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Biomedical subjects

G Hofer

Publications and source records attributed to G Hofer.

At least 19 recordsLinked to original sources
PubMed2000

Nitric oxide inhibits growth of glomerular mesangial cells: role of the transcription factor EGR-1.

UNLABELLED: Nitric oxide inhibits growth of glomerular mesangial cells: Role of the transcription factor Egr-1. BACKGROUND: In previous studies, we found a close link of early growth response gene-1 (Egr-1) expression to mesangial cell (MC) proliferation. Antiproliferative agents inhibited mitogen-induced Egr-1 expression. Here we investigated the effect of S-nitrosoglutathione (GSNO) on the proliferation of MCs, specifically asking how GSNO regulates the transcription factor Egr-1, which we have previously shown to be critical for the induction of MC mitogenesis. METHODS: The proliferation of MCs was measured by thymidine incorporation and cell counting. Egr-1 mRNA and protein levels were detected by Northern and Western blots. Electrophoretic mobility shift assays (EMSAs) and chloramphenicol acetyltransferase (CAT) assays were performed to test whether GSNO modulates DNA binding and transcriptional activation of Egr-1. RESULTS: GSNO strongly inhibited serum-induced MC proliferation (-84% at 1 mmol/L). A mild inhibition of serum-induced Egr-1 mRNA was observed at GSNO concentrations from 50 to 200 micromol/L, whereas mRNA levels increased again at concentrations above 500 micromol/L. This increased mRNA expression, however, was not translated into Egr-1 protein. Instead, Egr-1 protein induction was inhibited (-40%). EMSAs indicated that GSNO inhibited specific binding of Egr-1 to its DNA consensus sequence. Moreover, transcriptional activation by Egr-1 in CAT assays using a reporter plasmid bearing three Egr-1 binding sites was strongly suppressed by GSNO. CONCLUSIONS: Our data identify GSNO as a potent inhibitor of MC growth with potential beneficial effects in proliferative glomerular diseases. This antimitogenic property is mediated at least in part by inhibitory effects of GSNO on Egr-1 protein levels and by reducing the ability of Egr-1 to activate transcription by impairing its DNA binding activity.

Animals↗
PubMed1998

Lysophosphatidic acid-mediated signal-transduction pathways involved in the induction of the early-response genes prostaglandin G/H synthase-2 and Egr-1: a critical role for the mitogen-activated protein kinase p38 and for Rho proteins.

During inflammatory processes of the kidney, lesions of the glomerulus lead to aggregation of thrombocytes and infiltration of macrophages, which can release bioactive mediators. One of these important signalling molecules is lysophosphatidic acid (LPA). Incubation of rat mesangial cells with LPA induced mRNA and protein expression of the early-response genes pghs-2 (for prostaglandin G/H synthase-2/cyclo-oxygenase-2) and egr-1. As shown by antisense experiments, induction of egr-1 was related to the strong mitogenic effect of LPA. LPA-mediated gene expression was inhibited by pertussis toxin, indicating coupling to G-proteins of the Gi family. Specific inhibition of proteins of the small G-protein subfamily Rho with toxin B from Clostridium difficile led to changes in mesangial cell morphology without induction of apoptosis. LPA-mediated expression of pghs-2 and egr-1 was reduced to base-line levels by toxin B, indicating a role for Rho proteins in LPA-mediated gene induction. Of the two mitogen-activated protein kinase (MAPK) pathways investigated, the MAPK kinase-extracellular signal-regulated kinase pathway was involved in the induction of both pghs-2 and egr-1 mRNA expression, as shown by the inhibitory effect of PD98059. Activation of the MAPK p38, however, was only related to pghs-2 expression, whereas egr-1 expression was not affected by treatment of mesangial cells with the specific inhibitor SB203580. Taken together our data provide evidence that LPA-mediated activation of MAPK kinase and Rho proteins leads to the induction of the functionally distinct early-response genes pghs-2 and egr-1, whereas activation of MAPK p38 revealed considerable differences between the regulation of these two genes.

Animals↗
PubMed1997

[Egr-1 transcription factor regulates the growth of glomerular mesangium cells].

BACKGROUND: The transcriptional regulator Early growth response gene-1 (Egr-1) is rapidly and transiently induced by various mitogens in cultured rat mesangial cells (MCs). METHOD AND RESULTS: Here we show Egr-1 induction in an in vivo model of mesangioproliferative glomerulonephritis (GN). A 14.9-fold increase in Egr-1 mRNA was observed 6 days after disease induction. A concomitant increase in Egr-1 protein was demonstrated by immunocytochemistry. Egr-1 was mainly localized to the nuclei of cells in mesangial localization. To test whether Egr-1 directly regulated MC proliferation, we preincubated cultured MCs with antisense oligonucleotides directed against Egr-1. The platelet-derived growth factor (PDGF)-induced increase in Egr-1 mRNA and protein levels was inhibited by 75% and 74%, respectively. At the same time Egr-1 antisense oligonucleotides dose-dependently inhibited MC-proliferation as determined by thymidine-uptake by up to 75%. Control oligonucleotides were without effects on Egr-1 mRNA, protein or MC growth. CONCLUSION: We conclude that Egr-1 induction is a necessary step in the mitogenic signaling cascade in glomerular MCs.

Animals↗
PubMed1997

LDL-mediated interaction of Lp[a] with HepG2 cells: a novel fluorescence microscopy approach.

We studied the topography of Lp[a]-LDL-cell interactions by means of fluorescence microscopy, using fluorescence-labeled lipoproteins. In contrast to known methods which are based on noncovalent labeling of lipoproteins by positively charged amphiphiles, the protein moiety of LDL and Lp[a] was covalently labeled with either BODIP-succinimide-ester (green) or rhodamine X iodoacetamide (red). The interaction of the fluorescent lipoproteins with cultured HepG2 cells was studied using a confocal laser scanning fluorescence microscope. LDL and Lp[a], each labeled with a different dye, could be examined separately within a mixture of both lipoproteins during their interaction with HepG2 cells. At 4 degrees C, the majority of both fluorescent particles co-localized and only a few separate LDL- or Lp[a]-binding domains could be observed. Quantification of the amount of fluorescent lipoprotein associated with the cell surface at 4 degrees C showed that binding of Lp[a] was increased in the presence of LDL under these conditions, probably via formation of an Lp[a]-LDL complex. At 37 degrees C, LDL and Lp[a] were taken up by the cells within 10 min. Again the majority of LDL and Lp[a] particles co-localized intracellularly. Only minor amounts of LDL and Lp[a] could be observed separately. As the entire fluorescence of labeled Lp[a] co-localized with excess of LDL in cells, and taking into account the high tendency of LDL-Lp[a] association in solution and on cell surfaces, it is concluded that a significant portion of the internalized Lp[a] is taken up into the cells by the LDL receptor via LDL by a hitchhiking-like process.

Animals↗
PubMed1996

Transcription factor Egr-1 regulates glomerular mesangial cell proliferation.

Increase of glomerular mesangial cells (MCs) is a prominent histopathological finding in many types of glomerulonephritis. We have shown previously that expression of the zinc-finger transcription factor, early growth response gene-1 (egr-1), is closely correlated with the proliferation of cultured MCs. To elucidate whether Egr-1 is required for MC proliferation, we inhibited serum-induced Egr-1 expression by phosphothioate-modified antisense oligonucleotides (ODNs). Uptake of antisense ODNs into MCs was demonstrated, and five different egr-1 antisense ODNs were tested for their impact on serum-induced egr-1 mRNA and protein levels and on MC growth. The most potent egr-1 antisense ODN inhibited serum-induced egr-1 mRNA by 68%, protein induction by 58%, and MC replication as measured by [3H]thymidine uptake and cell counts by 78 and 46%, respectively. The effects of antisense ODNs on MC growth correlated closely with their ability to inhibit Egr-1 protein. ODNs acted in a dose-dependent manner, the minimal effective concentration being 1 microM. Control ODNs had no significant effects. In addition, antisense ODNs against egr-1 potently inhibited endothelin-1-induced Egr-1 expression and MC growth. Heparin, a known inhibitor of MC growth, suppressed serum-induced [3H]thymidine uptake by 39% and egr-1 mRNA expression by 44%. We conclude that Egr-1 is an essential part of the mitogenic signal transduction cascade in cultured MCs.

Animals↗
PubMed1996

Oxidation of fluorescent glycero- and sphingophospholipids in human plasma lipoproteins: alkenylacyl subclasses are preferred targets.

OBJECTIVES: Oxidation of polyunsaturated fatty acids in lipoproteins is supposed to play a crucial role at the early stages of atherogenesis. The polyunsaturated lipids (PUFAs) become oxidized and, thus, the degree and rate of lipid oxidation depend on their concentration and, probably, on the lipid moiety to which they are attached. DESIGN AND METHODS: To determine the relative oxidation susceptibilities of sphingo- and glycerolipid-bound fatty acyl chains, we used fluorescent analogs of the respective compounds, in which one natural fatty acyl chain was replaced by fluorescent diphenylhexatriene propionic acid. RESULTS: Oxidation susceptibilities of the fluorescent acyl chains in the presence of Cu2+ or AAPH depended, in general, on the phospholipid to which they were bound and the lipoprotein. Phospholipids were oxidized faster in HDL than in LDL or Lp(a). Plasmalogens were more susceptible to oxidation than phosphatidylcholine and sphingomyelin. CONCLUSION: Thus, HDL and plasmalogens may be considered as preferred targets of lipid oxidation before the bulk of polyunsaturated phospholipids (mainly phosphatidylcholine) in LDL is subject to free radical attack.

Amidines↗
PubMed1995

Concentrations of various arachidonic acid metabolites in menstrual fluid are associated with menstrual pain and are influenced by hormonal contraceptives.

In a pilot study we investigated the association between concentrations of various eicosanoids in menstrual blood with pain and oral contraceptive use. Menstrual fluid was collected on tampons by 12 women who did not use an oral contraceptive but suffered from slight primary dysmenorrhea and by three pain-free women who used an oral contraceptive. Eicosanoids (cyclooxygenase products: 6-ketoprostaglandin F1 alpha, thromboxane B2, prostaglandin E2, prostaglandin F2 alpha, 13,14-dihydro-15-ketoprostaglandin F2 alpha, 12-hydroxy-heptadecatrienoic acid; lipoxygenase products: 5-, 12-, 15-hydroxy-eicosatetraenoic acid (HETE), leukotriene B4, leukotriene C4, leukotriene D4, leukotriene E4) and female sex steroids (17 beta-estradiol and progesterone) were analyzed by the combined use of high-performance liquid chromatography and radioimmunoassay. 12-HETE was the main arachidonic acid metabolite. An increased metabolism of arachidonic acid was associated with pain, especially when synthesis of 12-HETE was elevated. Oral contraceptive use decreased the synthesis of prostaglandins as well as leukotrienes. The concordant changes of cyclooxygenase and lipoxygenase products in dysmenorrhea or in oral contraceptive use may be explained by an increased or decreased phospholipid metabolism, respectively.

12-Hydroxy-5,8,10,14-eicosatetraenoic Acid↗
PubMed1995

A new fluorescence method for the continuous determination of surface lipid oxidation in lipoproteins and plasma.

We report on a new method for the determination of lipid oxidation in lipoproteins and plasma. The biological lipid system is preloaded with a fluorescent analog of phosphatidylcholine containing diphenylhexatriene (DPH) propionic acid covalently linked to the sn-2 position. When externally added, the respective phospholipid label (DPHPC) localizes to the surface monolayer of a lipoprotein. Under oxidative conditions (e.g. in the presence of Cu2+ ions) the fluorophore undergoes decomposition, resulting in a continuous decrease of fluorescence intensity which reflects the oxidation of a chemically defined phospholipid molecule with well defined localization. When incorporated into LDL particles, the kinetics of the decrease in DPHPC fluorescence intensity upon exposure to Cu2+ us very similar to that of conjugated diene accumulation. Furthermore, our assay can be applied to follow the oxidation of lipids in diluted serum and may also be developed into a suitable test system for clinical studies of susceptibility of plasma lipids to oxidation.

Diphenylhexatriene↗
PubMed1995

Transfer of phospholipase A-resistant pyrene-dialkyl-glycerophosphocholine to plasma lipoproteins: differences between Lp[a] and LDL.

1-O-Hexadecyl-2-O-pyrenedecanyl-sn-glycero-3-phosphocholine, a non-hydrolyzable fluorescent diether analog of phosphatidylcholine (PC), was synthesized as a probe for studying phospholipid transfer to different lipoprotein classes with potential phospholipase activities. After incubation of total human plasma with the new probe at 37 degrees C for 4.5 h, a characteristic partition between the main lipoprotein fractions was observed. The fluorescent lipid was not degraded under these conditions and, therefore, served as a measure for choline glycerophospholipid distribution between plasma lipoproteins. In low density lipoprotein (LDL) and high density lipoprotein-3 (HDL3) the fluorescent PC analog showed only monomer fluorescence, whereas in Lp[a] and HDL2 monomer and excimer fluorescence were observed, indicating that the fluorescent phosphatidylcholine analog was incorporated into the respective lipoproteins to a different extent. According to the increased pyrene excimer fluorescence in Lp[a] compared with LDL the labeled phosphatidylcholine must be enriched and/or clustered in Lp[a]. Data from phospholipid and total fluorescence analyses are compatible with the assumption of higher label concentration in Lp[a]. On the other hand, transfer rates for serum protein-catalyzed lipid transport into isolated Lp[a] were slower as compared to LDL. It is suggested that slower lipid transfer to Lp[a] under these conditions is due to the decreased lipid mobility in the Lp[a] surface, whereas the higher extent of label partition into Lp[a] as observed in total plasma might be due to the higher affinity of apolipoproteins for phosphatidylcholine in Lp[a] (Sommer, A., et al. 1992. J. Biol. Chem. 267: 24217-24222). The use of a fluorescent dialkyl- instead of diacyl-glycerophosphocholine for transfer studies was mandatory, as we found that lipoproteins contained phospholipase A2 activity toward long-chain phosphatidylcholine. The lipoprotein-associated phospholipase A2 was three times more active in Lp[a] than in LDL. The degradation products formed by the phospholipase, fatty acids, and lyso-PC may add to the high atherogenic potential of Lp[a].

Biological Transport↗
PubMed1994

Insulin-responsive regions of the rat fatty acid synthase gene promoter.

Here we show that insulin may play a role in the diet-induced regulation of the rat fatty acid synthase (FAS; EC 2.3.1.85). Transient transfection of human and rat hepatoma cell lines with successively deleted FAS/CAT promoter fusion plasmids was used to determine the effect of insulin on FAS promoter activity. Our results indicate the existence of cis-acting insulin-responsive elements in the FAS promoter; the position of one of these is coincident with the position of a previously determined diet-induced DNAse I hypersensitive site (HSi-1) at approximately -500 bp relative to the transcription start site of FAS mRNA.

Animals↗
PubMed1993

Measurement of eicosanoids in menstrual fluid by the combined use of high pressure chromatography and radioimmunoassay.

Methods are described for the quantification of various eicosanoids (cyclooxygenase products: 6-KETO, TXB2, PGE2, PGF2 alpha, DHK; lipoxygenase products: 5-, 12-, 15-HETE, LTB4, LTC4, LTD4, LTE4) in menstrual blood collected by tampons. Samples were extracted with acidified ethanol. After purification by SEP-PACK C18 columns, the compounds were separated by reversed phase HPLC using a ternary gradient system. The eicosanoid concentrations of the fractionated eluents were measured by radioimmunoassay and corrected for recovery. 12-HETE was the most prominent metabolite of arachidonic acid in menstrual blood (mean: 1174 ng/g blood). With the exception of PGF2 alpha and TXB2 (mean: 343 and 212 ng/g blood, respectively) other eicosanoids were detected in remarkable lower concentrations.

Body Fluids↗
PubMed1992

Specific and non specific stimulation of prostaglandin release by human skin fibroblasts in culture.--Are changes of membrane fluidity involved?

In order to study a bidirectional relationship between changes of membrane fluidity and prostaglandin synthesis, the arachidonic acid cascade was stimulated in cultured human skin fibroblasts by unspecific stimuli (hypotonicity, low calcium concentrations) and by the specific stimulus, bradykinin. Fluorescence anisotropy of trimethylammoniumdiphenylhexatriene was used to measure membrane fluidity in cell monolayers. Hypotonicity or low calcium concentrations induce membrane fluidisation and prostaglandin synthesis. However, after specific stimulation of prostaglandins with bradykinin (at normocalcic and isotonic conditions) a rigidification of plasma membranes was observed in living cells. Fluidisation of membranes and bradykinin activate phospholipase A2 and induce prostaglandin synthesis. Although in cell membrane preparations increased phospholipase A2 activity leads to fluidisation, in our model a membrane fluidisation was not observed after stimulation of phospholipase with bradykinin. This suggests that in living cells a fluidizing effect of lysolecithin resulting from phospholipase A2 activation may be rapidly counteracted by its removal. A decrease of phosphatidylcholin content and consequently a rigidification of the membrane may ensue. Thus, the cell culture model using two different ways of stimulating phospholipase activity, helps to define the directional relationship between changes of membrane fluidity and activation of phospholipase and the arachidonic acid cascade in living human cells.

Bradykinin↗
PubMed1989

Radioimmunoassay for immunoreactive non-collagenous domain of type IV collagen (NC1) in serum: normal pregnancy and preeclampsia.

The use of a magnetic separation agent in a modified radioimmunoassay for the non-collagenous cross-linked region (NC1) of basement membrane collagen is described. The rabbit anti-NC1 serum employed in the assay revealed a binding behaviour similar to that reported recently (Mark et al. (1985) Eur. J. Biochem. 146, 555-562; Schuppan et al. (1986) J. Clin. Invest. 78, 241-248) with respect to the integrity of NC1-disulphide bridges, affinity to NC1 subunits and lack of reactivity with other determinants of type IV collagen. Immunoreactive serum NC1, which increased towards the end of gestation, showed a broad peak during the second trimester of pregnancy. In preeclampsia, immunoreactive serum-NC1 was slightly elevated.

Blotting, Western↗