Knut Rennert

Different functions of monocyte subsets in familial hypercholesterolemia: potential function of CD14+CD16+ monocytes in detoxification of oxidized LDL

The study was undertaken to investigate whether the two major monocyte subsets defined by the surface markers CD14+CD16+ and CD14++CD16− show differences in their responses to hypercholesterolemia. Monocytes were rapidly isolated from the blood of hypercholesterolemic, low‐density lipoprotein (LDL) receptor‐defective familial hypercholesterolemia (FH) patients and from control persons. Using flow cytometry and uptake, adhesion, and phagocytosis assays as well as laser scanning microscopy, we found significant differences between the monocyte subsets. FH‐CD14+CD16+ monocytes exhibit an increased uptake of oxidized LDL (oxLDL) via CD36, whereas FH‐CD14++CD16− monocytes preferentially take up native LDL (nLDL). FH‐CD14+CD16+ monocytes have an increased expression of surface proteins CD68, stabilin‐1, and CD11c and a higher adherence to activated endothelial cells in response to oxLDL and nLDL stimulation. In addition, all CD14+CD16+ monocytes have an increased ability for phagocytosis and a higher resistance to phagocytosis impairment by oxLDL compared with CD14++CD16− monocytes. We conclude that FH‐CD14+CD16+ monocytes have specialized functions in the uptake of oxLDL at activated endothelial cell surfaces, and we hypothesize that these functions are critical for the clearance of oxLDL deposits and apoptotic cells from the vessel wall under hyperlipidemic conditions.— Mosig, S., Rennert, K., Krause, S., Kzhyshkowska, J., Neunubel, K., Heller, R., Funke, H. Different functions of monocyte subsets in familial hypercholesterolemia: potential function of CD14+CD16+ monocytes in detoxification of oxidized LDL. FASEB J. 23, 866–874 (2009)

Live-cell imaging of endogenous Ras-GTP illustrates predominant Ras activation at the plasma membrane.

Ras‐GTP imaging studies using the Ras‐binding domain (RBD) of the Ras effector c‐Raf as a reporter for overexpressed Ras have produced discrepant results about the possible activation of Ras at the Golgi apparatus. We report that RBD oligomerization provides probes for visualization of endogenous Ras‐GTP, obviating Ras overexpression and the side effects derived thereof. RBD oligomerization results in tenacious binding to Ras‐GTP and interruption of Ras signalling. Trimeric RBD probes fused to green fluorescent protein report agonist‐induced endogenous Ras activation at the plasma membrane (PM) of COS‐7, PC12 and Jurkat cells, but do not accumulate at the Golgi. PM illumination is exacerbated by Ras overexpression and its sensitivity to dominant‐negative RasS17N and pharmacological manipulations matches Ras‐GTP formation assessed biochemically. Our data illustrate that endogenous Golgi‐located Ras is not under the control of growth factors and argue for the PM as the predominant site of agonist‐induced Ras activation.

Monocytes of patients with familial hypercholesterolemia show alterations in cholesterol metabolism

BackgroundElevated plasma cholesterol promotes the formation of atherosclerotic lesions in which monocyte-derived lipid-laden macrophages are frequently found. To analyze, if circulating monocytes already show increased lipid content and differences in lipoprotein metabolism, we compared monocytes from patients with Familial Hypercholesterolemia (FH) with those from healthy individuals.MethodsCholesterol and oxidized cholesterol metabolite serum levels of FH and of healthy, gender/age matched control subjects were measured by combined gas chromatography – mass spectroscopy. Monocytes from patients with FH and from healthy subjects were isolated by antibody-assisted density centrifugation. Gene expression profiles of isolated monocytes were measured using Affymetrix HG-U 133 Plus 2.0 microarrays. We compared monocyte gene expression profiles from FH patients with healthy controls using a Welch T-test with correction for multiple testing (p < 0.05; Benjamini Hochberg correction, False Discovery Rate = 0.05). The differential expression of FH associated genes was validated at the mRNA level by qRT-PCR and/or at the protein level by Western Blot or flow cytometry. Functional validation of monocyte scavenger receptor activities were done by binding assays and dose/time dependent uptake analysis using native and oxidized LDL.ResultsUsing microarray analysis we found in FH patients a significant up-regulation of 1,617 genes and a down-regulation of 701 genes compared to monocytes from healthy individuals. These include genes of proteins that are involved in the uptake, biosynthesis, disposition, and cellular efflux of cholesterol. In addition, plasma from FH patients contains elevated amounts of sterols and oxysterols. An increased uptake of oxidized as well as of native LDL by FH monocytes combined with a down-regulation of NPC1 and ABCA1 explains the lipid accumulation observed in these cells.ConclusionOur data demonstrate that circulating FH monocytes show differences in cell physiology that may contribute to the early onset of atherosclerosis in this disease.

Microfluidically supported biochip design for culture of endothelial cell layers with improved perfusion conditions

Hemodynamic forces generated by the blood flow are of central importance for the function of endothelial cells (ECs), which form a biologically active cellular monolayer in blood vessels and serve as a selective barrier for macromolecular permeability. Mechanical stimulation of the endothelial monolayer induces morphological remodeling in its cytoskeleton. For in vitro studies on EC biology culture devices are desirable that simulate conditions of flow in blood vessels and allow flow-based adhesion/permeability assays under optimal perfusion conditions. With this aim we designed a biochip comprising a perfusable membrane that serves as cell culture platform multi-organ-tissue-flow (MOTiF biochip). This biochip allows an effective supply with nutrition medium, discharge of catabolic cell metabolites and defined application of shear stress to ECs under laminar flow conditions. To characterize EC layers cultured in the MOTiF biochip we investigated cell viability, expression of EC marker proteins and cell adhesion molecules of ECs dynamically cultured under low and high shear stress, and compared them with an endothelial culture in established two-dimensionally perfused flow chambers and under static conditions. We show that ECs cultured in the MOTiF biochip form a tight EC monolayer with increased cellular density, enhanced cell layer thickness, presumably as the result of a rapid and effective adaption to shear stress by remodeling of the cytoskeleton. Moreover, endothelial layers in the MOTiF biochip express higher amounts of EC marker proteins von-Willebrand-factor and PECAM-1. EC layers were highly responsive to stimulation with TNFα as detected at the level of ICAM-1, VCAM-1 and E-selectin expression and modulation of endothelial permeability in response to TNFα/IFNγ treatment under flow conditions. Compared to static and two-dimensionally perfused cell culture condition we consider MOTiF biochips as a valuable tool for studying EC biology in vitro under advanced culture conditions more closely resembling the in vivo situation.

Gene expression in the detection of autologous blood transfusion in sports - a pilot study

The reinfusion of autologous blood to enhance performanceremains a significant problem in sports. Although allogeneicblood transfusions can be detected since 2003 [1], there is atpresent no detection method for autologous blood transfu-sions, although indirect approaches such as the biologicalpassport [2] might give indications on the illicit use of bloodtransfusions.It is well-documented that several molecular changesoccur in stored red blood cells (RBCs), commonly referred toas the ‘storage lesion’ [3–5]. We therefore hypothesize thatautologous transfusion will lead to a sudden exposure of celldetritus to the immune system causing a cellular and molecularimmune response including gene expression alterations ofwhite blood cells such as T-lymphocytes. Hence, the primaryobjective of this study was to investigate the transcriptionalresponse of T-lymphocytes after reinfusion of autologousRBCs in order to search for a theoretical model for an un-equivocal detection method of autologous blood doping. Themost significant Gene Ontology (GO) clusters of regulatedgenes at 72 h after autologous transfusion included leucocyteimmunoglobulin receptors, toll-like receptor (TLR) pathway[6], adaptive immune response and cell death/apoptosis aswell as regulation of endocytosis of surface receptors and theTLR pathway at 96 h, respectively. The quantitative reversetranscriptase polymerase chain reaction (qRT-PCR) confirmedsignificant up-regulation of TLR4, TLR5, TLR6, apoptosis-associated tyrosine kinase (AATK) [7,8] and low densitylipoprotein receptor related protein (LRP1) [9,10] at 72 h aswell as TLR6 at 96 h. Therefore, the main finding of our pilotstudy is the fact that the transfusion of autologous bloodtriggers a distinct immune reaction within the T-lymphocytesof the recipient and may aid in the development of a practicablemethod to detect autologous blood doping based on molecularimmune response measurements.

Ras activation in response to phorbol ester proceeds independently of the EGFR via an unconventional nucleotide-exchange factor system in COS-7 cells.

Ras is a major mediator of PE (phorbol ester) effects in mammalian cells. Various mechanisms for PE activation of Ras have been reported [Downward, Graves, Warne, Rayter and Cantrell (1990) Nature (London) 346, 719-723; Shu, Wu, Mosteller and Broek (2002) Mol. Cell. Biol. 22, 7758-7768; Roose, Mollenauer, Gupta, Stone and Weiss (2005) Mol. Cell. Biol. 25, 4426-4441; Grosse, Roelle, Herrlich, Höhn and Gudermann (2000) J. Biol. Chem. 275, 12251-12260], including pathways that target GAPs (GTPase-activating proteins) for inactivation and those that result in activation of GEFs (guanine nucleotide-exchange factors) Sos (son of sevenless homologue) or RasGRP (RAS guanyl releasing protein). However, a biochemical link between PE and GAP inactivation is missing and GEF stimulation is hard to reconcile with the observation that dominant-negative S17N-Ras does not compromise Ras-dependent ERK (extracellular-signal-regulated kinase) activation by PE. We have addressed this controversy and carried out an in-depth biochemical study of PE-induced Ras activation in COS-7 cells. Using a cell-permeabilization approach to monitor nucleotide exchange on Ras, we demonstrate that PE-induced Ras-GTP accumulation results from GEF stimulation. Nucleotide exchange stimulation by PE is prevented by PKC (protein kinase C) inhibition but not by EGFR [EGF (epidermal growth factor) receptor] blockade, despite the fact that EGFR inhibition aborts basal and PE-induced Shc (Src homology and collagen homology) phosphorylation and Shc-Grb2 (growth-factor-receptor-bound protein 2) association. In fact, EGFR inhibition ablates basal nucleotide exchange on Ras in growth-arrested COS-7 cells. These data disclose the existence of two separate GEF systems that operate independently from each other to accomplish PE-dependent formation of Ras-GTP and to maintain resting Ras-GTP levels respectively. We document that COS-7 cells do not express RasGRP and present evidence that the PE-responsive GEF system may involve PKC-dependent phosphorylation of Sos. More fundamentally, these observations shed new light on enigmatic issues such as the inefficacy of S17N-Ras in blocking PE action or the role of the EGFR in heterologous agonist activation of the Ras/ERK pathway.

Monocyte-induced recovery of inflammation-associated hepatocellular dysfunction in a biochip-based human liver model.

Liver dysfunction is an early event in sepsis-related multi-organ failure. We here report the establishment and characterization of a microfluidically supported in vitro organoid model of the human liver sinusoid. The liver organoid is composed of vascular and hepatocyte cell layers integrating non-parenchymal cells closely reflecting tissue architecture and enables physiological cross-communication in a bio-inspired fashion. Inflammation-associated liver dysfunction was mimicked by stimulation with various agonists of toll-like receptors. TLR-stimulation induced the release of pro- and anti-inflammatory cytokines and diminished expression of endothelial VE-cadherin, hepatic MRP-2 transporter and apolipoprotein B (ApoB), resulting in an inflammation-related endothelial barrier disruption and hepatocellular dysfunction in the liver organoid. However, interaction of the liver organoid with human monocytes attenuated inflammation-related cell responses and restored MRP-2 transporter activity, ApoB expression and albumin/urea production. The cellular events observed in the liver organoid closely resembled pathophysiological responses in the well-established sepsis model of peritoneal contamination and infection (PCI) in mice and clinical observations in human sepsis. We therefore conclude that this human liver organoid model is a valuable tool to investigate sepsis-related liver dysfunction and subsequent immune cell-related tissue repair/remodeling processes.

A human macrophage – hepatocyte co-culture model for comparative studies of infection and replication of Francisella tularensis LVS strain and subspecies holarctica and mediasiatica

BackgroundFrancisella tularensis, a gram-negative bacterium replicates intracellularly within macrophages and efficiently evades the innate immune response. It is able to infect and replicate within Kupffer cells, specialized tissue macrophages of the liver, and to modulate the immune response upon infection to its own advantage. Studies on Francisella tularensis liver infection were mostly performed in animal models and difficult to extrapolate to the human situation, since human infections and clinical observations are rare.ResultsUsing a human co-culture model of macrophages and hepatocytes we investigated the course of infection of three Francisella tularensis strains (subspecies holarctica – wildtype and live vaccine strain, and mediasiatica - wildtype) and analyzed the immune response triggered upon infection. We observed that hepatocytes support the intracellular replication of Franciscella species in macrophages accompanied by a specific immune response inducing TNFα, IL-1β, IL-6 and fractalkine (CX3CL1) secretion and the induction of apoptosis.ConclusionsWe could demonstrate that this human macrophage / hepatocyte co-culture model reflects strain-specific virulence of Francisella tularensis. We developed a suitable tool for more detailed in vitro studies on the immune response upon liver cell infection by F. tularensis.

An integrative microfluidically supported in vitro model of an endothelial barrier combined with cortical spheroids simulates effects of neuroinflammation in neocortex development

The development of therapeutic substances to treat diseases of the central nervous system is hampered by the tightness and selectivity of the blood-brain barrier. Moreover, testing of potential drugs is time-consuming and cost-intensive. Here, we established a new microfluidically supported, biochip-based model of the brain endothelial barrier in combination with brain cortical spheroids suitable to detect effects of neuroinflammation upon disruption of the endothelial layer in response to inflammatory signals. Unilateral perfusion of the endothelial cell layer with a cytokine mix comprising tumor necrosis factor, IL-1β, IFNγ, and lipopolysaccharide resulted in a loss of endothelial von Willebrand factor and VE-cadherin expression accompanied with an increased leakage of the endothelial layer and diminished endothelial cell viability. In addition, cytokine treatment caused a loss of neocortex differentiation markers Tbr1, Tbr2, and Pax6 in the cortical spheroids concomitant with reduced cell viability and spheroid integrity. From these observations, we conclude that our endothelial barrier/cortex model is suitable to specifically reflect cytokine-induced effects on barrier integrity and to uncover damage and impairment of cortical tissue development and viability. With all its limitations, the model represents a novel tool to study cross-communication between the brain endothelial barrier and underlying cortical tissue that can be utilized for toxicity and drug screening studies focusing on inflammation and neocortex formation.

Optimization of the transfection of human THP-1 macrophages by application of Nunc UpCell technology

We have established an electroporation protocol for transfection of premature adherent human THP-1 macrophages using Lonza Nucleofector technology. For efficient electroporation, detachment of adherent cells is necessary. We tested the Nunc UpCell product line of Thermo Fisher Scientific, which achieves detachment by a change of ambient temperature, as an alternative to enzymatic detachment. Here we present data verifying proper cell morphology and vitality and high transfection efficiency for macrophages cultured on UpCell plates. Appropriate macrophage behavior was confirmed by measuring markers of macrophage differentiation and polarization by reverse transcription quantitative polymerase chain reaction (RT-qPCR). In conclusion, Nunc UpCell materials are a viable alternative to enzymatic detachment.