high-resolution tandem-mass spectrometry. Following SM exposure, hTRPA1 channel modifications were found at Cys462 and Cys665, as well as at Asp339 and Glu341 described herein for the first time. Since Cys665 is a well-known target of hTRPA1 agonists and is involved in hTRPA1 activation, SM-induced modifications of cysteine, as well as aspartic acid and glutamic acid residues may play a role in hTRPA1 activation. Considering hTRPA1 as a target of other SM-related chemical warfare agents, analogous adducts may be predicted and identified applying the analytical approach described herein.

Capsaicin induces redox imbalance and ferroptosis through ACSL4/GPx4 signaling pathways in U87-MG and U251 glioblastoma cells.

Glioblastoma is one of the deadliest malignant gliomas. Capsaicin is a homovanillic acid derivative that can show anti-cancer effects by regulating various signaling pathways. The aim of this study is to investigate the effects of capsaicin on cell proliferation via ferroptosis in human U87-MG and U251 glioblastoma cells. Firstly, effects of capsaicin treatment on cell viability were determined by MTT analysis. Next, cellular-proliferation and cytotoxicity assays were determined by analyzing bromodeoxyuridine (BrdU) and lactate dehydrogenase (LDH) activity, respectively. Following, acyl-CoA synthetase long chain family member 4 (ACSL4), glutathione peroxidase 4 (GPx4), 5-hydroxyeicosatetraenoic acid (5-HETE), total oxidant status (TOS), malondialdehyde (MDA), total antioxidant status (TAS) and reduced glutathione (GSH) levels were determined by ELISA. Additionally, ACSL4 and GPx4 mRNA and protein levels were analyzed. Capsaicin showed a concentration-dependent anti-proliferative effects in U87-MG and U251 cells. Cell viability was decreased in the both cell lines treated with capsaicin concentrations above 50 µM, while LDH activity increased. Treatment of 121.6, 188.5, and 237.2 µM capsaicin concentrations for 24 h indicated an increase in ACSL4, 5-HETE, TOS and MDA levels in U87-MG and U251 cells (p < 0.05). On the other hand, we found that capsaicin administration caused a decrease in BrdU, GPx4, TAS and GSH levels in U87-MG and U251 cells (p < 0.05). Besides, ACSL4 mRNA and protein levels were increased in the glioblastoma cells treated with capsaicin, whereas GPx4 mRNA and protein levels were decreased. Finally, capsaicin might be used as a potential anticancer agent with ferroptosis-induced anti-proliferative effects in the treatment of human glioblastoma.

Paradoxical Induction of ALOX15/15B by Cortisol in Human Amnion Fibroblasts: Implications for Inflammatory Responses of the Fetal Membranes at Parturition.

Inflammation of the fetal membranes is an indispensable event of parturition, with increasing prostaglandin E2 (PGE2) synthesis as one of the ultimate products that prime labor onset. In addition to PGE2, the fetal membranes also boast a large capacity for cortisol regeneration. It is intriguing how increased PGE2 synthesis is achieved in the presence of increasing amounts of classical anti-inflammatory glucocorticoids in the fetal membranes at parturition. 15(S)-hydroxyeicosatetraenoic acid (15(S)-HETE) synthesized by lipoxygenase 15/15B (ALOX15/15B) has been shown to enhance inflammation-induced PGE2 synthesis in amnion fibroblasts. Here, we examined whether glucocorticoids could induce ALOX15/15B expression and 15(S)-HETE production to promote PGE2 synthesis in amnion fibroblasts at parturition. We found that cortisol and 15(S)-HETE abundance increased parallelly in the amnion at parturition. Cortisol induced ALOX15/15B expression and 15(S)-HETE production paradoxically in amnion fibroblasts. Mechanism study revealed that this paradoxical induction was mediated by p300-mediated histone acetylation and interaction of glucocorticoid receptor with transcription factors CREB and STAT3. Conclusively, cortisol regenerated in the fetal membranes can paradoxically induce ALOX15/15B expression and 15(S)-HETE production in human amnion fibroblasts, which may further assist in the induction of PGE2 synthesis in the inflammatory responses of the fetal membranes for parturition.

Monolayer autoxidation of arachidonic acid to epoxyeicosatrienoic acids as a model of their potential formation in cell membranes.

In light of the importance of epoxyeicosatrienoic acids (EETs) in mammalian pathophysiology, a nonenzymatic route that might form these monoepoxides in cells is of significant interest. In the late 1970s, a simple system of arranging linoleic acid molecules on a monolayer on silica was devised and shown to yield monoepoxides as the main autoxidation products. Here, we investigated this system with arachidonic acid and characterized the primary products. By the early stages of autoxidation (∼10% conversion of arachidonic acid), the major products detected by LC-MS and HPLC-UV were the 14,15-, 11,12-, and 8,9-EETs, with the 5,6-EET mainly represented as the 5-δ-lactone-6-hydroxyeicosatrienoate as established by 1H-NMR. The EETs were mainly the cis epoxides as expected, with minor trans configuration EETs among the products. 1H-NMR analysis in four deuterated solvents helped clarify the epoxide configurations. EET formation in monolayers involves intermolecular reaction with a fatty acid peroxyl radical, producing the EET and leaving an incipient and more reactive alkoxyl radical, which in turn gives rise to epoxy-hydro(pero)xides and other polar products. The monolayer alignment of fatty acid molecules resembles the arrangements of fatty acids in cell membranes and, under conditions of lipid peroxidation, this intermolecular mechanism might contribute to EET formation in biological membranes.

Ceramide kinase regulates acute wound healing by suppressing 5-oxo-ETE biosynthesis and signaling via its receptor OXER1.

The sphingolipid, ceramide-1-phosphate (C1P), has been shown to promote the inflammatory phase and inhibit the proliferation and remodeling stages of wound repair via direct interaction with group IVA cytosolic phospholipase A2, a regulator of eicosanoid biosynthesis that fine-tunes the behaviors of various cell types during wound healing. However, the anabolic enzyme responsible for the production of C1P that suppresses wound healing as well as bioactive eicosanoids and target receptors that drive enhanced wound remodeling have not been characterized. Herein, we determined that decreasing C1P activity via inhibitors or genetic ablation of the anabolic enzyme ceramide kinase (CERK) significantly enhanced wound healing phenotypes. Importantly, postwounding inhibition of CERK enhanced the closure rate of acute wounds, improved the quality of healing, and increased fibroblast migration via a "class switch" in the eicosanoid profile. This switch reduced pro-inflammatory prostaglandins (e.g., prostaglandin E2) and increased levels of 5-hydroxyeicosatetraenoic acid and the downstream metabolite 5-oxo-eicosatetraenoic acid (5-oxo-ETE). Moreover, dermal fibroblasts from mice with genetically ablated CERK showed enhanced wound healing markers, while blockage of the murine 5-oxo-ETE receptor (oxoeicosanoid receptor 1) inhibited the enhanced migration phenotype of these cell models. Together, these studies reinforce the vital roles eicosanoids play in the wound healing process and demonstrate a novel role for CERK-derived C1P as a negative regulator of 5-oxo-ETE biosynthesis and the activation of oxoeicosanoid receptor 1 in wound healing. These findings provide foundational preclinical results for the use of CERK inhibitors to shift the balance from inflammation to resolution and increase the wound healing rate.

Mitochondrial aldehyde dehydrogenase (ALDH2) rescues cardiac contractile dysfunction in an APP/PS1 murine model of Alzheimer's disease via inhibition of ACSL4-dependent ferroptosis.

Alzheimer's disease (AD) is associated with high incidence of cardiovascular events but the mechanism remains elusive. Our previous study reveals a tight correlation between cardiac dysfunction and low mitochondrial aldehyde dehydrogenase (ALDH2) activity in elderly AD patients. In the present study we investigated the effect of ALDH2 overexpression on cardiac function in APP/PS1 mouse model of AD. Global ALDH2 transgenic mice were crossed with APP/PS1 mutant mice to generate the ALDH2-APP/PS1 mutant mice. Cognitive function, cardiac contractile, and morphological properties were assessed. We showed that APP/PS1 mice displayed significant cognitive deficit in Morris water maze test, myocardial ultrastructural, geometric (cardiac atrophy, interstitial fibrosis) and functional (reduced fractional shortening and cardiomyocyte contraction) anomalies along with oxidative stress, apoptosis, and inflammation in myocardium. ALDH2 transgene significantly attenuated or mitigated these anomalies. We also noted the markedly elevated levels of lipid peroxidation, the essential lipid peroxidation enzyme acyl-CoA synthetase long-chain family member 4 (ACSL4), the transcriptional regulator for ACLS4 special protein 1 (SP1) and ferroptosis, evidenced by elevated NCOA4, decreased GPx4, and SLC7A11 in myocardium of APP/PS1 mutant mice; these effects were nullified by ALDH2 transgene. In cardiomyocytes isolated from WT mice and in H9C2 myoblasts in vitro, application of Aß (20 µM) decreased cell survival, compromised cardiomyocyte contractile function, and induced lipid peroxidation; ALDH2 transgene or activator Alda-1 rescued Aß-induced deteriorating effects. ALDH2-induced protection against Aß-induced lipid peroxidation was mimicked by the SP1 inhibitor tolfenamic acid (TA) or the ACSL4 inhibitor triacsin C (TC), and mitigated by the lipid peroxidation inducer 5-hydroxyeicosatetraenoic acid (5-HETE) or the ferroptosis inducer erastin. These results demonstrate an essential role for ALDH2 in AD-induced cardiac anomalies through regulation of lipid peroxidation and ferroptosis.

Lipids Alterations Associated with Metformin in Healthy Subjects: An Investigation Using Mass Spectrometry Shotgun Approach.

Metformin is an orally effective insulin-sensitizing drug widely prescribed for treating type 2 diabetes mellitus (T2DM). Metformin has been reported to alter lipid metabolism. However, the molecular mechanisms behind its impact on lipid metabolism remain partially explored and understood. In the current study, mass spectrometry-based lipid profiling was used to investigate the lipidomic changes in the serum of 26 healthy individuals after a single-dose intake of metformin. Samples were analyzed at five-time points: preadministration, before the maximum concentration of metformin (Cmax), Cmax, after Cmax, and 36 h post-administration. A total of 762 molecules were significantly altered between the five-time points. Based on a comparison between baseline level and Cmax, metformin significantly increased and decreased the level of 33 and 192 lipids, respectively (FDR ≤ 0.05 and fold change cutoff of 1.5). The altered lipids are mainly involved in arachidonic acid metabolism, steroid hormone biosynthesis, and glycerophospholipid metabolism. Furthermore, several lipids acted in an opposed or similar manner to metformin levels and included fatty acyls, sterol lipids, glycerolipids, and glycerophospholipids. The significantly altered lipid species pointed to fundamental lipid signaling pathways that could be linked to the pleiotropic effects of metformin in T2DM, insulin resistance, polycystic ovary syndrome, cancer, and cardiovascular diseases.

Catechin Reduces Blood Pressure in Spontaneously Hypertensive Rats through Modulation of Arachidonic Acid Metabolism.

(1) Background: hypertension affects approximately half of the adults in the United States (roughly 116 million). The cytochrome P450 (CYP)-mediated metabolism of arachidonic acid (AA) in the kidney has been found to play a major role in the pathogenesis of hypertension. This study examines the anti-hypertensive effect of the natural polyphenolic compound catechin (CAT) and investigates if it impacts the metabolism of AA in the kidney in comparison to captopril (CAP): a commonly used antihypertensive drug. (2) Methods: spontaneously hypertensive rats (SHR) were randomly divided into five groups. The treatment groups were administered CAT in drinking water at doses of 10 and 50 mg/kg. A positive control group received CAP at a dose of 10 mg/kg in the drinking water, and one group received both CAP and CAT at doses of 10 mg/kg and 50 mg/kg, respectively. Blood pressure was monitored weekly for five weeks. The activity of the two major enzymes involved in AA metabolism in the kidney, namely CYP4A and soluble epoxide hydrolase (sEH), were analyzed. (3) Results: CAP monotherapy was found to reduce blood pressure compared to the control untreated rats but did not demonstrate any effect on AA metabolism. Low- and high-dose CAT resisted the rise in blood pressure observed in the untreated SHR and significantly lowered blood pressure compared to the control group, respectively. Only rats treated with high CAT doses demonstrated significant inhibition of CYP4A and sEH enzyme activities. The coadministration of CAP and a high dose of CAT resulted in more pronounced blood pressure-lowering effects, but no more significant effects on AA metabolism were found compared to a high dose of CAT alone. (4) Conclusion: the modulation of AA metabolism in the kidney contributes, at least partially, to the blood pressure-lowering effect of CAT in SHR rats.

Inhibition of 20-hydroxyeicosatetraenoic acid biosynthesis by vitamin E analogs in human and bovine cytochrome P450 microsomes.

Dairy cattle are predisposed to disease around the time of calving due to dysfunctional inflammatory responses. Oxylipids are lipid-derived mediators that regulate all aspects of the inflammatory response, and shifts in oxylipid profiles are correlated with disease risk. For example, 20-hydroxyeicosatetraenoic acid (HETE) is an oxylipid derived from cytochrome P450 enzymes (CYP450) found at significantly greater concentrations around calving and during clinical disease. Biosynthesis of 20-HETE occurs almost exclusively from two specific CYP450 of which CYP450 family four sub-family F member two (CYP4F2) is the major contributor to 20-HETE production in humans. To further study the activities of 20-HETE and potentially reduce its production in vivo, mitigation methods must be explored. Additional substrates of CYP4F2, such as vitamin E, are known to both increase and decrease the metabolism of other CYP4F2 substrates. This study aimed to determine whether vitamin E analogs may reduce the production of 20-HETE through competition for CYP4F2 activity in human CYP4F2, bovine-kidney and bovine-mammary microsomes. Gamma-tocopherol reduced 20-HETE production from human and bovine-kidney microsomes (35.3% and 27.5%, respectively) whereas γ-tocotrienol only reduced 20-HETE production from human microsomes (40.1%). Finally, bovine-mammary microsomes did not produce a quantifiable amount of 20-HETE, suggesting basal mammary CYP4F2 activity may not be a significant contributor to 20-HETE found in milk. Together, these data show that analogs of vitamin E can reduce the production of 20-HETE, potentially through competition with arachidonic acid for metabolism by CYP4F2, posing a potential means for limiting 20-HETE production during clinical diseases of dairy cattle.

LUNG PROTECTIVE POTENTIAL EFFECT OF ZILEUTON DURING ENDOTOXAEMIA MODEL IN MALE MICE.

OBJECTIVE: The aim: This study was undertaken to investigatethe possible lung protective potential effect of zileuton during polymicrobial sepsis, through modulation of inflammatory and oxidative stress pathway. PATIENTS AND METHODS: Materials and methods: 24 adult male Swiss-albino mice aged 8-12 weeks, with a weight of 25-35g, were randomized into 4 equal groups n=6, sham (laparotomy without CLP), CLP (laparotomy with CLP), vehicle (equivalent volume of DMSO 1 hour prior to CLP), and Zileuton (5 mg/kg 1 hour prior to CLP) group. After 24 hrs. of sepsis, the lung tissue harvested and used to assess IL-6, IL-1B, IL-17, LTB-4,12(S) HETE and F2-isoprostane as well as histological examination. RESULTS: Results: Lung tissue inflammatory mediators IL-6, IL-1B, IL-17, LTB, 12 (S) HETE) and oxidative stress were carried out via ELISA. Lung tissue levels of IL-6, IL-1B, IL-17, LTB4, 12(S) HETE and oxidative stress (F2 isoprostan)level were significantly higher in sepsis group (p<0.05) as compared with sham group, while zileuton combination showed significant (p<0.05) lower level in these inflammatory mediators and oxidative stress as comparedto sepsis group. Histologically, All mice in sepsis group showed a significant (p<0.05) lung tissue injury, while in zileuton pretreated group showed significantly (p<0.05) reduced lung tissue injury. CONCLUSION: Conclusions: The results of the present study revealed that zileuton has the ability to attenuate lung dysfunction during CLP induced polymicrobial sepsis in male mice through their modulating effects on LTB4,12(S) HETE and oxidative stress downstream signaling pathways and subsequently decreased lungtissue levelsof proinflammatory cytokines (IL-1ß, and IL-6,IL-17).