Karlheinz Peter

Publications of Karlheinz Peter

• Implementation of proteomic biomarkers: making it work.

  Authors: Harald Mischak, John P A Ioannidis, Angel Argiles, Teresa K Attwood, Erik Bongcam-Rudloff, Mark Broenstrup, Aristidis Charonis, George P Chrousos, Christian Delles, Anna Dominiczak [......] Frederik Persson, Karlheinz Peter, Hans H Riese, Peter Rossing, Naveed Sattar, Goce Spasovski, Visith Thongboonkerd, Raymond Vanholder, Joost P Schanstra, Antonia Vlahou

  European journal of clinical investigation. 03/2012;

  Eur J Clin Invest 2012 ABSTRACT: While large numbers of proteomic biomarkers have been described, they are generally not implemented in medical practice. We have investigated the reasons for thisEur J Clin Invest 2012 ABSTRACT: While large numbers of proteomic biomarkers have been described, they are generally not implemented in medical practice. We have investigated the reasons for this shortcoming, focusing on hurdles downstream of biomarker verification, and describe major obstacles and possible solutions to ease valid biomarker implementation. Some of the problems lie in suboptimal biomarker discovery and validation, especially lack of validated platforms with well-described performance characteristics to support biomarker qualification. These issues have been acknowledged and are being addressed, raising the hope that valid biomarkers may start accumulating in the foreseeable future. However, successful biomarker discovery and qualification alone does not suffice for successful implementation. Additional challenges include, among others, limited access to appropriate specimens and insufficient funding, the need to validate new biomarker utility in interventional trials, and large communication gaps between the parties involved in implementation. To address this problem, we propose an implementation roadmap. The implementation effort needs to involve a wide variety of stakeholders (clinicians, statisticians, health economists, and representatives of patient groups, health insurance, pharmaceutical companies, biobanks, and regulatory agencies). Knowledgeable panels with adequate representation of all these stakeholders may facilitate biomarker evaluation and guide implementation for the specific context of use. This approach may avoid unwarranted delays or failure to implement potentially useful biomarkers, and may expedite meaningful contributions of the biomarker community to healthcare.

• Microparticles: major transport vehicles for distinct microRNAs in circulation.

  Authors: Philipp Diehl, Alba Fricke, Laura Sander, Johannes Stamm, Nicole Bassler, Nay Htun, Mark Ziemann, Thomas Helbing, Assam El-Osta, Jeremy B M Jowett, Karlheinz Peter

  Cardiovascular research. 03/2012; 93(4):633-44.

  Circulating microRNAs (miRNAs) have attracted major interest as biomarkers for cardiovascular diseases. Since RNases are abundant in circulating blood, there needs to be a mechanism protecting miRNAsCirculating microRNAs (miRNAs) have attracted major interest as biomarkers for cardiovascular diseases. Since RNases are abundant in circulating blood, there needs to be a mechanism protecting miRNAs from degradation. We hypothesized that microparticles (MP) represent protective transport vehicles for miRNAs and that these are specifically packaged by their maternal cells. Conventional plasma preparations, such as the ones used for biomarker detection, are shown to contain substantial numbers of platelet-, leucocyte-, and endothelial cell-derived MP. To analyse the widest spectrum of miRNAs, Next Generation Sequencing was used to assess miRNA profiles of MP and their corresponding stimulated and non-stimulated cells of origin. THP-1 (monocytic origin) and human umbilical vein endothelial cell (HUVEC) MP were used for representing circulating MP at a high purity. miRNA profiles of MP differed significantly from those of stimulated and non-stimulated maternal THP-1 cells and HUVECs, respectively. Quantitative reverse transcription-polymerase chain reaction of miRNAs which have been associated with cardiovascular diseases also demonstrated significant differences in miRNA profiles between platelets and their MP. Notably, the main fraction of miRNA in plasma was localized in MP. Furthermore, miRNA profiles of MP differed significantly between patients with stable and unstable coronary artery disease. Circulating MP represent transport vehicles for large numbers of specific miRNAs, which have been associated with cardiovascular diseases. miRNA profiles of MP are significantly different from their maternal cells, indicating an active mechanism of selective 'packaging' from cells into MP. These findings describe an interesting mechanism for transferring gene-regulatory function from MP-releasing cells to target cells via MP circulating in blood.

• An approach towards molecular imaging of activated platelets allows imaging of symptomatic human carotid plaques in a new model of a tissue flow chamber.

  Authors: Dominik von Elverfeldt, Mirko Meißner, Karlheinz Peter, Dominik Paul, Fabian Meixner, Irene Neudorfer, Annette Merkle, Andreas Harloff, Andreas Zirlik, Joachim Schöllhorn, Michael Markl, Jürgen Hennig, Christoph Bode, Constantin von Zur Muhlen

  Contrast media & molecular imaging. 03/2012; 7(2):204-213.

  The development of magnetic resonance imaging (MRI) contrast agents targeting epitopes in atherosclerosis is of general interest. In particular, early detection of activated platelets as key playersThe development of magnetic resonance imaging (MRI) contrast agents targeting epitopes in atherosclerosis is of general interest. In particular, early detection of activated platelets as key players in plaque rupture could provide improved triage of patients. However, so far the efficiency of contrast agents targeting human pathologies can only be examined in animal experiments, which do not necessarily reflect human in vivo conditions. We therefore describe application of a contrast agent targeting activated human platelets in an MRI tissue flow chamber, allowing detection and characterization of contrast agent binding. Microparticles of iron oxide (MPIO) were conjugated to an antibody targeting ligand-induced binding sites (LIBS) on the activated platelet glycoprotein IIb/IIIa-receptor or to control antibody, resulting in LIBS-MPIO or control-MPIO contrast agent. Human endarterectomy specimens from patients with acute stroke or transient ischemic attack were imaged ex vivo before and after contrast agent perfusion using a 9.4 T MRI system. Specimens were measured under static (n = 18) or flow conditions (n = 18) in a specially designed flow chamber setup, simulating physiological conditions in a stenosed vessel. A significant MPIO-induced negative contrast was achieved in MRI by LIBS-MPIO in specimens under static and flow conditions (LIBS-MPIO vs control-MPIO: p < 0.01), and the location of LIBS-MPIO binding corresponded well between histology and MRI (p < 0.05). The number of MPIOs per platelet area on endarterectomy specimens in histology was significantly higher with LIBS-MPIO (p < 0.001). Furthermore, the intensity of contrast agent binding and signal change showed the potential to reflect the severity of clinical symptoms. LIBS-MPIO allows the detection of activated platelets on the surface of symptomatic atherosclerotic human plaques using molecular MRI. Furthermore, the MRI tissue flow chamber setup described could help to evaluate binding properties of contrast agents, and might therefore be an interesting tool for contrast agent development from animal experiments towards clinical application. Copyright © 2012 John Wiley & Sons, Ltd.

• Urine proteome analysis reflects atherosclerotic disease in an ApoE-/- mouse model and allows the discovery of new biomarkers in mouse and human atherosclerosis.

  Authors: Constantin von Zur Muhlen, Eric Schiffer, Christine Sackmann, Petra Zürbig, Irene Neudorfer, Andreas Zirlik, Nay Htun, Alexander Iphöfer, Lothar Jänsch, Harald Mischak, Christoph Bode, Yung C Chen, Karlheinz Peter

  Molecular & cellular proteomics : MCP. 02/2012;

  Non-invasive diagnosis of atherosclerosis via single biomarkers has been attempted but remains elusive. However, a previous polymarker or pattern approach of urine polypeptides in humans reflectedNon-invasive diagnosis of atherosclerosis via single biomarkers has been attempted but remains elusive. However, a previous polymarker or pattern approach of urine polypeptides in humans reflected coronary artery disease with high accuracy. The aim of the current study is to use urine proteomics in ApoE-/- mice to discover proteins with pathophysiological roles in atherogenesis and to identify urinary polypeptide patterns reflecting early stages of atherosclerosis. Urine of ApoE-/- mice either on high fat diet (HFD) or chow diet (CD) was collected over 12 weeks; urine of wild type mice on HFD was used to exclude diet-related proteome changes. Capillary electrophoresis coupled to mass spectrometry (CE-MS) of samples identified 16 polypeptides specific for ApoE-/- mice on HFD. In a blinded test set, these polypeptides allowed identification of atherosclerosis at a sensitivity of 90% and specificity of 100%, as well as monitoring of disease progression. Sequencing of the discovered polypeptides identified fragments of α1-antitrypsin, EGF, kidney androgen regulated protein (KAP) and collagen. Using immunohistochemistry, α1-antitrypsin, EGF and collagen type I were shown to be highly expressed in atherosclerotic plaques of ApoE-/- mice on HFD. Urinary excretion levels of collagen and α1-antitrypsin fragments also significantly correlated with intraplaque collagen and α1-antitrypsin content, mirroring plaque protein expression in the urine proteome. To provide further confirmation that the newly identified proteins are relevant in humans, presence of collagen type I, α1-antitrypsin, and EGF was also confirmed in human atherosclerotic disease. Urine proteome analysis in mice exemplifies the potential of a novel multimarker approach for the non-invasive detection of atherosclerosis and monitoring of disease progression. Furthermore, this approach represents a novel discovery tool for the identification of proteins relevant in murine and human atherosclerosis and thus also defines potential novel therapeutic targets.

• CD40L Deficiency Attenuates Diet-Induced Adipose Tissue Inflammation by Impairing Immune Cell Accumulation and Production of Pathogenic IgG-Antibodies.

  Authors: Dennis Wolf, Felix Jehle, Alexandra Ortiz Rodriguez, Bianca Dufner, Natalie Hoppe, Christian Colberg, Andrey Lozhkin, Nicole Bassler, Benjamin Rupprecht, Ansgar Wiedemann, Ingo Hilgendorf, Peter Stachon, Florian Willecke, Mark Febbraio, Christoph J Binder, Christoph Bode, Andreas Zirlik, Karlheinz Peter

  PloS one. 01/2012; 7(3):e33026.

  Adipose tissue inflammation fuels the metabolic syndrome. We recently reported that CD40L - an established marker and mediator of cardiovascular disease - induces inflammatory cytokine production inAdipose tissue inflammation fuels the metabolic syndrome. We recently reported that CD40L - an established marker and mediator of cardiovascular disease - induces inflammatory cytokine production in adipose cells in vitro. Here, we tested the hypothesis that CD40L deficiency modulates adipose tissue inflammation in vivo. WT or CD40L(-/-) mice consumed a high fat diet (HFD) for 20 weeks. Inflammatory cell recruitment was impaired in mice lacking CD40L as shown by a decrease of adipose tissue macrophages, B-cells, and an increase in protective T-regulatory cells. Mechanistically, CD40L-deficient mice expressed significantly lower levels of the pro-inflammatory chemokine MCP-1 both, locally in adipose tissue and systemically in plasma. Moreover, levels of pro-inflammatory IgG-antibodies against oxidized lipids were reduced in CD40L(-/-) mice. Also, circulating low-density lipoproteins and insulin levels were lower in CD40L(-/-) mice. However, CD40L(-/-) mice consuming HFD were not protected from the onset of diet-induced obesity (DIO), insulin resistance, and hepatic steatosis, suggesting that CD40L selectively limits the inflammatory features of diet-induced obesity rather than its metabolic phenotype. Interestingly, CD40L(-/-) mice consuming a low fat diet (LFD) showed both, a favorable inflammatory and metabolic phenotype characterized by diminished weight gain, improved insulin tolerance, and attenuated plasma adipokine levels. We present the novel finding that CD40L deficiency limits adipose tissue inflammation in vivo. These findings identify CD40L as a potential mediator at the interface of cardiovascular and metabolic disease.

• HMGB1 signals danger in acute coronary syndrome: Emergence of a new risk marker for cardiovascular death?

  Authors: Karlheinz Peter, Alex Bobik

  Atherosclerosis. 12/2011;

• Binding of CD40L to Mac-1's I-domain involves the EQLKKSKTL motif and mediates leukocyte recruitment and atherosclerosis--but does not affect immunity and thrombosis in mice.

  Authors: Dennis Wolf, Jan-David Hohmann, Ansgar Wiedemann, Kamila Bledzka, Hermann Blankenbach, Timoteo Marchini, Katharina Gutte, Katharina Zeschky, Nicole Bassler, Natalie Hoppe [......] Florian Willecke, Daniel Duerschmied, Constantin von zur Muhlen, Dmitry A Soloviev, Li Zhang, Christoph Bode, Edward F Plow, Peter Libby, Karlheinz Peter, Andreas Zirlik

  Circulation research. 11/2011; 109(11):1269-79.

  CD40L figures prominently in chronic inflammatory diseases such as atherosclerosis. However, since CD40L potently regulates immune function and hemostasis by interaction with CD40 receptor and theCD40L figures prominently in chronic inflammatory diseases such as atherosclerosis. However, since CD40L potently regulates immune function and hemostasis by interaction with CD40 receptor and the platelet integrin GPIIb/IIIa, its global inhibition compromises host defense and generated thromboembolic complications in clinical trials. We recently reported that CD40L mediates atherogenesis independently of CD40 and proposed Mac-1 as an alternate receptor. Here, we molecularly characterized the CD40L-Mac-1 interaction and tested whether its selective inhibition by a small peptide modulates inflammation and atherogenesis in vivo. CD40L concentration-dependently bound to Mac-1 I-domain in solid phase binding assays, and a high-affinity interaction was revealed by surface-plasmon-resonance analysis. We identified the motif EQLKKSKTL, an exposed loop between the α1 helix and the β-sheet B, on Mac-1 as binding site for CD40L. A linear peptide mimicking this sequence, M7, specifically inhibited the interaction of CD40L and Mac-1. A cyclisized version optimized for in vivo use, cM7, decreased peritoneal inflammation and inflammatory cell recruitment in vivo. Finally, LDLr(-/-) mice treated with intraperitoneal injections of cM7 developed smaller, less inflamed atherosclerotic lesions featuring characteristics of stability. However, cM7 did not interfere with CD40L-CD40 binding in vitro and CD40L-GPIIb/IIIa-mediated thrombus formation in vivo. We present the novel finding that CD40L binds to the EQLKKSKTL motif on Mac-1 mediating leukocyte recruitment and atherogenesis. Specific inhibition of CD40L-Mac-1 binding may represent an attractive anti-inflammatory treatment strategy for atherosclerosis and other inflammatory conditions, potentially avoiding the unwanted immunologic and thrombotic effects of global inhibition of CD40L.

• Amyloid Plaques Dissociate Pentameric to Monomeric C-Reactive Protein: A Novel Pathomechanism Driving Cortical Inflammation in Alzheimer's Disease?

  Authors: Frederik Strang, Amelie Scheichl, Yung-Chih Chen, Xiaowei Wang, Nay-Min Htun, Nicole Bassler, Steffen U Eisenhardt, Jonathon Habersberger, Karlheinz Peter

  Brain pathology (Zurich, Switzerland). 09/2011;

  Beta-amyloid (Aβ) plaques and local inflammation are central to the pathogenesis of Alzheimer's disease. Although an association between circulating pentameric C-reactive protein (pCRP) andBeta-amyloid (Aβ) plaques and local inflammation are central to the pathogenesis of Alzheimer's disease. Although an association between circulating pentameric C-reactive protein (pCRP) and Alzheimer's disease has been reported no pathomechanistic link has been established. We hypothesized that Aβ plaques induce the dissociation of pCRP to individual monomers (mCRP), which possess strong pro-inflammatory properties not shared with pCRP and localizing inflammation to Alzheimer's plaques. pCRP was incubated with Aβ plaques generated in vitro and with non-aggregated Aβ(42) peptide. pCRP dissociation to mCRP was found only when co-incubated with Aβ plaques. Furthermore, sections of frontal cortex from brains of patients with and without Alzheimer's disease were stained with antibodies specific for mCRP and pCRP. There was significantly more mCRP in the cortex of Alzheimer's disease patients (P ≤ 0.01). In contrast, there was no significant difference in pCRP staining. These findings establish that Aβ plaques possess a previously unrecognized potential to dissociate pentameric CRP to monomeric CRP. The existence of mCRP but not pCRP in the brains of Alzheimer's disease patients strongly indicates that this newly described biological effect of Aβ plaques is relevant in Alzheimer pathobiology; potentially localizing and amplifying inflammation via the strong pro-inflammatory effects of locally generated mCRP.

• Evidence of platelet activation at medically used hypothermia and mechanistic data indicating ADP as a key mediator and therapeutic target.

  Authors: Andreas Straub, Stefanie Krajewski, Jan David Hohmann, Erik Westein, Fu Jia, Nicole Bassler, Carly Selan, Julia Kurz, Hans Peter Wendel, Shala Dezfouli, Yuping Yuan, Harshal Nandurkar, Shaun Jackson, Michael J Hickey, Karlheinz Peter

  Arteriosclerosis, thrombosis, and vascular biology. 07/2011; 31(7):1607-16.

  Hypothermia is used in various clinical settings to inhibit ischemia-related organ damage. However, prothrombotic effects have been described as potential side effects. This study aimed to elucidateHypothermia is used in various clinical settings to inhibit ischemia-related organ damage. However, prothrombotic effects have been described as potential side effects. This study aimed to elucidate the mechanism of hypothermia-induced platelet activation and subsequent prothrombotic events and to develop preventative pharmacological strategies applicable during clinically used hypothermia. Platelet function was investigated ex vivo and in vivo at clinically used hypothermia (28°C/18°C). Hypothermic mice demonstrated increased expression of platelet activation marker P-selectin, platelet-leukocyte aggregate formation, and thrombocytopenia. Intravital microscopy of FeCl(3)-injured murine mesenteric arteries revealed increased platelet thrombus formation with hypothermia. Ex vivo flow chamber experiments indicated increased platelet-fibrinogen adhesion under hypothermia. We show that hypothermia results in reduced ADP hydrolysis via reduction of CD39 (E-NTPDase1) activity, resulting in increased levels of ADP and subsequent augmented primary and secondary platelet activation. In vivo administration of ADP receptor P(2)Y(12) antagonists and recombinant soluble CD39 prevented hypothermia-induced thrombus formation and thrombocytopenia, respectively. The platelet agonist ADP plays a key role in hypothermia-induced platelet activation. Inhibition of receptor binding or hydrolysis of ADP has the potential to protect platelets against hypothermia-induced activation. Our findings provide a rational basis for further evaluation of novel antithrombotic strategies in clinically applied hypothermia.

• Measuring oxidative burden and predicting pharmacological response in coronary artery disease patients with a novel direct activator of haem-free/oxidised sGC.

  Authors: Ingo Ahrens, Jonathon Habersberger, Nadège Baumlin, Hongwei Qian, Belinda K Smith, Johannes-Peter Stasch, Christoph Bode, Harald H H W Schmidt, Karlheinz Peter

  Atherosclerosis. 06/2011; 218(2):431-4.

  The soluble guanylate cyclase (sGC) activator Cinaciguat (BAY 58-2667) represents a novel class of drugs that selectively activate oxidised sGC. The extent of oxidised sGC depends on the patient'sThe soluble guanylate cyclase (sGC) activator Cinaciguat (BAY 58-2667) represents a novel class of drugs that selectively activate oxidised sGC. The extent of oxidised sGC depends on the patient's oxidative burden. We here describe two platelet-based assays that allow determining the extent of oxidised sGC and thus provide a basis for an individualised pharmacotherapy. Platelets obtained from patients with (n=12) and without (n=12) coronary artery disease (CAD) were examined by flow cytometry (P-selectin expression), and Western blots (vasodilator associated phosphoprotein, VASP-phosphorylation). Results were compared to maximal oxidation of sGC achieved by the oxidising agent ODQ (1H-[1,2,4]oxadiazole[4,3-a]quinoxalin-1-one). Treatment of platelets with Cinaciguat resulted in differential activation of oxidised sGC. Platelet P-selectin expression and VASP-phosphorylation revealed significant differences (p=0.012, p=0.039, respectively) between CAD and non-CAD patients. We describe platelet-based assays that allow the determination of patients' oxidative status and thus allow the prediction of pharmacological response to direct sGC activators.

• An activation-specific platelet inhibitor that can be turned on/off by medically used hypothermia.

  Authors: Denijal Topcic, Wookhyun Kim, Jessica K Holien, Fu Jia, Paul C Armstrong, Jan David Hohmann, Andreas Straub, Guy Krippner, Carolyn A Haller, Helena Domeij, Christoph E Hagemeyer, Michael W Parker, Elliot L Chaikof, Karlheinz Peter

  Arteriosclerosis, thrombosis, and vascular biology. 06/2011; 31(9):2015-23.

  Therapeutic hypothermia is successfully used, for example, in cardiac surgery to protect organs from ischemia. Cardiosurgical procedures, especially in combination with extracorporeal circulation,Therapeutic hypothermia is successfully used, for example, in cardiac surgery to protect organs from ischemia. Cardiosurgical procedures, especially in combination with extracorporeal circulation, and hypothermia itself are potentially prothrombotic. Despite the obvious need, the long half-life of antiplatelet drugs and thus the risk of postoperative bleedings have restricted their use in cardiac surgery. We describe here the design and testing of a unique recombinant hypothermia-controlled antiplatelet fusion protein with the aim of providing increased safety of hypothermia, as well as cardiac surgery. An elastin-mimetic polypeptide was fused to an activation-specific glycoprotein (GP) IIb/IIIa-blocking single-chain antibody. In silico modeling illustrated the sterical hindrance of a β-spiral conformation of elastin-mimetic polypeptide preventing the single-chain antibody from inhibiting GPIIb/IIIa at 37°C. Circular dichroism spectra demonstrated reverse temperature transition, and flow cytometry showed binding to and blocking of GPIIb/IIIa at hypothermic body temperature (≤32°C) but not at normal body temperature. In vivo thrombosis in mice was selectively inhibited at hypothermia but not at 37°C. This is the first description of a broadly applicable pharmacological strategy by which the activity of a potential drug can be controlled by temperature. In particular, this drug steerability may provide substantial benefits for antiplatelet therapy.

• Molecular magnetic resonance imaging allows the detection of activated platelets in a new mouse model of coronary artery thrombosis.

  Authors: Daniel Duerschmied, Mirko Meisner, Karlheinz Peter, Irene Neudorfer, Freya Roming, Andreas Zirlik, Christoph Bode, Dominik von Elverfeldt, Constantin von Zur Muhlen

  Investigative radiology. 05/2011; 46(10):618-23.

  : The final event leading to myocardial infarction is adhesion and activation of platelets after rupture of an atherosclerotic plaque, ending in thrombotic occlusion of the coronary artery. Imaging: The final event leading to myocardial infarction is adhesion and activation of platelets after rupture of an atherosclerotic plaque, ending in thrombotic occlusion of the coronary artery. Imaging of imminent vessel occlusion may improve patient care. The feasibility of molecular magnetic resonance imaging (MRI) for the detection of coronary artery thrombosis in mice was examined. : The left anterior descending coronary artery was exposed by lateral thoracotomy and incubated with ferric chloride to induce nonocclusive thrombosis in C57Bl/6 mice. A single chain antibody targeting ligand-induced binding sites (LIBS) of the activated glycoprotein IIb/IIIa or control antibody was conjugated to 1 μm-sized microparticles of iron oxide (MPIOs), resulting in LIBS-MPIO or control-MPIO MRI contrast agent, and injected intravenously. Hearts were subjected to histology and ex vivo MRI at 9.4 Tesla. : Thrombus size was comparable in mice injected with control-MPIO and LIBS-MPIO in histology. Significant binding of MPIOs to thrombi was observed in LIBS-MPIO-injected animals while no binding was observed in control animals (P < 0.05). In MRI, LIBS-MPIO binding to thrombi of the left anterior descending coronary artery resulted in significant MPIO-induced signal void compared with controls (P < 0.05). MRI signal void and the amount of bound contrast agent particles in histology showed a significant positive linear correlation (r = 0.939, P < 0.001). : We established a new mouse model of nonocclusive coronary artery thrombosis. LIBS-MPIO contrast agent binds to activated platelets in this model, allowing molecular MRI of coronary thrombosis. This could have important implications on the timely noninvasive detection of arterial thrombosis, helping to initiate early therapeutic interventions.

• A proteomic analysis of C-reactive protein stimulated THP-1 monocytes

  Authors: Steffen Eisenhardt, Jonathon Habersberger, Karen Oliva, Graeme Lancaster, Mustafa Ayhan, Kevin Woollard, Holger Bannasch, Greg Rice, Karlheinz Peter

  Proteome Science. 01/2011;

  Abstract Background C-reactive protein (CRP) is a predictor of cardiovascular risk. It circulates as a pentameric protein in plasma. Recently, a potential dissociation mechanism from the disc-shapedAbstract Background C-reactive protein (CRP) is a predictor of cardiovascular risk. It circulates as a pentameric protein in plasma. Recently, a potential dissociation mechanism from the disc-shaped pentameric CRP (pCRP) into single monomers (monomeric or mCRP) has been described. It has been shown that mCRP has strong pro-inflammatory effects on monocytes. To further define the role of mCRP in determining monocyte phenotype, the effects of CRP isoforms on THP-1 protein expression profiles were determined. The hypothesis to be tested was that mCRP induces specific changes in the protein expression profile of THP-1 cells that differ from that of pCRP. Methods Protein cell lysates from control and mCRP, pCRP or LPS-treated THP-1 cells were displayed using 2-dimensional SDS PAGE and compared. Differentially expressed proteins were identified by MALDI-TOF MS and confirmed by Western blotting. Results mCRP significantly up-regulates ubiquitin-activating enzyme E1, a member of the ubiquitin-proteasome system in THP-1 monocytes. Furthermore, HSP 70, alpha-actinin-4 (ACTN4) and alpha-enolase/enolase 1 were upregulated. The proteomic profile of LPS and pCRP treated monocytes differ significantly from that of mCRP. Conclusion The data obtained in this study support the hypothesis that isoform-specific effects of CRP may differentially regulate the phenotype of monocytes.

• A proteomic analysis of C-reactive protein stimulated THP-1 monocytes.

  Authors: Steffen U Eisenhardt, Jonathon Habersberger, Karen Oliva, Graeme I Lancaster, Mustafa Ayhan, Kevin J Woollard, Holger Bannasch, Greg E Rice, Karlheinz Peter

  Proteome science. 01/2011; 9(1):1.

  C-reactive protein (CRP) is a predictor of cardiovascular risk. It circulates as a pentameric protein in plasma. Recently, a potential dissociation mechanism from the disc-shaped pentameric CRPC-reactive protein (CRP) is a predictor of cardiovascular risk. It circulates as a pentameric protein in plasma. Recently, a potential dissociation mechanism from the disc-shaped pentameric CRP (pCRP) into single monomers (monomeric or mCRP) has been described. It has been shown that mCRP has strong pro-inflammatory effects on monocytes. To further define the role of mCRP in determining monocyte phenotype, the effects of CRP isoforms on THP-1 protein expression profiles were determined. The hypothesis to be tested was that mCRP induces specific changes in the protein expression profile of THP-1 cells that differ from that of pCRP. Protein cell lysates from control and mCRP, pCRP or LPS-treated THP-1 cells were displayed using 2-dimensional SDS PAGE and compared. Differentially expressed proteins were identified by MALDI-TOF MS and confirmed by Western blotting. mCRP significantly up-regulates ubiquitin-activating enzyme E1, a member of the ubiquitin-proteasome system in THP-1 monocytes. Furthermore, HSP 70, alpha-actinin-4 (ACTN4) and alpha-enolase/enolase 1 were upregulated. The proteomic profile of LPS and pCRP treated monocytes differ significantly from that of mCRP. The data obtained in this study support the hypothesis that isoform-specific effects of CRP may differentially regulate the phenotype of monocytes.

• Successful in vitro expansion and differentiation of cord blood derived CD34+ cells into early endothelial progenitor cells reveals highly differential gene expression.

  Authors: Ingo Ahrens, Helena Domeij, Denijal Topcic, Izhak Haviv, Ruusu-Maaria Merivirta, Alexander Agrotis, Ephraem Leitner, Jeremy B Jowett, Christoph Bode, Martha Lappas, Karlheinz Peter

  PloS one. 01/2011; 6(8):e23210.

  Endothelial progenitor cells (EPCs) can be purified from peripheral blood, bone marrow or cord blood and are typically defined by a limited number of cell surface markers and a few functional tests.Endothelial progenitor cells (EPCs) can be purified from peripheral blood, bone marrow or cord blood and are typically defined by a limited number of cell surface markers and a few functional tests. A detailed in vitro characterization is often restricted by the low cell numbers of circulating EPCs. Therefore in vitro culturing and expansion methods are applied, which allow at least distinguishing two different types of EPCs, early and late EPCs. Herein, we describe an in vitro culture technique with the aim to generate high numbers of phenotypically, functionally and genetically defined early EPCs from human cord blood. Characterization of EPCs was done by flow cytometry, immunofluorescence microscopy, colony forming unit (CFU) assay and endothelial tube formation assay. There was an average 48-fold increase in EPC numbers. EPCs expressed VEGFR-2, CD144, CD18, and CD61, and were positive for acetylated LDL uptake and ulex lectin binding. The cells stimulated endothelial tube formation only in co-cultures with mature endothelial cells and formed CFUs. Microarray analysis revealed highly up-regulated genes, including LL-37 (CAMP), PDK4, and alpha-2-macroglobulin. In addition, genes known to be associated with cardioprotective (GDF15) or pro-angiogenic (galectin-3) properties were also significantly up-regulated after a 72 h differentiation period on fibronectin. We present a novel method that allows to generate high numbers of phenotypically, functionally and genetically characterized early EPCs. Furthermore, we identified several genes newly linked to EPC differentiation, among them LL-37 (CAMP) was the most up-regulated gene.

• GPVI and GPIbα mediate staphylococcal superantigen-like protein 5 (SSL5) induced platelet activation and direct toward glycans as potential inhibitors.

  Authors: Houyuan Hu, Paul C J Armstrong, Elie Khalil, Yung-Chih Chen, Andreas Straub, Min Li, Juliana Soosairajah, Christoph E Hagemeyer, Nicole Bassler, Dexing Huang, Ingo Ahrens, Guy Krippner, Elizabeth Gardiner, Karlheinz Peter

  PloS one. 01/2011; 6(4):e19190.

  Staphylococcus aureus (S. aureus) is a common pathogen capable of causing life-threatening infections. Staphylococcal superantigen-like protein 5 (SSL5) has recently been shown to bind to plateletStaphylococcus aureus (S. aureus) is a common pathogen capable of causing life-threatening infections. Staphylococcal superantigen-like protein 5 (SSL5) has recently been shown to bind to platelet glycoproteins and induce platelet activation. This study investigates further the interaction between SSL5 and platelet glycoproteins. Moreover, using a glycan discovery approach, we aim to identify potential glycans to therapeutically target this interaction and prevent SSL5-induced effects. In addition to platelet activation experiments, flow cytometry, immunoprecipitation, surface plasmon resonance and a glycan binding array, were used to identify specific SSL5 binding regions and mediators. We independently confirm SSL5 to interact with platelets via GPIbα and identify the sulphated-tyrosine residues as an important region for SSL5 binding. We also identify the novel direct interaction between SSL5 and the platelet collagen receptor GPVI. Together, these receptors offer one mechanistic explanation for the unique functional influences SSL5 exerts on platelets. A role for specific families of platelet glycans in mediating SSL5-platelet interactions was also discovered and used to identify and demonstrate effectiveness of potential glycan based inhibitors in vitro. These findings further elucidate the functional interactions between SSL5 and platelets, including the novel finding of a role for the GPVI receptor. We demonstrate efficacy of possible glycan-based approaches to inhibit the SSL5-induced platelet activation. Our data warrant further work to prove SSL5-platelet effects in vivo.

• Activated platelets in carotid artery thrombosis in mice can be selectively targeted with a radiolabeled single-chain antibody.

  Authors: Timo Heidt, Friederike Deininger, Karlheinz Peter, Jürgen Goldschmidt, Annette Pethe, Christoph E Hagemeyer, Irene Neudorfer, Andreas Zirlik, Wolfgang A Weber, Christoph Bode, Philipp T Meyer, Martin Behe, Constantin von Zur Mühlen

  PloS one. 01/2011; 6(3):e18446.

  Activated platelets can be found on the surface of inflamed, rupture-prone and ruptured plaques as well as in intravascular thrombosis. They are key players in thrombosis and atherosclerosis. In thisActivated platelets can be found on the surface of inflamed, rupture-prone and ruptured plaques as well as in intravascular thrombosis. They are key players in thrombosis and atherosclerosis. In this study we describe the construction of a radiolabeled single-chain antibody targeting the LIBS-epitope of activated platelets to selectively depict platelet activation and wall-adherent non-occlusive thrombosis in a mouse model with nuclear imaging using in vitro and ex vivo autoradiography as well as small animal SPECT-CT for in vivo analysis. LIBS as well as an unspecific control single-chain antibody were labeled with (111)Indium ((111)In) via bifunctional DTPA ( = (111)In-LIBS/(111)In-control). Autoradiography after incubation with (111)In-LIBS on activated platelets in vitro (mean 3866 ± 28 DLU/mm(2), 4010 ± 630 DLU/mm(2) and 4520 ± 293 DLU/mm(2)) produced a significantly higher ligand uptake compared to (111)In-control (2101 ± 76 DLU/mm(2), 1181 ± 96 DLU/mm(2) and 1866 ± 246 DLU/mm(2)) indicating a specific binding to activated platelets; P<0.05. Applying these findings to an ex vivo mouse model of carotid artery thrombosis revealed a significant increase in ligand uptake after injection of (111)In-LIBS in the presence of small thrombi compared to the non-injured side, as confirmed by histology (49630 ± 10650 DLU/mm(2) vs. 17390 ± 7470 DLU/mm(2); P<0.05). These findings could also be reproduced in vivo. SPECT-CT analysis of the injured carotid artery with (111)In-LIBS resulted in a significant increase of the target-to-background ratio compared to (111)In-control (1.99 ± 0.36 vs. 1.1 ± 0.24; P < 0.01). Nuclear imaging with (111)In-LIBS allows the detection of platelet activation in vitro and ex vivo with high sensitivity. Using SPECT-CT, wall-adherent activated platelets in carotid arteries could be depicted in vivo. These results encourage further studies elucidating the role of activated platelets in plaque pathology and atherosclerosis and might be of interest for further developments towards clinical application.

• High-mobility group box protein 1 neutralization reduces development of diet-induced atherosclerosis in apolipoprotein e-deficient mice.

  Authors: Peter Kanellakis, Alex Agrotis, Tin Soe Kyaw, Christine Koulis, Ingo Ahrens, Shuji Mori, Hideo K Takahashi, Keyue Liu, Karlheinz Peter, Masahiro Nishibori, Alex Bobik

  Arteriosclerosis, thrombosis, and vascular biology. 11/2010; 31(2):313-9.

  High-mobility group box protein 1 (HMGB1) is a DNA-binding protein and cytokine highly expressed in atherosclerotic lesions, but its pathophysiological role in atherosclerosis is unknown. WeHigh-mobility group box protein 1 (HMGB1) is a DNA-binding protein and cytokine highly expressed in atherosclerotic lesions, but its pathophysiological role in atherosclerosis is unknown. We investigated its role in the development of atherosclerosis in ApoE-/- mice. Apolipoprotein E-deficient (ApoE-/-) mice fed a high-fat diet were administered a monoclonal anti-HMGB1 neutralizing antibody, and the effects on lesion size, immune cell accumulation, and proinflammatory mediators were assessed using Oil Red O, immunohistochemistry, and real-time polymerase chain reaction. As with human atherosclerotic lesions, lesions in ApoE-/- mice expressed HMGB1. Treatment with the neutralizing antibody attenuated atherosclerosis by 55%. Macrophage accumulation was reduced by 43%, and vascular cell adhesion molecule-1 and monocyte chemoattractant protein-1 expression was attenuated by 48% and 72%, respectively. CD11c+ dendritic cells were reduced by 65%, and the mature (CD83+) population was reduced by 60%. Treatment also reduced CD4+ cells by nearly 50%. mRNAs in lesions encoding tumor necrosis factor-α and interleukin-1β tended to be reduced. Mechanistically, HMGB1 stimulated macrophage migration in vitro and in vivo; in vivo, it markedly augmented the accumulation of F4/80+Gr-1(Ly-6C)+ macrophages and also increased F4/80+CD11b+ macrophage numbers. HMGB1 exerts proatherogenic effects augmenting lesion development by stimulating macrophage migration, modulating proinflammatory mediators, and encouraging the accumulation of immune and smooth muscle cells.

• Urinary proteomic diagnosis of coronary artery disease: identification and clinical validation in 623 individuals.

  Authors: Christian Delles, Eric Schiffer, Constantin von Zur Muhlen, Karlheinz Peter, Peter Rossing, Hans-Henrik Parving, Jane A Dymott, Ulf Neisius, Lukas U Zimmerli, Janet K Snell-Bergeon, David M Maahs, Roland E Schmieder, Harald Mischak, Anna F Dominiczak

  Journal of hypertension. 11/2010; 28(11):2316-22.

  We studied the urinary proteome in a total of 623 individuals with and without coronary artery disease (CAD) in order to characterize multiple biomarkers that enable prediction of the presence ofWe studied the urinary proteome in a total of 623 individuals with and without coronary artery disease (CAD) in order to characterize multiple biomarkers that enable prediction of the presence of CAD. Urine samples were analyzed by capillary electrophoresis coupled online to micro time-of-flight mass spectrometry. We defined a pattern of 238 CAD-specific polypeptides from comparison of 586 spot urine samples from 408 individuals. This pattern identified patients with CAD in a blinded cohort of 138 urine samples (71 patients with CAD and 67 healthy individuals) with high sensitivity and specificity (area under the receiver operator characteristic curve 87%, 95% confidence interval 81-92) and was superior to previously developed 15-marker (area under the receiver operator characteristic curve 68%, P < 0.0001) and 17-marker panels (area under the receiver operator characteristic curve 77%, P < 0.0001). The sequences of the discriminatory polypeptides include fragments of alpha-1-antitrypsin, collagen types 1 and 3, granin-like neuroendocrine peptide precursor, membrane-associated progesterone receptor component 1, sodium/potassium-transporting ATPase gamma chain and fibrinogen-alpha chain. Several biomarkers changed significantly toward the healthy signature following 2-year treatment with irbesartan, whereas short-term treatment with irbesartan did not significantly affect the polypeptide pattern. Urinary proteomics identifies CAD with high confidence and might also be useful for monitoring the effects of therapeutic interventions.

• Naturally occurring human urinary peptides for use in diagnosis of chronic kidney disease.

  Authors: David M Good, Petra Zürbig, Angel Argilés, Hartwig W Bauer, Georg Behrens, Joshua J Coon, Mohammed Dakna, Stéphane Decramer, Christian Delles, Anna F Dominiczak [......] Joost P Schanstra, Eric Schiffer, Jens-Uwe Stolzenburg, Lise Tarnow, Dan Theodorescu, Visith Thongboonkerd, Raymond Vanholder, Eva M Weissinger, Harald Mischak, Philippe Schmitt-Kopplin

  Molecular & cellular proteomics : MCP. 11/2010; 9(11):2424-37.

  Because of its availability, ease of collection, and correlation with physiology and pathology, urine is an attractive source for clinical proteomics/peptidomics. However, the lack of comparable dataBecause of its availability, ease of collection, and correlation with physiology and pathology, urine is an attractive source for clinical proteomics/peptidomics. However, the lack of comparable data sets from large cohorts has greatly hindered the development of clinical proteomics. Here, we report the establishment of a reproducible, high resolution method for peptidome analysis of naturally occurring human urinary peptides and proteins, ranging from 800 to 17,000 Da, using samples from 3,600 individuals analyzed by capillary electrophoresis coupled to MS. All processed data were deposited in an Structured Query Language (SQL) database. This database currently contains 5,010 relevant unique urinary peptides that serve as a pool of potential classifiers for diagnosis and monitoring of various diseases. As an example, by using this source of information, we were able to define urinary peptide biomarkers for chronic kidney diseases, allowing diagnosis of these diseases with high accuracy. Application of the chronic kidney disease-specific biomarker set to an independent test cohort in the subsequent replication phase resulted in 85.5% sensitivity and 100% specificity. These results indicate the potential usefulness of capillary electrophoresis coupled to MS for clinical applications in the analysis of naturally occurring urinary peptides.