Hyperlipidemic Conditions Research Articles

The emerging role of xanthine oxidase inhibition for suppression of breast cancer cell migration and metastasis associated with hypercholesterolemia.

Hypercholesterolemia is reported to increase reactive oxygen species (ROS) and to promote breast cancer progression. ROS play an important role in tumor biology, and xanthine oxidase (XO) is an enzyme that generates ROS. The effects of febuxostat (FBX), an XO inhibitor, on breast cancer cell migration under LDL stimulation in vitro and metastasis of breast cancer associated with hypercholesterolemia in vivo were studied. In vitro, FBX significantly inhibited LDL-induced ROS production and cell migration. Treatment of small interfering RNA against XO was consistent with the findings of FBX treatment. In vivo, a significant increase of tumor growth and pulmonary metastasis was observed in a xenograft mouse model with 4T1 cells on a high cholesterol diet (HCD), both of which were markedly inhibited by FBX or allopurinol treatment. Moreover, ERK represented the main target-signaling pathway that was affected by FBX treatment in a xenograft mouse model on an HCD evaluated by NanoString nCounter analysis. Consistently, MEK/ERK inhibitors directly decreased the LDL-induced cell migration in vitro. In conclusion, FBX mitigates breast cancer cell migration and pulmonary metastasis in the hyperlipidemic condition, associated with decreased ROS generation and MAPK phosphorylation. The inhibition of ERK pathways is likely to underlie the XO inhibitor-mediated suppression of breast cancer cell migration.-Oh, S.-H., Choi, S.-Y., Choi, H.-J., Ryu, H.-M., Kim, Y.-J., Jung, H.-Y., Cho, J.-H., Kim, C.-D., Park, S.-H., Kwon, T.-H., Kim, Y.-L. The emerging role of xanthine oxidase inhibition for suppression of breast cancer cell migration and metastasis associated with hypercholesterolemia.

Hypercholesterolemia Induces a Mast Cell-CD4+ T Cell Interaction in Atherosclerosis.

Mast cells (MCs) are potent innate immune cells that aggravate atherosclerosis through the release of proinflammatory mediators inside atherosclerotic plaques. Similarly, CD4+ T cells are constituents of the adaptive immune response and accumulate within the plaques following lipid-specific activation by APCs. Recently it has been proposed that these two cell types can interact in a direct manner. However, no indication of such an interaction has been investigated in the context of atherosclerosis. In our study, we aimed to examine whether MCs can act as APCs in atherosclerosis, thereby modulating CD4+ T cell responses. We observed that MCs increased their MHC class II expression under hyperlipidemic conditions both in vivo and in vitro. Furthermore, we showed that MCs can present Ags in vivo via MHC class II molecules. Serum from high-fat diet-fed mice also enhanced the expression of the costimulatory molecule CD86 on cultured MCs, whereas OVA peptide-loaded MCs increased OT-II CD4+ T cell proliferation in vitro. The aortic CD4+ and TH1 cell content of atherosclerotic mice that lack MCs was reduced as compared with their wild-type counterparts. Importantly, we identified MCs that express HLA-DR in advanced human atheromata, indicating that these cells are capable of Ag presentation within human atherosclerotic plaques. Therefore, in this artice, we show that MCs may directly modulate adaptive immunity by acting as APCs in atherosclerosis.

Isoquercetin upregulates antioxidant genes, suppresses inflammatory cytokines and regulates AMPK pathway in streptozotocin-induced diabetic rats

Lifestyle and genetic factors contribute to the initiation of oxidative stress and inflammation in diabetes mellitus (DM). Oxidative stress and lipid peroxidation worked in an orchestrated manner and reported to be strongly associated with the formation of the hyperlipidemic condition in DM patients. Isoquercetin, a bioactive constituent isolated from guava leaves has attracted considerable attention because of its antidiabetic activity. The antidiabetic activity of guava leaves may be due to the presence of isoquercetin at a significant level. However, how isoquercetin regulates different pathways in DM is insufficiently studied. We have selected versatile regulators of oxidative stress and inflammatory pathways to fully analyze if isoquercetin effectively modulated the genes of these pathways. At the end of our experimental duration, rats were dissected and analyzed for the oxidative stress, lipid peroxidation, inflammatory and lipid markers. The nuclear factor erythroid 2–related factor 2 (Nrf2) pathway is believed to be the key regulator of expression of various antioxidant enzyme genes and it is directly or indirectly related to nuclear factor Kappa- B (NF-kB) and AMP-activated protein kinase (AMPK) pathways. Therefore, we tend to study the effects of STZ on Nrf2, NF-kB and AMPK pathway and how the isoquercetin treatment performs at a molecular level to overcome the burden of DM. The results of our study provided convincing evidence of significant pharmacological properties of isoquercetin in context of its ability to inhibit the oxidative stress elicited by the STZ through generation of the free radicals and regulation of the expression of Nrf2 pathway-associated proteins and genes and it also reduced the burden of hyperlipidemia and inflammation. By taking the above results into consideration isoquercetin can be studied further to elucidate its antidiabetic effects at various levels.

ANTIHYPERLIPIDEMIC ACTIVITY OF ECLIPTA ALBA IN HIGH FAT DIET-INDUCED HYPERLIPIDEMIC ALBINO RATS

Objective: The objective is to study the antihyperlipidemic activity of Eclipta alba in hyperlipidemic albino rats and the use of herbal plants and their parts such as seeds, berries, leaves, roots, barks, or flowers for the development of drugs for therapeutic purposes which have an effective role in recent years.
 Methods: A total of 30 adult albino rats of Wistar strain weighing 165–215 g were utilized. Animals were treated with high fat diet for 8 weeks followed by post-treatment of E. alba for 1 week, 2 weeks, and 3 weeks, respectively. After 12 h of fasting on the last day of experiment, serum blood samples were collected in EDTA vials and processed for biochemical analysis.
 Results: A significant decrease in levels of total cholesterol and triglycerides was noted on animals treated with E. alba compared to high fat diet animals. Treatment of hypercholesterolemic rats with E. alba showed a marked decrease of serum low-density lipoprotein (LDL) and very LDL-cholesterol concentrations compared to the hypercholesterolemic rats. High fat diet feeding worsened the levels of serum glutamic oxaloacetic transaminase, serum glutamic pyruvic transaminase, and alkaline phosphatase enzymes, whereas the same markers were significantly improved by supplementation with E. alba compared to normal group.
 Conclusion: E. alba acts as antihyperlipidemic agent in hyperlipidemic conditions and helps for better health.

MiR-652-3p inhibition enhances endothelial repair and reduces atherosclerosis by promoting Cyclin D2 expression.

BackgroundAtherosclerosis is a hyperlipidemia-induced condition affecting the arterial wall that damages healthy endothelial cell (EC) function, leading to enhanced risk of atherothrombotic events. Certain microRNAs regulate EC dysfunction in response to hyperlipidemia and may be suitable therapeutic targets to combat atherosclerosis.MethodsmiRNA expression in human ECs was analyzed under various conditions to identify key microRNAs. High-cholesterol diet (HCD)-fed Mir652−/−Apoe−/− (Mir652−/−) mice and matching Mir652+/+Apoe−/− (Mir652+/+) mice were subjected to carotid injury to analyze the effects of miR-652 knockdown on endothelial repair. In silico analysis followed by in vitro and in vivo experiments were applied to identify miR-652's target gene Ccnd2 and investigate the pair's effects on ECs. miR-652-5p and miR-652-3p antagomir therapies were tested in Mir652+/+ mice under normal and HCD diet to assess their effect on endothelial repair.FindingsmiR-652-3p, which is upregulated in human and murine atherosclerotic plaques, suppresses expression of the endothelial repair gene Ccnd2, thereby enhancing atherosclerotic lesion formation. Post-denudation recovery of ECs was promoted in Mir652−/− mice due to enhanced EC proliferation attributable to de-repression of miR-652-3p's (but not miR-652-5p's) regulation of Ccnd2 expression. Under hyperlipidemic conditions at non-predilection sites, miR-652-3p produces anti-proliferative effects in ECs, such that Mir652−/− mice display reduced atherosclerotic progression. In contrast, neither miR-652-3p nor Ccnd2 displayed significant effects on the endothelium at predilection sites or under disturbed flow conditions. Administration of a miR-652-3p antagomir rescued the proliferation of ECs in vivo, thereby limiting atherosclerotic development.InterpretationmiR-652-3p blockade may be a potential therapeutic strategy against atherosclerosis.

Identification of a novel function of hepatic long-chain acyl-CoA synthetase-1 (ACSL1) in bile acid synthesis and its regulation by bile acid-activated farnesoid X receptor

Long-chain acyl-CoA synthetase 1 (ACSL1) plays a pivotal role in fatty acid β‑oxidation in heart, adipose tissue and skeletal muscle. However, key functions of ACSL1 in the liver remain largely unknown. We investigated acute effects of hepatic ACSL1 deficiency on lipid metabolism in adult mice under hyperlipidemic and normolipidemic conditions. We knocked down hepatic ACSL1 expression using adenovirus expressing a ACSL1 shRNA (Ad-shAcsl1) in mice fed a high-fat diet or a normal chow diet. Hepatic ACSL1 depletion generated a hypercholesterolemic phenotype in mice fed both diets with marked elevations of total cholesterol, LDL-cholesterol and free cholesterol in circulation and accumulations of cholesterol in the liver. Furthermore, SREBP2 pathway in ACSL1 depleted livers was severely repressed with a 50% reduction of LDL receptor protein levels. In contrast to the dysregulated cholesterol metabolism, serum triglycerides, free fatty acid and phospholipid levels were unaffected. Mechanistic investigations of genome-wide gene expression profiling and pathway analysis revealed that ACSL1 depletion repressed expressions of several key enzymes for bile acid biosynthesis, consequently leading to reduced liver bile acid levels and altered bile acid compositions. These results are the first demonstration of a requisite role of ACSL1 in bile acid biosynthetic pathway in liver tissue. Furthermore, we discovered that Acsl1 is a novel molecular target of the bile acid-activated farnesoid X receptor (FXR). Activation of FXR by agonist obeticholic acid repressed the expression of ACSL1 protein and mRNA in the liver of FXR wild-type mice but not in FXR knockout mice.

Identification of the Genetic Variant Responsible for Variable Platelet CD36 Expression By Massively Parallel Reporter Assay

CD36 is a platelet membrane glycoprotein whose engagement with oxidized low density lipoprotein (oxLDL) results in platelet activation. Deletion of CD36 in mice fed a high fat diet results in attenuation of the pro-thrombotic state and platelet hyper-activity. The CD36 gene has been associated with platelet count, platelet volume, as well as lipid levels and CVD risk. Platelet CD36 expression is highly variable with 3-8% of non-white populations displaying a complete lack of expression. Platelet CD36 expression levels have been shown to be associated with both the platelet oxLDL response and an elevated risk of thrombo-embolism. Several genomic variants have been identified as associated with platelet CD36 levels, however none have been conclusively demonstrated to be causative.We have previously reported platelet expression Quantitative Trait Loci (eQTLs) that associate single nucleotide polymorphisms (SNPs) with platelet RNA levels, indicating genetic influence on gene expression. CD36 is one of the 612 platelet-expressed RNAs whose abundance has significant genotypic associations, suggesting a mechanism by which CD36 level is regulated. 86 eQTL SNPs located within a +/- 100kb window of the CD36 gene are associated with platelet CD36 mRNA levels at a significance of P<1x10-6, spanning a range of 125kb. Rather than prioritizing these 86 variants for functional testing based on genomic annotation, we opted to use an approach termed a Massively Parallel Reporter Array (MPRA), in which all 86 SNPs were tested simultaneously. A library of plasmids was generated in which both alleles of the variants including 150bp of surrounding genomic context were cloned 5' to a minimal promoter and unique barcode. For statistical robustness, 40 barcodes were constructed per allele, resulting in a library containing a total of ~5000 plasmids that were generated and transfected into K562 erythroleukemia cells. RNA-Seq was then conducted on the transfected cells and the abundance of individual barcodes was quantified, normalized to the DNA plasmid library, and then compared across variant alleles.The MPRA analysis led to identification of 5 SNPs located within a 35kb region 5‘ to the CD36 transcription start site that had significantly different barcode production between alleles. These 5 SNPs are in linkage disequilibrium with one another, with variants reported to be associated with CD36 level, and with variants reported to be associated with platelet count and volume by GWAS. To verify the functionality of these variants, we conducted luciferase assays in which the genomic sequence surrounding the variants were inserted 5‘ to the reporter gene. The genomic fragment containing the alternate alleles of two of these SNPs, rs2366739 and rs1194196, located within 29bp of each other, demonstrated 25-40% lower transcriptional activity than the reference alleles. Next, to determine if these two variants altered the interaction of DNA-binding proteins with the locus, we conducted electromobility-shift assays (EMSA) using probes derived from both alleles of the genomic sequence. As with the MPRA and luciferase assays, the alternative alleles of rs2366739 and rs1194196 resulted in lower levels of DNA:protein complexes, indicating an altered affinity for regulatory complexes. Finally, to further confirm the role of this genomic locus in regulating CD36 expression levels, we deleted a 596bp region containing these two SNPs from the K562 erythroleukemia cell genome using CRISPR/Cas. Deletion of this region resulted in an approximately 10-fold increase in CD36 RNA in these cells, confirming a role for this loci in the regulation of CD36 expression.These data indicate that we have identified a regulatory locus for CD36 expression, which has important implications for platelet function in cardiovascular disease. These variants are located within a retrotransposon long-terminal repeat and alter a putative GATA1 binding site, suggesting that these variants may alter binding of that transcription factor to the locus. Platelet CD36 expression level has been linked to the platelet response to oxLDL and may modulate the thrombotic response in hyperlipidemic conditions. Therefore, the mechanism described here, in which a genetic variant alters platelet CD36 levels, and thus the platelet response to hyperlipidemic conditions, may serve as an important biomarker for increased thrombotic potential. DisclosuresNo relevant conflicts of interest to declare.

Inhibition of protein kinase R protects against palmitic acid-induced inflammation, oxidative stress, and apoptosis through the JNK/NF-kB/NLRP3 pathway in cultured H9C2 cardiomyocytes.

Double-stranded RNA-dependent protein kinase (PKR) is a critical regulator of apoptosis, oxidative stress, and inflammation under hyperlipidemic and insulin resistance conditions. Saturated free fatty acids, such as palmitic acid (PA), are known inducers of apoptosis in numerous cell types. However, the underlying molecular mechanism is not fully understood. The aim of the present study was to examine the effect of PA on cultured rat H9C2 cardiac myocytes cells and to investigate the PKR mediated harmful effects of PA in vitro in cultured cardiomyocytes. PKR expression was determined by immunofluorescence and immunoblotting. Oxidative stress and apoptosis were determined by flow cytometry and assay kits. The expression of different gene markers of apoptosis, oxidative stress, and inflammation were measured by Western blot analysis and reverse transcription polymerase chain reaction. PKR expression, reactive oxygen species levels as well as apoptosis were increased in PA-treated cultured H9C2 cardiomyocytes. The harmful effects of PA were attenuated by a selective PKR inhibitor, C16. Moreover, we observed that upregulation of c-Jun N-terminal kinase (JNK), nuclear factor-kB (NF-kB) and NACHT, LRR and PYD domains-containing protein 3 (NLRP3) pathways is associated with increased expression of interleukin 6 and tumor necrosis factor-α in PA-treated cardiomyocytes and attenuation by a selective PKR inhibitor. Our study reports, for the first time, that PKR-mediated harmful effects of PA in cultured cardiomyocytes via activation of JNK, NF-kB, and NLRP3 pathways. Inhibition of PKR is one of the possible mechanistic approaches to inhibit inflammation, oxidative stress, and apoptosis in lipotoxicity-induced cardiomyocyte damage.

Acclimation of C2C12 myoblasts to physiological glucose concentrations for in vitro diabetes research

AimsThe interplay between hyper-glycemia and -lipidemia in diabetes mellitus (DM) is important in simulating diabetic conditions. However, cell culture media typically contain supraphysiological levels of glucose to stimulate cellular growth, which also desensitizes cells to elevated glucose levels. Moreover, creating hyperlipidemic conditions in vitro requires specialized carriers because unbound lipids form micelles when introduced to liquid media. This study sought to develop a novel method for simulating DM conditions in vitro. Materials and methodsWe acclimated the C2C12 mouse myoblasts to culture medium with 5.6 mM glucose, which mimics physiological levels, and created a bovine serum albumin-palmitic acid conjugate for lipid transport to explore the effects of hyperlipidemia. We simulated diabetic conditions in vitro by using both hyper–glycemic and –lipidemic conditions and compared the results to that of only hyperglycemic or hyperlipidemic conditions. Key findingsAcclimated cells exposed to these hyper-glycemic (15 mM glucose) and/or -lipidemic (0.25 mM palmitate) conditions for 2 h showed increased mitochondrial fragmentation and membrane potential as well as elevated reactive oxygen species production compared to control cells. These findings suggest altered mitochondrial morphology and function, which have been confirmed using isolated rat flexor digitorum brevis myofibers. Hyper-glycemic and/or -lipidemic stimulations for 24 h significantly increased mitogen-activated protein kinase kinase MEK 1/2 protein expression, upregulated the early pro-apoptotic transcription factor C/EBP homologous protein (CHOP), and induced apoptosis. SignificanceOur results further support and confirm the utility of this method which will allow for subsequent investigations studying the effects of hyper-glycemia and/or -lipidemia in vitro.

Development of an in vitro model to study hepatitis C virus effects on hepatocellular lipotoxicity and lipid metabolism

Hepatic steatosis is common in patients infected with hepatitis C virus (HCV). Particularly in patients infected with non-genotype 3 HCV, hepatic steatosis is closely related to factors of the metabolic syndrome such as hyperlipidemia. However, the molecular mechanisms involved in this “metabolic” steatosis in non-3 genotype HCV infections are not well understood. Here, we aimed to develop an in vitro model to study the effect of genotype 1 HCV infection on hepatic lipotoxicity and lipid metabolism. Cellular lipid accumulation was induced in Huh-7 hepatoma cells transfected with HCV genotype 1b replicon (HCV+) by incubation with increasing doses of palmitic acid (C16:0) or oleic acid (C18:1 n-9) complexed to albumin mimicking hyperlipidemic conditions. Mock transfected hepatoma cells (HCV−) were used as controls. Incubation with oleic acid concentrations as high as 0.5 mM did not induce toxic effects in HCV+ or HCV− cells. In contrast, incubation with palmitic acid caused dose-dependently cytotoxic effects which were more pronounced in HCV+ compared to HCV− cells. Further analysis with subtoxic palmitic and oleic acid concentrations revealed a higher uptake of fatty acids and intracellular triglyceride accumulation in HCV+ compared to HCV− cells. Carnitine palmitoyltransferase I (CPT1) expression, indicative of mitochondrial beta-oxidation, was markedly stimulated by lipid exposure in HCV+ but not in HCV− cells. Furthermore, heme oxygenase 1 (HMOX1) expression levels increased in FA stimulated cells, and this increase was significantly higher in HCV+ compared to HCV− cells. In contrast, expression of the key enzymes of hepatic de novo lipogenesis fatty acid synthase (FASN) and stearoyl-CoA desaturase (SCD-1) was significantly reduced upon oleate exposure in HCV− but not in HCV+ cells.In summary, our newly developed cell culture model revealed effects of HCV genotype 1b infection on metabolic susceptibility to lipid accumulation and toxicity particularly to saturated lipids. These results may indicate that HCV (genotype 1b) infected individuals with hyperlipidemia may benefit from dietary or pharmacological intervention.

Fetuin A—A Major Player in Lipid-Induced Islet Dysfunction and ß-Cell Apoptosis

Fetuin-A is a glycoprotein that is secreted from both liver and adipose tissue. Circulating Fetuin-A (FetA) is elevated in type 2 diabetes and causes impaired insulin sensitivity. Few recent findings suggested that FetA is responsible for palmitate induced inflammatory injury to the β-cell via TLR4 pathway. Yet it is unknown whether FetA is associated with β-cell death and reduction of functional islet mass that causes dearth of circulatory insulin in chronic hyperlipidemic condition. To validate this hypothesis MIN6 cells (mice insulinoma cells) were treated with various concentrations of palmitate and /or FetA for 48h and subjected to MTT assay and flowcytometry analysis. The results depicted higher mortality of cells (30-40%) with increasing dose of palmitate (0.25-0.75 mM) and/ or FetA (5-20 mM). Palmitate-FetA treated cells demonstrated substantial reduction in the cell proliferative markers (Akt, cyclin D1) while higher expression of apoptotic markers (Bax, caspase 3 and PARP). Silencing of NF-kB or TLR4 in MIN6 cells restore cell viability on palmitate-FetA treatment indicating significance of TLR4-NF-kB axis in β-cell mortality. Our recent findings demonstrated FetA secretion from MIN6 cells on stimulation of palmitate. Silencing of fetA in MIN6 cells, reduced cell mortality with improved GSIS (1.5 fold). Treatment of MIN6 cells or mice islets with cytokines, such as IL-1β and TNF-α, results β-cell apoptosis mimicking the inflamed conditions of the islets. Co-culture experiments of MIN6 cells with adipocytes or macrophages in presence of palmitate showed elevated levels of cytokines (IL-1β, IL6, TNF-α) in the media and significant number of apoptotic MIN6 cells (20-30%). In vivo analysis of islets structure and function in high fat diet (HFD) fed mice model for a period of 20 weeks demonstrated atrophied islets and accumulation of macrophages. Our present investigation therefore indicated FetA to be a major player in lipid induced pancreatic-β cell death and dysfunction in type 2 diabetes. Disclosure R. Kundu: None. A. Mukhuty: None.

Obesity-induced overexpression of miRNA-24 regulates cholesterol uptake and lipid metabolism by targeting SR-B1

Scavenger Receptor B1 (SR-B1) is an 82 kDa integral membrane glycoprotein that mediates selective uptake of high-density lipoprotein cholesteryl ester (CE) in vitro and in vivo. Previously, we defined several kinds of regulatory mechanisms of SR-B1 expression and function. Here, we have dissected the function of a novel miR-24 on SR-B1 expression, HDL uptake and lipid metabolism. We showed that miR-24 was upregulated in HepG2 cells cultured in the mimicked hyperlipidemic condition and in the livers of dietary induced and genetic obesity mice. Overexpression of miR-24 inhibited SR-B1 expression by directly targeting SR-B1 3′ UTR and repressed HDL uptake and steroidogenesis in steroidogenic cells. HepG2 cells with miR-24 showed attenuation of TG levels and lipid accumulation. Moreover, we validated that overexpression of miR-24 downregulated the expression of certain genes involved in lipogenesis, FASN, ACLY and SCD1, and increased the expression of genes of cholesterol synthesis, HMGCR, DHCR24 and SREBP2. Taken together, we demonstrated that obesity induced miR-24 repressed HDL uptake, steroid hormone synthesis and lipid metabolism by targeting SR-B1.

Zingiber officinale extract administration diminishes steroyl-CoA desaturase gene expression and activity in hyperlipidemic hamster liver by reducing the oxidative and endoplasmic reticulum stress

BackgroundStearoyl CoA desaturases (SCD) are enzymes that convert saturated to monounsaturated fatty acids and have increased activity in hepatic steatosis. PurposeWe aimed to investigate the potential of ginger extract (GIN) to modulate the liver SCD1 expression and activity in hyperlipidemic (HL) conditions, in order to lower lipid accumulation in the steatotic liver. Study design/MethodsMale Golden Syrian hamsters were divided in three groups: (i) fed with standard chow (N), (ii) fed with standard chow plus 3% cholesterol and 15% butter for 21 weeks (HL), (iii) HL treated with GIN (800 µg/kg body weight/day) in the last 5 weeks of fat diet (HL-GIN). Cholesterol (C), triglycerides (TG), non-esterified fatty acids (NEFA), SCD1 estimated activity (C16:1n7/C16:0; C18:1n9/C18:0) and gene expression, acetyl-CoA carboxylase (ACC), thiobarbituric acid reactive substances (TBARS), paraoxonase1 (PON1) and myeloperoxidase (MPO) were determined in the plasma and liver of all hamsters. We measured protein expression of endoplasmic reticulum stress (ERS) markers, gene and protein expression of liver X receptor α/β (LXRα/β), peroxisome proliferator-activated receptor γ (PPARγ), ATP-binding cassette sub-family G member 5/8 (ABCG5/G8) and 7α-hydroxylase1 (CYP7A1) in all hamsters’ livers. ResultsIn plasma, in HL-GIN versus HL hamsters, SCD1 estimated activity was lower (27%; 15%, p < 0.05), NEFA levels decreased by 91%, p < 0.001, while C and TG levels did not vary; the oxidative stress expressed as MPO and TBARS levels decreased (15%; 11%, p < 0.01), while PON1 protein increased (75%, p < 0.05). In the liver of HL-GIN versus HL, C, TG, NEFA, MPO and TBARS levels decreased (8–40%, p < 0.05) and PON1 protein levels increased (30%, p < 0.05), SCD1 estimated activity decreased (8%; 9%, p < 0.05), in parallel with the reduced gene expression of SCD1 and ACC (70–80%, p < 0.05). The protein expression of the ERS sensors decreased (30–65%, p < 0.05), while that of ABCG5/G8, CYP7A1, LXRα/β and PPARγ increased in HL-GIN (20–30%, p < 0.05) versus HL liver. ConclusionGIN reduces SCD1 estimated activity and expression, as well as the lipids accumulated in the livers of HL hamsters. This is achieved through a mechanism involving the decrease of the oxidative and ERS, and the enhancement of cholesterol efflux.

Vascular peroxide 1 promotes ox-LDL-induced programmed necrosis in endothelial cells through a mechanism involving β-catenin signaling

Background and aimsVascular peroxidase 1 (VPO1) plays a key role in mediation of cardiovascular oxidative injury. This study aims to determine whether VPO1 can promote programmed necrosis of endothelial cells and the underlying mechanisms. Methods and resultsHuman umbilical vein endothelial cells (HUVECs) were incubated with oxidized low-density lipoprotein (ox-LDL, 100 μg/mL) for 48 h to induce cell injury, which showed an elevation in cell necrosis (reflected by the increased propidium iodide (PI) positive-staining cells, LDH release and decreased cell viability), concomitant with an increase in programmed necrosis-relevant proteins including receptor-interacting protein kinase 1/3 (RIPK1/3), p-RIPK3 and mixed lineage kinase domain like (MLKL); these phenomena were attenuated by necrostatin-1(Nec-1) and RIPK3 siRNA. Meanwhile, VPO1 was up-regulated in ox-LDL-treated endothelial cells accompanied by a decrease in GSK-3β activity and p-β-catenin levels, and an elevation of β-catenin levels; these phenomena were reversed in the presence of VPO1 siRNA or hypochlorous acid (HOCl) inhibitor; replacement of ox-LDL with HOCl could also induce endothelial programmed necrosis and activate the β-catenin signaling; β-catenin inhibitor could also suppress ox-LDL-induced RIPK-dependent necrosis. In hyperlipidemic patients, the plasma level of VPO1 was obviously increased concomitant with an elevation in plasma levels of RIPK1, RIPK3 and MLKL, and they were positively correlated. ConclusionsVPO1 plays an important role in promotion of endothelial programmed necrosis under hyperlipidemic conditions through activation of β-catenin signaling. It may serve as a novel therapeutic target for prevention of endothelial dysfunction in hyperlipidemia.

Abstract 212: Chronic Systemic Inflammation Accelerates Endothelial Cholesterol Crystal Formation and Lysosomal Dysfunction

Objective: Atherogenesis is an inflammatory process accelerated in psoriasis, a chronic inflammatory skin disease associated with premature cardiovascular diseases (CVD). Cholesterol crystal (CC) formation is implicated at onset and progression of atherosclerotic CVD, and we have recently shown CC formation to be upregulated in a murine model of psoriasis-related atherosclerosis. Therefore, we investigated inflammatory CC production in this psoriasis mouse model and also using psoriasis patient derived samples. Methods: K14-Rac1V12 psoriasis mouse aortas were examined for CC formation and structural abnormalities, such as subendothelial spaces, via electron microscopy and histology. Human aortic endothelial cells (HAoEC) were stimulated with sera from healthy and psoriatic volunteers (n=5 each), inflammatory markers TNFα+IFNγ, LDL or combination of TNFα+IFNγ+LDL. The effect on CC formation and lysosomal function was determined by advanced microscopy and flow cytometry. HAoECs were also treated with TNFα+IFNγ+LDL combined with β-cyclodextrin (CD), a stimulant for cholesterol release from cells, and analyzed for CC formation and lysosomal function. Results: Psoriatic mouse aortas showed enlarged subendothelial spaces and 40% increased CC formation under normolipidemic conditions compared to littermate controls. Treatment of HAoECs with psoriasis patient-sera (severe skin disease, low CVD risk score) or TNFα+IFNγ+LDL showed a 2-fold increase in CC formation and 30% increased lysosomal frequency compared to controls. Despite an increase in lysosome frequency, TNFα+IFNγ+LDL treatment decreased overall pH-related lysosomal activity. CD treatment diminished TNFα+IFNγ+LDL-induced CC formation (compared to vehicle), and restored lysosomal function without affecting lysosome frequency. Conclusion: Psoriasis mice displayed accelerated CC formation in the absence of hyperlipidemic conditions suggesting chronic systemic inflammation accelerates CC formation in vivo and in vitro , accompanied by lysosomal dysfunction in ECs. These findings suggest that dyslipidemia and systemic inflammation in psoriasis are important contributors to early atherogenesis and should be treatment targets for CVD risk mitigation.

Phytochemical composition, antilipidemic and antihypercholestrolemic perspectives of Bael leaf extracts

BackgroundIn recent times, focus on plant research has improved all over the world and essential parts of plants provide bioactive compounds in human diet. The bael (Aegle marmelos) has enormous traditional uses in the treatment of chronic diarrhea, dysentery, peptic ulcers and as a laxative. The main focus of this study was characterization of bael leaf extract for its bioactive constituents, antihypercholestrolemic and antilipidemic perspectives.MethodsAfter proximate composition of bael powder, the aqueous extract of bael leaf was used for phytochemical profiling (alkaloids, total phenolic content and total flavonoid content). Afterwards, normal rats group G0 was administrated basal diet while G1 and G2 normal rat groups were fed diets containing bael leaf extract 125 mg and 250 mg, respectively for consecutive 60 days. In a similar way, hyperlipidemic rats group Gh0 was administrated basal diet while Gh1 and Gh2 hyperlipidemic rat groups were fed diets containing bael leaf extract 125 mg and 250 mg, respectively for consecutive 60 days. The blood drawn on day 0, day 30 and day 60 was analyzed for serum parameters, such as total cholesterol, high-density lipoprotein cholesterol, low–density lipoprotein cholesterol, triglycerides concentration and free and ester cholesterol.ResultsBael leaf powder is a rich source of crude fiber (14.50 ± 0.10 g/100 g). Aqueous extract of bael leaf contains alkaloids (15.58 ± 0.05 mg/g), flavonoids (64.00 ± 0.05 mg/g), phenolics (30.34 ± 0.01 GAEmg/g). From the In vivo studies, the lowest weight gain was observed in group G2 and in Gh2 as compared to control of both groups. The decrease in serum TC for G1–15.06%, G2–17.27% while in Gh1–22.46% and Gh2–34.82% after day 60, respectively. The maximum decrease was observed in group G2 (− 14.33%) and in Gh2 (− 24.79%) for triglycerides after 60 days. For HDL-cholesterol, significant increase (11.20%) in G2 and (49.83%) in Gh2 was observed of after 60 days. A trend in decrease of serum LDL–cholesterol in G2 (− 9.63%) and in Gh2 (− 44.65%) was also observed at day 60, and − 19.05% and − 30.06% decrease was noted in G2 and Gh2, respectively and decreasing trend was observed in free and total cholesterol − 22.30% and − 81.49% for groups G2 and Gh2 after day 60.ConclusionsThe results of the present study demonstrated that the extract contents of bael leaf provide protective role against hypercholesterolemic and hyperlipidemic conditions.

Maresin 1 attenuates NAFLD by suppression of endoplasmic reticulum stress via AMPK–SERCA2b pathway

Maresin 1 (MAR1), which is derived from docosahexaenoic acid biosynthesized by macrophages, has been reported to improve insulin resistance. Recently, it has been documented that MAR1 could ameliorate inflammation and insulin resistance in obese mice. These findings led us to investigate the effects of MAR1 on hepatic lipid metabolism. We found that MAR1 could stimulate AMP-activated protein kinase (AMPK), thereby augmenting sarcoendoplasmic reticulum Ca2+-ATPase 2b (SERCA2b) expression. This stimulation suppressed lipid accumulation by attenuating the endoplasmic reticulum (ER) stress in hepatocytes under hyperlipidemic conditions. Attenuation was mitigated by knockdown of AMPK or thapsigargin, a SERCA2b inhibitor. We also demonstrated that MAR1 administration resulted in increased hepatic AMPK phosphorylation and Serca2b mRNA expression, whereas hepatic ER stress was reduced in high-fat diet (HFD)-fed mice. Moreover, MAR1 treatment suppressed hepatic lipid synthesis, thereby attenuating hepatic steatosis in HFD-fed mice. In conclusion, our results suggest that MAR1 ameliorates hepatic steatosis via AMPK/SERCA2b-mediated suppression of ER stress. Therefore, MAR1 may be an effective therapeutic strategy for treating non-alcoholic fatty liver disease (NAFLD) via regulation of ER stress-induced hepatic lipogenesis.

CD1d deficiency inhibits the development of abdominal aortic aneurysms in LDL receptor deficient mice.

An abdominal aortic aneurysm (AAA) is a dilatation of the abdominal aorta leading to serious complications and mostly to death. AAA development is associated with an accumulation of inflammatory cells in the aorta including NKT cells. An important factor in promoting the recruitment of these inflammatory cells into tissues and thereby contributing to the development of AAA is angiotensin II (Ang II). We demonstrate that a deficiency in CD1d dependent NKT cells under hyperlipidemic conditions (LDLr-/-CD1d-/- mice) results in a strong decline in the severity of angiotensin II induced aneurysm formation when compared with LDLr-/- mice. In addition, we show that Ang II amplifies the activation of NKT cells both in vivo and in vitro. We also provide evidence that type I NKT cells contribute to AAA development by inducing the expression of matrix degrading enzymes in vSMCs and macrophages, and by cytokine dependently decreasing vSMC viability. Altogether, these data prove that CD1d-dependent NKT cells contribute to AAA development in the Ang II-mediated aneurysm model by enhancing aortic degradation, establishing that therapeutic applications which target NKT cells can be a successful way to prevent AAA development.

What Do Experimental Models Teach Us About Comorbidities in Stroke?

There is a general consensus that animal models do not perfectly represent human conditions. This is not derived from any specific event, such as an ischemic episode, but rather reflects inherent differences in the physiology, anatomy, and metabolism among different species. Disparities among species also arise from differences in assessing functional recovery in stroke. Humans place a priority in regaining function in speech, cognition, and limb control, but these functions are not always readily translatable in animal models. Moreover, because functional recovery depends on the stroke type and location, induction of an ischemic lesion in a predefined area of an animal model, at best, reflects only a subtype of human stroke. Nevertheless, certain clinical pathophysiology features of stroke, such as acute outcomes and inflammatory/immune responses, are shared in animal models.1 Combined with complementary knowledge from in vitro studies, animal models can provide valuable insight into stroke pathology at a systems level. Analyses of translational inefficiencies in stroke model systems have led to the identification of underlying issues, which include, but are not limited to, rigor in experimental design, internal and external validity, negative bias, and insufficient statistical power. Together, these inefficiencies have steered collaborative international efforts in preclinical research to overcome these challenges.2 An additional issue of importance is the insufficient understanding of stroke pathophysiology beyond the acute phase and the primary reliance on infarct volume in reporting acute outcomes. Together with a lack of inclusion of comorbidities in animal models of stroke, the development of clinical protocols based on findings from normal metabolic animals may contribute to translational failures. In light of a recent concerted effort to address the future of translational stroke research,3 this review focuses on underlying translational issues and discusses insights gained from stroke models inclusive of comorbidities in the preclinical literature. ### Stroke Pathology: A Moving Target With Multiple Components Despite …

High Fat Diet Upregulates Fatty Acid Oxidation and Ketogenesis via Intervention of PPAR-γ

Background/Aims: Systemic hyperlipidemia and intracellular lipid accumulation induced by chronic high fat diet (HFD) leads to enhanced fatty acid oxidation (FAO) and ketogenesis. The present study was aimed to determine whether activation of peroxisome proliferator-activated receptor-γ (PPAR-γ) by surplus free fatty acids (FA) in hyperlipidemic condition, has a positive feedback regulation over FAO and ketogenic enzymes controlling lipotoxicity and cardiac apoptosis. Methods: 8 weeks old C57BL/6 wild type (WT) or PPAR-γ-/- mice were challenged with 16 weeks 60% HFD to induce obesity mediated type 2 diabetes mellitus (T2DM) and diabetic cardiomyopathy. Treatment course was followed by echocardiographic measurements, glycemic and lipid profiling, immunoblot, qPCR and immunohistochemistry (IHC) analysis of PPAR-γ and following mitochondrial metabolic enzymes 3-hydroxy-3-methylglutaryl-CoA synthase (HMGCS2), mitochondrial β- hydroxy butyrate dehydrogenase (BDH1) and pyruvate dehydrogenase kinase isoform 4 (PDK4). In vivo model was translated in vitro, with neonatal rat cardiomyocytes (NRCM) treated with PPAR-γ agonist/antagonist and PPAR-γ overexpression adenovirus in presence of palmitic acid (PA). Apoptosis was determined in vivo from left ventricular heart by TUNEL assay and immunoblot analysis. Results: We found exaggerated circulating ketone bodies production and expressions of the related mitochondrial enzymes HMGCS2, BDH1 and PDK4 in HFD-induced diabetic hearts and in PA-treated NRCM. As a mechanistic approach we found HFD mediated activation of PPAR-γ is associated with the above-mentioned mitochondrial enzymes. HFD-fed PPAR-γ-/-mice display decreased hyperglycemia, hyperlipidemia associated with increased insulin responsiveness as compared to HFD-fed WT mice PPAR-γ-/–HFD mice demonstrated a more robust functional recovery after diabetes induction, as well as significantly reduced myocyte apoptosis and improved cardiac function. Conclusions: PPAR-γ has been described previously to regulate lipid metabolism and adipogenesis. The present study suggests for the first time that increased PPAR-γ expression by HFD is responsible for cardiac dysfunction via upregulation of mitochondrial enzymes HMGCS2, BDH1 and PDK4. Targeting PPAR-γ and its downstream mitochondrial enzymes will provide novel strategies in preventing metabolic and myocardial dysfunction in diabetes mellitus.