Receptor Gamma Research Articles

Theobromine suppresses adipocyte differentiation through the MAPK pathway in 3T3‐L1 preadipocytes (1037.11)

Theobromine (TB) is an alkaloid belonging to the methylxanthines. It is mainly found in cocoa beans. In this study, we show that the inhibitory effect of TB on adipocyte differentiation in 3T3‐L1 cells by reducing adipogenic gene expression. To investigate the effects of TB on adipocyte differentiation, 3T3‐L1 cells were induced to differentiate using 3‐isobutyl‐1‐methylzanthine, dexamethasone, and insulin (MDI) for 8 days in the presence of 50, 100, and 150 μg/ml TB. TB attenuated the accumulation of lipid droplets stained with Oil Red O in dose‐dependent manner. We also found the effect of TB on the inhibition of adipocyte differentiation occurred during early stage of adipogenesis. To elucidate the anti‐adipogenic mechanism of TB, adipogenic transcription factors, the phosphorylation levels of MAPK, and adipogenic gene expression were analyzed by Western blotting. Treatment of cells with TB resulted in a decrease in the protein level of (Peroxisome proliferator‐activated receptor gamma) PPARγ, (CCAAT/enhancer binding protein beta) C/EBPβ, and phosphorylation of ERK and JNK. These results demonstrate that TB inhibits adipogenic differentiation via suppression of the ERK and JNK signaling pathway during the early stages of adipogenesis.

Is obesity an absolute evil? Increase in adipose tissue does not always decrease insulin sensitivity.

Is obesity an absolute evil? Increase in adipose tissue does not always decrease insulin sensitivity.

Progesterone increases ex vivo testosterone production and decreases the expression of progestin receptors and steroidogenic enzymes in the fathead minnow (Pimephales promelas) ovary

Progesterone (P4) is a metabolic precursor for a number of steroids, including estrogens and androgens. P4 also has diverse roles within the vertebrate ovary that include oocyte growth and development. The objectives of this study were to measure the effects of P4 on testosterone (T) and 17β-estradiol (E2) production in the fathead minnow (FHM) ovary and on the mRNA abundance of transcripts involved in steroidogenesis and steroid receptor signaling. Ovary explants were treated with P4 (10−6M) for 6 and 12h. P4 administration significantly increased T production ∼3-fold at both 6 and 12h, whereas E2 production was not affected, consistent with the hypothesis that excess P4 is not converted to terminal estrogens in the mature ovary. Nuclear progesterone receptor mRNA was decreased at 6h and membrane progesterone receptor gamma-2 mRNA was significantly down-regulated at both 6 and 12h; however there was no change in membrane progesterone receptor alpha or beta mRNA levels. Androgen receptor (ar) and estrogen receptor 2a (esr2a) mRNA were significantly reduced at 6h with P4 treatment, but there was no change in esr2b mRNA at either time point. Transcripts for enzymes in the steroid pathway (star, hsd11b2) were significantly lower at 6h compared to controls, whereas cyp17a and cyp19a mRNA abundance did not change with treatments at either time point. These data suggest that P4 incubation can lead to increased T production in the FHM ovary without a concomitant change in E2, and that the membrane bound progestin receptors are differentially regulated by P4 in the teleost ovary. As environmental progestins have received increased attention due to their suspected role as endocrine disruptors, mechanistic data on the role of exogenous P4 treatments in the male and female gonad is warranted.

A Moderate Zinc Deficiency Does Not Impair Gene Expression of PPARα, PPARγ, and Mitochondrial Enoyl-CoA Delta Isomerase in the Liver of Growing Rats

The aim of the study was to investigate the impact of a moderate zinc deficiency and a high intake of polyunsaturated fat on the mRNA expression of peroxisome-proliferator-activated receptor alpha (PPARα), peroxisome-proliferator-activated receptor gamma (PPARγ), and mitochondrial Δ3Δ2-enoyl-CoA isomerase (ECI) in the liver. Weanling rats were assigned to five groups (eight animals each) and fed semi-synthetic, low-carbohydrate diets containing 7 or 50 mg Zn/kg (low-Zn (LZ) or high-Zn (HZ)) and 22% cocoa butter (CB) or 22% safflower (SF) oil for four weeks. One group each was fed the LZ-CB, LZ-SF, or HZ-SF diet free choice, and one group each was fed the HZ-CB and HZ-SF diets in restricted amounts according to intake of the respective LZ diets. The LZ diets markedly lowered growth and zinc concentrations in plasma and femur. Hepatic mRNA levels of PPARα, PPARγ, and ECI were not reduced by the moderate zinc deficiency. Overall, ECI-mRNA abundance was marginally higher in the SF-fed than in the CB-fed animals.

222 PRODUCTION OF KNOCKOUT RATS BY USING ZINC FINGER NUCLEASES AND TAL EFFECTOR NUCLEASES

The rat has been used as an important animal for understanding human diseases. Genetically engineered rat strains are used as a human disease model in various research fields. Genetically engineered rat strains are now being routinely produced, not only as transgenic animals but also using gene knockout techniques. Recently, zinc finger nucleases (ZFN) and TAL effector nucleases (TALEN) have enabled editing targeted genes without using embryonic stem cells. These techniques have been applied for production of the knockout and knockin animals. We here studied that the effects of gene targeting by ZFN and TALEN introduced into rat embryos for efficient production of knockout rats. We custom-designed ZFN and TALEN plasmids targeted rat interleukin 2 receptor gamma (Il2rg) gene. Each mRNA was transcribed in vitro from these plasmids. Final concentration of mRNA was adjusted at 10 ng μL–1 in sterilized water for microinjection. Messenger RNA was injected into rat pronuclear stage embryos. The embryos were then cultured in vitro to the 2-cell stage, and were transferred into oviducts of pseudopregnant females. The rate of development of offspring of embryos and effects of editing targeted genes were examined. Of 41 two-cell embryos introduced ZFN after embryo transfer, 9 embryos (22%) developed to offspring. Three offspring (33%) had an edited targeted gene locus. In the embryos introduced TALEN, 30% (6 offspring) of embryos developed to offspring after embryo transfer and all offspring had an edited targeted gene locus. This study demonstrated that the ZFN and TALEN mRNA was active after introduction into rat embryos. Knockout rats could be produced by introduction of ZFN and TALEN into rat embryos. ZFN and TALEN will provide a powerful new approach for targeted gene editing not only in rats but also in other animal species.

Peroxisome proliferator-activated receptors alpha and gamma2 polymorphisms in nonalcoholic fatty liver disease: A study in Brazilian patients

BackgroundNon-alcoholic fatty liver disease (NAFLD) refers to the accumulation of hepatic steatosis in the absence of excess alcohol consumption. The pathogenesis of fatty liver disease and steatohepatitis (NASH) is not fully elucidated, but the common association with visceral obesity, hyperlipidemia, hypertension and type 2 diabetes mellitus (T2DM) suggests that it is the hepatic manifestation of metabolic syndrome. Peroxisome proliferator-activated receptor PPARα and PPARγ are members of a family of nuclear receptors involved in the metabolism of lipids and carbohydrates, adipogenesis and sensitivity to insulin. The objective of this study was to analyze the polymorphisms Leu162Val of PPARα and Pro12Ala of PPARγ as genetic risk factors for the development and progression of NAFLD. MethodsOne hundred and three NAFLD patients (89 NASH, 14 pure steatosis) and 103 healthy volunteers were included. Single nucleotide polymorphisms (SNPs) Leu162Val and Pro12Ala were analyzed by polymerase chain reaction (PCR) and restriction fragment length polymorphism (RFLP). ResultsNASH patients presented higher BMI, AST and prevalence of T2DM than patients with pure steatosis. A higher prevalence of 12Ala allele was observed in the NASH Subgroup when compared to Control Group. When we grouped NASH and Steatosis Subgroups (NAFLD), we found lower serum glucose and more advanced fibrosis in the Leu162Val SNP. On the other hand, there was no statistical difference in clinical, laboratorial and histological parameters according to the Pro12Ala SNP. ConclusionsWe documented a lower prevalence of 12Ala allele of gene PPARγ in the NASH Subgroup when compared to Control Group. In NAFLD patients, there were no associations among the occurrence of Pro12Ala SNP with clinical, laboratorial and histological parameters. We also documented more advanced fibrosis in the Leu162Val SNP. The obtained data suggest that Pro12Ala SNP may result in protection against liver injury and that Leu162Val SNP may be involved in the progression of NAFLD.

Low Osteogenic Differentiation Potential of Placenta-Derived Mesenchymal Stromal Cells Correlates with Low Expression of the Transcription Factors Runx2 and Twist2

Recent studies indicated that mesenchymal stromal cells from bone marrow (bmMSC) differ in their osteogenic differentiation capacity compared to MSC from term placenta (pMSC). We extended these studies and investigated the expression of factors involved in regulation of bone metabolism in both cell types. To this end, MSC were expanded in vitro and characterized. The total transcriptome was investigated by microarrays, and for selected genes, the differences in gene expression were explored by quantitative reverse transcriptase-polymerase chain reaction, immunocytochemistry, and flow cytometry. We report that bmMSC and pMSC share expression of typical lineage surface markers, including CD73, CD90, CD105, and lack of CD14, CD34, and CD45. However, according to transcriptome analyses, they differ significantly in their expression of more than 590 genes. Factors involved in bone metabolism, including alkaline phosphatase (P<0.05), osteoglycin (P<0.05), osteomodulin (P<0.05), runt-related transcription factor 2 (Runx2) (P<0.04), and WISP2 (P<0.05), were expressed at significantly lower levels in pMSC, but twist-related protein 2 (Twist2) (P<0.0002) was expressed at significantly higher levels. The osteogenic differentiation capacity of pMSC was very low. The adipogenic differentiation was somewhat more prominent in bmMSC, while the chondrogenic differentiation seemed not to differ between bmMSC and pMSC, as determined by histochemical staining. However, expression and induction of peroxisome proliferator-activated receptor gamma-2 (PPARγ2) and Sox9, factors involved in early adipogenesis and chondrogenesis, respectively, were higher in bmMSC. We conclude that despite many similarities between bmMSC and pMSC, when expanded under identical conditions, they vary considerably with respect to their in vitro differentiation potential. For regenerative purposes, the choice of MSC may therefore influence the outcome of a treatment considerably.

Does endothelial peroxisome proliferator‐activated receptor gamma (ePPAR‐γ) protect baroreflex function during obesity?

We tested the hypothesis that ePPAR‐γ protects baroreflex function during obesity by determining whether a long term (10–12 weeks, LT) or short term (1–2 weeks, ST) high fat diet (HFD, 45% kcal fat) impairs baroreflex gain (BRG) in mice with a dominant‐negative mutation of ePPAR‐γ (E‐V290M) more than wild‐type mice (WT). Arterial pressure and HR were measured by telemetry for 24hrs in mice on a 12:12 dark (D): light (L) cycle. Spontaneous BRG (sBRG; msec/mmHg) was derived using the sequence method. sBRG was lower in lean E‐V290M compared to WT during L, but not D. The LT‐HFD attenuated sBRG in WT and E‐V290M during both L and D, with no difference between groups. During L, the ST‐HFD also blunted sBRG in WT, and sBRG remained lower in E‐V290M mice. During D, ST‐HFD only lowered sBRG in E‐V290M. However, the ST‐HFD‐induced decreases in sBRG were not greater in E‐V290M compared to WT mice during L (WT: −0.30±0.05 vs E‐V290M: −0.17±0.04) or D (WT: −0.06±0.05 vs E‐V290M: −0.17±0.04), indicating that there was not an interactive effect between obesity and the ePPAR‐γ mutation on sBRG. These data suggest that while ePPAR‐γ supports BRG in lean mice, it does not protect the baroreflex during obesity. sBRG (msec/mmHg) Lean L D WT (n=11) 1.63 ± 0.08 1.18 ± 0.06 E‐V290M (n=11) 1.28 ± 0.07† 1.08 ± 0.07 LT‐HFD WT (n=5) 0.96 ± 0.18 # 0.84± 0.10 # E‐V290M (n=3) 0.88 ± 0.19 # 0.69 ± 0.08 # ST‐HFD WT HFD (n=5) 1.36 ± 0.05# 1.10 ± 0.06 E‐V290M HFD (n=7) 1.12 ± 0.05† 0.89 ± 0.06†# Data are mean ± SE. P&lt;0.05 group within diet, P&lt;0.05 diet within group

Human Mammary Epithelial Cell Gene Expression and Product Secretion of Milk Lipids during Secretory Activation

BackgroundThe gene expression of lipid biosynthetic enzymes during initiation of lactation in humans in unknown.ObjectiveTo study mRNA expression of lipid metabolic enzymes in human mammary epithelial cell (MEC) in conjunction with the measurement of milk fatty acid (FA) composition during secretory activation.SubjectsGene expression from mRNA isolated from milk fat globule (MFG), a reflection of gene expression in MEC, and milk FA composition were measured from 6h to 42d postpartum in 7 normal women.ResultsOver the first 96h postpartum, gene expression of all aspects of lipid metabolism and milk FA production increased several folds, including: lipolysis at the MEC membrane, FA uptake from blood, intracellular FA transport, de novo FA synthesis, FA and glycerol activation, FA elongation, FA desaturation, synthesis of triglycerides, cholesterol synthesis and lipid droplet formation. Gene expression of the key lipid synthesis regulator, sterol regulatory element‐binding transcription factor1 (SREBF1), increased 2.0‐fold by 36h and remained elevated over the study duration. The mRNA expression of thyroid hormone responsive (THRSP) and insulin‐induced2 (INSIG2) genes increased progressively to plateau by 96h. In contrast, mRNA expression of peroxisome proliferator‐activated receptor gamma (PPARG) decreased several folds. With the onset of lactation, de novo synthesis of FAs (C6‐C12) was the most prominent change in milk FA composition and mirrored the expression of FA synthesis genes.ConclusionMilk fat synthesis and secretion in humans is a complex process requiring the orchestration of a wide variety of pathways of which SREBF1 may play a primary role.Grant Funding Source: USDA/ARS Grant # 6250–51000

Hemin‐Induced miR‐27a Reduces PPARγ Expression in Sickle Cell Disease with Pulmonary Hypertension

The hallmark of sickle cell disease (SCD) is hemolysis, vaso‐occlusion, and oxidative stress. Pulmonary hypertension (PH) is a serious complication of SCD that causes significant morbidity and mortality. We previously demonstrated that: 1) activation of the nuclear hormone receptor, peroxisome proliferator‐activated receptor gamma (PPARγ attenuated hypoxia‐induced Nox4 and endothelin‐1 (ET‐1) expression and PH in mice and 2) that levels of Nox4 and ET‐1 were increased in the lungs of 8–10 week old SCD transgenic mice compared to controls whereas PPARγ levels were reduced. The current study further examines mechanisms of PH in SCD. Human pulmonary arterial endothelial cells (HPAECs) were treated with the hemolysis product, hemin (5 μM), for 72 h. Hemin reduced PPARγ and increased Nox4 and ET‐1 expression and HPAEC proliferation. MicroRNA‐27a (miR‐27a), which negatively regulates PPARγ, was increased in hemin‐treated HPAECs. In contrast, treating HPAECs with the PPARγ rosiglitazone (10 μM) for the final 24 h of hemin exposure attenuated increased HPAEC proliferation and miR‐27a levels. These findings suggest that hemin increases miR‐27a to reduce PPARγ and increase Nox4, ET‐1, and PAEC proliferation. These results suggest novel pathways for SCD‐PH pathogenesis and therapy. Supported by Atlanta VA Merit Review, NIH HL102167, and Emory/Childrens Healthcare CEB F16788–00.

Dietary DHA promotes an anti‐oxidant response in mice exposed to environmental pollutants

Consumption of fish oil is associated with improved coronary health outcomes, but the mechanism of protection is not well understood. Environmental stressors, such as polychlorinated biphenyls (PCBs), contribute to the development of cardiovascular diseases. We study the protective benefits of nutrients such as the omega‐3 fatty acid, docosahexaenoic acid (DHA), from fish oil in reducing PCB inflammatory response. To understand the protective mechanism of DHA, we fed C57BL/6 mice DHA‐enriched or safflower oil control diet for 4 weeks before administering PCB 126. Liver samples were analyzed to examine the interactive effects of PCBs and DHA on the nuclear factor (erythroid‐derived 2)‐like 2 (Nrf‐2) antioxidant response pathway. Microarray data revealed that animals exposed to DHA and PCBs had decreased expression of kelch‐like ECH‐associated protein 1 (KEAP1), an inhibitor of Nrf‐2 activity. In addition, peroxisome proliferator‐activated receptor gamma (PPARγ), an Nrf‐2 co‐activator, and its chaperone PPARγ co‐activator 1a (PPARγc1a) had increased expression. Real‐time PCR showed increased expression of Nrf‐2 activated antioxidant enzymes, NAD(P)H dehydrogenase (quinone 1) (NQO1) and hemeoxygenase 1 (HO‐1) in PCB treated, DHA fed mice. Data suggest that DHA promotes an antioxidant response in mice exposed to environmental pollutants such as PCBs. Supported by NIEHS, NIH P42ES007380 and the UK AES.

Loss of PPARγ promotes NF‐κB activation, Nox4 induction, and proliferation of human pulmonary artery smooth muscle cells

Hypoxia stimulates pulmonary hypertension (PH) in part by increasing the proliferation of pulmonary vascular wall cells. Recent evidence suggests that signaling events involved in hypoxia‐induced cell proliferation include sustained Nuclear Factor‐kappaB (NF‐κB) activation, increased NADPH oxidase4 (Nox4) expression, and downregulation of peroxisome proliferator‐activated receptor gamma (PPARγ) levels. To further understand the role of reduced PPARγ levels in PH pathobiology, siRNA was employed to reduce PPARγ levels in human pulmonary artery smooth muscle cells (HPASMC) in vitro. PPARγ siRNA reduced PPARγ levels to an extent comparable to those observed under hypoxic conditions. PPARγ depletion activated NF‐κB as determined by phosphorylation of RelA/p65 (NF‐κB) at Ser536 and degradation of its inhibitory protein, I‐kappaB alpha. PPARγ depletion also induced the expression of the NF‐κB target gene, Nox4 and increased HPASMC proliferation as determined by MTT assay and elevated levels of proliferating cell nuclear antigen. Furthermore, treatment with PEG‐catalase attenuated HPASMC proliferation caused by PPARγ depletion. Taken together, these findings provide novel evidence that reductions in PPARγ levels are sufficient to promote HPASMC proliferation via NF‐κB activation, Nox4 induction, and H2O2 generation.Supported by: Atlanta VA Research Service, NIH DK074518 and HL102167, and AHA‐SDG

Prevalence of Gly482Ser polymorphism of PPARGC1A in Cuban Americans

Type 2 Diabetes (T2D) has grown to be a major public health issue worldwide. The fundamental clinical characteristic of T2D is high blood glucose levels which may be caused by anomalies in one or more different molecular pathways regulated by certain genes. The Gly482Ser polymorphism in human peroxisome proliferator‐activated receptor gamma coactivator‐1 alpha (PPARGC1A) has been associated with T2D. We genotyped Gly482Ser polymorphism in the PPARGC1A gene from 315 Cuban Americans (164 without T2D / 151 with T2D) using ABI TaqMan assays. Descriptive statistical analysis was performed using SPSS software. The genotype distribution for rs8192678 (Gly482Ser) polymorphism was found to be 0.42 for Gly/Gly, 0.47 for Gly/Ser and 0.10 for Ser/Ser with T2D and 0.47, 0.41 and 0.11 for Gly/Gly, Gly/Ser and Ser/Ser respectively without T2D. The findings in individuals without T2D demonstrate presence of homozygous genotype Gly/Gly to be highest. However, the prevalence of heterozygous genotype Gly/Ser was found to be relatively higher in individuals with T2D than without T2D. The clinical importance of the results has to be established in further studies. Funding was provided by an NIH grant.

Inhibition of the receptor of advanced glycation reverses pulmonary arterial hypertension

Pulmonary arterial (PA) hypertension (PAH) is a vasculopathy characterized by enhanced PA smooth muscle cells (PASMC) proliferation and suppressed apoptosis. A recent proteomic analysis of PAH lungs demonstrated that the receptor of advanced glycation endproducts (RAGE) is one of the most upregulated proteins in PAH patients. We hypothesized that RAGE could play a critical role in PAH etiology and progression. We demonstrated a specific RAGE overexpression in PAH lungs. In human PASMC, we showed that RAGE was implicated in the proliferative and apoptosis‐resistant phenotype. Recent studies demonstrated an altered BMPR2 (Bone Morphogenetic Protein Receptor 2)/PPARγ (Peroxisome Proliferator‐Activated Receptor gamma) axis in PAH. We showed that RAGE is accountable for BMPR2/PPARγ downregulation, thus regulating proliferation and apoptosis. In vivo, RAGE inhibition (siRNA nebulization) in rats with Sugen‐induced PAH showed decreased PA pressure and right ventricular hypertrophy, associated with a significant improvement in lung perfusion and decreased PA wall thickness. To conclude, we demonstrated the implication of RAGE in PAH etiology. Thus, RAGE inhibitors, which are in clinical phase II for Alzheimer's disease, represent potential therapeutic agents for PAH treatment.A Canadian Thoracic Society scholarship to JM and Canadian Institutes of Health Research grants to SB supported this work.

NRF‐1 and its target genes are increased by exercise: Potential role of NRF‐1 in type 2 diabetes

Nuclear respiratory factor (NRF)‐1 is a mitochondrial transcriptional factor whose function of late shown to be involved in glucose transport (Ramachandran et al., 2008, Baar et al., 2003) and set it as a potential therapeutic modality in the treatment and management of type 2 diabetes. In this study we sought to access NRF‐1 and its target genes expression during exercise which are crucial in glucose transport and lipid oxidation.Five to six weeks old male Wistar rats were exercised to identify the time points for optimum increase in the levels of NRF‐1 and its target genes. Gastrocnemius muscle was harvested after 0, 2, 4, 6, 8, 10, 12 and 15 hours (h) post exercise and non‐exercise rats (control). Primers were used to amplify the region of following genes: Nrf‐1, Glut 4, Carnitine palmitoyltransferase (Cpt‐1&amp; Cpt‐2), Peroxisome proliferator‐activated receptor gamma co‐activator 1 (Pgc‐1), Mef2a, Acetyl‐CoA Carboxylase‐1 (Acc‐1). Relative mRNA expression was normalized to actin reference gene.Cpt‐1, Nrf‐1, Mef2a, Glut4, Cpt2 and Pgc‐1 showed 2.5, 8, 1.2, 4.1, 4.6, 3.5 fold increases respectively after 8h post exercise compared to control whereas Acc‐1 showed 3.1 fold decrease in gene expression ratio after 6 h post exercise compared to control. NRF‐1 binding to cpt‐1 and mef‐2 increased 3 and 3.5 fold respectively 6h post exercise compared to controls.These results show that NRF‐1 was increased by exercise and also its binding to target genes which has huge implication in emeliorating type 2 diabetes and insulin resistance.

Critical Role for the Advanced Glycation End‐Products Receptor in Pulmonary Arterial Hypertension Etiology

BackgroundPulmonary arterial hypertension (PAH) is a vasculopathy characterized by enhanced pulmonary artery smooth muscle cell (PASMC) proliferation and suppressed apoptosis. This results in both increase in pulmonary arterial pressure and pulmonary vascular resistance. Recent studies have shown the implication of the signal transducer and activator of transcription 3 (STAT3)/bone morphogenetic protein receptor 2 (BMPR2)/peroxisome proliferator‐activated receptor gamma (PPARγ) in PAH. STAT3 activation induces BMPR2 downregulation, decreasing PPARγ, which both contribute to the proproliferative and antiapoptotic phenotype seen in PAH. In chondrocytes, activation of this axis has been attributed to the advanced glycation end‐products receptor (RAGE). As RAGE is one of the most upregulated proteins in PAH patients' lungs and a strong STAT3 activator, we hypothesized that by activating STAT3, RAGE induces BMPR2 and PPARγ downregulation, promoting PAH‐PASMC proliferation and resistance to apoptosis.Methods and ResultsIn vitro, using PASMCs isolated from PAH and healthy patients, we demonstrated that RAGE is overexpressed in PAH‐PASMC (6‐fold increase), thus inducing STAT3 activation (from 10% to 40% positive cells) and decrease in BMPR2 and PPARγ levels (>50% decrease). Pharmacological activation of RAGE in control cells by S100A4 recapitulates the PAH phenotype (increasing RAGE by 6‐fold, thus activating STAT3 and decreasing BMPR2 and PPARγ). In both conditions, this phenotype is totally reversed on RAGE inhibition. In vivo, RAGE inhibition in monocrotaline‐ and Sugen‐induced PAH demonstrates therapeutic effects characterized by PA pressure and right ventricular hypertrophy decrease (control rats have an mPAP around 15 mm Hg, PAH rats have an mPAP >40 mm Hg, and with RAGE inhibition, mPAP decreases to 20 and 28 mm Hg, respectively, in MCT and Sugen models). This was associated with significant improvement in lung perfusion and vascular remodeling due to decrease in proliferation (>50% decrease) and BMPR2/PPARγ axis restoration (increased by ≥60%).ConclusionWe have demonstrated the implications of RAGE in PAH etiology. Thus, RAGE constitutes a new attractive therapeutic target for PAH.

Association of RXR-Gamma Gene Variants with Familial Combined Hyperlipidemia: Genotype and Haplotype Analysis

Background. Familial combined hyperlipidemia (FCHL), the most common genetic form of hyperlipdemia, is characterized by a strong familial clustering and by premature coronary heart disease. The FCHL locus has been mapped to human chromosome 1q21-q23. This region includes the retinoid X receptor gamma (RXRG), a nuclear factor member of the RXR superfamily, which plays important roles in lipid homeostasis. Objective. To investigate the possible role of the RXRG gene in the genetic susceptibility to FCHL. Methods. Variations in RXRG gene were searched by direct sequencing, and the identified SNPs were genotyped by PCR-RFLP in 192 FCHL individuals from 74 families and in 119 controls. Results. We identified 5 polymorphisms in the RXRG gene (rs1128977, rs2651860, rs2134095, rs283696, and rs10918169). Genotyping showed that the A-allele of rs283696 SNP was significantly associated with FCHL (corrected P, P c < 0.01). Also the alleles of the rs10918169 and of the rs2651860 SNP were more frequent in FCHL subjects compared to those in controls, although not significantly after correction. When the clinical characteristics of the FCHL subjects were stratified by allele carrier status for each SNP, the rs2651860 SNP was significantly associated with increased levels of LDL-cholesterol and of Apo-B in T-allele carriers (P < 0.04). Finally, haplotypes analysis with all 5 SNPs confirmed the significant association of RXRG gene with FCHL. Specifically, the haplotype containing all 3 “at-risk” alleles, significantly associated with FCHL (A-allele of rs283696, G-allele of rs10918169, and T-allele of rs2651860), showed an OR (Odds Ratio) of 2.02, P c < 0.048. Conversely, the haplotype without all these 3 alleles was associated with a reduced risk for FCHL (OR = 0.39, P c < 0.023). The “at-risk” haplotype CTTAG was also associated with higher LDL-C (P < 0.015). In conclusion, variation in the RXRG gene may contribute to the genetic dyslipidemia in FCHL subjects.

The Pro12Ala Polymorphism in the Peroxisome Proliferator-Activated Receptor Gamma-2 Gene (PPARγ2) Is Associated with Increased Risk of Coronary Artery Disease: A Meta-Analysis

BackgroundContradictory results have been reported regarding the association between Pro12Ala polymorphism of PPARγ2 and coronary artery disease (CAD). We sought to estimate the inconsistent results by performing a comprehensive meta-analysis.MethodsStudies in English or Chinese publications were identified by screening MEDLINE, Embase, CNKI, Wanfang and CBM. 22 studies including 8948 cases and 14427 controls were selected. A random-effects model was applied to combine the divergent outcomes of the individual studies, while addressing between-study heterogeneity and publication bias.ResultsThe Pro12Ala polymorphism of control population followed Hardy-Weinberg equilibrium for all studies (P>0.05). Overall, a marginal increased risk of CAD under the recessive genetic model (AlaAla vs ProAla+ProPro: P = 0.04, OR = 1.31, 95%CI 1.01–1.69, Pheterogeneity = 0.67, I2 = 0%) and the homozygote comparison (AlaAla vs ProPro: P = 0.04,OR = 1.30, 95%CI 1.01–1.68, Pheterogeneity = 0.68, I2 = 0%) was observed. In the subgroup analysis by ethnicity, carriers of AlaAla homozygotes had a significant increased risk for CAD among Caucasians (AlaAla vs ProAla+ProPro: P = 0.01, OR = 1.45, 95%CI 1.08–1.96, Pheterogeneity = 0.48, I2 = 0%; AlaAla vs ProPro: P = 0.02,OR = 1.44, 95%CI 1.07–1.93, Pheterogeneity = 0.46, I2 = 0%). After dividing into population source, the CAD risk magnitude of hospital-based studies was distinctly strengthened under the recessive model (P = 0.03,OR = 1.85,95%CI 1.07–3.19, Pheterogeneity = 0.87,I2 = 0%) and the homozygote comparison (P = 0.03,OR = 1.83, 95%CI 1.06–3.16, Pheterogeneity = 0.88, I2 = 0%). There was no observable publication bias as reflected by funnel plot and Egger’s linear regression test (t = -0.12, P = 0.91).Conclusion:Our results demonstrated that the PPARγ2 Pro12Ala polymorphism might be risk-conferring locus for the progression of CAD among Caucasians, but not among Asians.

Genetic Polymorphisms of the Main Transcription Factors for Adiponectin Gene Promoter in Regulation of Adiponectin Levels: Association Analysis in Three European Cohorts

Adiponectin serum concentrations are an important biomarker in cardiovascular epidemiology with heritability etimates of 30–70%. However, known genetic variants in the adiponectin gene locus (ADIPOQ) account for only 2%–8% of its variance. As transcription factors are thought to play an under-acknowledged role in carrying functional variants, we hypothesized that genetic polymorphisms in genes coding for the main transcription factors for the ADIPOQ promoter influence adiponectin levels. Single nucleotide polymorphisms (SNPs) at these genes were selected based on the haplotype block structure and previously published evidence to be associated with adiponectin levels. We performed association analyses of the 24 selected SNPs at forkhead box O1 (FOXO1), sterol-regulatory-element-binding transcription factor 1 (SREBF1), sirtuin 1 (SIRT1), peroxisome-proliferator-activated receptor gamma (PPARG) and transcription factor activating enhancer binding protein 2 beta (TFAP2B) gene loci with adiponectin levels in three different European cohorts: SAPHIR (n = 1742), KORA F3 (n = 1636) and CoLaus (n = 5355). In each study population, the association of SNPs with adiponectin levels on log-scale was tested using linear regression adjusted for age, sex and body mass index, applying both an additive and a recessive genetic model. A pooled effect size was obtained by meta-analysis assuming a fixed effects model. We applied a significance threshold of 0.0033 accounting for the multiple testing situation. A significant association was only found for variants within SREBF1 applying an additive genetic model (smallest p-value for rs1889018 on log(adiponectin) = 0.002, β on original scale = −0.217 µg/ml), explaining ∼0.4% of variation of adiponectin levels. Recessive genetic models or haplotype analyses of the FOXO1, SREBF1, SIRT1, TFAPB2B genes or sex-stratified analyses did not reveal additional information on the regulation of adiponectin levels. The role of genetic variations at the SREBF1 gene in regulating adiponectin needs further investigation by functional studies.

Defects of mtDNA Replication Impaired Mitochondrial Biogenesis During Trypanosoma cruzi Infection in Human Cardiomyocytes and Chagasic Patients: The Role of Nrf1/2 and Antioxidant Response

BackgroundMitochondrial dysfunction is a key determinant in chagasic cardiomyopathy development in mice; however, its relevance in human Chagas disease is not known. We determined if defects in mitochondrial biogenesis and dysregulation of peroxisome proliferator-activated receptor gamma (PPARγ) coactivator-1 (PGC-1)–regulated transcriptional pathways constitute a mechanism or mechanisms underlying mitochondrial oxidative-phosphorylation (OXPHOS) deficiency in human Chagas disease.Methods and ResultsWe utilized human cardiomyocytes and left-ventricular tissue from chagasic and other cardiomyopathy patients and healthy donors (n>6/group). We noted no change in citrate synthase activity, yet mRNA and/or protein levels of subunits of the respiratory complexes were significantly decreased in Trypanosoma cruzi–infected cardiomyocytes (0 to 24 hours) and chagasic hearts. We observed increased mRNA and decreased nuclear localization of PGC-1-coactivated transcription factors, yet the expression of genes for PPARγ-regulated fatty acid oxidation and nuclear respiratory factor (NRF1/2)–regulated mtDNA replication and transcription machinery was enhanced in infected cardiomyocytes and chagasic hearts. The D-loop formation was normal or higher, but mtDNA replication and mtDNA content were decreased by 83% and 40% to 65%, respectively. Subsequently, we noted that reactive oxygen species (ROS), oxidative stress, and mtDNA oxidation were significantly increased, yet NRF1/2-regulated antioxidant gene expression remained compromised in infected cardiomyocytes and chagasic hearts.ConclusionsThe replication of mtDNA was severely compromised, resulting in a significant loss of mtDNA and expression of OXPHOS genes in T cruzi–infected cardiomyocytes and chagasic hearts. Our data suggest increased ROS generation and selective functional incapacity of NRF2-mediated antioxidant gene expression played a role in the defects in mtDNA replication and unfitness of mtDNA for replication and gene expression in Chagas disease.