W. Graier - Academia.edu (original) (raw)

Papers by W. Graier

Biomedicines

By addressing the mechanisms involved in transcription, signaling, stress reaction, apoptosis and... more By addressing the mechanisms involved in transcription, signaling, stress reaction, apoptosis and cell-death, cellular structure and cell-to-cell contacts, adhesion, migration as well as inflammation; HBO upregulates processes involved in repair while mechanisms perpetuating tissue damage are downregulated. Many experimental and clinical studies, respectively, cover wound healing, regeneration of neural tissue, of bone and cartilage, muscle, and cardiac tissue as well as intestinal barrier function. Following acute injury or in chronic healing problems HBO modulates proteins or molecules involved in inflammation, apoptosis, cell growth, neuro- and angiogenesis, scaffolding, perfusion, vascularization, and stem-cell mobilization, initiating repair by a variety of mechanisms, some of them based on the modulation of micro-RNAs. HBO affects the oxidative stress response via nuclear factor erythroid 2-related factor 2 (Nrf2) or c-Jun N-terminal peptide and downregulates inflammation by t...

Scientific Reports, 2016

Histone acetylation depends on the abundance of nucleo-cytoplasmic acetyl-CoA. Here, we present a... more Histone acetylation depends on the abundance of nucleo-cytoplasmic acetyl-CoA. Here, we present a novel route for cytoplasmic acetyl-CoA production in brown adipocytes. N-acetylaspartate (NAA) is a highly abundant brain metabolite catabolized by aspartoacylase yielding aspartate and acetate. The latter can be further used for acetyl-CoA production. Prior to this work, the presence of NAA has not been described in adipocytes. Here, we show that accumulation of NAA decreases the brown adipocyte phenotype. We increased intracellular NAA concentrations in brown adipocytes via media supplementation or knock-down of aspartoacylase and measured reduced lipolysis, thermogenic gene expression and oxygen consumption. Combinations of approaches to increase intracellular NAA levels showed additive effects on lipolysis and gene repression, nearly abolishing the expression of Ucp1, Cidea, Prdm16 and Ppara. Transcriptome analyses of aspartoacylase knock-down cells indicate deficiencies in acetyl-C...

PKD family members control the secretion of pro-inflammatory cytokines and chemokines. BV-2 cells... more PKD family members control the secretion of pro-inflammatory cytokines and chemokines. BV-2 cells were cultured on 12-well plates; serum-starved o/n and the supernatants were collected after incubation with DMSO, DMSO plus LPA (1 μM) or LPA (1 μM) plus CRT (1 μM). ELISAs were used to quantitate IL-6, IL-1β, CXCL10 (IP-10), TNF-α, CXCL2 (MIP-2), and CCL5 (RANTES) concentrations. Results shown represent mean + SD from three independent experiments performed in triplicate (*p

The phosphorylation of pro-inflammatory transcription factors is under PKD family control. BV-2 c... more The phosphorylation of pro-inflammatory transcription factors is under PKD family control. BV-2 cells, serum-starved overnight and treated with LPA (1 μM) or LPA (1 μM) in the presence of (A) CRT0066101 (1 μM) for the indicated time periods. Cells incubated only with 0.1% BSA or CRT (1 μM) were used as negative control. The phosphorylation state of p65-NF-κB, STAT1, STAT3, and c-Jun was detected by western blotting. One representative blot is shown. (B) Densitometric analysis of western blots (N = 3). Results are presented as mean values + SEM (**p

TCLPA5 inhibits the phosphorylation of pro-inflammatory transcription factors. BV-2 microglia cel... more TCLPA5 inhibits the phosphorylation of pro-inflammatory transcription factors. BV-2 microglia cells were cultured in 6-well plates and serum-starved overnight. Cells were treated with LPA (1 μM) or LPA (1 μM) in the presence of (A) TCLPA5 (5 μM) for the indicated time periods. Cells incubated only with 0.1% BSA or TCLPA5 (5 μM) were used as negative control. The phosphorylation of p65-NF-κB, STAT1, STAT3, and c-Jun was detected using western blotting and one representative blot is shown. (B) Densitometric analysis of western blots (N = 3). Results represent mean values + SEM (*p

LPA promotes activation of pro-inflammatory transcription factors in BV-2 cells. Serum-starved (A... more LPA promotes activation of pro-inflammatory transcription factors in BV-2 cells. Serum-starved (A) BV-2 cells were treated with 0.1% BSA (control) or LPA (1 μM) for the indicated time periods, the cellular protein lysates were collected and phosphorylation state of IKKα/β, IκBα, p65-NF-κB, STAT1, STAT3, and c-Jun was detected using immunoblotting. One representative blot out of N = 3 experiments is shown. Actin was used as loading control. (B) Densitometric analysis of western blots show the significance of changes in the protein expression and represent mean values + SEM (*p

PKD family inhibition abrogates LPA-mediated downstream signaling. (A) BV-2 microglia cells were ... more PKD family inhibition abrogates LPA-mediated downstream signaling. (A) BV-2 microglia cells were cultured in 6-well plates, serum-starved overnight and preincubated with CRT0066101 ('CRT', 1 μM) for the indicated time periods before incubation with LPA (1 μM) or LPA (1 μM) plus CRT. Cells incubated only with 0.1% BSA or CRT (1 μM) were used as negative control. The phosphorylation states of PKDs, JNK, AKT, ERK1/2, and p38 were detected by immunoblotting and one representative blot for each protein is shown. (B) Densitometric analysis of western blots (N = 3). Results are presented as mean values + SEM (***p

LPAR5 controls LPA-mediated downstream signaling. (A) BV-2 microglia cells were cultured in 6-wel... more LPAR5 controls LPA-mediated downstream signaling. (A) BV-2 microglia cells were cultured in 6-well plates and serum-starved overnight. The cells were preincubated with TCLPA5 (5 μM) for the indicated times and then incubated with LPA (1 μM) or LPA (1 μM) plus TCLPA5. Cells incubated only with 0.1% BSA or TCLPA5 (5 μM) were used as negative control. The phosphorylation states of PKDs, JNK, AKT, ERK1/2, and p38 were detected using western blotting. One representative blot is shown. (B) Densitometric analysis of western blots (N = 3). Results represent mean values + SEM (***p

Clinical and Experimental Pharmacology and Physiology, 2002

1. This work was designed to introduce human uterine arteries as a new model for cardiovascular r... more 1. This work was designed to introduce human uterine arteries as a new model for cardiovascular research. Advantages of this model include considerable availability of tissue because of the appearance of uterus myomatosus in post-menopausal women who undergo surgery and the chance to work on dysfunctional and healthy vessels. 2. Histamine evoked relaxation of the uterine artery that was prevented by removal of the endothelium or by the presence of N G-nitro-L-arginine. 3. Receptor antagonists for histamine H1 (mepyramine) and H2 (ranitidine) receptors increased the EC50 of histamine by 112-and 67-fold, respectively. 4. Remarkably, isolated uterine arteries could be stored in incubators for 5 days without any change in contractility to phenylephrine and endothelium-dependent relaxation to acetylcholine and histamine. 5. Endothelial cells could be isolated and cultured in high purity, as demonstrated by histochemical staining of factor VIII, low CD45-RO for macrophages and no smooth muscle ␣-actin. In addition, cultured human uterine artery endothelial cells could be used for single cell Ca 2+ measurements. 6. In agreement with our findings in the intact vessel, histamine-initiated elevation of the intracellular free Ca 2+ concentration was reduced in the presence of mepyramine and ranitidine by 59 and 55%, respectively. 7. These data indicate that, in the human uterine artery, H1 and H2 receptors are involved in histamine-induced endothelium-dependent relaxation that is mediated by nitric oxide. 8. In addition, this vessel can be stored for possible virus-mediated gene expression for 5 days without any loss of reagibility. 9. Finally, endothelial cells can be isolated and cultured from the human uterine artery and maintain their reactivity to histamine in culture.

Biochemical and Biophysical Research Communications, 1992

Oxidatively modified LDL (LDLox) reduces the response of soluble guanylyl cyclase to nitrovasodil... more Oxidatively modified LDL (LDLox) reduces the response of soluble guanylyl cyclase to nitrovasodilators. We now demonstrate that this desensitization can be antagonized by HDL. Similar to its protective effect against LDLox, HDL also inhibited the lysolecithin-induced desensitization of soluble guanylyl cyclase. Since the lysolecithin content of LDLox correlated with the amount of lysolecithin necessary to diminish stimulation of soluble guanylyl cyclase, our data support the hypothesis that lysolecithin may be responsible for the inhibitory effect of LDLox on smooth muscle relaxation and provide evidence that the antagonistic effect of HDL against desensitization of soluble guanylyl cyclase by atherogenic compounds could be responsible for the protective role of HDL in atherosclerosis.

Biochemical and Biophysical Research Communications, 1990

Arteriosclerosis, Thrombosis, and Vascular Biology, 2004

Objective— Myeloperoxidase, a heme enzyme that is present and active in human atherosclerotic les... more Objective— Myeloperoxidase, a heme enzyme that is present and active in human atherosclerotic lesions, provides a source for the generation of proinflammatory chlorinated reactants contributing to endothelial dysfunction. Modification of high-density lipoprotein (HDL) by hypochlorous acid/hypochlorite (HOCl/Oce − )—generated in vivo by the myeloperoxidase-hydrogen peroxide-chloride system of activated phagocytes—forms a proatherogenic lipoprotein particle that binds to and is internalized by endothelial cells. Methods and Results— Here we show that HDL, modified with physiologically relevant HOCl concentrations, attenuates the expression and activity of vasculoprotective endothelial nitric oxide synthase. HOCl-HDL promotes dislocalization of endothelial nitric oxide synthase from the plasma membrane and perinuclear location of human umbilical venous endothelial cells. We could identify 2-chlorohexadecanal as the active component mediating this inhibitory activity. This chlorinated f...

British Journal of Pharmacology, 2002

The role of smooth muscle-derived lipoprotein lipase (LPL) that translocates to the endothelium s... more The role of smooth muscle-derived lipoprotein lipase (LPL) that translocates to the endothelium surface on vascular dysfunction during atherogenesis is unclear. Thus, the role of vascular LPL on blood vessel reactivity was assessed in transgenic mice that speci®cally express human LPL in the circulatory system. 2 Aortic free fatty acids (FFAs) were increased by 69% in the transgenic mice expressing human LPL in aortic smooth muscle cells (L2LPL) compared with their non-transgenic littermates (L2). 3 Contractility to KCl was increased by 33% in aortae of L2LPL mice. Maximal contraction to phenylephrine (PE) was comparable in L2 and L2LPL animals, while the frequency of tonus oscillation to PE increased by 104% in L2LPL mice. 4 In L2LPL animals, .NO mediated relaxation to acetylcholine (ACh) and ATP was reduced by 47 and 32%, respectively. In contrast, endothelium-independent relaxation to sodium nitroprusside (SNP) was not dierent in both groups tested. 5 ATP-initiated Ca 2+ elevation that triggers .NO formation was increased by 41% in single aortic endothelial cells freshly isolated from L2LPL animals. 6 In aortae from L2LPL mice an increased .O 2 7 release occurred that was normalized by removing the endothelium and by the NAD(P)H oxidase inhibitor DPI and the PKC inhibitor GF109203X. 7 The reduced ACh-induced relaxation in L2LPL animals was normalized in the presence of SOD, indicating that the reduced relaxation is due, at least in part, to enhanced .NO scavenging by .O 2 7. 8 These data suggest that despite normal lipoprotein levels increased LPL-mediated FFAs loading initiates vascular dysfunction via PKC-mediated activation of endothelial NAD(P)H oxidase. Thus, vascular LPL activity might represent a primary risk factor for atherosclerosis independently from cholesterol/LDL levels.

Atherosclerosis, 2014

ABSTRACT Figure optionsDownload full-size imageDownload high-quality image (119 K)Download as Pow... more ABSTRACT Figure optionsDownload full-size imageDownload high-quality image (119 K)Download as PowerPoint slide

Journal of Cell Science

In addition to their central role in triglyceride storage, fat cells are a primary depot of unest... more In addition to their central role in triglyceride storage, fat cells are a primary depot of unesterified cholesterol (FC) in the body. In comparison, peripheral cells contain very little FC. This difference in adipocytes versus peripheral tissues is inconsistent with the current theory of cholesterol homeostasis. Attempting to resolve this discrepancy, we examined intracellular storage sites of FC in murine 3T3-F442A adipocytes. Using the cholesterol-binding antibiotic, filipin, in combination with high resolution fluorescence microscopy, intense fluorescent staining characteristically decorated the periphery of triglyceride droplets (TGD) as well as the plasma membrane (PM) of fat cells. Filipin-staining was not visible inside the lipid droplets. Purification of TGD by subcellular fractionation demonstrated that the rise in total FC content of adipocytes upon differentiation was attributable to an increase in TGD-FC, which contributed up to one third of the total cellular FC. The p...

Lancet, 1995

H. Candiloros ah, N. Zeghari b, O. Ziegler b, M. Donner b, P. Drouin b, Hermann Toplak cdefg, Wol... more H. Candiloros ah, N. Zeghari b, O. Ziegler b, M. Donner b, P. Drouin b, Hermann Toplak cdefg, Wolfgang Graier cdefg, Peter Dittrich cdefg, UlrichN. Wiesmann cdefg, ThomasC. Wascher cdefg, AnnaF. Dominiczak ah, Lucilla Poston ijk, Gordon Murray ijk, Michael ...

European Heart Journal

Background Heart failure (HF) with preserved ejection fraction (HFpEF) accounts for about 50% of ... more Background Heart failure (HF) with preserved ejection fraction (HFpEF) accounts for about 50% of all cases of HF and there are currently no effective therapies. Purpose To assess the effects of histone deacetylase (HDAC) inhibition on cardiac and mitochondrial function and the plasma metabolome in a large mammalian model of slow-progressive pressure overload with features of HFpEF. Methods Male domestic short hair cats (n=26, aged 2mo), underwent either sham (S) procedures (n=5) or aortic constriction with a customized pre-shaped band (n=21), resulting in slow progressive pressure overload during growth. 2 months post-banding, animals were treated daily with either 10mg/kg suberoylanilide hydroxamic acid (b+SAHA) (n=8), a pan-HDAC inhibitor, or vehicle (b+veh) (n=8) for 2 months. Serial in-vivo cardiopulmonary phenotyping was performed monthly, and invasive hemodynamic and gas exchange parameters were evaluated 4 months post-banding. Ex-vivo myofibril mechanical studies and blood-ba...

Journal of Neuroinflammation

Background: Extracellular lysophosphatidic acid (LPA) species transmit signals via six different ... more Background: Extracellular lysophosphatidic acid (LPA) species transmit signals via six different G protein-coupled receptors (LPAR1-6) and are indispensible for brain development and function of the nervous system. However, under neuroinflammatory conditions or brain damage, LPA levels increase, thereby inducing signaling cascades that counteract brain function. We describe a critical role for 1-oleyl-2-hydroxy-sn-glycero-3-phosphate (termed "LPA" throughout our study) in mediating a motile and pro-inflammatory microglial phenotype via LPAR5 that couples to protein kinase D (PKD)-mediated pathways. Methods: Using the xCELLigence system and time-lapse microscopy, we investigated the migrational response of microglial cells. Different M1 and M2 markers were analyzed by confocal microscopy, flow cytometry, and immunoblotting. Using qPCR and ELISA, we studied the expression of migratory genes and quantitated the secretion of pro-inflammatory cytokines and chemokines, respectively. Different transcription factors that promote the regulation of pro-inflammatory genes were analyzed by western blot. Reactive oxygen species (ROS) and nitric oxide (NO) production, phagocytosis, and microglial cytotoxicity were determined using commercially available assay kits. Results: LPA induces MAPK family and AKT activation and pro-inflammatory transcription factors' phosphorylation (NF-κB, c-Jun, STAT1, and STAT3) that were inhibited by both LPAR5 and PKD family antagonists. LPA increases migratory capacity, induces secretion of pro-inflammatory cytokines and chemokines and expression of M1 markers, enhances production of ROS and NO by microglia, and augments cytotoxicity of microglial cell-conditioned medium towards neurons. The PKD family inhibitor blunted all of these effects. We propose that interference with this signaling axis could aid in the development of new therapeutic approaches to control neuroinflammation under conditions of overshooting LPA production. Conclusions: In the present study, we show that inflammatory LPA levels increased the migratory response of microglia and promoted a pro-inflammatory phenotype via the LPAR5/PKD axis. Interference with this signaling axis reduced microglial migration, blunted microglial cytotoxicity, and abrogated the expression and secretion of proinflammatory mediators.

Atherosclerosis Supplements, 2003