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...
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
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...
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...
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 ...
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...
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...
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
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...
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...
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 ...
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...
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