Significant Decrease In Gene Expression Research Articles

Selective Blockade of Periostin Exon 17 Preserves Cardiac Performance in Acute Myocardial Infarction.

We previously reported that overexpression of full-length periostin, Pn-1, resulted in ventricular dilation with enhanced interstitial collagen deposition in a rat model. However, other reports have documented that the short-form splice variants Pn-2 (lacking exon 17) and Pn-4 (lacking exons 17 and 21) promoted cardiac repair by angiogenesis and prevented cardiac rupture after acute myocardial infarction. The apparently differing findings from those reports prompted us to use a neutralizing antibody to selectively inhibit Pn-1 by blockade of exon 17 in a rat acute myocardial infarction model. Administration of Pn neutralizing antibody resulted in a significant decrease in the infarcted and fibrotic areas of the myocardium, which prevented ventricular wall thinning and dilatation. The inhibition of fibrosis by Pn neutralizing antibody was associated with a significant decrease in gene expression of fibrotic markers, including collagen I, collagen III, and transforming growth factor-β1. Importantly, the number of α-smooth muscle actin-positive myofibroblasts was significantly reduced in the hearts of animals treated with Pn neutralizing antibody, whereas cardiomyocyte proliferation and angiogenesis were comparable in the IgG and neutralizing antibody groups. Moreover, the level of Pn-1 expression was significantly correlated with the severity of myocardial infarction. In addition, Pn-1, but not Pn-2 or Pn-4, inhibited fibroblast and myocyte attachment, which might account for the cell slippage observed during cardiac remodeling. Collectively, these results indicate that therapeutics that specifically inhibit Pn exon-17, via a neutralizing antibody or drug, without suppressing other periostin variants might offer a new class of medication for the treatment of acute myocardial infarction patients.

583. Nonionic Block Copolymers Guide Macrophages To Transfect Skeletal Muscle Cells Upon Direct i. m. Injections

Purpose: Skeletal muscle offers a clinically feasible site for i. m. delivery of genes for therapeutic proteins expression and prophylactic DNA vaccine purposes. Though much research has been focused on development of cationic lipids and polymers for encapsulating plasmid DNA (pDNA) for in vitro transfections, direct i. m. injection of pDNA still results in higher muscle transfection levels. In comparison to existing technologies such as viral vectors there still is a need to further improve muscle transfection levels with pDNA injections. Non-ionic block copolymers, Pluronic P85 and SP1017 (mixture of L61 and F127), when mixed with pDNA, enhance skeletal muscle transfection in euthymic (BALB/c and C57BL/6) mouse and not athymic (BALB/c nu/nu) mouse. Therefore, we studied role of immune cells in muscle transfection.Methods and Results: pDNA alone or an admix of pDNA+Pluronic were injected into tibialis anterior (TA) muscle in presence or absence of local inflammation to demonstrate the effect of immune cells in muscle transfection. Local-inflammation drastically enhanced DNA, RNA and gene expression levels when pDNA was co-delivered with P85 or SP1017 and not pDNA alone. Pluronics enhanced in vitro gene expression in MPs to upto 4 orders in magnitude and not muscle cells. Moreover, adoptive transfer of ex vivo transfected MPs with GFP DNA to ischemic mice resulted in GFP expression in muscle fibers along with MPs. Therefore, in vitro muscle inflammation model was developed by co-culturing DNA transfected macrophages (MPs) with myotubes (MTs) to see the effect of P85 on gene transfer to MTs. Treatment of co-culture (pDNA transfected MPs with MTs) with P85 resulted in increase in total and muscle specific gene expression ca. 30 and 120 times respectively, confirming pDNA transfer from MPs to MTs. Moreover, in vivo depletion of MPs resulted in significant decrease in gene expression confirming the role of MPs in Pluronic based muscle transfection. Finally, pre-injection with P85 on day 1.5 and not day 5 at subsequent sites of DNA injections, recruited MPs to the site of injections and further enhanced the gene expression levels of muscles injected with pDNA+P85 and not pDNA alone. View Large Image | Download PowerPoint SlideConclusions: Pluronic block copolymers utilize immune cells, specifically MPs, to deliver genes to otherwise very hard to transfect skeletal muscle fibers and hence constitute a novel class of biopharmaceuticals or a platform technology for in vivo gene delivery.

Maneb disturbs expression of superoxide dismutase and glutathione peroxidase, increases reactive oxygen species production, and induces genotoxicity in liver of adult mice.

Maneb (MB), a fungicide largely used in agriculture throughout the world including Tunisia, protects many vegetables, fruits and field crops against a wide spectrum of fungal diseases. However there is a lack of informations regarding the risks arising from MB exposure on non target organisms, especially mammals. The aim of this study was to investigate the morphological, biochemical and molecular aspects of liver injury after exposure of mice to MB. Four doses of MB corresponding to 1/8 (group D1), 1/6 (group D2), 1/4 (group D3), and 1/2 (group D4) of lethal dose (DL50 = 1500 mg/kg body weight) were administered to adult mice. Oxidative stress parameters were also objectified by molecular and histological endpoints in the liver. Maneb caused hepatotoxicity as characterized by the significant increase in the levels of malondialdehyde and protein oxidation marker, advanced oxidation protein products (AOPP). The activities of catalase, glutathione peroxidase, superoxide dismutase and the levels of glutathione decreased significantly in all treated mice, while vitamin C levels decreased only in group D4. We also noted a significant decrease in gene expression of superoxide dismutase and glutathione peroxidase enzymes. Maneb caused nucleic acids degradation testifying its genotoxicity. Yet, biochemical markers in plasma showed a decrease in total protein and an increase in aspartate, alanine amino transferases and bilirubin levels in all treatment groups. Moreover, plasma levels of cholesterol, triglycerides and low density lipoprotein-cholesterol significantly increased, while those of high density lipoprotein-cholesterol decreased. These biochemical alterations were correlated with significantly histological changes. Our data showed, for the first time, that intraperitoneal injection of very high non environmentally relevant MB concentrations to adult mice resulted in oxidative stress leading to hepatotoxicity and the impairment of defense systems, confirming the pro-oxidant and genotoxic effects of this fungicide.

Glucose metabolism disorder is a risk factor in ethanol exposure induced malformation in embryonic brain

Prenatal exposure to ethanol has been reported to cause developmental defects in the brain. During brain development, a sufficient energy source is deemed essential and glucose is regarded as the primary energy source for neurons. In this study, the impact of ethanol on embryonic malformation and cerebral glucose metabolism in developing embryo was investigated. Different doses of ethanol (0, 10, 20, 40 mg/egg) were administrated to chicken embryos after 36 h incubation. Embryonic brain weight was found significantly decreased. Moreover, we observed an obvious reduction of neurofilament expression in the central nervous system (CNS) by immunostaining assay. All the above indicated that ethanol exposure caused obvious CNS damages and resulted malformations in the developing brain. Mechanism research showed that cerebral glucose and lactic acid contents, activities of hexokinase, pyruvate kinase and lactic dehydrogenase were decreased dose dependently. Meanwhile, mRNA levels of glucose transporter 1, glucose transporter 3 and insulin-like growth factor I in the brain demonstrated a significant decrease in gene expression after ethanol exposure. These results suggested that glucose metabolism disorder is an important risk factor in ethanol exposure induced malformation in embryonic brain.

Effects of Gravitational Mechanical Unloading in Endothelial Cells: Association between Caveolins, Inflammation and Adhesion Molecules

Mechanical forces including gravity affect endothelial cell (ECs) function, and have been implicated in vascular disease as well as physiologic changes associated with low gravity environments. The goal of this study was to investigate the impact of gravitational mechanical unloading on ECs phenotype as determined by patterns of gene expression. Human umbilical vascular endothelial cells were exposed to 1-gravity environment or mechanical unloading (MU) for 24 hours, with or without periods of mechanical loading (ML). MU led to a significant decrease in gene expression of several adhesion molecules and pro-inflammatory cytokines. On the contrary, eNOS, Caveolin-1 and -2 expression were significantly increased with MU. There was a decrease in the length and width of the cells with MU. Addition of ML during the MU period was sufficient to reverse the changes triggered by MU. Our results suggest that gravitational loading could dramatically affect vascular endothelial cell function.

Mannose Receptor Ligands Regulate the Gene Expression of Toll-like Receptors in Chicken Monocytes

Pattern recognition receptors play an important role in distinguishing foreign organisms and triggering host defense responses. In this study, we present evidence for differential immune responses in chicken monocytes exposed to the mannose receptor (MR) ligands mannan and horseradish peroxidase (HRP). In our study, mannan significantly up-regulated the expression of the MR gene at a high concentration of 200 μg/ml, but HRP did not. In stimulation experiments, neither mannan nor HRP induced nitric oxide (NO) production or stimulated cytokine (IL1β, IL-6, and IL-10) gene expression. A concentration of 200 μg/ml mannan induced a significant increase in Tolllike receptor 2 (TLR2) gene expression. The gene expression of TLR1 and 3 was significantly up-regulated by different HRP concentrations. In contrast to TLR1 and 3, TLR4 exhibited a significant decrease in gene expression in a dose-dependent manner. These results may suggest that MR ligands affect the innate immune response of TLRs in chicken monocytes.

Abstract 9035: Epigenetic Modifications are Associated with Ascending Thoracic Aneurysm Formation in Patients with Bicuspid Aortic Valve

Objective: Bicuspid aortic valve (BAV) disease predisposes to early and more frequent ascending thoracic aortic aneurysm formation, but the mechanisms underlying this aortopathy remain incompletely characterized. The objective of this study was to identify genes predisposing to ascending aneurysm formation in patients with BAV aortopathy using a multipronged molecular approach. Methods: Ascending aortic aneurysm tissue samples were collected at the time of ascending aortic replacement in subjects with bicuspid (N=16) and trileaflet (N=16) aortic valves. All patients in the trileaflet valve group had degenerative aneurysms and those with connective tissue disorders were excluded. Using the aortic tissue samples, genome-wide DNA methylation status was determined using Illumina 450K methylation chips. Gene expression was determined using Illumina Whole-Genome DASL arrays. Gene methylation and expression were compared between bicuspid and trileaflet groups using Wilcoxon Rank Sum tests. Results: Twenty-six genes were differentially methylated between the bicuspid and trileaflet groups, and six of these genes also had significantly different gene expression between groups. Protein tyrosine phosphatase, non-receptor type 22 ( PTPN22 ) was strongly hypermethylated (beta: bicuspid 0.68 vs. trileaflet 0.55, P=4x10 -5 ) and had a resultant highly significant decrease in gene expression (log 2 gene expression intensity: bicuspid 5.1 vs. trileaflet 7.9, P=2x10 -5 ). Conclusions: In patients with ascending thoracic aortic aneurysms, there exist differential DNA methylation and gene expression signatures between those with BAV aortopathy versus acquired degenerative aneurysms and trileaflet aortic valves. The strongest finding was for PTPN22 , a gene involved in T-cell receptor signaling and associated with various autoimmune disorders. These differences may highlight novel mechanisms of aneurysm development in the BAV population, potentially serving as biomarkers and/or pharmacologic targets.

Chromatin H3K27me3/H3K4me3 histone marks define gene sets in high-grade serous ovarian cancer that distinguish malignant, tumour-sustaining and chemo-resistant ovarian tumour cells

In embryonic stem (ES) cells, bivalent chromatin domains containing H3K4me3 and H3K27me3 marks silence developmental genes, while keeping them poised for activation following differentiation. We have identified gene sets associated with H3K27me3 and H3K4me3 marks at transcription start sites in a high-grade ovarian serous tumour and examined their association with epigenetic silencing and malignant progression. This revealed novel silenced bivalent marked genes, not described previously for ES cells, which are significantly enriched for the PI3K (P<10(-7)) and TGF-β signalling pathways (P<10(-5)). We matched histone marked gene sets to gene expression sets of eight normal fallopian tubes and 499 high-grade serous malignant ovarian samples. This revealed a significant decrease in gene expression for the H3K27me3 and bivalent gene sets in malignant tissue. We then correlated H3K27me3 and bivalent gene sets to gene expression data of ovarian tumour 'stem cell-like' sustaining cells versus non-sustaining cells. This showed a significantly lower expression for the H3K27me3 and bivalent gene sets in the tumour-sustaining cells. Similarly, comparison of matched chemo-sensitive and chemo-resistant ovarian cell lines showed a significantly lower expression of H3K27me3/bivalent marked genes in the chemo-resistant compared with the chemo-sensitive cell line. Our analysis supports the hypothesis that bivalent marks are associated with epigenetic silencing in ovarian cancer. However it also suggests that additional tumour specific bivalent marks, to those known in ES cells, are present in tumours and may potentially influence the subsequent development of drug resistance and tumour progression.

PS204. Mechanical (or “Gravitational”) Unloading Reduces Inflammatory and Cell Adhesion Molecule Gene Expression in Human Endothelial Cells

Mechanical forces including gravity affect mechanotransduction and cell function. The goal of this study was to investigate the impact of mechanical unloading (MU) and loading (ML) of endothelial cells (ECs) with microgravity and hypergravity respectively, with the hypothesis that MU alters expression of inflammatory and adhesion molecule gene expression and these changes are reversed by ML. Human umbilical vascular ECs grown to confluency were studied. A desktop random positioning machine and a gravitational cell-loading apparatus provided MU and ML. The experimental conditions included: 1) controls exposed to 1-gravity environment for 24 h (CL), 2) MU for 24 hours, 3) MU for 24 hours with three 30-minutes periods of ML of 12-gravity (MU/ML). Gene expression was studied with reverse transcription followed by real-time quantitative polymerase chain reaction (qRTPCR). MU led to a significant decrease in gene expression of the adhesion molecules ICAM-1, VCAM-1, E-Selectin, as well as TNF-α, IL-6 and VEGF. In contrast, NOS-3, Caveolin-1 and −2 were significantly increased with MU. The changes observed in gene expression with MU were reversed by gravitational mechanical loading (MU/ML). Gravitational MU decreases inflammatory and adhesion molecule gene expression and these changes are reversed by short periods of ML. This points towards the importance of gravitational loading in ECs function and cellular interactions.

Abstract 353: Mechanical (or “Gravitational”) Unloading Reduces Inflammatory and Cell Adhesion Molecule Gene Expression in Human Endothelial Cells

Objectives: Mechanical forces including gravity affect mechanotransduction and subsequent cell function. The goal of this study was to investigate the impact of mechanical unloading (MU) and loading (ML) of endothelial cells (ECs) with microgravity and hypergravity respectively, with the hypothesis that MU alters expression of inflammatory and adhesion molecule gene expression and these changes are reversed by ML. Methods: Human umbilical vascular endothelial cells (HUVECs) grown to confluency were studied. A desktop random positioning machine and a gravitational cell-loading apparatus provided MU and ML conditions, respectively. The experimental conditions included: 1) controls exposed to 1-gravity environment for 24 h (CL), 2) MU for 24 hours, 3) MU for 24 hours with three 30-minutes periods of ML of 12-gravity (MU/ML). Gene expression was studied with reverse transcription followed by real-time quantitative polymerase chain reaction (qRTPCR). Results: MU led to a significant decrease in gene expression of the adhesion molecules ICAM-1, VCAM-1, E-Selectin, as well as TNF-α, IL-6 and VEGF. In contrast, NOS-3, Caveolin-1 and -2 were significantly increased with MU. The changes observed in gene expression with MU were reversed by gravitational mechanical loading (MU/ML). Conclusions: Gravitational MU decreases inflammatory and adhesion molecule gene expression and these changes are reversed by short periods of ML. This points towards the importance of gravitational loading in ECs function and cellular interactions.

Effect of semax and its C-terminal peptide PGP on expression of neurotrophins and their receptors in rat brain during incomplete global ischemia

Neurotrophins regulate key functions of nervous tissue cells. Analysis of neurotrophin mRNA expression is an appropriate tool to assess therapeutic efficiency of antistroke drugs. We have analyzed the effect of synthetic peptide semax and its C-terminal Pro-Gly-Pro tripeptide on mRNA expression of neurotrophins Ngf, Bdnf, and Nt-3 and their receptors TrkA, TrkB, TrkC, and p75 in rat frontal cortex, hippocampus, and cerebellum after bilateral common carotid artery occlusion. The animals were decapitated at 30 min and 1, 2, 4, 8, 12, and 24 h after the operation. The mRNA expression of neurotrophins and their receptors was assessed by relative quantification using real-time RT-PCR. Our results demonstrated that ischemia caused a significant decrease in gene expression in the hippocampus. Semax and PGP treatment affected the expression of neurotrophins and their receptors predominantly in the frontal cortex and hippocampus of the ischemized animals. In the frontal cortex, Semax treatment resulted in a decrease of mRNA level of neurotrophin receptors, while PGP treatment increased the level of these mRNA. Maximal neuroprotective effect of both peptides was observed in the hippocampus 12 h after occlusion. A decrease of gene expression of neurotrophins and their receptors caused by the occlusion was overcome by Semax and PGP. These results clarify the mechanism of Semax action and reveal certain features of mRNA expression of neurotrophins and their receptors under experimental conditions.

Abstract PL05-04: Epigenetic marks as biomarkers and their mechanisms of maintenance as targets in ovarian cancer.

Abstract PL05-04: Epigenetic marks as biomarkers and their mechanisms of maintenance as targets in ovarian cancer.

Mouse CSB protein is important for gene expression in the presence of a single-strand break in the non-transcribed DNA strand

CSB protein is required for strand-specific repair of bulky DNA lesions in transcribed genes and mediates transcription recovery after exposure to DNA-damaging agents. We enzymatically generated DNA single-strand breaks (SSBs) with 3′-OH and 5′-phosphate termini in defined positions of a plasmid-borne gene and measured their effect on transcription in cell lines with different statuses of the Csb gene. A single SSB in the transcribed region of the gene caused significant decrease of gene expression. In all tested cell lines of mouse and human origin, a SSB in the transcribed DNA strand was less harmful for gene expression than a SSB situated in the opposing DNA strand. CSB deficiency exhibited no effect on the expression of the nicked DNA in human fibroblasts immortalised by SV40 large T-antigen but caused a very strong decrease of gene expression in spontaneously transformed mouse embryonic fibroblasts (MEFs). Compared to the corresponding CSB-proficient MEFs, the effect was on average 6.7-fold stronger for a defined SSB located in the non-transcribed DNA strand, but only 2.4-fold for a SSB in the transcribed strand and 1.7-fold for a SSB located in the non-genic region. At the same time, CSB deficiency did not compromise the overall efficiency of repair of SSBs generated by treatment of the cells with hydrogen peroxide. The gene expression data thus indicate that CSB prevents irreversible transcription failures at the sites of DNA damage, acting preferentially at SSBs located in the non-transcribed DNA strand of the transcribed genes. We further conclude that SSBs in the non-transcribed DNA strand are commonly more harmful for transcription than those situated in the transcribed strand.

Insulin-like Growth Factor Binding Protein-3 Induces G1 Cell Cycle Arrest with Inhibition of Cyclin-dependent Kinase 2 and 4 in MCF-7 Human Breast Cancer Cells

Insulin-like growth factor binding protein (IGFBP)-3 has been shown to potently inhibit proliferation of various cell types in an insulin-like growth factor (IGF)-independent manner. We have previously shown that IGFBP-3 induces apoptosis in an IGF-independent manner through the activation of caspases involved in a death receptor-mediated pathway in MCF-7 human breast cancer cells. In the present study, we present further evidence that IGFBP-3 inhibits cell proliferation through the induction of cell cycle arrest in the same cell line. Induction of IGFBP-3 in MCF-7 cells inhibited cell proliferation whereas presence of small interfering RNA against IGFBP-3 abolished cell inhibitory effect of IGFBP-3, suggesting that the observed growth inhibition is specific. Flow cytometry analysis showed that induced expression of IGFBP-3 led to an arrest of the cell cycle in G1-S phase. Western immunoblot analysis showed a significant decrease in the levels of the cell cycle-regulated proteins such as cyclin D1, cyclin D3, cyclin E, cyclin A, cyclin-dependent kinase (CDK) 2, CDK4, retinoblastoma protein (pRB), and phosph-pRB, suggesting a possible mechanism for cell cycle arrest by IGFBP-3. Northern blot analysis and real-time quantitative PCR demonstrated a significant decrease in gene expression of cyclin D1. Additional phosphorylation assay showed that IGFBP-3 decreased the phosphorylation activity of CDK2 and CDK4. These results show that cellular production of IGFBP-3 leads to G1 cell cycle arrest with inhibition of CDK2 and CDK4. Taken together, IGFBP-3 exerts its growth inhibitory action through not only induction of apoptosis but also the G1 cell cycle arrest in human breast cancer cells.

Di-(2 ethylhexyl) phthalate and flutamide alter gene expression in the testis of immature male rats

We previously demonstrated that the androgenic and anti-androgenic effects of endocrine disruptors (EDs) alter reproductive function and exert distinct effects on developing male reproductive organs. To further investigate these effects, we used an immature rat model to examine the effects of di-(2 ethylhexyl) phthalate (DEHP) and flutamide (Flu) on the male reproductive system. Immature male SD rats were treated daily with DEHP and Flu on postnatal days (PNDs) 21 to 35, in a dose-dependent manner. As results, the weights of the testes, prostate, and seminal vesicle and anogenital distances (AGD) decreased significantly in response to high doses of DEHP or Flu. Testosterone (T) levels significantly decreased in all DEHP- treated groups, whereas luteinizing hormone (LH) plasma levels were not altered by any of the two treatments at PND 36. However, treatment with DEHP or Flu induced histopathological changes in the testes, wherein degeneration and disorders of Leydig cells, germ cells and dilatation of tubular lumen were observed in a dose-dependent manner. Conversely, hyperplasia and denseness of Leydig, Sertoli and germ cells were observed in rats given with high doses of Flu. The results by cDNA microarray analysis indicated that 1,272 genes were up-regulated by more than two-fold, and 1,969 genes were down-regulated in response to DEHP, Flu or both EDs. These genes were selected based on their markedly increased or decreased expression levels. These genes have been also classified on the basis of gene ontology (e.g., steroid hormone biosynthetic process, regulation of transcription, signal transduction, metabolic process, biosynthetic process...). Significant decreases in gene expression were observed in steroidogenic genes (i.e., Star, Cyp11a1 and Hsd3b). In addition, the expression of a common set of target genes, including CaBP1, Vav2, Plcd1, Lhx1 and Isoc1, was altered following exposure to EDs, suggesting that they may be marker genes to screen for the anti-androgenic or androgenic effects of EDs. Overall, our results demonstrated that exposure to DEHP, Flu or both EDs resulted in a alteration of gene expression in the testes of immature male rats. Furthermore, the toxicological effects of these EDs on the male reproductive system resulted from their anti-androgenic effects. Taken together, these results provide a new insight into the molecular mechanisms underlying the detrimental impacts of EDs, in regards to anti-androgenic effects in humans and wildlife.

Mitochondrial development and the influence of its dysfunction during rat adipocyte differentiation

Mitochondrial biogenesis is inherent to adipocyte differentiation. Mitochondrial dysfunction leads to abnormal lipid accumulation or the deterioration of the differentiation process. The aim of this study is to investigate the mitochondrial development during the differentiation of rat primary adipocytes and the effect of mitochondrial dysfunction on this process. We found, for the first time, that the number of mitochondria markedly increased during adipocyte differentiation by transmission electron microscopy. By immunofluorescence staining that the protein content of Cyt c increased in differentiated adipocyte in comparison with preadipocyte. The mRNA expression levels of mitochondrial gene including cytochromes c (Cyt c), malate dehydrogenases (MDH), and peroxisome proliferator activated receptor (PPAR) gamma coactivator-1beta (PGC-1beta) significantly increased along with the proceeding of adipocyte differentiation. The damage to mitochondrial respiratory chain function by rotenone caused significant decrease in gene expressions including mitochondrial MDH and PGC-1beta, and PPARgamma, CAAT/enhancer binding protein alpha (C/EBPalpha) and sterol regulatory element binding protein-1c (SREBP-1c), which are known as transcription factors of differentiation, and differentiation marker gene named fatty acid synthetase. Moreover, an apparent decrease was found in the synthesis of triglyceride and ATP due to the damage to mitochondria by rotenone. Based on the above results, our present study revealed that the density and oxidative capacity of mitochondrial markedly increased during primary adipocyte differentiation, and on the other hand, we suggested that mitochondria dysfunction might inhibit the differentiation process.

The effect of dehydroepiandrosterone on lipogenic gene mRNA expression in cultured primary chicken hepatocytes

AbstractThe effects of dehydroepiandrosterone (DHEA) on lipogenic gene mRNA expression were examined using cultured primary chicken hepatocytes. Cell samples were equilibrated to culture conditions for 24 h and then exposed to DHEA (1, 10 and 100 µM) dissolved in DMSO. The expression of sterol regulatory element‐binding protein‐1 (SREBP‐1) mRNA in cultured primary hepatocytes did not vary with time, except for a significant decrease in gene expression when cells were treated with 10 or 100 µM DHEA for either 1 or 2 h. A similar tendency toward decreased gene expression was evident for acetyl CoA carboxylase. The expression of peroxisome proliferator‐activated receptor α (PPARα) and carnitine palmitoyl transferase I mRNA was dramatically enhanced subsequent to treatment with 10 or 100 µM DHEA for periods of time from 6 to 48 h, while there was a decrease in the expression of PPARα in the presence of 10 µM DHEA after 1 and 2 h of exposure, and in the presence of 100 µM DHEA after 2 h of exposure. Hepatocytes treated with 10 or 100 µM DHEA contained more mitochondria and mitochondria with a higher electron density than did untreated hepatocytes. Furthermore, cell survival was significantly inhibited by treatment with 100 µM DHEA at 24, 48 and 72 h. In contrast, 1 µM DHEA administration significantly increased cell survival after 72 h; however, 10 µM DHEA treatment had no pronounced effect on cell survival. Overall, the results reported here indicate that DHEA accelerates lipid catabolism by direct regulation of hepatic gene expression and by induction of changes in hepatocyte mitochondria.

PREVENTION OF HEMORRHAGIC SHOCK-INDUCED INTESTINAL TISSUE INJURY BY GLUTAMINE VIA HEME OXYGENASE-1 INDUCTION

Hemorrhagic shock (HS) is an oxidative stress that causes intestinal tissue injury. Heme oxygenase 1 (HO-1) is induced by oxidative stress and is thought to play an important role in the protection of tissues from oxidative injury. We previously reported the ileum to be the most susceptible to HS-induced tissue injury site in the intestine because HO-1 induction is the lowest at this site. We also previously demonstrated that glutamine (GLN) significantly induced HO-1 in the lower intestinal tract. In the present study, we investigated whether GLN pretreatment improves HS-induced intestinal tissue injury in the ileum by HO-1 induction. Treatment of rats with GLN (0.75 g/kg, i.v.) markedly induced functional HO-1 protein in mucosal epithelial cells in the ileum. Glutamine treatment before HS (MAP of 30 mmHg for 60 min) significantly ameliorated HS-induced mucosal inflammation and apoptotic cell death in the ileum, as judged by significant decreases in gene expression of TNF-alpha, iNOS, intercellular adhesion molecule 1, and vascular cell adhesion molecule 1, myeloperoxidase activity, the number of infiltrated neutrophils, DNA fragmentation by in situ oligo ligation assay, and activated caspase-3 expression, and by increases in gene expression of IL-10 and Bcl-2. In contrast, treatment with tin mesoporphyrin, a specific inhibitor of HO activity, abolished the beneficial effect of GLN pretreatment. These findings indicate that GLN pretreatment significantly ameliorated tissue injury in the ileum after HS by inducing HO-1. Glutamine treatment may thus protect mucosal cells from HS-induced oxidative damage via the anti-inflammatory and antiapoptotic properties of HO-1.

Monocyte Chemoattractant Protein-1 (CCL-2) Integrates Mechanical and Endocrine Signals That Mediate Term and Preterm Labor

Recent evidence suggests that leukocytes infiltrate uterine tissues at or around the time of parturition, implicating inflammation as a key mechanism of human labor. MCP-1 (also known as C-C chemokine motif ligand 2, CCL-2) is a proinflammatory cytokine that is up-regulated in human myometrium during labor. Myometrium was collected from pregnant rats across gestation and at labor. Total RNA and proteins were subjected to real-time PCR and ELISA, respectively. Ccl-2 gene and protein expression was significantly up-regulated in the gravid rat myometrium before and during labor, which might suggest that it is regulated positively by mechanical stretch of the uterus imposed by the growing fetus and negatively by physiological withdrawal of progesterone (P4). We confirmed in vivo that: 1) administration of P4 receptor antagonist RU486 induced an increase in Ccl-2 mRNA and preterm labor, whereas 2) artificial maintenance of elevated P4 levels at late gestation caused a significant decrease in gene expression and blocked labor; 3) Ccl-2 was elevated specifically in the gravid horn of unilaterally pregnant rats suggesting that mechanical strain imposed by the growing fetus controls its expression in the myometrium; 4) in vitro static mechanical stretch of primary rat myometrial smooth muscle cells (25% elongation) induced a release of Ccl-2 protein, which was repressed by pretreatment with P4 (1 microM); and 5) stretch enhanced their monocyte chemoattractant activity. These data indicate that Ccl-2 protein serves to integrate mechanical and endocrine signals contributing to uterine inflammation and the induction of labor and thus may represent a novel target for therapeutic prevention of preterm labor in humans.

A High Resolution Epigenomic Map of Myelofibrosis Reveals Multiple Chromosomal Deletions and Amplifications Accompanied by a High Level of Functionally Important Methylation.

Chronic idiopathic myelofibrosis (MF) is a clonal hematopoietic disorder that leads to progressive marrow fibrosis and peripheral cytopenias. Very little is known about the role of chromosomal alterations and DNA methylation in the pathobiology of this disease. We used a combination of gene expression analysis, high density array based comparative genomic hybridization (aCGH) and genome wide methylation analysis to perform an integrated genomic analysis of MF. Gene expression analysis was performed using 37K oligo maskless arrays and high density aCGH was performed at 6Kb resolution using Nimblegen platform. Whole genome methylation was analyzed by a recently described novel method ( Khulan et al, Genome Res. 2006 Aug;16(8)) that uses differential methylation specific digestion by HpaII and MspI followed by pcr amplification, two color labeling and hybridization to quantitatively determine individual promoter CpG island methylation. aCGH revealed a very high number of microdeletions (range 622–1148, mean ± SD 555±144 ) and amplifications (range 463–770, mean ± SD 781±246) in a pilot study conducted in 4 patients with MF. Twenty three common regions were found to amplified in all patients which included regions of chr3p25, chr8p21, chr12q24, chr14q32, chr17p13 and chr17q12, which code for a novel set of genes including B-cell CLL/lymphoma 7A, GTPase activating Rap, BRF1 and others. Five regions were found to be commonly deleted in all samples (chr1q31, chr5q12, chr20p13, chrXq21, chrXq28). Thirty eight DNA segments were found to be deleted and 142 amplified in 75% of the samples. Several potential pathogenic genes encoding for transcription factors, cytokines and cytokine receptors were found to be coded by these segments. A custom human oligo array was used to determine methylation by calculating HpaII/MspI cut fragment intensity ratio. All patient samples had a very high level of methylation (range 64–82%, mean 72% ± 8.6%). Expression was found to be significantly decreased for the genes that were methylated (p<.0001, T test) demonstrating the functional relevance of this assay. Analysis of common differentially methylated genes (when compared to normal samples) and their validation are ongoing. Microdeletions and amplifications seen on aCGH did not correlate with changes in global expression of the involved genes. Interestingly, when data from all platforms were combined, methylation of the genes with altered copy number led to significant decreases in gene expression (p=.03, T test). This result suggests pathogenic changes in gene expression in MF result from an interplay between DNA copy number alterations and methylation of the remaining alleles. The high rate of methylation demonstrated in MF suggests that epigenetic silencing of genes may play an important role in pathogenesis and points to the potential utility of hypomethylating agents in this disease.