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Biomedical subjects

Andreas Schober

Publications and source records attributed to Andreas Schober.

At least 19 recordsLinked to original sources
PubMed2007

An active site tyrosine residue is essential for amidohydrolase but not for esterase activity of a class 2 histone deacetylase-like bacterial enzyme.

HDACs (histone deacetylases) are considered to be among the most important enzymes that regulate gene expression in eukaryotic cells acting through deacetylation of epsilon-acetyl-lysine residues within the N-terminal tail of core histones. In addition, both eukaryotic HDACs as well as their bacterial counterparts were reported to also act on non-histone targets. However, we are still far from a comprehensive understanding of the biological activities of this ancient class of enzymes. In the present paper, we studied in more detail the esterase activity of HDACs, focussing on the HDAH (histone deacetylase-like amidohydrolase) from Bordetella/Alcaligenes strain FB188. This enzyme was classified as a class 2 HDAC based on sequence comparison as well as functional data. Using chromogenic and fluorogenic ester substrates we show that HDACs such as FB188 HDAH indeed have esterase activity that is comparable with those of known esterases. Similar results were obtained for human HDAC1, 3 and 8. Standard HDAC inhibitors were able to block both activities with similar IC(50) values. Interestingly, HDAC inhibitors such as suberoylanilide hydroxamic acid (SAHA) also showed inhibitory activity against porcine liver esterase and Pseudomonas fluorescens lipase. The esterase and the amidohydrolase activity of FB188 HDAH both appear to have the same substrate specificity concerning the acyl moiety. Interestingly, a Y312F mutation in the active site of HDAH obstructed amidohydrolase activity but significantly improved esterase activity, indicating subtle differences in the mechanism of both catalytic activities. Our results suggest that, in principle, HDACs may have other biological roles besides acting as protein deacetylases. Furthermore, data on HDAC inhibitors affecting known esterases indicate that these molecules, which are currently among the most promising drug candidates in cancer therapy, may have a broader target profile requiring further exploration.

Alcaligenes↗
PubMed2006

GDNF applied to the MPTP-lesioned nigrostriatal system requires TGF-beta for its neuroprotective action.

GDNF is a potent neurotrophic factor for nigrostriatal dopaminergic neurons in vitro and in animal models of Parkinson's disease (PD), but has largely failed when tested in therapeutic applications in human PD. We report here that GDNF requires transforming growth factor-beta (TGF-beta) to elicit its neurotrophic activity. Lesioning the mouse nigrostriatal system with MPTP significantly upregulates striatal TGF-beta2 mRNA levels. As expected, GDNF protects against the destructive effects of MPTP, including losses of TH-ir nigral neurons, striatal dopamine and TH-ir fibers. Application of antibodies neutralizing all three TGF-beta isoforms to the MPTP-lesioned striatum abolishes the neurotrophic effect of GDNF. We show that TGF-beta antibodies are not toxic and do not interfere with retrograde transport of iodinated GDNF, suggesting that TGF-beta antibodies do not impair internalization and retrograde trafficking of GDNF. We conclude that striatal TGF-beta may be essential for permitting exogenous GDNF to act as a neuroprotective factor.

1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine↗
PubMed2006

Indium-111 oxine labelling affects the cellular integrity of haematopoietic progenitor cells.

PURPOSE: Cell-based therapy by transplantation of progenitor cells has emerged as a promising development for organ repair, but non-invasive imaging approaches are required to monitor the fate of transplanted cells. Radioactive labelling with (111)In-oxine has been used in preclinical trials. This study aimed to validate (111)In-oxine labelling and subsequent in vivo and ex vivo detection of haematopoietic progenitor cells. METHODS: Murine haematopoietic progenitor cells (10(6), FDCPmix) were labelled with 0.1 MBq (low dose) or 1.0 MBq (high dose) (111)In-oxine and compared with unlabelled controls. Cellular retention of (111)In, viability and proliferation were determined up to 48 h after labelling. Labelled cells were injected into the cavity of the left or right cardiac ventricle in mice. Scintigraphic images were acquired 24 h later. Organ samples were harvested to determine the tissue-specific activity. RESULTS: Labelling efficiency was 75 +/- 14%. Cellular retention of incorporated (111)In after 48 h was 18 +/- 4%. Percentage viability after 48 h was 90 +/- 1% (control), 58 +/- 7% (low dose) and 48 +/- 8% (high dose) (p<0.0001). Numbers of viable cells after 48 h (normalised to 0 h) were 249 +/- 51% (control), 42 +/- 8% (low dose) and 32 +/- 5% (high dose) (p<0.0001). Cells accumulated in the spleen (86.6 +/- 27.0% ID/g), bone marrow (59.1 +/- 16.1% ID/g) and liver (30.3 +/- 9.5% ID/g) after left ventricular injection, whereas most of the cells were detected in the lungs (42.4 +/- 21.8% ID/g) after right ventricular injection. CONCLUSION: Radiolabelling of haematopoietic progenitor cells with (111)In-oxine is feasible, with high labelling efficiency but restricted stability. The integrity of labelled cells is significantly affected, with substantially reduced viability and proliferation and limited migration after systemic transfusion.

Animals↗
PubMed2006

Expression pattern and functional characterization of connexin29 in transgenic mice.

Using newly generated transgenic mice in which the coding region of the connexin29 (Cx29) gene was replaced by the lacZ reporter gene, we confirmed previous immunochemical results that Cx29 is expressed in Schwann cells, oligodendrocytes and Bergmann glia cells. In addition, we detected lacZ/Cx29 in Schwann cells of the sciatic nerve and in particular of the spiral ganglion in the inner ear, as well as at low abundance in the stria vascularis. Furthermore, we found lacZ/Cx29 expression in nonmyelinating Schwann cells of the adrenal gland, in chondrocytes of intervertebral discs and the epiphysis of developing bones. Electron microscopic analyses of myelin sheaths in the central and peripheral nervous system of Cx29-deficient mice detected no abnormalities. The nerve conduction in the sciatic nerve of adult Cx29-deficient mice and the auditory brain stem response as well as visually evoked potentials in 4- to 10-week-old Cx29-deficient mice were not different from wild-type littermate controls. Thus, in contrast to connexin32 and connexin47, which are also expressed in myelinating cells, Cx29 does not contribute to the function of myelin in adult mice.

Adrenal Medulla↗
PubMed2006

Substrate and inhibitor specificity of class 1 and class 2 histone deacetylases.

Histone deacetylases (HDACs) are key enzymes in the transcriptional regulation of gene expression in eukaryotic cells. In recent years HDACs have attracted considerable attention as promising new targets in anticancer therapy. Currently, different histone deacetylase subtypes are divided into four groups denoted as classes 1-4. Here, we compare in more detail representatives of class 1 HDACs and FB188 HDAH as a close bacterial homologue of class 2 HDAC6, in regard of substrate and inhibitor specificity. Structure comparison is used to identify candidate regions responsible for observed specificity differences. Knowledge of these structural elements expedite studies on the biochemical role of different HDAC subtypes as well as the development of highly selective HDAC inhibitors as antitumor agents.

Amino Acid Sequence↗
PubMed2006

Loss of leukemia inhibitory factor receptor beta or cardiotrophin-1 causes similar deficits in preganglionic sympathetic neurons and adrenal medulla.

Leukemia inhibitory factor (LIF) receptor beta (LIFRbeta) is a receptor for a variety of neurotrophic cytokines, including LIF, ciliary neurotrophic factor (CNTF), and cardiotrophin-1 (CT-1). These cytokines play an essential role for the survival and maintenance of developing and postnatal somatic motoneurons. CNTF may also serve the maintenance of autonomic, preganglionic sympathetic neurons (PSNs) in the spinal cord, as suggested by its capacity to prevent their death after destruction of one of their major targets, the adrenal medulla. Although somatic motoneurons and PSNs share a common embryonic origin, they are distinct in several respects, including responses to lesions. We have studied PSNs in mice with targeted deletions of the LIFRbeta or CT-1 genes, respectively. We show that LIF, CNTF, and CT-1 are synthesized in embryonic adrenal gland and spinal cord and that PSNs express LIFRbeta. In embryonic day 18.5 LIFRbeta (-/-) and CT-1 (-/-) mice, PSNs were reduced by approximately 20%. PSNs projecting to the adrenal medulla were more severely affected (-55%). Although LIFRbeta (-/-) mice revealed normal numbers of adrenal chromaffin cells and axons terminating on chromaffin cells, levels of adrenaline and numbers of adrenaline-synthesizing cells were significantly reduced. We conclude that activation of LIFRbeta is required for normal development of PSNs and one of their prominent targets, the adrenal medulla. Thus, both somatic motoneurons and PSNs in the spinal cord depend on LIFRbeta signaling for their development and maintenance, although PSNs seem to be overall less affected than somatic motoneurons by LIFRbeta deprivation.

Actins↗
PubMed2006

Reduction of the aortic inflammatory response in spontaneous atherosclerosis by blockade of macrophage migration inhibitory factor (MIF).

Atherosclerosis is an inflammatory response of the arterial wall to "injury", which is prominently driven by cytokines. The inflammatory mediator macrophage migration inhibitory factor (MIF) is a unique cytokine that was recently associated with atherogenesis. Here, we have investigated whether MIF has a role in spontaneous atherosclerosis by studying apolipoprotein E-deficient (ApoE(-/-)) mice treated with neutralizing anti-MIF monoclonal antibody and comparison with isotype IgG-treated controls. After 14 weeks, the aortas and heart valves were analyzed for inflammatory status, macrophage content and plaque areas. MIF expression in the aortic wall was elevated upon spontaneous atherogenesis, with foam cells representing a major source. Of note, MIF blockade led to a marked reduction in intimal Mac-1-positive macrophages. Similarly, treatment with anti-MIF antibody led to a reduction of a variety of inflammatory mediators typically associated with atherosclerosis including the circulating levels of fibrinogen, MIF and IL-6. Importantly, the local aortic expression of ICAM-1, MMP-2, TNF, IL-12, and CD40L was reduced by MIF blockade, as were the levels of the phospho-c-Jun and C/EBPbeta transcription factors. The observed strong reduction of inflammatory parameters by anti-MIF treatment was associated with a small, yet non-significant, reduction in aortic plaque area. Thus, although MIF's role is not directly linked to plaque volume expansion, in this mouse model of spontaneous atherogenesis, MIF plays an important role in intimal inflammation.

Animals↗
PubMed2006

SDF-1alpha-mediated tissue repair by stem cells: a promising tool in cardiovascular medicine?

Circulating smooth muscle progenitor cells have been identified as a source for neointimal smooth muscle cells after various types of injuries to the vessel wall contributing to neointimal hyperplasia, implying a fundamental role of these progenitor cells in the vascular response to injury. Recent studies have provided insight into the molecular mechanisms of mobilization and local recruitment of smooth muscle progenitor cells. The CXC chemokine SDF-1alpha and its receptor CXCR4 have been identified as the central signaling axis regulating the homing of smooth muscle progenitor cells into the injured vessel wall. This review discusses the unique biologic functions of this chemokine and its contribution to stem-cell-based vascular repair and disease.

Animals↗
PubMed2005

Peripheral CD34+ cells and the risk of in-stent restenosis in patients with coronary heart disease.

In-stent restenosis represents the major limitation of percutaneous coronary revascularization. The underlying neointimal hyperplasia mainly consists of smooth muscle cells (SMCs), which can be derived from bone marrow cells. We hypothesized that changes in the peripheral progenitor cell counts after coronary stenting may predict the development of restenosis. We prospectively studied men with atherosclerotic coronary artery disease who had undergone successful elective stenting of solitary target lesions (n = 17). Peripheral blood samples were drawn at baseline (before stenting) and 1 day after stenting. The CD34+ cell count was determined by flow cytometry. Follow-up quantitative coronary angiography was performed after 8.1 +/- 2.6 months. Except for longer primary lesions in patients with angiographic restenosis, no significant differences in patient and lesion characteristics were seen. The rate of restenosis (75% vs 11%, p = 0.015) and the extent of diameter stenosis at follow-up (56.9 +/- 26.9% vs 26.5 +/- 16.5%, p = 0.012) were higher in patients with a postprocedural increase in CD34+ cells than in those with a decrease in CD34+ cells. Postprocedural CD34+ cell counts were increased in patients with restenosis but decreased in those without restenosis (p = 0.002). A robust correlation was seen between the change in CD34+ cells and late lumen loss (r = 0.65, p <0.005). In a multivariate regression model, the change in CD34+ cells, lesion length, and preprocedural minimal lumen diameter independently predicted for late lumen loss. In conclusion, an increase in circulating CD34+ cells after coronary stenting constitutes an independent risk factor predicting in-stent restenosis and may be suggestive of their involvement in neointimal hyperplasia.

Adult↗
PubMed2005

SDF-1alpha/CXCR4 axis is instrumental in neointimal hyperplasia and recruitment of smooth muscle progenitor cells.

Recent evidence infers a contribution of smooth muscle cell (SMC) progenitors and stromal cell-derived factor (SDF)-1alpha to neointima formation after arterial injury. Inhibition of plaque area and SMC content in apolipoprotein E-deficient mice repopulated with LacZ+ or CXCR4-/- BM or lentiviral transfer of an antagonist reveals a crucial involvement of local SDF-1alpha and its receptor CXCR4 in neointimal hyperplasia via recruitment of BM-derived SMC progenitors. After arterial injury, SDF-1alpha expression in medial SMCs is preceded by apoptosis and inhibited by blocking caspase-dependent apoptosis. SDF-1alpha binds to platelets at the site of injury, triggers CXCR4- and P-selectin-dependent arrest of progenitor cells on injured arteries or matrix-adherent platelets, preferentially mobilizes and recruits c-kit-/platelet-derived growth factor receptor (PDGFR)-beta+/lineage-/sca-1+ progenitors for neointimal SMCs without being required for their differentiation. Hence, the SDF-1alpha/CXCR4 axis is pivotal for vascular remodeling by recruiting a subset of SMC progenitors in response to apoptosis and in concert with platelets, epitomizing its importance for tissue repair and identifying a prime target to limit lesion development.

Animals↗
PubMed2005

Involvement of JAM-A in mononuclear cell recruitment on inflamed or atherosclerotic endothelium: inhibition by soluble JAM-A.

OBJECTIVE: The junctional adhesion molecule (JAM)-A on endothelium contributes to the inflammatory recruitment of mononuclear cells involving engagement of its integrin receptor lymphocyte function-associated antigen (LFA)-1. It is unknown whether these functions can be inhibited by soluble forms of JAM-A, whether JAM-A is expressed on atherosclerotic endothelium, and whether it participates in atherogenic recruitment of mononuclear cells. METHODS AND RESULTS: Adhesion assays revealed that LFA-1-mediated binding of mononuclear cells to intercellular adhesion molecule (ICAM)-1 or cytokine-costimulated endothelium was dose-dependently inhibited by soluble JAM-A.Fc (sJAM-A.Fc). Similarly, sJAM-A.Fc reduced stromal cell-derived factor (SDF)-1alpha-triggered transendothelial chemotaxis of activated T cells and their SDF-1alpha-triggered arrest on cytokine-costimulated endothelium under flow conditions. Immunofluorescence analysis revealed an upregulation of JAM-A on early atherosclerotic endothelium of carotid arteries from apolipoprotein E-deficient (apoE-/-) mice fed an atherogenic diet. In ex vivo perfusion assays, pretreatment of mononuclear cells with sJAM-A.Fc inhibited their very late antigen (VLA)-4-independent accumulation on atherosclerotic endothelium of these arteries. CONCLUSIONS: Soluble forms of JAM-A can be effectively applied to inhibit distinct steps of mononuclear cell recruitment on inflamed or atherosclerotic endothelium. In conjunction with its expression on atherosclerotic endothelium, this suggests a functional contribution of JAM-A to atherogenesis.

Animals↗
PubMed2005

MPTP treatment impairs tyrosine hydroxylase immunopositive fibers not only in the striatum, but also in the amygdala.

Parkinson's disease (PD) is characterized by a triad of symptoms (tremor, rigidity, and bradykinesia). Aside from this, emotional deficits are known to be associated with PD. A key structure of emotional processing is the amygdala. Emotional deficits seen in PD might be due to alterations in the catecholaminergic innervation of this limbic structure. We therefore examined whether 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) applied to C57/BL6 mice (an animal model of PD) affects the density of tyrosine hydroxylase (TH) immunoreactive fibers in the amygdala as it does in the striatum. MPTP treatment caused a prominent reduction in dopamine levels (about -70%) in the striatum (determined by high-performance liquid chromatography and electrochemical detection), accompanied by massive losses of TH-positive fibers in the striatum (-48.3%). Moreover, MPTP treatment caused prominent reductions of TH-positive fiber densities in the basolateral, lateral and central nucleus of the amygdala (about -20%). These results may provide the morphological basis for behavioral studies analyzing altered emotional responses in animal models of PD.

1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine↗
PubMed2004

Oxidized phospholipids trigger atherogenic inflammation in murine arteries.

OBJECTIVE: Lipoprotein-derived phospholipid oxidation products have been implicated as candidate triggers of the inflammatory process in atherosclerosis. However, in vivo evidence regarding the impact of oxidized phospholipids on the artery wall thus far has been elusive. Therefore, the aim of this study was to investigate if structurally defined oxidized phospholipids induce expression of atherogenic chemokines and monocyte adhesion in intact murine arteries. METHODS AND RESULTS: To model the accumulation of oxidized phospholipids in the arterial wall, oxidized 1-palmitoyl-2-arachidonoyl-sn-3-glycero-phosphorylcholine (OxPAPC) was topically applied to carotid arteries in mice using pluronic gel. Using quantitative reverse-transcriptase polymerase chain reaction (PCR) and immunohistochemistry, we show that OxPAPC induced a set of atherosclerosis-related genes, including monocyte chemotactic protein 1 (MCP-1) and keratinocyte-derived chemokine (KC), tissue factor (TF), interleukin 6 (IL-6), heme oxygenase 1 (HO-1), and early growth response 1 (EGR-1). OxPAPC-regulated chemokines were also expressed in atherosclerotic lesions of apolipoprotein E-deficient (ApoE-/-) mice. In isolated perfused carotid arteries, OxPAPC triggered rolling and firm adhesion of monocytes in a P-selectin and KC-dependent manner. CONCLUSIONS: Oxidized phospholipids contribute to vascular inflammation in murine arteries in vivo, initiating atherogenic chemokine expression that leads to monocyte adhesion; therefore, they can be regarded as triggers of the inflammatory process in atherosclerosis.

Animals↗
PubMed2004

Crucial role of the CCL2/CCR2 axis in neointimal hyperplasia after arterial injury in hyperlipidemic mice involves early monocyte recruitment and CCL2 presentation on platelets.

Monocyte chemoattractant protein-1 (also known as CC chemokine ligand 2 [CCL2]) and its receptor CC chemokine receptor 2 (CCR2) play a central role in the inflammatory response and neointimal formation after vascular injury. In the context of hyperlipidemia, this appears to involve neointimal monocyte infiltration. Hence, we investigated the function of the CCL2/CCR2 axis in early monocyte recruitment to injured arteries. Wire-induced injury of the carotid artery in apoE-/- mice caused a rapid increase of JE/CCL2 protein in the vessel wall peaking at 24 hours after injury, whereas serum JE/CCL2 was increased solely at 6 hours and blood cell-associated levels were unaltered, as demonstrated by enzyme-linked immunosorbent assay. Immunohistochemistry revealed intense staining for JE/CCL2 in smooth muscle cells (SMCs) and in association with platelets adherent to the denuded vessel wall 24 hours after injury. In vitro, exogenous or SMC-derived JE/CCL2 binds to the platelet surface and triggers monocyte arrest on adherent platelets but not on SMCs in flow assays. Accordingly, monocyte arrest in ex vivo perfused apoE-/- carotid arteries isolated 24 hours after injury was profoundly inhibited by pretreatment with a JE/CCL2 antibody. In CCR2-/-/apoE-/- mice, neointimal plaque area was reduced by 47% compared with CCR2+/+/apoE-/- mice. Moreover, CCR2 deletion markedly decreased neointimal macrophage content while expanding SMC content. Vascular JE/CCL2 expressed by SMCs and immobilized by adherent platelets after endothelial denudation is crucial for mediating early monocyte recruitment to injured arteries in hyperlipidemic mice. This mechanism may explain reduced neointimal macrophage infiltration and lesion formation in CCR2-deficient apoE-/- mice.

Animals↗
PubMed2004

Involvement of growth differentiation factor-15/macrophage inhibitory cytokine-1 (GDF-15/MIC-1) in oxLDL-induced apoptosis of human macrophages in vitro and in arteriosclerotic lesions.

Growth differentiation factor-15/macrophage inhibitory cytokine-1 (GDF-15/MIC-1) is a new member of the transforming growth factor beta (TGF-beta) superfamily, which has most recently been found in activated macrophages (MPhi). We have now investigated GDF-15/MIC-1 in human MPhi after exposure to oxidized low-density lipoproteins (oxLDL) related mediators in vitro and in arteriosclerotic carotid arteries. Using RT-PCR and Western blotting a pronounced induction of GDF-15/MIC-1 expression by oxLDL, C6-ceramide, tumor necrosis factor (TNFalpha) and hydrogen peroxide (H2O2) was found in cultured human MPhi. In 11 human arteriosclerotic carotid arteries, immunohistochemical analyses supported by computer-assisted morphometry and regression analyses demonstrated a significant colocalization of GDF-15/MIC-1 immunoreactivity (IR) with oxLDL IR and manganese superoxide dismutase (MnSOD) IR in CD68 immunoreactive (ir) MPhi, which were also expressing AIF-IR (apoptosis-inducing factor), caspase-3-IR (CPP32), PARP-IR, c-Jun/AP-1-IR and p53-IR. Our data suggest that GDF-15/MIC-1 is inducible in human MPhi by oxLDL and its mediators in vitro and is supposed to contribute to oxidative stress dependent consequences in arteriosclerotic plaques, e.g. modulating apoptosis and inflammatory processes in activated MPhi.

Antigens, CD↗
PubMed2004

Blockade of keratinocyte-derived chemokine inhibits endothelial recovery and enhances plaque formation after arterial injury in ApoE-deficient mice.

OBJECTIVE: We evaluated the involvement of keratinocyte-derived chemokine (KC) in neointimal hyperplasia and endothelial repair after arterial injury. METHODS AND RESULTS: Expression of KC was detected by immunohistochemistry in carotid arteries of apolipoprotein E-deficient (apoE-/-) mice not earlier than 2 weeks after wire-injury. Double immunofluorescence staining revealed a colocalization of KC with Mac-2-positive macrophages. Immunoreactivity for KC and its receptor CXCR2 was detectable in regenerating CD31-positive endothelial cells. Treatment of apoE-/- mice with a blocking monoclonal antibody (mAb) to KC after carotid injury for 3 weeks substantially increased neointimal plaque area compared with isotype control-treated or untreated mice. As assessed by luminal CD31 or VE-cadherin and Evans blue staining, neutralization of KC inhibited endothelial recovery in injured arteries, whereas macrophage and smooth muscle cell content were unaffected. In vitro, treatment with KC mAb, a blocking CXCR2 mAb, or the CXCR2 antagonist 8-73GRO-alpha delayed KC-mediated endothelial cell chemotaxis and wound repair of endothelial monolayers after scratch injury. Conversely, addition of exogenous KC accelerated wound repair in a CXCR2-dependent manner. CONCLUSIONS: Neutralization of KC increased plaque formation and delayed endothelial recovery after arterial injury, without affecting neointimal monocyte infiltration. As an underlying mechanism, KC was involved in promoting CXCR2-mediated endothelial chemotaxis and wound repair.

Animals↗