Siegfried Krause

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)

Activation of AMP-activated Protein Kinase by Vascular Endothelial Growth Factor Mediates Endothelial Angiogenesis Independently of Nitric-oxide Synthase

AMP-activated protein kinase (AMPK) is a sensor of cellular energy state and a regulator of cellular homeostasis. In endothelial cells, AMPK is stimulated via the upstream kinases LKB1 and Ca2+/calmodulin-dependent protein kinase kinase β (CaMKKβ). Previously, AMPK has been reported to activate endothelial nitric-oxide synthase (eNOS). Using genetic and pharmacological approaches, we show that vascular endothelial growth factor (VEGF) stimulates AMPK in human and mice endothelial cells via CaMKKβ. VEGF-induced AMPK activation is potentiated under conditions of energy deprivation induced by 2-deoxyglucose. To investigate the role of AMPK in endothelial function, CaMKKβ, AMPKα1, or AMPKα2 was down-regulated by RNA interference, and studies in AMPKα1−/− mice were performed. We demonstrate that AMPK does not mediate eNOS phosphorylation at serine residue 1177 or 633, NO- dependent cGMP generation, or Akt phosphorylation in response to VEGF. Using inhibitors of eNOS or soluble guanylyl cyclase and small interfering RNA against eNOS, we show that NO does not act upstream of AMPK. Taken together, these data indicate that VEGF-stimulated AMPK and eNOS pathways act independently of each other. However, acetyl-CoA carboxylase, a key enzyme in the regulation of fatty acid oxidation, was phosphorylated in response to VEGF in an AMPKα1- and AMPKα2-dependent manner. Our results show that AMPKα1 plays an essential role in VEGF-induced angiogenesis in vitro (tube formation and sprouting from spheroids) and in vivo (Matrigel plug assay). In contrast, AMPKα2 was not involved in VEGF-triggered sprouting. The data suggest that AMPKα1 promotes VEGF-induced angiogenesis independently of eNOS, possibly by providing energy via inhibition of acetyl-CoA carboxylase.

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.

Increased generation of reactive oxygen species in mononuclear blood cells from hypercholesterolemic patients

Hypercholesterolemia is a major risk factor of atherosclerosis [11* Accumulation of cholesterol esters in monocyte-derived macrophages within the arterial intima is an early sign of an atherosclerotic lesion [2]. It is believed that oxidative modification of low density lipoproteins (LDL) increases the cholesterol accumulation since the modified LDL are more susceptible for the uptake by macrophages [3,4]. Evidence has been provided that reactive oxygen species (ROS) produced by monocytes/macrophages may be involved in LDL modification [5,6].

Contact-induced modulation of neutrophil elastase secretion and phagocytic activity by platelets.

It has been reported that platelets stimulate generation of reactive oxygen species in neutrophils and monocytes by a mechanism that requires mutual cell-cell contact and the presence of P-selectin on the platelet surface. In the present study we investigated the effect of platelet-neutrophil contacts on neutrophil elastase secretion and phagocytic activity. Non-activated or thrombin-activated platelets were fixed with formaldehyde, washed and incubated with neutrophils in the absence or presence of various neutrophil agonists. Elastase secretion was determined by measuring the enzyme activity in cell-free supernatants using a chromogenic substrate. Platelet-neutrophil adhesion and ingestion of zymosan particles by neutrophils were quantitated by light microscopy. Platelets significantly reduced elastase secretion from neutrophils but had no effect on the elastase activity in the supernatant of neutrophil lysates. When neutrophils were stimulated with the ionophore A23187 or the chemotactic peptide FMLP, thrombin-activated platelets were more potent to inhibit elastase secretion when compared with non-activated platelets. Neutrophils that were not able to bind platelets to their surface had a significantly lower phagocytic activity when compared with neutrophil with adherent platelets or neutrophils that were incubated in the absence of platelets. The results indicate that platelet-neutrophil contacts may also lead to an inhibition of neutrophil functions and that such inhibition could be due to a transient contact rather than due to a firm platelet-neutrophil adhesion.

Generation of reactive oxygen species and activity of platelet-activating factor acetylhydrolase in human monocyte-derived macrophages

Monocytes were prepared from healthy human volunteers and were allowed to differentiate into macrophages by adhesion to plastic surface and cultured over 7 days in presence of either 10% fetal calf serum (FCS), human control serum or serum from hyperlipaemic patients. Hyperlipaemic serum stimulated the differentiation (measured as an increase in cellular protein and DNA content) to a higher extent when compared to control serum and FCS. With all sera a marked increase of the cellular activity of the enzyme platelet-activating factor acetylhydrolase (PAF-AH) and a tremendous decrease in the capacity of cells to generate reactive oxygen species (ROS) was observed. After seven days of culture the increase in PAH-AH activity was about 19-fold with hyperlipaemic serum, 11-fold with control serum and 6-fold with FCS. During the same period of time ROS generation measured as zymosan-induced chemiluminescence decreased by about 98% and no significant differences between the three types of serum were found. The results indicate that the activity of PAF-AH and the capacity of ROS generation which are both assumed to play an important role in the oxidation of low-density lipoproteins (LDL) and thus in the development of atherosclerosis, change in opposite direction during the differentiation of blood monocytes into macrophages, and that hyperlipaemic serum stimulates PAF-AH activity but not ROS generation.

Monitoring the effects of platelet glycoprotein IIb/IIIa antagonists with a microtiter plate method for detection of platelet aggregation

Measurement of platelet aggregation in platelet-rich plasma (PRP) is a fundamental tool in platelet studies, despite the fact that the technique required for this is time-consuming, may need large volumes of blood, and require particular skill and special equipment. The use of a microplate reader seems useful to perform platelet aggregation more rapidly and with less material. So, the aim of the present study was to validate a simple and rapid method which enables performance of kinetic measurements of platelet aggregation directly in a microtiter plate reader. Platelet aggregation was carried out in 96-well, flat-bottomed microtiter plates. Samples of PRP (140 w l/well) were placed in a microtiter plate. Agonists (10 w l/well) were added using an electronic multichannel dispenser directly before the reading was started. Measurements of the optical density were performed at 650 nm using a THERMOmax™ microplate reader (Molecular Devices, Sunnyvale, USA). During the run time the plate was incubated at 37°C and was mixed with the automix function of the reader. The technique was verified by comparing dose-response curves of platelet agonists and glycoprotein IIb/IIIa antagonists, obtained with the standard aggregometer and with the microtiter plate reader. Platelet aggregation in microtiter plates is simple and rapid. It offers the advantages of lowering the test volumes and the possibility to perform about 90 tests simultaneously. The method was successfully applied to measure platelet inhibition by glycoprotein IIb/IIIa antagonists.

Functional behaviour of mononuclear blood cells from patients with hypercholesterolemia

Mononuclear cells were prepared from venous blood obtained from 20 patients with a newly diagnosed hypercholesterolemia and without clinical signs of vascular disease, and from 19 age and sex matched controls. Adhesiveness to plastic surface, phagocytic activity measured as ingestion of zymosan particles, and spontaneous motility of mononuclear cells from patients were significantly higher by 57%, 19% and 50%, respectively, when compared to controls. In controls chemotaxis induced by the chemotactic peptide FMLP was slightly higher than spontaneous motility measured in absence of FMLP, whereas in patients FMLP significantly inhibited cell motility by about 47%. With the exception of FMLP-induced chemotaxis the results indicate that mononuclear cells are hyperreactive in hypercholesterolemia.

Inhibition of leukocyte chemiluminescence by platelets: role of platelet-bound fibrinogen.

Tethering of PMNL by platelets via CD62P has been shown to cause PMNL activation. Co-incubation of purified PMNL with platelets that were activated with thrombin and then fixed and washed, resulted in the formation of platelet-PMNL conjugates as well as in a generation of reactive oxygen species that were measured as luminol-enhanced chemiluminescence. When platelets were thrombin activated in the presence of RGDS to prevent binding of fibrinogen to membrane receptors, they had a reduced capacity to adhere to PMNL, but ROS generation was enhanced. In samples of citrated whole blood RGDS as well as the more specific platelet fibrinogen receptor antagonist GR144053F or a dissociation of the platelet glycoprotein IIb/IIIa complex markedly enhanced ROS generation that was induced by stirring the samples for 10 min at 1000 rpm, by 175%, 95% and 138%, respectively. Removal of platelets from the whole blood samples also resulted in an enhancement of stirring-induced ROS generation, which was inversely correlated to the platelet count. These data provide some evidence that platelets are capable of inhibiting ROS generation in PMNL by a mechanism that involves platelet-bound fibrinogen and probably depends on fibrinogen-mediated platelet-PMNL contact.

Activation of leukocytes in whole blood samples by N-formyl-methionyl-leucyl-phenylalanine (FMLP) enhances platelet aggregability but not platelet P-selectin exposure and adhesion to leukocytes

Adhesion of platelets to neutrophils and monocytes is believed to play an important role in intercellular communication. Evidence has been provided that such heterotypic cell-cell contacts via adhesion molecules may be directly involved in intercellular signal transduction as well as facilitate the action of soluble signal transmitters, e.g. cathepsin G, PAF or nitric oxide. With respect to platelet activation, stimulatory and inhibitory effects of leukocytes have been reported, and the results obtained seem to be influenced by the experimental conditions. We investigated the effect of leukocyte stimulation on platelet behaviour in samples of human citrated whole blood. Adding the chemotactic peptide FM LP, which stimulates neutrophils and monocytes but not lymphocytes and platelets, to stirred whole blood samples resulted in a significant enhancement ( P < 0.01) of spontaneous as well as ADP-induced platelet aggregation (25 vs 33% and 66 vs 69% , respectively). In contrast stirring-induced as well as ADP-induced increase of P-selectin exposure (33 and 107% , respectively) was not affected by FMLP. In unstirred whole blood samples, about 10 to 20% of neutrophils and monocytes had bound platelets to their surfaces, and the number of these heterotypic conjugates was enhanced about twofold during spontaneous platelet aggregation. Addition of FMLP significantly reduced the stirring-induced formation of platelet-neutrophil conjugates but not of platelet-monocyte conjugates. These results indicate that neutrophil and/or monocyte activation in whole blood may enhance platelet aggregation, but not secretion (CD62P exposure) and formation of heterotypic platelet-leukocyte conjugates.