Pro-inflammatory gene expression profile in obese adults with high plasma GIP levels.

BACKGROUND: Glucose-dependent insulinotropic peptide (GIP) provides a novel link between the immune system and the gut, although results from different experimental and observational studies are contradictory, ranging from anti-inflammatory, through neutral to pro-inflammatory action of GIP. Thus, the aim of this study was to analyze inflammatory pathways on the level of gene expression and circulating inflammatory markers in relation to plasma GIP level. SUBJECTS/METHODS: The study included 128 obese adults. Two groups of obese subjects were created according to fasting GIP levels, with cutoff point at the 66th percentile and compared in respect with molecular and circulating markers of inflammation. GIP, interleukin (IL)-6 and adipokines: leptin, adiponectin, visfatin were measured by enzyme-linked immunosorbent assay. Inflammatory markers: monocyte chemoattractant protein-1 (MCP-1), sE-Selectin, sVCAM-1, sPECAM-1 were studied at fasting and after nutrient challenges. Gene expression in blood cells was determined by human gene microarray. RESULTS: Obese patients with high GIP levels had elevated fasting glucose (Q2 (Q1-Q3): 5.6 (5.0-6.0) vs 5.0 (4.8-5.4), P<0.001), homeostasis model assessment of insulin resistance (Q2 (Q1-Q3): 3.68 (2.72-5.42) vs 2.70 (2.13-4.33), P=0.021), thus increased markers of insulin resistance as well as elevated inflammatory markers Il-6 (Q2 (Q1-Q3): 1.34 (1.0-2.04) vs 1.12 (0.76-1.64), P=0.045), MCP-1 (Q2 (Q1-Q3): 363 (287-447) vs 323 (263-389), P=0.026). Leptin to adiponectin ratio was significantly associated with fasting plasma GIP levels (ß (95% CI): 0.84 (0.10-1.59)) independently of glucose levels. sE-Selectin was found to be a factor influencing GIP response to oral glucose intake (ß (95% CI): 0.47 (0.14-0.81)) and sVCAM was found to be a factor influencing GIP response to high-fat meal intake (ß (95% CI): 0.19 (0.01-0.37)). We identified 32 genes of inflammatory pathways differentially expressed in subjects with a high plasma GIP level compared to low GIP. Most upregulated genes play a role in leukocyte chemotaxis and tissue infiltration. CONCLUSIONS: These findings support the hypothesis that increased GIP signaling has a role in chronic low-grade inflammation.

The human immunodeficiency virus (HIV1) protease inhibitor sanquinavir activates autophagy and removes lipids deposited in lipid droplets.

Reduction in mortality and increased average life span of the human immunodeficiency virus (HIV)-infected patients treated with antiretroviral therapy (ART) are associated with the risk of unwanted effects, such as insulin resistance and dyslipidemia with cardiovascular complications. Antiretroviral therapy may also be associated with lipodystrophy characterized as peripheral lipoatrophy with central fat accumulation. Understanding the molecular mechanisms of lipodystrophy caused by ART is important for therapeutic strategy and the prediction of side-effects. Influence of protease inhibitor saquinavir (SQV) on preadipocyte differentiation was analyzed in in vitro human Chub-S7 cell line model. For measurement of the effects of SQV the drug was added to differentiated or non-differentiated cells. The influence of SQV on changes in the profile of gene expression was verified by microarray and changes in lipid species content were analyzed using GC-MS/MS. Results were confirmed by real-time PCR and analysis of autophagy. Addition of SQV to differentiated Chub-S7 cells lead to removal of lipids deposited in lipid droplets, down-regulation of expression of transcription factors and markers of adipocyte differentiation. Antiviral activity of SQV based on its non-selective inhibition of proteases resulted in proteasome inhibition, induction of endoplasmic reticulum stress and induction of macroautophagy. This activity was accompanied by an increase in PI, PEPL, PC lipid species especially with MUFA and PUFA. Additionally up-regulation of miR-100-3p, miR-222-5p, miR-483-5p were found, which correlated with obesity, insulin resistance, increasing insulin secretion and activation of lipolysis. Our results indicated that SQV, by inhibition of proteasome protein degradation, activated the unfolded protein response resulting in autophagic breakdown of lipids deposited in adipose tissue causing lipodystrophy.

Modulation of endothelial cell proliferation and capillary network formation by the ox-LDL component: 1-palmitoyl-2-archidonoyl-sn-glycero-3-phosphocholine (ox-PAPC).

Atherosclerotic plaque formation is often associated with pathological angiogenesis. Modified phospholipids, including oxidized lipoproteins such as LDL, are found to induce adhesion of the monocytes to the endothelial cells and to stimulate their chemotaxis. Effects of oxidized 1-palmitoyl-2-archidonoyl-sn-glycero-3-phosphocholine (ox-PAPC) mimic actions of minimally modified LDL in vivo. Interleukin-8 (IL-8) and interleukin-15 (IL-15) are known to induce both inflammation and angiogenesis. The goal of our study was to analyze a potential synergism between ox-PAPC and IL-15 in the in vitro model of angiogenesis carried out in the human endothelial cells (HUVECs). Increasing IL-15 concentrations led to formation of the tube-like structures in the matrigel 3D-model of angiogenesis (P < 0.05), in contrast to ox-PAPC that inhibited this process. HUVECs incubation with ox-PAPC led to reduced IL-15 gene basal expression (P = 0.033) along with parallel increase, however statistically insignificant, of basal gene expression of IL-8 (P = 0.086). Our findings point to the ox-PAPC opposite effects on the IL-8- and IL-15-mediated angiogenic responses that contribute to pathological angiogenesis induced by ox-LDL.

The adipose tissue gene expression in mice with different nitric oxide availability.

Mice with the knockout of endothelial nitric oxide synthase (eNOS ko) demonstrate symptoms resembling the human metabolic syndrome. NO has been recently demonstrated to be deeply involved in regulation of not only blood flow and angiogenesis, but also in modulation of mammalian basal energy substrate metabolism. Asymmetric dimethylarginine (ADMA) is an endogenous competitive inhibitor of NOS. The enzyme dimethylarginine dimethylaminohydrolase (DDAH) catabolizes ADMA, what leads to increase of endogenous NO bioavailability. This study was aimed to compare the brown (BAT) and white (WAT) adipose tissue gene expression of age matched mice with decreased (eNOS ko) and increased (overexpressing DDAH) endogenous NO generation. The 19 week old eNOS ko mice demonstrated significantly lower weight, higher circulating glucose, insulin, leptin and cholesterol concentrations. The adiponectin as well as fasting triglyceride concentrations were not significantly altered. Animals with DDAH knock in, presented significantly increased angiogenic activity than eNOS ko mice. The microarray analysis pointed to activation of adipogenesis-related genes in eNOS ko mice in WAT, what was in contrast with the inhibition observed in the DDAH overexpressing mice. The angiogenesis related gene expression was down-regulated in both models in comparison to WT animals. This study support the multipotential role of endogenous NO in maintaining homeostasis of energy substrate catabolism.

The MAPK-dependent regulation of the Jagged/Notch gene expression by VEGF, bFGF or PPAR gamma mediated angiogenesis in HUVEC.

The Jagged-Notch signalling, plays a crucial role in cell differentiation. Angiogenesis, is regulated by VEGF, bFGF as well as by the free fatty acid metabolites , which are regulators of transcription factors such as peroxisome proliferation activating receptors (PPARs). The study analyzed the signalling pathways involved in the regulation of Jagged-1/Notch-4 expression in endothelial cells (HUVECs) in response to VEGF, bFGF and PPAR-gamma exogenous activator - ciglitazone. HUVECs were incubated with investigated substances for 24 hours, with or without the presence of the MAP-kinases inhibitors were used. Jagged-1 and Notch-4 gene expression was determined using quantitative Real-Time PCR. The Jagged-1/Notch-4 protein expression was compared by flow cytometry, when the phosphorylation-dependent activation of kinases was estimated by Western-blot method. The opposite effect of VEGF, bFGF, or ciglitazone on the Jagged-1/Notch-4 expression on HUVEC was connected with the different activation of MAPKs. Ciglitazone, activated p38 MAPK pathway and simultaneously inhibited phosphorylation of p42/44 MAPK. The pro-angiogenic: bFGF and VEGF, also activated the p38 MAPK, but they did not attenuate the p42/44 MAPK phosphorylation. Maintaining of the Jagged/Notch interactions by VEGF, when down-regulation by bFGF and ciglitazone, seems to be dependent on the different effect on p38 MAPK and p42/44 MAPK pathway regulation.

Differentiation of human adipose tissue SVF cells into cardiomyocytes.

Progenitor cells have been extensively studied and therapeutically applied in tissue reconstructive therapy. Stromal vascular fraction (SVF) cells, which are derived from adipose tissue, may represent a potential source of the cells which undergo phenotypical differentiation into many lineages both in vitro as well as in vivo. The goal of this study was to check whether human SVF cells may differentiate into cardiomyocyte-like entities. Human SVF cells were induced to differentiate by their incubation in Methocult medium in the presence of SCF, IL-3 and IL-6. Morphological transformation of the cells was monitored using optical light microscope, whereas changes in expression of the genes typical for cardiac phenotype were measured by qRT-PCR. Incubation of the human SVF cells in the medium that promotes cardiomyocyte differentiation in vitro resulted in formation of myotubule-like structures accompanied by up-regulation of the myocardium-characteristic genes, such as GATA, MEF2C, MYOD1, but not ANP. Human SVF cells differentiate into cardiomyocyte-like cells in the presence of the certain set of myogenesis promoting cytokines.

Human adipose tissue stromal vascular fraction cells differentiate depending on distinct types of media.

OBJECTIVES: Angiogenesis, the process of formation of blood vessels, is essential for many physiological as well as pathological processes. It has been shown that human adipose tissue contains a population of non-characterized cells, called stromal-vascular fraction (SVF) cells, which are able to differentiate into several lineages. The aim of this study was to determine conditions for promoting differentiation of human adipose tissue progenitors towards endothelial cells, as well as to show that SVF cells cooperate with differentiated endothelium in capillary network formation. MATERIALS AND METHODS: Stromal vascular fraction cells were isolated according to modified Hauner's method and after adaptation they were cultured in pro-angiogenic or pro-adipogenic medium. Cells were characterized by presence of surface antigens by flow cytometry, and by expression of genes characteristic for endothelial cells or for adipocytes, quantitative real-time polymerase chain reaction. A number of tests were performed to verify their differentiation. RESULTS: Differentiation of human SVF cells towards endothelium was stimulated by the presence of serum and absence of adipogenic factors, documented by the pattern of gene expression as well as different functional in vitro assays. SVF cells were found to work together with human umbilical vein endothelial cells to form capillary networks. CONCLUSIONS: Here, we show that differentiation of SVF cells to endothelial cells or adipocyte-like cells depended on the medium used. Our work provides a clear model for analysing the differentiation capacity of SVF cells.

Beta-carotene and arachidonic acid induced DNA methylation and the regulation of pro-chemotactic activity of endothelial cells and its progenitors.

DNA methylation is one of the important mechanisms regulating gene expression. Since beta-carotene (BC) was shown to have pro-chemotactic activity and stimulates expression of pro-angiogenic genes, this study was undertaken to define the possible changes in DNA methylation in endothelial cell and its progenitors in the presence of BC. The culture medium for human umbilical vein endothelial cells (HUVEC) and endothelial progenitor cells (EPC) was supplemented with BC (1 - 10 microM) with the presence of arachidonic acid (AA) (3 microM). Global DNA methylation tended to be lower in both endothelial cell lines, after incubation with BC and AA. HUVEC incubated with AA demonstrated the lowest DNA methylation. The decrease of DNA methylation in EPC, induced by BC, was concentration-dependent. The microarray study revealed, that the angiogenesis and homing-related genes were mostly influenced by BC and AA in investigated cells. Our results indicate that BC and AA-induced DNA hypomethylation in EPC and HUVEC, might be a mechanism which may alter gene expression in endothelial cells what in certain conditions may be connected with the suggested pro-malignant effect of this compounds.

Influence of ghrelin on gastric and duodenal growth and expression of digestive enzymes in young mature rats.

UNLABELLED: Ghrelin, a nature ligand for the growth hormone secretagogue receptor (GHS-R), stimulates a release of growth hormone, prolactin and adrenocorticotropic hormone. Also, ghrelin increases food intake in adult rats and humans and exhibits gastroprotective effect against experimental ulcers induced by ethanol or stress. The aim of present study was to examine the influence of ghrelin administration on gastric and duodenal growth and expression of pepsin and enterokinase in young mature rats with intact or removed pituitary. METHODS: Two week after sham operation or hypophysectomy, eight week old Wistar male rats were treated with saline (control) or ghrelin (4, 8 or 16 nmol/kg/dose) i.p. twice a day for 4 days. Expression of pepsin in the stomach and enterokinase in the duodenum was evaluated by real-time PCR. RESULTS: In animals with intact pituitary, treatment with ghrelin increased food intake, body weight gain and serum level of growth hormone and insulin-like growth factor-1 (IGF-1). These effects were accompanied with stimulation of gastric and duodenal growth. It was recognized as the significant increase in gastric and duodenal weight and mucosal DNA synthesis. In both organs, ghrelin administered at the dose of 8 nmol/kg caused maximal growth-promoting effect. In contrast to these growth-promoting effects, administration of ghrelin reduced expression of mRNA for pepsin in the stomach and was without effect on expression of mRNA for enterokinase in the duodenum. Hypophysectomy alone lowered serum concentration of growth hormone under the detection limit and reduced serum level of IGF-1 by 90%. These effects were associated with reduction in daily food intake, body weight gain and gastroduodenal growth. In hypophysectomized rats, administration of ghrelin was without significant effect on food intake, body weight gain or growth of gastroduodenal mucosa. Also, serum concentration of growth hormone or IGF-1 was not affected by ghrelin administration in rats with removed pituitary. CONCLUSION: Administration of ghrelin stimulates gastric and duodenal growth in young mature rats with intact pituitary, but inhibits expression of mRNA for pepsin in the stomach. Growth hormone and insulin-like growth factor-1 play an essential role in growth-promoting effects of ghrelin in the stomach and duodenum.

Long-term individual housing in C57BL/6J and DBA/2 mice: assessment of behavioral consequences.

The aim of the present study was to investigate the effects of individual housing on mouse behavior. The male mice of the C57BL/6J and DBA/2 strains were separated at the age of 4 weeks and kept in individual housing for 7 weeks until behavioral testing began. Their behavior was compared to the group-housed mice in a battery of tests during the following 7 weeks. The single-housed mice were hyperactive and displayed reduced habituation in the tests assessing activity and exploration. Reduced anxiety was established in the elevated plus-maze, but an opposite effect was observed in the dark-light (DL) and hyponeophagia tests. Immobility in the forced swimming test was reduced by social isolation. The DBA mice displayed higher anxiety-like behavior than the B6 mice in the plus-maze and DL exploration test, but hyponeophagia was reduced in the DBA mice. Moreover, all effects of individual housing on the exploratory and emotional behavior were more evident in the DBA than in the B6 mice. Novel object recognition and fear conditioning (FC) were significantly impaired in the single-housed mice, whereas water-maze (WM) learning was not affected. Marked strain differences were established in all three learning tests. The B6 mice performed better in the object recognition and FC tasks. Initial spatial learning in the WM was faster and memory retention slightly enhanced in the B6 mice. The DBA mice displayed lower preference to the new and enhanced preference to the old platform location than the B6 mice after reversal learning in the WM. We conclude that individual housing has strong strain- and test-specific effects on emotional behavior and impairs memory in certain tasks.

Proangiogenic activity of beta-carotene is coupled with the activation of endothelial cell chemotaxis.

Endothelial cells play an important role in angiogenesis (formation of new vessels from preexisting ones), which is essential for organogenesis, tissue remodeling but also inflammatory response, carcinogenesis in all periods of our life. Beta-carotene (BC) in non-toxic concentrations (up to 3 microM) had no detectable effect on HUVECs (human umbilical vein endothelial cells) proliferation or apoptosis, despite significant changes of the expression patterns of pro- and anti-apoptotic genes. However beta-carotene did not change the tubulogenic activity of HUVEC in the in vitro angiogenesis model, it potently accelerated the bFGF-induced development of microcapillaries, as well as the migration of endothelial cells, in matrigel plug injected subcutaneously to mice. Potent activation of endothelial cell migration in the in vitro model of chemotaxis was also observed. According to the microarray data, genes involved in cell/cell and cell/matrix adhesion, matrix reorganization, activation of chemotaxis, the G-protein regulated intracellular signaling as well as genes involved in the rapid remodeling of protein cytoskeleton were the most affected by BC in HUVEC. We conclude that beta-carotene in the physiological concentration range stimulates early steps of angiogenesis by the activation of cellular migration as well as matrix reorganization and decrease of cell adhesion.

[Is atherosclerosis an autoimmunological process?]. / Miazdzyca procesem autoimmunologicznym?

The theories formulated to explain atherogenesis evolved from simple vessel wall lipid accumulation assumption to endothelial dysfunction with adverse vascular wall remodelling hypothesis. The theory that has been accepted lately integrates the former hypotheses and allows for introducing the local immunological activation concept. This immunological activation is initiated by negatively charged and oxidatively modified lipids (e.g. oxPAPC) and their complexes with proteins (like beta 2-GP I). Antibodies and cellular response against chaperonins: HSP 60 and their analogues from bacterial pathogens such as HSP 65, GroEi etc.) as well as release of cytokines, adhesion molecules and inflammatory mediators (CD 40/CD 40-L, IL 15, IFN gamma, IL 1 beta, TNF alpha) also take part in the process. Another important element of atherogenesis is the pathological angiogenic response within the plaque connected with the expression of angiogenic growth factors (such as VEGF, bFGF and PDGF), metallo-proteinases and local hemostasis regulators. This complex activation of local inflammatory and immunological process initiates such phenomena as development of unstable plaque, vascular remodelling, vessel lumen constriction and ischemic, thromboembolic complications of atherosclerosis.

Regulation of inducible nitric oxide synthase (iNOS) and GTP cyclohydrolase I (GTP-CH I) gene expression by ox-LDL in rat vascular smooth muscle cells.

The generation of nitric oxide is regulated by several factors, including the substrates and cofactors supplementation. Decreased expression and activity of nitric oxide synthase as well as diminished amount of L-arginine or enzyme cofactors results in the inhibition of nitric oxide generation in vascular wall cells. GTP cyclohydrolase 1 is a key enzyme involved in the synthesis of tetrahydrobiopterin, one of the most important cofactors of NO synthases. We have demonstrated that oxidized LDL inhibit not only inducible nitric oxide synthase gene expression but also GTP cyclohydrolase I gene expression in interleukin-1 beta activated rat vascular smooth muscle cells in vitro. It is postulated that diminished availability of tetrahydrobiopterin may additionally impair the generation of nitric oxide in atherosclerosis.

Influence of insulin on cholesterol removal from macrophages and cholesterol ester uptake by HepG2 cells.

The fact that an increased blood insulin level is observed in patients with coronary artery disease (CAD) confirms the hypothesis that insulin promotes the development of atherosclerosis. The low high-density lipoprotein (HDL) concentration observed in such patients may contribute to alteration in reverse cholesterol transport and promote the accumulation of sterols in vascular tissue. We examined the effect of insulin (20-1000 microU mL-1) on cholesterol efflux into HDL3 particles from human blood monocyte/macrophages and rat peritoneal macrophages preloaded with labelled cholesterol esters, and the influence of insulin on the accumulation of sterols by rat liver cells and HepG2 cell line in vitro models. Insulin at concentrations up to 250 microU mL-1 inhibited the efflux of cholesterol from rat macrophages and promoted high uptake of sterols by both types of hepatic cells. Pharmacological concentrations higher than 250 microU mL-1 exerted the opposite effect. In the case of human macrophages, an insulin concentration of 20 microU mL-1 increased cholesterol removal, whereas 100-200 microU mL-1 insulin inhibited cholesterol removal from cells, and very high concentrations (> 350 microU mL-1) again increased cholesterol removal. We have shown that insulin excess counteracts the beneficial effects of HDL in removing cellular cholesterol and, therefore, may promote development of atherogenesis.

Driver behaviour and accident records at unsignalized urban intersections.

The main purpose of the study was to investigate whether Stop signs differ from Yield signs in terms of their associated accident rates, and whether or not an increase in level or traffic control is necessarily beneficial to traffic safety at all hazardous urban intersections. The study is based on a "before" and "after" evaluation of accident rates at several unsignalized intersections, where the level of control was changed because of their accident history. It was found that any increase in the level of control at such intersections tended to cause more vehicle accidents and fewer pedestrian accidents, although most of the changes were statistically insignificant. Several possible explanations to the findings are evaluated and discussed. It is concluded that increasing the control level at unsignalized intersections will not necessarily result in an overall reduction in accidents.