Christoph Kaun

HMG CoA reductase inhibitors affect the fibrinolytic system of human vascular cells in vitro: a comparative study using different statins

The results of several clinical studies investigating the effect of statin therapy on the fibrinolytic system in vivo are inconclusive. We compared the effect of six different statins (atorvastatin, cerivastatin, fluvastatin, lovastatin, pravastatin, simvastatin) on components of the fibrinolytic system expressed by human vascular endothelial cells and smooth muscle cells and by the human hepatoma cell line HepG2. All statins used except pravastatin significantly decreased PAI‐1 production in human endothelial and smooth muscle cells. This effect was also seen in the presence of IL‐1α and TNF‐α. All statins except pravastatin increased t‐PA production in human smooth muscle cells. On a molar basis cerivastatin was the most effective HMG CoA reductase inhibitor used. Only simvastatin and lovastatin increased t‐PA production in endothelial cells. The effects on the fibrinolytic system were reversed by mevalonate. Statins decreased mRNA levels for PAI‐1 in endothelial and smooth muscle cells and increased mRNA levels for t‐PA in smooth muscle cells. Statins did not affect PAI‐1 expression in HepG2 cells. Cell viability was not influenced by statins in endothelial cells and HepG2 cells whereas in smooth muscle cells a cytotoxic effect was seen at high concentrations. If the effects on the fibrinolytic system of vascular cells in vitro shown in this study are also operative in vivo one could speculate that by increasing t‐PA and decreasing PAI‐1 at sites of vascular lesions statins might reduce fibrin formation and thrombus development. Such an effect might contribute to the clinically proven benefits of statin therapy.

Vascular Endothelial Growth Factor Is Induced by the Inflammatory Cytokines Interleukin-6 and Oncostatin M in Human Adipose Tissue In Vitro and in Murine Adipose Tissue In Vivo

Objectives—It is believed that adipose tissue acts as an endocrine organ by producing inflammatory mediators and thereby contributes to the increased cardiovascular risk seen in obesity. A link between adipose tissue mass and angiogenesis has been suggested. Vascular endothelial growth factor (VEGF) seems to be implicated in this process. Members of the glycoprotein (gp)130 ligand family regulate VEGF expression in other cells. Methods and Results—We used tissue explants as well as primary cultures of preadipocytes and adipocytes from human subcutaneous and visceral adipose tissue to investigate whether the gp130 ligands oncostatin M (OSM), interleukin-6 (IL-6), leukemia inhibitory factor (LIF), and cardiotrophin-1 (CT-1) regulate VEGF expression in human adipose tissue. Human subcutaneous and visceral adipose tissue responded to treatment with IL-6 and OSM with a significant increase in VEGF production. Human preadipocytes were isolated from subcutaneous and visceral adipose tissue. Adipocyte-differentiation was induced by hormone-supplementation. All cell types responded to IL-6 and OSM with a robust increase in VEGF protein production and a similar increase in VEGF-specific mRNA. Furthermore, IL-1&bgr; synergistically enhanced the effect of OSM on VEGF production. AG-490, a JAK/STAT inhibitor, abolished the OSM-dependent VEGF induction almost completely. In mice, IL-6 and OSM increased serum levels of VEGF and VEGF mRNA and vessel density in adipose tissue. Conclusion—We speculate that the inflammatory cytokines IL-6 and OSM might support angiogenesis during adipose tissue growth by upregulating VEGF.

Clinical and experimental evidence of prostaglandin E1-induced angiogenesis in the myocardium of patients with ischemic heart disease

OBJECTIVEnProstaglandin E1 (PGE-1) is a potent vasodilative agent which has been used to bridge patients with chronic heart failure listed for heart transplantation (HTX). In various experimental settings PGE-1 appears to stimulate angiogenesis by inducing vascular endothelial growth factor expression. This observational clinical study sought to investigate the angiogenic effects of PGE-1 in the failing human heart.nnnMETHODSnNeovascularization was investigated in 14 explanted hearts from patients with ischemic cardiomyopathy (ICMP) who had been bridged to HTX with PGE-1 (8+/-1 mg/kg/min, 97+/-75.6 days) and compared with 14 hearts who did not receive PGE-1 prior to HTX. In three sectional areas obtained from the left ventricular wall CD34, von Willebrand factor (vWf), nuclear Ki67 (MIB-1), and VEGF were quantified by immunohistochemistry to estimate capillary density and endothelial cell proliferation. Additionally, to investigate a possible angiogenic effect of PGE-1 in vitro, cultured human coronary artery smooth muscle cells (HCASMCs) were treated with PGE-1.nnnRESULTSnPGE-1-treated patients had significantly more CD34- and vWf-positive cells in the subepicardium (both P<0.01), myocardium (both P<0.0001) and subendocardium (P<0.01 and P<0.001) as compared to the nonPGE-1 group. Proliferative endothelial activity expressed by the presence of MIB-1- and VEGF-positive cells (both P<0.0001 in all layers) was increased more than twofold. Addition of PGE-1 to HCASMCs in cell culture resulted in a significant increase in VEGF production (164.0+/-19.7 pg/10(5) cells/24 h, P<0.005) as compared to the control cell line (66.6+/-8.7 pg/10(5) cells/24 h, P<0.005).nnnCONCLUSIONSnOur data demonstrate that PGE-1 is a potent stimulator of angiogenesis via upregulation of VEGF expression. The induction of therapeutic angiogenesis in patients with severe ICMP might explain the favorable clinical outcome in PGE-1 treated patients until HTX.

Polymorphic Membrane Protein (PMP) 20 and PMP 21 of Chlamydia pneumoniae Induce Proinflammatory Mediators in Human Endothelial Cells In Vitro by Activation of the Nuclear Factor-κB Pathway

We tested whether polymorphic membrane proteins (PMPs) of Chlamydia pneumoniae might play a role in triggering an inflammatory response in human endothelial cells. Of 15 purified, recombinant chlamydial PMPs tested, 2 (PMP 20 and PMP 21) dose-dependently increased the production of the inflammatory mediators interleukin (IL)-6 and monocyte chemoattractant protein-1 (MCP-1), in cultured human endothelial cells; production of IL-8 was also increased. When endothelial cells were infected by live C. pneumoniae, an increase in the production of IL-6, IL-8, and MCP-1 was seen. We used adenovirus-induced overexpression of IkappaBalpha-an inhibitor of nuclear factor (NF)-kappaB-to demonstrate that PMP 20 and PMP 21 increase the production of IL-6 and MCP-1 in human endothelial cells by activation of the NF-kappaB pathway, because, in cells overexpressing IkappaBalpha, treatment with the respective PMP did not result in increased production of IL-6 and MCP-1. Thus, C. pneumoniae could, by interactions of its PMPs with the endothelium, contribute to the process of vascular injury during the development and progression of atherosclerotic lesions.

Effect of Glycoprotein IIb/IIIa Antagonist Abciximab on Monocyte-Platelet Aggregates and Tissue Factor Expression

Objective—Activated platelets rapidly adhere to monocytes and upregulate the expression of tissue factor (TF), the major trigger of the coagulation cascade. In this study, we examined the effect of abciximab, a nonselective glycoprotein IIb/IIIa-receptor antagonist, on monocyte TF expression in thrombin receptor activator–stimulated whole blood in vitro. Methods and Results—Abciximab (50 &mgr;g/mL) reduced the mass of platelets attached to monocytes, measured by the mean fluorescence intensity (MFI) of CD42b on CD14+ cells, 1 (CD42b, 471±197 versus 1073±217 MFI, mean±SD, P <0.05), 5, and 10 minutes after thrombin receptor activator stimulation of whole blood to the same extent as anti–P-selectin (50 &mgr;g/mL; 288±177 MFI, P <0.05) when determined by flow cytometry. In parallel, the expression of the platelet activation marker P-selectin colocalized with CD14+ monocytes was reduced up to 25% by abciximab at the same time points. Expression of monocyte TF antigen (CD14+/TF+, 39.9±8.7% versus 66.3±19.9%, P <0.05), chromogenic TF-activity (TF, 8.4±1.9 versus 13.2±2.8 U, arbitrary units, P <0.05), and TF mRNA was suppressed in the presence of abciximab as a consequence of reduced platelet mass attached to monocytes. Conclusions—Our data suggest that heterotypic monocyte-platelet aggregates are a target for abciximab, which suppresses monocyte TF because of a reduction of monocyte-platelet cross talk.

Chlamydia pneumoniae in Carotid Artery Atherosclerosis A Comparison of Its Presence in Atherosclerotic Plaque, Healthy Vessels, and Circulating Leukocytes From the Same Individuals

Background and Purpose— There is growing clinical and experimental evidence that infections with Chlamydia pneumoniae might contribute to the development and progression of atherosclerosis. However, studies detecting the pathogen in atherosclerotic lesions examined either only atherosclerotic vessels or control vessels without atherosclerosis obtained from a different group of individuals. We analyzed atherosclerotic plaques of the carotid artery, samples of apparently healthy greater saphenous veins, and circulating leukocytes from the same individual patients for the presence of C pneumoniae. Methods— From each of 46 patients undergoing carotid endarterectomy for symptomatic carotid artery stenosis, these samples were analyzed by nested polymerase chain reaction for C pneumoniae–specific DNA. Furthermore, we determined IgA and IgG titers specific for the pathogen and plasma levels of C-reactive protein in these patients. Results— C pneumoniae DNA was detected in 86.9% of the leukocytes and in 82.6% of the atherosclerotic plaques but in only 6.5% of the saphenous veins. In 85% of patients who also had leukocytes positive for C pneumoniae, the atherosclerotic plaques were positive and the saphenous veins were negative. The presence of C pneumoniae–specific DNA in leukocytes significantly coincided with the presence of the respective DNA in the plaques of the carotid arteries (P =0.0002). No association between the presence of C pneumoniae and specific IgA or IgG levels was seen. C-reactive protein levels were significantly higher in patients with chlamydia-positive atherosclerotic plaques and with positive leukocytes than in patients with negative plaques of the carotid arteries or negative leukocytes, respectively (P <0.01, P <0.05). Conclusions— Our observation of >80% incidence of C pneumoniae in atherosclerotic plaques of the carotid artery does not prove causality between an infection with the pathogen and the development of atherosclerosis. It must be emphasized, however, that >90% of apparently healthy saphenous veins were negative for C pneumoniae. Given the structural and functional differences between veins and arteries, careful interpretation of our results regarding a possible causative role of C pneumoniae seems warranted.

Urokinase type plasminogen activator receptor is involved in insulin-like growth factor-induced migration of rhabdomyosarcoma cells in vitro.

Urokinase‐type plasminogen activator (uPA) binds to its receptor, uPAR, on the surface of cancer cells, leading to the formation of plasmin. Rhabdomyosarcoma (RMS) cell lines secrete high levels of insulin‐like growth factor II (IGF‐II), suggesting autocrine IGFs play a major role in the unregulated growth and metastasis of RMS. In vitro, IGF‐II and IGF‐I increased migration of RD cells to 124u2009±u20099% (Pu2009<u20090.01) and 131u2009±u20098% (Pu2009<u20090.05) of control, respectively. IGF‐II‐induced migration was abolished by insulin‐like growth factor binding protein‐6 (IGFBP‐6) (Pu2009<u20090.01), a relatively specific inhibitor of IGF‐II, and by plasminogen activator inhibitor type 1 (PAI‐1) (Pu2009<u20090.05). Aprotinin, a plasmin inhibitor, and mannosamine, which inhibits the synthesis of glycosylphosphatidylinositol (GPI), thereby preventing anchorage of GPI‐linked proteins such as uPAR to the cell membrane, also decreased IGF‐II‐ (Pu2009<u20090.05 for both) but not IGF‐I‐induced migration. [Arg54,Arg55]IGF‐II and [Leu27]IGF‐II, which preferentially bind to the IGF‐I and IGF‐II/mannose‐6‐phosphate receptors (IGF‐II/M6PR), respectively, both induced RD cell migration to 146u2009±u20098% (Pu2009<u20090.01) and 120u2009±u20097% (Pu2009<u20090.05) of control, respectively. An anti‐uPAR anti‐serum reduced IGF‐II‐ and IGF‐I‐induced migration (Pu2009<u20090.05 for both). An anti‐low density lipoprotein‐related protein (LRP) anti‐serum reduced IGF‐I‐induced migration (Pu2009<u20090.05). IGF‐I and ‐II both increased specific 125I‐single chain uPA (scuPA) binding to RD cells in a dose‐dependent manner (Pu2009<u20090.01). These results suggest involvement of the PA/plasmin system in IGF‐induced migration and indicate important roles these systems may have in RMS metastasis. J. Cell. Physiol. 197: 131–138, 2003© 2003 Wiley‐Liss, Inc.

Glycoprotein 130 ligand oncostatin-M induces expression of vascular endothelial growth factor in human adult cardiac myocytes

OBJECTIVEnIn murine and rat cardiac myocytes the gp130 system transduces survival as well as hypertrophic signals and via induction of the expression of the potent angiogenic factor VEGF in these cells also indirectly contributes to cardiac repair processes through the development of new blood vessels. There are, however, species differences in receptor specificity and receptor crossreactivity in the gp130-gp130 ligand system. We asked whether gp130 signaling is also involved in the regulation of VEGF in human cardiac myocytes and if so which gp130 ligands are critical for such an effect.nnnMETHODSnHuman adult cardiac myocytes (HACMs) were isolated from myocardial tissue and characterised by positive staining for myocardial actin, troponin-I and cardiotin. HACMs were treated with the gp130 ligands CT-1, IL-6, LIF or OSM and VEGF-1 was determined by a specific ELISA in the conditioned media of these cells. RT-PCR and Western blot analysis was used in order to detect gp130, IL-6-receptor, LIF-receptor or OSM-receptor specific protein and mRNA in human adult cardiac myocytes and for detection of VEGF-1 specific mRNA in cardiac myocytes after incubation with OSM. Pieces of myocardial tissue were incubated ex vivo in the presence and absence of OSM and VEGF was determined in supernatants of these cultures and immunohistochemistry was performed on the tissue using specific antibodies for VEGF-1. Immunohistochemistry was also employed to detect VEGF in sections from a healthy human heart and in a heart from a patient suffering from acute myocarditis.nnnRESULTSnOSM, but not CT-1, IL-6 or LIF increased VEGF-1 production in human adult cardiac myocytes dose-dependently derived from five different donors. This selective stimulation of VEGF by gp130 ligands was also reflected by a specific receptor expression on these cells. We detected high levels of mRNA for gp130 and the OSM receptor in freshly isolated human cardiac myocytes but only low amounts of mRNA for the IL-6 receptor whereas mRNA for the LIF receptor was hardly detectable by RT-PCR. OSM receptor and IL-6 receptor were also detectable by Western blotting whereas LIF receptor was only present as a faint band. OSM also increased the expression of VEGF-1 mRNA in cardiac myocytes. When pieces of human myocardial tissue were incubated with the gp130 ligands in an ex vivo model only OSM resulted in an increase in VEGF-1 in the supernatants of these cultures. Furthermore, VEGF increased in tissue samples treated with OSM in cardiac myocytes as evidenced by immunohistochemistry. In addition, we found increased VEGF-1 expression in myocardial tissue from a patient suffering from acute myocarditis.nnnCONCLUSIONnThe gp130-gp130 ligand system is also involved in VEGF regulation in human cardiac myocytes and OSM is the gp130 ligand responsible for this effect in the human system whereas LIF and CT-1 which had been shown to regulate VEGF expression in mouse and rat cardiac myocytes had no effect. Thus we have added OSM, which is produced by activated T lymphocytes and monocytes, to the list of regulatory molecules of VEGF production in the human heart. Our results lend further support to the notion that besides hypoxia, inflammation via induction of VEGF through autocrine or paracrine pathways plays a key role in (re)vascularisation of the myocardium.

Anisodamine Counteracts Lipopolysaccharide-Induced Tissue Factor and Plasminogen Activator Inhibitor-1 Expression in Human Endothelial Cells: Contribution of the NF-κB Pathway

In this study we aimed to investigate whether the therapeutic efficacy of anisodamine in the treatment of bacteraemic shock could – at least in part – be brought about by its direct interference with the lipopolysaccharide (LPS)-induced activation of endothelial cells. Thus, we investigated the effect of anisodamine on LPS-induced expression of plasminogen activator inhibitor-1 (PAI-1) and tissue factor (TF), two major markers of endothelial activation. PAI-1 was measured in the conditioned media of human umbilical vein endothelial cells (HUVEC) by a specific enzyme-linked immunosorbent assay (ELISA) whereas TF activity was measured in the lysates of these cells by using a single step clotting assay. Results obtained in these assays were confirmed on the level of specific mRNA expression by Northern blotting using specific probes for human PAI-1 or TF. In order to evaluate a possible contribution of the NF-ĸB pathway on the effects observed, electrophoretic mobility shift assays (EMSA) were performed using nuclear extracts from HUVEC and NF-ĸB-binding oligonucleotides. When HUVEC were treated with 1 µg/ml LPS a significant increase in PAI-1 and TF activity was observed compared with cells incubated without LPS. Anisodamine dose-dependently inhibited this LPS-induced upregulation of PAI-1 and TF. Anisodamine alone had no effect on the constitutive expression of PAI-1 and TF in these cells. These effects were also confirmed on the level of specific PAI-1 and TF mRNA expression by Northern blotting. Furthermore, we could show by EMSA that anisodamine completely abolished LPS-induced NF-ĸB DNA binding activity in nuclear extracts from HUVEC treated with LPS together with anisodamine. Thus, we provide evidence that anisodamine counteracts endothelial cell activation by inhibiting LPS-induced PAI-1 and TF expression in these cells. Its interference with the NF-ĸB pathway might – at least in part – contribute to this effect. The ability of anisodamine to counteract LPS effects on endothelial cells might be one underlying mechanism explaining its efficacy in the treatment of bacteraemic shock.

The inflammatory mediator oncostatin M induces angiopoietin 2 expression in endothelial cells in vitro and in vivo

Summary.u2002 Objectives: Members of the glycoprotein 130 (gp130) receptor–gp130 ligand family play a role in angiogenesis in different tissues. We tested the effect of this cytokine family on the angiopoietin (Ang)–Tie system, which is involved in blood vessel maturation, stabilization, and regression. Results: Oncostatin M (OSM) increased Ang2 expression in human umbilical vein endothelial cells via Janus kinase/signal transducer and activator of transcription (JAK/STAT) and mitogen‐activated protein (MAP) kinase activation. Furthermore, OSM induced Ang2 expression in macrovascular endothelial cells isolated from the human aorta and in microvascular endothelial cells isolated from human heart. Our in vivo experiments revealed that mRNA expression of Ang2 in hearts of mice injected with OSM increased significantly, and levels of OSM mRNA significantly correlated with mRNA levels of Ang2 in human hearts. In addition, OSM increased the expression of its own receptors, gp130 and OSM receptor, in endothelial cells in vitro and in mice in vivo, and levels of OSM mRNA significantly correlated with mRNA levels of gp130 and OSM receptor in human hearts. Conclusion: Our data, showing the effects of OSM on the Ang–Tie system in endothelial cells, in hearts of mice, and in human heart tissue, provide yet another link between inflammation and angiogenesis.