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Biomedical subjects

Gerd Schmitz

Publications and source records attributed to Gerd Schmitz.

At least 19 recordsLinked to original sources
PubMed2006

Isomer specific effects of Conjugated Linoleic Acid on macrophage ABCG1 transcription by a SREBP-1c dependent mechanism.

Conjugated Linoleic Acids (CLAs) are minor components of the diet with many reported biological activities. Our aim was to examine the function of the single trans-9,trans-11 (t9,t11), cis-9,trans-11 (c9,t11), and trans-10,cis-12 (t10,c12) isomers on gene expression in human macrophages. Therefore we incubated in vitro MCSF differentiated monocyte derived macrophages from three healthy donors and THP-1 macrophages with these CLA-isomers and analyzed whole genome transcripts with Affymetrix U133 Plus 2.0 DNA-microarrays and real-time RT-PCR. We found that t9,t11-CLA, but not c9,t11- and t10,c12-CLA activates target genes of SREBP, SREBP-1, and ABCG1. Gene reporter assays with deletion constructs of the ABCG1 regulatory region and cotransfections with SREBP-1a and SREBP-1c expression plasmids in RAW 264.7 macrophages showed that t9,t11-CLA activates ABCG1 via SREBP-1c. These results indicate that positional and geometrical isomers of CLAs have specific effects on gene expression of human macrophages and that t9,t11-CLA activates ABCG1 by a SREBP-1c-dependent mechanism.

ATP Binding Cassette Transporter, Subfamily G, Mem↗
PubMed2006

High molecular weight adiponectin reduces apolipoprotein B and E release in human hepatocytes.

Low circulating levels of high molecular weight adiponectin (HMW-Apm) have been linked to dyslipidaemia and systemic HMW-Apm negatively correlates with very low density lipoprotein (VLDL), apolipoprotein B (ApoB), and ApoE and is positively associated with ApoA-I. Therefore, it was investigated whether HMW-Apm alters the hepatic synthesis of ApoB, ApoE, and ApoA-I or the activity of the hepatic ATP-binding cassette transporter A1 (ABCA1), as the main determinant of plasma HDL. HMW-Apm reduces hepatic ApoB and ApoE release whereas ABCA1 protein, activity and ApoA-I were not altered. Global gene expression analysis revealed that hepatic nuclear factor 4-alpha (HNF4-alpha) and HNF4-alpha regulated genes like ApoB are downregulated by HMW-Apm and this was confirmed at the mRNA and protein level. Therefore it is concluded that HMW-adiponectin may ameliorate dyslipidaemia by reducing the hepatic release of ApoB and ApoE, whereas ABCA1 function and ApoA-I secretion are not influenced.

Adiponectin↗
PubMed2006

Aberrant intestinal expression and allelic variants of mucin genes associated with inflammatory bowel disease.

Loss of intestinal mucosa integrity is an important factor in the pathogenesis of inflammatory bowel disease (IBD). The aim of this study was to characterize expression changes and allelic variants of genes related to intestinal epithelial barrier function in this disease. Therefore, ileal and colonic mucosal biopsies from nonaffected regions of patients with ulcerative colitis (UC) and Crohn's disease (CD), as well as non-IBD probands, were subjected to Affymetrix DNA-microarray analysis. Real-time reverse transcription polymerase chain reaction was used for verification in larger IBD sample numbers. Disturbed mRNA expression was identified for several mucin genes in both disease groups and tissues. A significant downregulation in the colon was obtained for MUC2 in CD and MUC12 in CD and UC. Expression analysis of all dysregulated mucins in a broad human tissue panel revealed dominant epithelial tissue-specific transcription. In silico analysis of the regulatory regions of these mucins indicated nuclear factor kappaB (NFkappaB) binding sites in each promoter. Furthermore, NFkappaB was overrepresented in mucin promoters and a component of a specific combination of transcription factors (composite module). In vivo stimulation experiments in the adenocarcinoma cell line LS174T showed inducible mucin expression by the cytokines tumor necrosis factor-alpha and transforming growth factor-beta, which could be blocked by NFkappaB signaling inhibitors. Allelic discrimination screening obtained statistically significant associations for the MUC2-V116M (P = 0.003) polymorphism with CD and for MUC4-A585S (P = 0.025), as well as MUC13-R502S (P = 0.0003) with UC. These data suggest that the disturbed expression of mucin genes and the connection to the NFkappaB pathway may influence the integrity of the intestine and therefore contribute to the pathophysiology of IBD.

Adult↗
PubMed2006

Lipidomic strategies to study structural and functional defects of ABC-transporters in cellular lipid trafficking.

The majority of the human ATP-binding cassette (ABC)-transporters function in cellular lipid trafficking and in the regulation of membrane lipid composition associating their dysfunction with human disease phenotypes related to sterol, phospholipid and fatty acid homeostasis. Based on findings from monogenetic disorders, animal models, and in vitro systems, major clues on the expression, function and cellular localization of human ABC-transporters have been gained. Here we review novel lipidomic technologies including quantitative mRNA expression monitoring by realtime RT-PCR and DNA-microarrays, lipid mass spectrometry, cellular fluorescence imaging and flow cytometry as promising tools to further define regulatory networks, lipid species patterns and subcellular domains important for ABC-transporter-mediated lipid trafficking.

ATP-Binding Cassette Transporters↗
PubMed2006

Acute myocardial infarction and a new ABCC6 mutation in a 16-year-old boy with pseudoxanthoma elasticum.

Pseudoxanthoma elasticum is caused by mutations of the ABCC6 gene. We hereby report a case of pseudoxanthoma elasticum with clinical features dominated by early coronary involvement in addition to typical skin and ocular abnormalities. A 16-year-old survivor of acute myocardial infarction with 3-vessel coronary artery disease exhibited compound heterozygosity for the well-known nonsense mutation (c.3421C>T; R1141X) in exon 24 and a novel missense mutation (c.3662G>A; R1221H) in exon 26 of the ABCC6 gene.

Adolescent↗
PubMed2006

Metabolic profiling of glycerophospholipid synthesis in fibroblasts loaded with free cholesterol and modified low density lipoproteins.

Currently, the detailed regulation of major pathways of glycerophospholipid synthesis upon cholesterol loading is largely unknown. Therefore, a detailed lipid metabolic profiling using stable isotope-labeled choline, ethanolamine, and serine was performed by quantitative electrospray ionization tandem mass spectrometry (ESI-MS/MS) in free cholesterol (FC), oxidized (Ox-LDL) and enzymatically modified LDL (E-LDL)-loaded primary human skin fibroblasts. As previously described, an adaptive induction of phosphatidylcholine (PC) synthesis via CDP-choline was found upon FC loading. In contrast to PC, CDP-ethanolamine-mediated phosphatidylethanolamine (PE) synthesis was inhibited by FC incubation. Furthermore, FC induced a shift toward polyunsaturated PE and PC species, which was mediated primarily by PE biosynthesis but not PE remodeling, whereas PC species were shifted mainly by fatty acid (FA) remodeling of existing PC. Modified lipoprotein incubation revealed rather different effects on glycerophospholipid synthesis. E-LDL greatly enhanced PC synthesis, whereas Ox-LDL did not change PC synthesis. Addition of different free FAs (FFA) with and without FC coincubation, as major components of E-LDL, clearly indicated an incorporation of FFA into newly synthesized PC and PE species as well as FFA as important driving force for PC synthesis. Because FC and FFA are known to affect lipid membrane properties including membrane curvature, these data support that CTP:phosphocholine cytidylyl-transferase activity and consequently PC synthesis are regulated by modulation of membrane characteristics at the cellular level. In conclusion, the application of high throughput metabolic profiling of major glycerophospholipid pathways by ESI-MS/MS is a powerful tool to unravel mechanisms underlying the regulation of cellular lipid metabolism.

Cholesterol↗
PubMed2006

Alteration of the pulmonary surfactant system in full-term infants with hereditary ABCA3 deficiency.

RATIONALE: ABCA3 mutations are known to cause fatal surfactant deficiency. OBJECTIVE: We studied ABCA3 protein expression in full-term newborns with unexplained respiratory distress syndrome (URDS) as well as the relevance of ABCA3 mutations for surfactant homeostasis. METHODS: Lung tissue of infants with URDS was analyzed for the expression of ABCA3 in type II pneumocytes. Coding exons of the ABCA3 gene were sequenced. Surfactant protein expression was studied by immunohistochemistry, immunoelectron microscopy, and Western blotting. RESULTS: ABCA3 protein expression was found to be greatly reduced or absent in 10 of 14 infants with URDS. Direct sequencing revealed distinct ABCA3 mutations clustering within vulnerable domains of the ABCA3 protein. A strong expression of precursors of surfactant protein B (pro-SP-B) but only low levels and aggregates of mature surfactant protein B (SP-B) within electron-dense bodies in type II pneumocytes were found. Within the matrix of electron-dense bodies, we detected precursors of SP-C (pro-SP-C) and cathepsin D. SP-A was localized in small intracellular vesicles, but not in electron-dense bodies. SP-A and pro-SP-B were shown to accumulate in the intraalveolar space, whereas mature SP-B and SP-C were reduced or absent, respectively. CONCLUSION: Our data provide evidence that ABCA3 mutations are associated not only with a deficiency of ABCA3 but also with an abnormal processing and routing of SP-B and SP-C, leading to severe alterations of surfactant homeostasis and respiratory distress syndrome. To identify infants with hereditary ABCA3 deficiency, we suggest a combined diagnostic approach including immunohistochemical, ultrastructural, and mutation analysis.

ATP-Binding Cassette Transporters↗
PubMed2006

Relation between charge-based apolipoprotein B-containing lipoprotein subfractions and remnant-like particle cholesterol levels.

OBJECTIVE: Both mildly modified LDL subfraction that carries a more-negative electric charge and remnant-like particles (RLP) are closely related to triglyceride (TG) levels. We examined the relation between the RLP-cholesterol (C) level and charge-based apolipoprotein (apo) B-containing lipoprotein subfractions as determined by capillary isotachophoresis (cITP) in patients with hypercholesterolemia. METHODS AND RESULTS: cITP apo B lipoprotein subfractions were identified by analyzing plasma depleted of the related lipoproteins. While fast-migrating triglyceride-rich lipoprotein (fTRL) subfraction contained both chylomicrons and VLDL fraction, slow TRL (sTRL) only contained VLDL. cITP fLDL also contained VLDL fraction, i.e., beta-VLDL. Levels of cITP fTRL and sTRL were significantly correlated with serum levels of TG, RLP-C, apo C-II, and C-III. Levels of cITP sTRL were also correlated with apo E. Levels of cITP fLDL were positively correlated with not only LDL-C levels but also levels of TG, RLP-C, apo C-II, C-III, and E. CONCLUSION: cITP fast LDL correlated with RLP-C levels and modified the relation between RLP-C and TG levels.

Adult↗
PubMed2006

Expression of tumor necrosis factor alpha and its receptors during cellular differentiation.

Tumor necrosis factor alpha (TNFalpha) is a potent proinflammatory cytokine also involved in cellular differentiation processes. TNFalpha and both of its receptors (TNFR1 and TNFR2) can be co-expressed on the same cell, allowing for local signaling. This study has examined the expression of all components necessary for autocrine cytokine regulation during human hematopoietic, epithelial, and mesenchymal models of cellular differentiation. Macrophage and dendritic differentiation of human peripheral blood monocytes decreased their TNFalpha and TNFR2 expression while increasing the TNFR1 mRNA. In colon epithelial cell lines (HT-29 and Caco-2) TNFalpha-, TNFR1-, and TNFR2-expression was decreased upon differentiation. No changes, however, were seen during human skin keratinocyte differentiation. TNFR1 expression was unchanged in all three mesenchymal lineages (adipogenesis, chondrogenesis, osteogenesis) tested. Differentiation decreases the TNFalpha message in adipocytes and the TNFR2 mRNA in adipocytes and osteocytes. Our results demonstrate that there is no general principle for TNFalpha signaling during conversion of cells from progenitor to a more differentiated phenotype. Paracrine signaling by TNFalpha to orchestrate different cell types during tissue development and remodeling, therefore, probably overrides the autocrine regulation of differentiation by TNFalpha. Non-signaling TNF-receptors may protect chondrocytes and osteocytes from the anti-differentiation effects of local TNFalpha production.

Adipocytes↗
PubMed2006

Induction of Raf kinase inhibitor protein contributes to macrophage differentiation.

Differential gene expression analysis of human blood monocytes has identified the Raf kinase inhibitor protein (RKIP) as a continuously upregulated gene in macrophage and dendritic cell maturation. Using realtime RT-PCR and Western blot analysis we were able to confirm the initial DNA-microarray findings of RKIP induction on mRNA and protein levels. RKIP upregulation in primary cells and overexpression in THP-1 cells did not alter ERK activity but strongly reduced the amount of the NFkappaB subunit p65 in the nucleus. mRNA levels and cell surface expression of maturation markers including the integrin CD11c and the scavenger receptor CD36 were significantly increased in RKIP transfected THP-1 cells. Our data show for the first time that RKIP is upregulated during macrophage and dendritic cell differentiation on mRNA and protein levels and we conclude that RKIP contributes to the monocytic differentiation process via inhibition of the NFkappaB signaling cascade independent from the canonical Ras/Raf/MEK/ERK pathway.

Androgen-Binding Protein↗
PubMed2006

Decreased frequencies of ABCA1 polymorphisms R219K and V771M in Hungarian patients with cerebrovascular and cardiovascular diseases.

BACKGROUND AND PURPOSE: Genetic polymorphisms in ABC transporter A1 (ABCA1) may alter the regulation of plasma high-density lipoprotein (HDL), promoting or protecting from vascular diseases. METHODS: We investigated 244 unrelated, consecutively enrolled patients with ischemic stroke, 150 patients with coronary heart disease (CHD) and 193 blood donors for allele frequencies (AFs) of three common ABCA1 polymorphisms (R219K, V771M and I883M). RESULTS: Compared to controls (30.8 +/- 4.7 and 4.9 +/- 2.2%, respectively), decreased AFs were found in both patient groups for R219K and V771M (28.7 +/- 4.1 and 3.1 +/- 1.6% in stroke, and 25.7 +/- 5.0%; 1.3 +/- 1.3% in CHD patients, respectively). In a subset of stroke patients younger than 50, both variants occurred in significantly lower frequencies (22.4 +/- 5.5 and 1.8 +/- 1.7%, respectively). Similarly, among CHD patients younger than 60, AFs of R219K and V771M (22.6 +/- 7.5 and 0 +/- 1.6%, respectively) were decreased. V771M was almost exclusively (35/36) found in individuals carrying the R219K allele. CONCLUSIONS: Our data confirm earlier observations that ABCA1 R219K and V771M polymorphisms may be associated with a protective role against CHD and extend those to another important pathologic condition, namely stroke.

ATP Binding Cassette Transporter 1↗
PubMed2006

High throughput quantification of cholesterol and cholesteryl ester by electrospray ionization tandem mass spectrometry (ESI-MS/MS).

Analysis of free cholesterol (FC) is not well suited for electrospray ionization (ESI); however, cholesteryl ester (CE) form ammonium adducts in positive ion mode and generate a fragment ion of m/z 369 upon collision-induced fragmentation. In order to allow parallel analysis of FC and CE using ESI tandem mass spectrometry (ESI-MS/MS), we developed an acetyl chloride derivatization method to convert FC to cholesteryl acetate (CE 2:0). Derivatization conditions were chosen to provide a quantitative conversion of FC to CE 2:0 without transesterification of naturally occurring CE species. FC and CE were analyzed by direct flow injection analysis using a fragment of m/z 369 in a combination of selected reaction monitoring (SRM) and precursor ion scan for FC and CE, respectively. Quantification was achieved using deuterated D(7)-FC and CE 17:0/CE 22:0 as internal standards as well as calibration lines generated by addition of FC and naturally occurring CE species to the respective sample matrix. The developed assay showed a precision and detection limit sufficient for routine analysis. A run time of 1.3 min and automated data analysis allow high throughput analysis. Loading of human skin fibroblast and monocyte derived macrophages with stable isotope labeled FC showed a potential application of this method in metabolism studies. Together with existing mass spectrometry methodologies for lipid analysis, the present methodology will provide a useful tool for clinical and biochemical studies and expands the lipid spectrum that can be analyzed from one lipid sample on a single instrumental platform.

Cholesterol↗
PubMed2006

Alterations in intestinal fatty acid metabolism in inflammatory bowel disease.

Inflammatory bowel disease (IBD) constitutes a severe intestinal disorder in developed countries with increasing incidence worldwide. Upcoming evidence indicates an important role of intestinal epithelial barrier function in the development of IBD. Fatty acids exert nutritional and protective effects on enterocytes, serve as activators of transcription and constitute precursors of inflammatory mediators. The aim of this study was to investigate differential regulation of genes involved in fatty acid uptake and endogenous fatty acid biosynthesis in IBD. Mucosal biopsy specimens from non-affected regions of the intestine were subjected to DNA microarray analysis. Gene array analysis revealed a variety of genes involved in fatty acid uptake and synthesis to be differentially expressed in ileum and colon of selected IBD patients. To verify these results, real-time RT-PCR was performed for selected regulated candidate genes in larger IBD sample numbers. In single biopsy analysis long chain acyl-CoA synthetase (ACSL) 1 and 4 were upregulated in IBD (P<0.05), while a significant decrease in fatty acid synthase expression was found in ileum and colon of ulcerative colitis patients (P<0.001). Expression of the transcription factor liver X receptor (LXR) which was previously shown to induce fatty acid synthase gene expression was not altered on mRNA level in IBD. However, in cell culture experiments using the human intestinal cell line LS174T induction of fatty acid synthase by the LXR ligand T0901317 was inhibited by TNFalpha. Moreover, these experiments indicated a decrease of LXR protein levels by TNFalpha treatment. These data suggest that the decrease of fatty acid synthase expression in ulcerative colitis patients could be at least partially due to a loss of LXR expression and function in the presence of pro-inflammatory cytokines. Observed alterations in expression of genes of fatty acid metabolism may contribute to the pathophysiology of ulcerative colitis.

Animals↗
PubMed2006

High density lipoprotein modulates platelet function.

BACKGROUND: Platelet activation by atherogenic lipoproteins can be antagonized by high density lipoprotein (HDL), probably via interaction with the ATP-binding cassette transporter A1 (ABCA1). METHODS: ABCA1 expression and its association with cholesterol rich membrane domains was analyzed by mRNA and Western blot analysis. HDL effects on platelet receptor clustering were analyzed by flow cytometric analysis of fluorescence resonance energy transfer between fluorochrome-labeled antibodies. RESULTS: ABCA1 expression increased upon megakaryocytic differentiation of human stem cells and ABCA1 protein partially associated to LubroIWX-resistant membrane domains. Plasma HDL-cholesterol in healthy donors negatively correlated to the platelet membrane cholesterol content. Receptor cluster analysis revealed a decrease in the association of Gplb and FcgammaRII upon incubation of platelets with HDL3. CONCLUSION: Our results suggest that HDL modulates platelet reactivity by altering lipid raft associated receptor clustering.

ATP Binding Cassette Transporter 1↗
PubMed2006

Ezetimib influences the expression of raft-associated antigens in human monocytes.

Aminopeptidase N (CD13) was recently identified as a molecular target of the cholesterol absorption inhibitor Ezetimib. Regarding that CD13 is expressed in lipid rafts of monocytic cells, we have investigated whether Ezetimib influences raft function in these cells. Expression of raft-associated antigens (CD11b, CD13, CD14, CD16, CD36, and CD64) was followed by flow cytometry and/or immunoblot in human monocyte-derived macrophages in response to in vitro administration of Ezetimib. Cellular redistribution of CD13 was assessed by confocal imaging. Ezetimib significantly decreased the surface expression of CD13, CD16, CD64, and CD36; furthermore, it induced a shift of CD13 from plasma membrane to intracellular vesicles, and thus it quite likely modulated monocytic raft-assembly.

Anticholesteremic Agents↗
PubMed2006

E-LDL and Ox-LDL differentially regulate ceramide and cholesterol raft microdomains in human Macrophages.

Atherosclerosis is characterized by the generation of lipid-loaded macrophage-derived foam cells. To study the effect of different types of atherogenic lipoproteins, human macrophages were loaded with enzymatically degraded low density lipoprotein (E-LDL) or oxidized LDL (Ox-LDL). Cellular cholesterol content was increased by E-LDL, whereas Ox-LDL increased the ceramide content. Cell surface expression analysis by flow cytometry and confocal microscopy revealed that Ox-LDL increased ceramide and lactosylceramide expression compared to E-LDL loading and induced ceramide rafts, whereas loading with E-LDL induced cholesterol-rich microdomains. Formation of different rafts may have consequences for raft associated signaling in cholesterol homeostasis and apoptosis in human macrophages.

Antigens, CD↗
PubMed2006

Regulation of surface CD163 expression and cellular effects of receptor mediated hemoglobin-haptoglobin uptake on human monocytes and macrophages.

Local dysregulation of iron metabolism is suggested to contribute to atherosclerotic lesion development through hemoglobin scavenging pathways. We evaluated the effects of CD163-mediated uptake of hemoglobin-haptoglobin (HbHp) complexes on surface CD163 and intracellular heme oxygenase-1 expression and the secretion of pro- and antiinflammatory cytokines by macrophages. We found that increased availability of HbHp complexes triggers the upregulation of surface CD163, and also results in a dose-dependent secretion of IL-6 and IL-10.

Antigens, CD↗