Detection of differentially expressed genes in synovial fibroblasts by restriction fragment differential display

Objective. To identify differentially expressed genes in synovial fibroblasts and examine the effect on gene expression of exposure to TNF-alpha and IL-1beta. Methods. Restriction fragment differential display was used to isolate genes using degenerate primers complementary to the lysophosphatidic acid acyl transferase gene family. Differential gene expression was confirmed by reverse transcription-polymerase chain reaction and immunohistochemistry using a variety of synovial fibroblasts, including cells from patients with osteoarthritis and self-limiting parvovirus arthritis. Results. Irrespective of disease process, synovial fibroblasts constitutively produced higher levels of IL-6 and monocyte chemoattractant protein 1 (MCP-1) (CCL2) than skin fibroblasts. Seven genes were differentially expressed in synovial fibroblasts compared with skin fibroblasts. Of these genes, four [tissue factor pathway inhibitor 2 (TFPI2), growth regulatory oncogene beta (GRObeta), manganese superoxide dismutase (MnSOD) and granulocyte chemotactic protein 2 (GCP-2)] were all found to be constitutively overexpressed in synoviocytes derived from patients with osteoarthritis. These four genes were only weakly expressed in other synovial fibroblasts (rheumatoid and self-limiting parvovirus infection). However, expression in all types of fibroblasts was increased after stimulation with TNF-alpha and IL-1beta. Three other genes (aggrecan, biglycan and caldesmon) were expressed at higher levels in all types of synovial fibroblasts compared with skin fibroblasts even after stimulation with TNF-alpha and IL-1. Conclusions. Seven genes have been identified with differential expression patterns in terms of disease process (osteoarthritis vs rheumatoid arthritis), state of activation (resting vs cytokine activation) and anatomical location (synovium vs skin). Four of these genes, TFPI2, GRObeta (CXCL2), MnSOD and GCP-2 (CXCL6), were selectively overexpressed in osteoarthritis fibroblasts rather than rheumatoid fibroblasts. While these differences may represent differential behaviour of synovial fibroblasts in in vitro culture, these observations suggest that TFPI2, GRObeta (CXCL2), MnSOD and GCP-2 (CXCL6) may represent new targets for treatments specifically tailored to osteoarthritis.

Pioglitazone increases renal tubular cell albumin uptake but limits proinflammatory and fibrotic responses

Background. Peroxisome proliferator-activated receptor gamma (PPARgamma) agonists. which are known to be critical factors in lipid metabolism, have also been reported to reduce proteinuria. The mechanism and its relevance to progressive nephropathy have not been determined. The aims of this study were to assess the direct effects of a PPARgamma agonist on tubular cell albumin uptake, proinflammatory and profibrotic markers of renal pathology, using an opossum kidney model of proximal tubular cells. Methods. Cells were exposed to pioglitazone (10 mumol/L) in the presence and absence of low-density lipoprotein (LDL) 100 mug/mL +/- exposure to albumin 1 mg/mL. Results were expressed relative to control (5 mmol/L glucose) conditions. Results. Pioglitazone caused a dose-dependent increase in tubular cell albumin uptake (P < 0.0001). Despite the increase in albumin reabsorption, no concurrent increase in inflammatory or profibrotic markers were observed. Exposure to LDL increased monocyte chemoattractant protein-1 (MCP-1) (P < 0.05) and transforming growth factor-beta1 (TGF-beta1) (P < 0.05) production. which were reversed in the presence of pioglitazone. LDL induced increases in MCP-1 and TGF-β1 were independent of nuclear factor-κB (NF-κB) transcriptional activity. In contrast. tubular exposure to albumin increased tubular protein uptake, in parallel with an increase in MCP-1 (P = 0.05): TGF-β1 (P < 0.02) and NF-kappaB transcriptional activity (P < 0.05). which were unaffected by concurrent exposure to pioglitazone. Conclusion. These findings suggest that dyslipidemia potentiates renal pathology through mechanisms that may be modified PPARγ activation independent of NF-κB transcriptional activitv. In contrast, tubular exposure to protein induces renal damage through NF-κB-dependent mechanisms that are Unaffected by PPARγ activation.

Plasma cytokine changes in relation to exercise intensity and muscle damage

The purpose of this study was to compare the effects of exercise intensity and exercise-induced muscle damage on changes in anti-inflammatory cytokines and other inflammatory mediators. Nine well-trained male runners completed three different exercise trials on separate occasions: ( 1) level treadmill running at 60% VO2max (moderate-intensity trial) for 60 min; (2) level treadmill running at 85% VO2max (high-intensity trial) for 60 min; (3) downhill treadmill running ( - 10% gradient) at 60% VO2 max (downhill running trial) for 45 min. Blood was sampled before, immediately after and 1 h after exercise. Plasma was analyzed for interleukin-1 receptor antagonist (IL-1ra), IL-4, IL-5, IL-10, IL-12p40, IL-13, monocyte chemotactic protein-1 (MCP-1), prostaglandin E-2, leukotriene B-4 and heat shock protein 70 (HSP70). The plasma concentrations of IL-1ra, IL-12p40, MCP-1 and HSP70 increased significantly (P< 0.05) after all three trials. Plasma prostaglandin E-2 concentration increased significantly after the downhill running and high-intensity trials, while plasma IL-10 concentration increased significantly only after the high-intensity trial. IL-4 and leukotriene B4 did not increase significantly after exercise. Plasma IL-1ra and IL-10 concentrations were significantly higher ( P< 0.05) after the high-intensity trial than after both the moderate-intensity and downhill running trials. Therefore, following exercise up to 1 h duration, exercise intensity appears to have a greater effect on anti-inflammatory cytokine production than exercise-induced muscle damage.

Zebrafish as a model for caveolin-associated muscle disease; caveolin-3 is required for myofibril organization and muscle cell patterning

Caveolae are an abundant feature of many animal cells. However, the exact function of caveolae remains unclear. We have used the zebrafish, Danio rerio, as a system to understand caveolae function focusing on the muscle-specific caveolar protein, caveolin-3 (Cav3). We have identified caveolin-1 (alpha and beta), caveolin-2 and Cav3 in the zebrafish. Zebrafish Cav3 has 72% identity to human CAV3, and the amino acids altered in human muscle diseases are conserved in the zebrafish protein. During embryonic development, cav3 expression is apparent by early segmentation stages in the first differentiating muscle precursors, the adaxial cells and slightly later in the notochord. cav3 expression appears in the somites during mid-segmentation stages and then later in the pectoral fins and facial muscles. Cav3 and caveolae are located along the entire sarcolemma of late stage embryonic muscle fibers, whereas beta-dystroglycan is restricted to the muscle fiber ends. Down-regulation of Cav3 expression causes gross muscle abnormalities and uncoordinated movement. Ultrastructural analysis of isolated muscle fibers reveals defects in myoblast fusion and disorganized myofibril and membrane systems. Expression of the zebrafish equivalent to a human muscular dystrophy mutant, CAV3P104L, causes severe disruption of muscle differentiation. In addition, knockdown of Cav3 resulted in a dramatic up-regulation of eng1a expression resulting in an increase in the number of muscle pioneer-like cells adjacent to the notochord. These studies provide new insights into the role of Cav3 in muscle development and demonstrate its requirement for correct intracellular organization and myoblast fusion.

Antifibrotic activity of an inhibitor of group IIA secretory phospholipase A(2) in young spontaneously hypertensive rats

The development of fibrosis in the chronically hypertensive heart is associated with infiltration of inflammatory cells and cardiac hypertrophy. In this study, an inhibitor of the proinflammatory enzyme, group IIA human secretory phospholipase A(2) (sPLA(2)-IIA), has been found to prevent collagen deposition as an important component of cardiovascular remodeling in a rat model of developing chronic hypertension. Daily treatment of young male spontaneously hypertensive rats (SHR) with an sPLA2-IIA inhibitor (KH064, 5-(4-benzyloxyphenyl)-4S-(phenyl-heptanoylamino)-pentanoic acid, 5 mg/kg/day p.o.) prevented increases in the content of perivascular,(SHR 20.6 +/- 0.9%, n = 5; SHR+KH064 14.0 +/- 1.2%, n = 5) and interstitial (SHR 7.9 +/- 0.3%, n = 6; SHR+KH064 5.4 +/- 0.7%, n = 6) collagen in the left ventricle of rat hearts, but did not affect numbers of infiltrating monocytes/macrophages, left ventricular hypertrophy (SHR 2.88 +/- 0.08, n = 12; SHR+KH064 3.09 +/- 0.08 mg/g body weight, n = 9), increased systolic blood pressure, or thoracic aortic responses. This selective antifibrotic activity suggests that sPLA2-IIA may have an important but specific role in cardiac fibrosis, and that its inhibitors could be useful in dissecting molecular pathways leading to fibrotic conditions.

The β domain is required for Vps4p oligomerization into a functionally active ATPase

Endocytic and biosynthetic trafficking pathways to the lysosome/vacuole converge at the prevacuolar endosomal compartment. During transport through this compartment, integral membrane proteins that are destined for delivery to the lysosome/vacuole lumen undergo multivesicular body (MVB) sorting into internal vesicles formed by invagination of the endosomal limiting membrane. Vps4 is an AAA family ATPase which plays a key role in MVB sorting and facilitates transport through endosomes. It possesses an N-terminal microtubule interacting and trafficking domain required for recruitment to endosomes and an AAA domain with an ATPase catalytic site. The recently solved 3D structure revealed a P domain, which protrudes from the AAA domain, and a final C-terminal alpha-helix. However, the in vivo roles of these domains are not known. In this study, we have identified motifs in these domains that are highly conserved between yeast and human Vps4. We have mutated these motifs and studied the effect on yeast Vps4p function in vivo and in vitro. We show that the P domain of the budding yeast Vps4p is not required for recruitment to endosomes, but is essential for all Vps4p endocytic functions in vivo. We also show that the P domain is required for Vps4p homotypic interaction and for full ATPase activity. In addition, it is required for interaction with Vta1p, which works in concert with Vps4p in vivo. Our studies suggest that assembly of a Vps4p oligomeric complex with full ATPase activity that interacts with Vta1p is essential for normal endosome function.

The PhD Finger Protein VRN5 Functions in the Epigenetic Silencing of Arabidopsis FLC

Vernalization, the acceleration of flowering by the prolonged cold of winter, ensures that plants flower in favorable spring conditions. During vernalization in Arabidopsis, cold temperatures repress FLOWERING LOCUS C (FLC) expression [1,2] in a mechanism involving VERNALIZATION INSENSITIVE 3 (VIN3) [3], and this repression is epigenetically maintained by a Polycomb-like chromatin regulation involving VERNALIZATION 2 (VRN2), a Su(z)12 homolog, VERNALIZATION 1 (VRN1), and LIKE-HETEROCHROMATIN PROTEIN 1 [4,5,6,7,8]. In order to further elaborate how cold repression triggers epigenetic silencing, we have targeted mutations that result in FLC misexpression both at the end of the prolonged cold and after subsequent development. This identified VERNALIZATION 5 (VRN5), a PHD finger protein and homolog of VIN3. Our results suggest that during the prolonged cold, VRN5 and VIN3 forma heterodimer necessary for establishing the vernalization-induced chromatin modifications, histone deacetylation, and H3 lysine 27 trimethylation required for the epigenetic silencing of FLC. Double mutant and FLC misexpression analyses reveal additional VRN5 functions, both FLC-dependent and -independent, and indicate a spatial complexity to FLC epigenetic silencing with VRN5 acting as a common component in multiple pathways.

Extracellular loops 1 and 3 and their associated transmembrane regions of the calcitonin receptor-like receptor are needed for CGRP receptor function

The first and third extracellular loops (ECL) of G protein-coupled receptors (GPCRs) have been implicated in ligand binding and receptor function. This study describes the results of an alanine/leucine scan of ECLs 1 and 3 and loop-associated transmembrane (TM) domains of the secretin-like GPCR calcitonin receptor-like receptor which associates with receptor activity modifying protein 1 to form the CGRP receptor. Leu195Ala, Val198Ala and Ala199Leu at the top of TM2 all reduced aCGRP-mediated cAMP production and internalization; Leu195Ala and Ala199Leu also reduced aCGRP binding. These residues form a hydrophobic cluster within an area defined as the "minor groove" of rhodopsin-like GPCRs. Within ECL1, Ala203Leu and Ala206Leu influenced the ability of aCGRP to stimulate adenylate cyclase. In TM3, His219Ala, Leu220Ala and Leu222Ala have influences on aCGRP binding and cAMP production; they are likely to indirectly influence the binding site for aCGRP as well as having an involvement in signal transduction. On the exofacial surfaces of TMs 6 and 7, a number of residues were identified that reduced cell surface receptor expression, most noticeably Leu351Ala and Glu357Ala in TM6. The residues may contribute to the RAMP1 binding interface. Ile360Ala impaired aCGRP-mediated cAMP production. Ile360 is predicted to be located close to ECL2 and may facilitate receptor activation. Identification of several crucial functional loci gives further insight into the activation mechanism of this complex receptor system and may aid rational drug design.

A comparative study of cationic liposome and niosome-based adjuvant systems for protein subunit vaccines:characterisation, environmental scanning electron microscopy and immunisation studies in mice

Vesicular adjuvant systems composing dimethyldioctadecylammonium (DDA) can promote both cell-mediated and humoral immune responses to the tuberculosis vaccine fusion protein in mice. However, these DDA preparations were found to be physically unstable, forming aggregates under ambient storage conditions. Therefore there is a need to improve the stability of such systems without undermining their potent adjuvanticity. To this end, the effect of incorporating non-ionic surfactants, such as 1-monopalmitoyl glycerol (MP), in addition to cholesterol (Chol) and trehalose 6,6′-dibehenate (TDB), on the stability and efficacy of these vaccine delivery systems was investigated. Differential scanning calorimetry revealed a reduction in the phase transition temperature (T c) of DDA-based vesicles by ∼12°C when MP and cholesterol (1:1 molar ratio) were incorporated into the DDA system. Transmission electron microscopy (TEM) revealed the addition of MP to DDA vesicles resulted in the formation of multi-lamellar vesicles. Environmental scanning electron microscopy (ESEM) of MP-Chol-DDA-TDB (16:16:4:0.5 μmol) indicated that incorporation of antigen led to increased stability of the vesicles, perhaps as a result of the antigen embedding within the vesicle bilayers. At 4°C DDA liposomes showed significant vesicle aggregation after 28 days, although addition of MP-Chol or TDB was shown to inhibit this instability. Alternatively, at 25°C only the MP-based systems retained their original size. The presence of MP within the vesicle formulation was also shown to promote a sustained release of antigen in-vitro. The adjuvant activity of various systems was tested in mice against three subunit antigens, including mycobacterial fusion protein Ag85b-ESAT-6, and two malarial antigens (Merozoite surface protein 1, MSP1, and the glutamate rich protein, GLURP). The MP- and DDA-based systems induced antibody responses at comparable levels whereas the DDA-based systems induced more powerful cell-mediated immune responses. © 2006 The Authors.

Expression of uncoupling proteins-1,-2 and-3 mRNA is induced by an adenocarcinoma-derived lipid-mobilizing factor

The abnormalities of lipid metabolism observed in cancer cachexia may be induced by a lipid-mobilizing factor produced by adenocarcinomas. The specific molecules and metabolic pathways that mediate the actions of lipid-mobilizing factor are not known. The mitochondrial uncoupling proteins-1, -2 and -3 are suggested to play essential roles in energy dissipation and disposal of excess lipid. Here, we studied the effects of lipid-mobilizing factor on the expression of uncoupling proteins-1, -2 and -3 in normal mice. Lipid-mobilizing factor isolated from the urine of cancer patients was injected intravenously into mice over a 52-h period, while vehicle was similarly given to controls. Lipid-mobilizing factor caused significant reductions in body weight (-10%, P=0.03) and fat mass (-20%, P<0.01) accompanied by a marked decrease in plasma leptin (-59%, P<0.01) and heavy lipid deposition in the liver. In brown adipose tissue, uncoupling protein-1 mRNA levels were elevated in lipid-mobilizing factor-treated mice (+96%, P<0.01), as were uncoupling proteins-2 and -3 (+57% and +37%, both P<0.05). Lipid-mobilizing factor increased uncoupling protein-2 mRNA in both skeletal muscle (+146%, P<0.05) and liver (+142%, P=0.03). The protein levels of uncoupling protein-1 in brown adipose tissue and uncoupling protein-2 in liver were also increased with lipid-mobilizing factor administration (+49% and +67%, both P=0.02). Upregulation by lipid-mobilizing factor of uncoupling proteins-1, -2 and -3 in brown adipose tissue, and of uncoupling protein-2 in skeletal muscle and liver, suggests that these uncoupling proteins may serve to utilize excess lipid mobilized during fat catabolism in cancer cachexia.

Effect of maternal protein restriction during pregnancy and postweaning high-fat feeding on diet-induced thermogenesis in adult mouse offspring

Purpose: Prenatal undernutrition followed by postweaning feeding of a high-fat diet results in obesity in the adult offspring. In this study, we investigated whether diet-induced thermogenesis is altered as a result of such nutritional mismatch. Methods: Female MF-1 mice were fed a normal protein (NP, 18 % casein) or a protein-restricted (PR, 9 % casein) diet throughout pregnancy and lactation. After weaning, male offspring of both groups were fed either a high-fat diet (HF; 45 % kcal fat) or standard chow (C, 7 % kcal fat) to generate the NP/C, NP/HF, PR/C and PR/HF adult offspring groups (n = 7-11 per group). Results: PR/C and NP/C offspring have similar body weights at 30 weeks of age. Postweaning HF feeding resulted in significantly heavier NP/HF offspring (P <0.01), but not in PR/HF offspring, compared with their chow-fed counterparts. However, the PR/HF offspring exhibited greater adiposity (P <0.01) v the NP/HF group. The NP/HF offspring had increased energy expenditure and increased mRNA expression of uncoupling protein-1 and β-3 adrenergic receptor in the interscapular brown adipose tissue (iBAT) compared with the NP/C mice (both at P <0.01). No such differences in energy expenditure and iBAT gene expression were observed between the PR/HF and PR/C offspring. Conclusions: These data suggest that a mismatch between maternal diet during pregnancy and lactation, and the postweaning diet of the offspring, can attenuate diet-induced thermogenesis in the iBAT, resulting in the development of obesity in adulthood. © 2014 Springer-Verlag Berlin Heidelberg.

Resveratrol inhibits the release of soluble fms-like tyrosine kinase (sFlt-1) from human placenta

Objective - Soluble vascular endothelial growth factor receptor–1 (also know as soluble fms-like tyrosine kinase [sFlt]-1) is a key causative factor of preeclampsia. Resveratrol, a plant phytoalexin, has antiinflammatory and cardioprotective properties. We sought to determine the effect of resveratrol on sFlt-1 release. Study Design - Human umbilical vein endothelial cells, transformed human trophoblast-8 (HTR/SVneo)-8/SVneo trophoblast cells, or placental explants were incubated with cytokines and/or resveratrol. Conditioned media were assayed for sFlt-1 by enzyme-linked immunosorbent assay and cell proteins used for Western blotting. Results - Resveratrol inhibited cytokine-induced release of sFlt-1 from normal placental explants and from preeclamptic placental explants. Preincubation of human umbilical vein endothelial cells or HTR-8/SVneo cells with resveratrol abrogated sFlt-1 release. Resveratrol prevented the up-regulation of early growth response protein-1 (Egr-1), a transcription factor necessary for induction of the vascular endothelial growth factor receptor–1 gene and caused up-regulation of heme oxygenase–1, a cytoprotective enzyme found to be dysfunctional in preeclampsia. Conclusion - In summary, resveratrol can inhibit sFlt-1 release and up-regulate heme oxygenase–1; thus, may offer therapeutic potential in preeclampsia.

Can emerging biomarkers of myocardial remodelling identify asymptomatic hypertensive patients at risk for diastolic dysfunction and diastolic heart failure?

AIMS: Hypertension is one of the main drivers of the heart failure (HF) epidemic. The aims of this study were to profile fibro-inflammatory biomarkers across stages of the hypertensive heart disease (HHD) spectrum and to examine whether particular biochemical profiles in asymptomatic patients identify a higher risk of evolution to HF.

METHODS AND RESULTS: This was a cross-sectional observational study involving a population of 275 stable hypertensive patients divided into two different cohorts: Group 1, asymptomatic hypertension (AH) (n= 94); Group 2, HF with preserved ejection fraction (n= 181). Asymptomatic hypertension patients were further subdivided according to left atrial volume index ≥34 mL/m(2) (n= 30) and <34 mL/m(2) (n= 64). Study assays involved inflammatory markers [interleukin 6 (IL6), interleukin 8 (IL8), monocyte chemoattractant protein 1 (MCP1), and tumour necrosis factor α], collagen 1 and 3 metabolic markers [carboxy-terminal propeptide of collagen 1, amino-terminal propeptide of collagen 1, amino-terminal propeptide of collagen 3 (PIIINP), and carboxy-terminal telopeptide of collagen 1 (CITP)], extra-cellular matrix turnover markers [matrix metalloproteinase 2 (MMP2), matrix metalloproteinase 9 (MMP9), and tissue inhibitor of metalloproteinase 1 (TIMP1)], and the brain natriuretic peptide. Data were adjusted for age, sex, systolic blood pressure, and creatinine. Heart failure with preserved ejection fraction was associated with an increased inflammatory signal (IL6, IL8, and MCP1), an increased fibrotic signal (PIIINP and CITP), and an increased matrix turnover signal (MMP2 and MMP9). Alterations in MMP and TIMP enzymes were found to be significant indicators of greater degrees of asymptomatic left ventricular diastolic dysfunction.

CONCLUSION: These data define varying fibro-inflammatory profiles throughout different stages of HHD. In particular, the observations on MMP9 and TIMP1 raise the possibility of earlier detection of those at risk of evolution to HF which may help focus effective preventative strategies.

Epigenetics, the epicenter of the hypoxic response

It is becoming increasingly apparent that epigenetics plays a crucial role in the cellular response to hypoxia. Such epigenetic regulation may work hand in hand with the hypoxia-induced transcription factor (HIF) family or may contribute in a more substantial way to the maintenance of a hypoxia-adapted cellular phenotype long after HIF has initiated the immediate response pathways. In this article we discuss the current research implicating epigenetic mechanisms in the cellular response to hypoxic environments. This includes; the role of epigenetics in both the stabilization and binding of HIF to its transcriptional targets, the role of histone demethylase enzymes following direct HIF transactivation, and finally, the impact of hypoxic environments on global patterns of histone modifications and DNA methylation.

Generation of an epigenetic signature by chronic hypoxia in prostate cells

Increasing levels of tissue hypoxia have been reported as a natural feature of the aging prostate gland and may be a risk factor for the development of prostate cancer. In this study, we have used PwR-1E benign prostate epithelial cells and an equivalently aged hypoxia-adapted PwR-1E sub-line to identify phenotypic and epigenetic consequences of chronic hypoxia in prostate cells. We have identified a significantly altered cellular phenotype in response to chronic hypoxia as characterized by increased receptor-mediated apoptotic resistance, the induction of cellular senescence, increased invasion and the increased secretion of IL-1 beta, IL6, IL8 and TNFalpha cytokines. In association with these phenotypic changes and the absence of HIF-1 alpha protein expression, we have demonstrated significant increases in global levels of DNA methylation and H3K9 histone acetylation in these cells, concomitant with the increased expression of DNA methyltransferase DMNT3b and gene-specific changes in DNA methylation at key imprinting loci. In conclusion, we have demonstrated a genome-wide adjustment of DNA methylation and histone acetylation under chronic hypoxic conditions in the prostate. These epigenetic signatures may represent an additional mechanism to promote and maintain a hypoxic-adapted cellular phenotype with a potential role in tumour development.