Age intrinsic loss of telomere protection via TRF1 reduction in endothelial cells.

Aging is a major factor predisposing for multiple diseases. Telomeres at the ends of chromosomes protect the integrity of chromosomal DNA. A specialized six-protein complex termed shelterin protects the telomere from unwanted interaction with DNA damage pathways. The aim of our study was to evaluate the integrity of telomeres and the stability of telomere protection during aging in endothelial cells (EC). We describe that aging EC can be characterized by an increased cell size (40%, p=0.02) and increased expression of PAI 1 (4 fold, p=0.02), MCP1 (10 fold, p=0.001) and GMCSF (15 fold, p=0.004). Telomeric state in aging cells is defined by an increased telomere oxidation (27%, p=0.01), reduced telomere length (62%, p=0.02), and increased DNA damage foci formation (5% in young EC versus 16% in aged EC, p=0.003). This telomeric dysfunction is accompanied by a reduction in the shelterin component TRF1 (33% mRNA, p=0.001; 24% protein, p=0.007). Overexpression of TRF1 in aging EC reduced telomere-associated DNA damage foci to 5% (p=0.02) and reduced expression levels of MCP1 (18% reduction, p=0.008). Aged EC have increased telomere damage and an intrinsic loss of telomere protection. Reestablishing telomere integrity could therefore be a target for rejuvenating endothelial cell function.

Reduced Ang2 expression in aging endothelial cells.

Aging endothelial cells are characterized by increased cell size, reduced telomere length and increased expression of proinflammatory cytokines. In addition, we describe here that aging reduces the migratory distance of endothelial cells. Furthermore, we observe an increase of the quiescence protein Ang1 and a decrease of the endothelial activation protein Ang2 upon aging. Supplementing Ang2 to aged endothelial cells restored their migratory capacity. We conclude that aging shifts the balance of the Ang1/Ang2 network favouring a quiescent state. Activation of endothelial cells in aging might be necessary to enhance wound healing capacities.

Neutrophil Gelatinase-Associated Lipocalin (NGAL) is Associated with Symptomatic Carotid Atherosclerosis and Drives Pro-inflammatory State In Vitro.

OBJECTIVE: Neutrophil gelatinase-associated lipocalin (NGAL), a protein found in activated neutrophils, is expressed in kidney tubule cells in response to noxious stimuli, and is thus recognized as a marker of acute kidney injury. Recent studies have suggested that NGAL could also have pathophysiological importance in cardiovascular diseases. The aim of the present study was to examine NGAL expression in human carotid endarterectomy tissues ex vivo as well as the effects of NGAL in the main cell types involved in atherogenesis, namely in human macrophages, endothelial cells, and smooth muscle cells in vitro. METHODS: NGAL protein was analyzed in human endarterectomy samples from patients with asymptomatic and symptomatic carotid stenosis by immunofluorescence, and NGAL mRNA expression was detected using RealTime-PCR. Human monocyte derived macrophages (MDM), human coronary artery smooth muscle cells (HCASMC), and human umbilical vein endothelial cells (HUVEC) were treated with recombinant human (rh) NGAL at different concentrations. Interleukin (IL)-6, IL-8, and monocyte chemo-attractant protein-1 (MCP-1) were determined by specific enzyme linked immunosorbent assays (ELISAs) in culture supernatants of such treated cells. RESULTS: Expression of NGAL protein was demonstrated by macrophages, smooth muscle cells, and endothelial cells in human carotid atherosclerotic tissue. NGAL mRNA expression was detected at a higher rate in atherosclerotic tissue of patients with symptomatic carotid stenosis (in 70%; n = 19) compared with asymptomatic patients (in 37%; n = 20, p < .001). Treatment of MDM, HCASMC, and HUVEC with rhNGAL led to a significant (p < 0.05) and concentration dependent increase of pro-inflammatory cytokines IL-6, IL-8, and MCP-1 in all cell types analyzed. CONCLUSION: By induction of pro-inflammatory mediators in human macrophages, smooth muscle cells and endothelial cells, NGAL, which is predominantly expressed in atherosclerotic plaques of symptomatic patients, could be involved in creating the local and systemic pro-inflammatory environment characteristic for atherosclerosis.

Cyclic and constant hyperoxia cause inflammation, apoptosis and cell death in human umbilical vein endothelial cells.

BACKGROUND: Perioperative high-dose oxygen (O2 ) exposure can cause hyperoxia. While the effect of constant hyperoxia on the vascular endothelium has been investigated to some extent, the impact of cyclic hyperoxia largely remains unknown. We hypothesized that cyclic hyperoxia would induce more injury than constant hyperoxia to human umbilical vein endothelial cells (HUVECs). METHODS: HUVECs were exposed to cyclic hyperoxia (5-95% O2 ) or constant hyperoxia (95% O2 ), normoxia (21% O2 ), and hypoxia (5% O2 ). Cell growth, viability (Annexin V/propidium iodide and 3-(4,5-dimethythiazol-2-yl)-2,5-diphenyl tetrazolium bromide, MTT) lactate dehydrogenase (LDH), release, cytokine (interleukin, IL and macrophage migration inhibitory factor, MIF) release, total antioxidant capacity (TAC), and superoxide dismutase activity (SOD) of cell lysate were assessed at baseline and 8, 24, and 72 h. A signal transduction pathway finder array for gene expression analysis was performed after 8 h. RESULTS: Constant and cyclic hyperoxia-induced gradually detrimental effects on HUVECs. After 72 h, constant or cyclic hyperoxia exposure induced change in cytotoxic (LDH +12%, P = 0.026; apoptosis +121/61%, P < 0.01; alive cells -15%, P < 0.01; MTT -16/15%, P < 0.01), inflammatory (IL-6 +142/190%, P < 0.01; IL-8 +72/43%, P < 0.01; MIF +147/93%, P < 0.01), or redox-sensitive (SOD +278%, TAC-25% P < 0.01) markers. Gene expression analysis revealed that constant and cyclic hyperoxia exposure differently activates oxidative stress, nuclear factor kappa B, Notch, and peroxisome proliferator-activated receptor pathways. CONCLUSIONS: Extreme hyperoxia exposure induces inflammation, apoptosis and cell death in HUVECs. Although our findings cannot be transferred to clinical settings, results suggest that hyperoxia exposure may cause vascular injury that could play a role in determining perioperative outcome.

Severe obesity increases adipose tissue expression of interleukin-33 and its receptor ST2, both predominantly detectable in endothelial cells of human adipose tissue.

OBJECTIVE: Obesity is associated with chronic inflammation of the adipose tissue, which contributes to obesity-associated complications such as insulin resistance and type 2 diabetes. Interleukin (IL)-33 acts via its receptor ST2 and is involved in the pathogenesis of inflammatory disorders including atherosclerosis and heart disease. IL-33 has been demonstrated to promote endothelial cell inflammatory response, but also anti-inflammatory and protective actions such as TH2 and M2 polarization of T cells and macrophages, respectively. IL-33 and ST2 have been shown to be expressed in human and murine adipose tissue. Our objective was to investigate alterations in obesity and a possible role of IL-33 in adipose tissue inflammation. SUBJECTS AND METHODS: We investigated severely obese patients (BMI>40 kg m(-2), n=20) and lean to overweight controls (BMI<30 kg m(-2); n=20) matched for age and sex, as well as diet-induced obese and db/db mice, in order to determine the impact of obesity on IL-33 and ST2 gene and protein expression levels in adipose tissue and blood, and their correlation with inflammatory and metabolic parameters. Furthermore, we examined the cellular source and location of IL-33 and ST2 in situ. RESULTS: IL-33 and ST2 expression levels were markedly elevated in omental and subcutaneous adipose tissue of severely obese humans and in diet-induced obese mice, but not in leptin receptor-deficient db/db mice. In addition, soluble ST2, but not IL-33 serum levels, were elevated in obesity. The main source for IL-33 in adipose tissue were endothelial cells, which, in humans, exclusively expressed ST2 on their surface. IL-33 expression strongly correlated with leptin expression in human adipose tissue. CONCLUSIONS: Expression of IL-33 and its receptor ST2 in human adipose tissue is predominantly detectable in endothelial cells and increased by severe obesity indicating an autocrine action. Thus, the adipose tissue microvasculature could participate in obesity-associated inflammation and related complications via IL-33/ST2.

Transtemporal ultrasound application potentially elevates brain temperature: results of an anthropomorphic skull model.

PURPOSE: Transtemporal sonothrombolysis is a tool for a more effective treatment in acute stroke patients. However, some reports revealed side effects, which might be potentially connected to temperature elevation. To gain better insight into cerebral temperature changes during transtemporal sonication, diagnostic and therapeutic ultrasound (US) applications were evaluated using an anthropomorphic skull model. MATERIALS AND METHODS: The impact of diagnostic (PW-Doppler, 1.8-MHz, 0.11 W/cm², TIC 1.2) and therapeutic (1-MHz and 3-MHz, 0.07 - 0.71 W/cm², continuous and pulsed mode) US application on temperature changes was evaluated at the level of muscle/temporal bone (TB), TB/brain, brain and at the middle cerebral artery (MCA) using 4 miniature thermocouples along the US beam. Sonication lasted 120 minutes. RESULTS: Diagnostic ultrasound revealed a maximum temperature increase of 1.45°/0.60°/0.39°/0.41°C (muscle/TB, TB/brain, brain, MCA) after 120 minutes. Therapeutic-1-MHz ultrasound raised temperature by 4.33°/2.02°/1.05 °C/0.81°C (pulsed 1:20) and by 10.38°/4.95°/2.43°/2.08°C (pulsed 1:5) over 120 minutes. Therapeutic-3-MHz US raised temperature by 4.89°/2.56°/1.24/1.25°C (pulsed 1:20) and by 14.77°/6.59°/3.56°/2.86°C (pulsed 1:5) over 120 minutes, respectively. Continuous application of therapeutic US (1-MHz and 3-MHz) led to a temperature increase of 13.86°/3.63°/1.66°/1.48°C and 17.09°/4.28°/1.38/0.99°C within 3 minutes. CONCLUSION: Diagnostic PW-Doppler showed only a moderate temperature increase and can be considered as safe. Therapeutic sonication is very powerful in delivering energy so that even pulsed application modes resulted in significant and potentially harmful temperature increases.

The inflammatory mediator oncostatin M induces stromal derived factor-1 in human adult cardiac cells.

Stromal derived factor 1 (SDF-1) is a CXC chemokine important in the homing process of stem cells to injured tissue. It has been implicated in healing and tissue repair. Growing evidence suggests that the glycoprotein-130 (gp130) ligand family is involved in repair processes in the heart. The aim of our study was to determine whether gp130 ligands could affect SDF-1 expression in cardiac cells. Human adult cardiac myocytes (HACMs) and fibroblasts (HACFs) were treated with gp130 ligands. Protein and mRNA levels of SDF-1 were determined using ELISA and RT-PCR, respectively. mRNA levels of SDF-1 were determined in human and mouse heart samples by RT-PCR. HACMs and HACFs constitutively express SDF-1, which was significantly up-regulated by the gp130 ligand oncostatin M (OSM). This effect was counteracted by a p38 inhibitor and to a lesser extent by a PI3K inhibitor. mRNA expression of SDF-1 in hearts of mice injected with OSM increased significantly. Levels of OSM and SDF-1 mRNA correlated significantly in human failing hearts. Our data, showing that OSM induces SDF-1 protein secretion in human cardiac cells in vitro and murine hearts in vivo, suggest that OSM via the induction of SDF-1 might play a key role in repair and tissue regeneration.

Roux-en-Y-Bariatric Surgery Reduces Markers of Metabolic Syndrome in Morbidly Obese Patients.

BACKGROUND: Obesity is closely linked to increased markers of metabolic syndrome and development of diabetes. Roux-en-Y bariatric surgery reduces hyperinsulinemia and improves insulin sensitivity and hence benefits morbidly obese patients. AIM: To determine changes in markers of metabolic syndrome, pancreatic function, and hepatic insulin sensitivity in patients before and 1 year after undergoing Roux-en-Y gastric bypass surgery. METHODS: We enrolled 43 consecutive patients in a single center. Markers for metabolic syndrome included proinsulin, insulin, C-peptide, liver enzymes, and serum levels of selected microRNAs hsa-miR-122, hsa-miR-130, hsa-miR-132, and hsa-miR-375. RESULTS: After surgery, all patients showed a significant 37% drop of body mass index (p < 0.001). Furthermore, proinsulin (59% reduction, p < 0.001), insulin (76% reduction, p < 0.001), and C-peptide (56% reduction, p < 0.001) were all reduced 1 year after surgery. Using the hepatic insulin clearance score, we determined a significant increase in hepatic insulin clearance after surgery (76% increase, p < 0.001). Especially diabetic patients showed a marked 2.1-fold increase after surgery. Hepatic enzymes ALT (35% reduction, p = 0.002) and γGT (48% reduction, p < 0.001) were significantly reduced in all patients with similar improvement in diabetic and non-diabetic patients. miRNAs hsa-miR-122, hsa-miR-130, and hsa-miR-132 were all significantly reduced whereas hsa-miR-375 was increased after gastric bypass surgery (p < 0.001 for all miRNAs). CONCLUSION: Both liver and pancreatic stress parameters were reduced significantly 1 year after Roux-en-Y gastric bypass surgery suggesting an overall amelioration of the metabolic syndrome in all patients regardless of previous health status.

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-1beta 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.

Selective interface transparency in graphene nanoribbon based molecular junctions.

A clear understanding of electrode-molecule interfaces is a prerequisite for the rational engineering of future generations of nanodevices that will rely on single-molecule coupling between components. With a model system, we reveal a peculiar dependence on interfaces in all graphene nanoribbon-based carbon molecular junctions. The effect can be classified into two types depending on the intrinsic feature of the embedded core graphene nanoflake (GNF). For metallic GNFs with |NA - NB| = 1, good/poor contact transparency occurs when the core device aligns with the center/edge of the electrode. The situation is reversed when a semiconducting GNF is the device, where NA = NB. These results may shed light on the design of real connecting components in graphene-based nanocircuits.

Reduction of Premature Aging Markers After Gastric Bypass Surgery in Morbidly Obese Patients.

BACKGROUND: Obesity is considered to be a major comorbidity. Obese patients suffer from an increased proinflammatory state associated with a premature aging phenotype including increased secretion of senescence-associated secretory proteins (SASP) and reduced telomere length. Micro-ribonucleic acids (miRNAs) are non-coding RNA molecules that could modify the post-transcriptional process. Several studies have reported associations between miRNAs and metabolic unhealthy conditions. AIM: To determine if bariatric surgery and the resulting weight loss could reverse the premature aging phenotype. METHODS: We enrolled 58 morbidly obese patients undergoing bariatric surgery. Markers of premature aging including the SASP IL-6, CRP and PAI-1, 7 miRNAs, as well as telomere length and telomere oxidation in mononuclear cells were evaluated. RESULTS: Patients showed a significant drop of body mass index (BMI; 43.98 ± 3.5 versus 28.02 ± 4.1, p < 0.001). We observed a significant reduction in SASP including a reduction of 55% of plasma IL-6 levels (p = 0 < 0.001), 83% of CRP levels (p = 0.001) and 15% of plasma PAI-1 levels (p < 0.001). Telomere length doubled in the patient cohort (p < 0.001) and was accompanied by a reduction in the telomere oxidation index by 70% (p < 0.001). Telomere length was inversely correlated with telomere oxidation. The aging-associated miRNA miR10a_5p was upregulated significantly (p = 0.039), while the other tested miRNAs showed no difference. CONCLUSION: Our data indicate a significant reduction of the proinflammatory SASP after bariatric surgery. We observed an increase in telomere length and reduced oxidative stress at telomeres. miR10a_5p which is downregulated during aging was upregulated after surgery. Overall, bariatric surgery ameliorated the premature aging phenotype.

Macrophage colony stimulating factor expression in human cardiac cells is upregulated by tumor necrosis factor-alpha via an NF-kappaB dependent mechanism.

INTRODUCTION: Macrophage colony stimulating factor (M-CSF) is a key factor for monocyte and macrophage survival and proliferation. M-CSF has been implicated in cardiac healing and repair after myocardial infarction. METHODS AND RESULTS: We show by immunohistochemistry and Western blotting analysis that M-CSF protein is present in human heart tissue. Cultured human adult cardiac myocytes (HACM) and human adult cardiac fibroblasts (HACF) isolated from human myocardial tissue constitutively express M-CSF. When HACM and HACF were treated with tumor necrosis factor-alpha (TNF-alpha) M-CSF protein production and M-CSF mRNA expression, determined by ELISA or by using RT-PCR, respectively, was significantly increased. To determine a possible role of nuclear factor kappaB (NF-kappaB) and activating protein 1 (AP-1) in M-CSF regulation, blockers to both pathways and an adenovirus overexpressing a dominant negative (dn) form of IkappaB kinase 2 (IKK2) were used. Only the NF-kappaB blocker dimethylfumarate and the dn IKK2, but not januskinase inhibitor-1 (JNK-I), were able to block the TNF-alpha-induced increase in M-CSF production in these cells, suggesting that the induction of M-CSF through TNF-alpha is mainly dependent on the activation of the NF-kappaB pathway. The monocyte activation marker CD11b was significantly increased after incubating U937 cells with conditioned medium from HACM or HACF as determined by FACS analysis. CONCLUSIONS: Our in vitro data taken together with our immunohistochemistry data suggest that human cardiac cells constitutively express M-CSF. This expression of M-CSF in the human heart and its upregulation by TNF-alpha might contribute to monocyte and macrophage survival and differentiation.

Statins decrease TNF-alpha-induced osteoprotegerin production by endothelial cells and smooth muscle cells in vitro.

Recent reports have implicated osteoprotegerin (OPG) in cardiovascular disease processes. Endothelial and smooth muscle cells produce OPG and its expression in these cells is upregulated by inflammatory mediators. Statins, which besides their lipid lowering properties have various vasculoprotective effects, have been shown to regulate OPG expression in osteoblasts. We investigated whether statins affect the expression of OPG in human endothelial and smooth muscle cells. Using an ELISA we could demonstrate that statins reduce tumor necrosis factor-alpha (TNF-alpha)-induced OPG production in cultured human endothelial cells and smooth muscle cells. Atorvastatin also downregulated interleukin-1alpha (IL-1alpha)-induced OPG production in endothelial cells. A significant reduction of TNF-alpha-induced OPG was seen when statins were used in the nanomolar range. These results were confirmed at the level of specific mRNA expression by real-time-PCR. Using LDH leakage as a marker of cell damage we show that cell viability was not affected by statins at concentrations used in our study. The effect of statins on TNF-alpha-induced OPG production was reversed by mevalonate and geranyl-geranyl pyrophosphate at the level of protein production and at the level of mRNA expression, suggesting that it was brought about by inhibition of the mevalonic acid pathway and protein prenylation. Through our results we have added OPG to the list of molecules whose TNF-alpha-induced upregulation is counteracted by statins. If such an effect is also operative in the in vivo setting, one could postulate a role for statins in the modulation of cardiovascular disease processes possibly regulated by OPG.

Nilotinib-induced vasculopathy: identification of vascular endothelial cells as a primary target site.

The BCR/ABL1 inhibitor Nilotinib is increasingly used to treat patients with chronic myeloid leukemia (CML). Although otherwise well-tolerated, Nilotinib has been associated with the occurrence of progressive arterial occlusive disease (AOD). Our objective was to determine the exact frequency of AOD and examine in vitro and in vivo effects of Nilotinib and Imatinib on endothelial cells to explain AOD-development. In contrast to Imatinib, Nilotinib was found to upregulate pro-atherogenic adhesion-proteins (ICAM-1, E-selectin, VCAM-1) on human endothelial cells. Nilotinib also suppressed endothelial cell proliferation, migration and tube-formation and bound to a distinct set of target-kinases, relevant to angiogenesis and atherosclerosis, including angiopoietin receptor-1 TEK, ABL-2, JAK1 and MAP-kinases. Nilotinib and siRNA against ABL-2 also suppressed KDR expression. In addition, Nilotinib augmented atherosclerosis in ApoE-/- mice and blocked reperfusion and angiogenesis in a hindlimb-ischemia model of arterial occlusion, whereas Imatinib showed no comparable effects. Clinically overt AOD-events were found to accumulate over time in Nilotinib-treated patients. After a median observation-time of 2.0 years, the AOD-frequency was higher in these patients (29.4%) compared to risk factor- and age-matched controls (<5%). Together, Nilotinib exerts direct pro-atherogenic and anti-angiogenic effects on vascular endothelial cells, which may contribute to development of AOD in patients with CML.

Inflammatory cytokines interleukin-6 and oncostatin m induce plasminogen activator inhibitor-1 in human adipose tissue.

BACKGROUND: Adipose tissue is a prominent source of plasminogen activator inhibitor-1 (PAI-1), the primary physiological inhibitor of plasminogen activation. Increased PAI-1 expression acts as a cardiovascular risk factor, and plasma levels of PAI-1 strongly correlate with body mass index (BMI). Elevated serum levels of interleukin-6 (IL-6), an inflammatory cytokine and a member of the glycoprotein 130 (gp130) ligand family, are found in obese patients and might indicate low-grade systemic inflammation. Another gp130 ligand, oncostatin M (OSM), upregulates PAI-1 in cardiac myocytes, astrocytes, and endothelial cells. We used tissue explants and primary cultures of preadipocytes and adipocytes from human subcutaneous and visceral adipose tissue to investigate whether IL-6 and OSM affect PAI-1 expression in fat. METHODS AND RESULTS: Human subcutaneous and visceral adipose tissue responded to treatment with IL-6 and OSM with a significant increase in PAI-1 production. Human preadipocytes were isolated from subcutaneous and visceral adipose tissue. Adipocyte differentiation was induced by hormone supplementation. All cell types expressed receptors for IL-6 and OSM and produced up to 12-fold increased levels of PAI-1 protein and up to 9-fold increased levels of PAI-1 mRNA on stimulation with IL-6 and OSM. AG-490, a janus kinase/signal transducer and activator of transcription inhibitor, abolished the OSM-dependent PAI-1 induction almost completely. CONCLUSIONS: We have for the first time established a link between the gp130 ligands, the proinflammatory mediators IL-6 and OSM, and the expression of PAI-1 in human adipose tissue. Thus, we speculate that IL-6 and OSM, by upregulating PAI-1 in adipose tissue, can contribute to the increased cardiovascular risk of obese patients.

The complement component C5a induces the expression of plasminogen activator inhibitor-1 in human macrophages via NF-kappaB activation.

BACKGROUND: Atherosclerosis is considered to be a chronic inflammatory disorder. Activation of the complement cascade is a major aspect of chronic inflammatory diseases. Complement components were identified in atherosclerotic plaques, and a correlation between adverse events and C5a plasma levels was found. These findings support the notion that complement activation contributes to development and progression of atherosclerotic lesions. OBJECTIVES: We investigated whether complement components C3a and C5a regulate plasminogen activator inhibitor (PAI-1) in human macrophages. METHODS: Human monocyte-derived macrophages (MDM) and human plaque macrophages were cultured and incubated with the complement component C5a. RESULTS: C5a increased PAI-1 up to 11-fold in human MDM and up to 2.7-fold in human plaque macrophages. These results were confirmed at the mRNA level using real time-polymerase chain reaction. Pertussis toxin or anti-C5aR/CD88 antibody completely abolished the effect of recombinant human C5a on PAI-1 production, suggesting a role of the C5a receptor. Experiments with antitumor necrosis factor (TNF)-alpha antibodies and tiron showed that the effect of C5a was not mediated by TNF-alpha or oxidative burst. Furthermore C5a induced NF-kappaB binding to the cis element in human macrophages and the C5a-induced increase in PAI-1 was completely abolished by an NF-kappaB inhibitor. CONCLUSIONS: We conclude that C5a upregulates PAI-1 in macrophages via NF-kappaB activation. We hypothesize that - if operative in vivo- this effect could favor thrombus development and thrombus stabilization in the lesion area. On the other hand one could speculate that C5a-induced upregulation of PAI-1 in plaque macrophages could act as a defense mechanism against plaque destabilization and rupture.

No evidence for a direct role of Helicobacter pylori and Mycoplasma pneumoniae in carotid artery atherosclerosis.

BACKGROUND: That infections with certain pathogens, by initiating an inflammatory response, may contribute to the development of atherosclerosis is suggested by clinical and experimental evidence. AIM: To analyse atherosclerotic plaques of the carotid artery, samples of apparently healthy greater saphenous veins and circulating leucocytes from the same individual patients for the presence of Helicobacter pylori and Mycoplasma pneumoniae. METHODS: Samples from 36 patients undergoing carotid endarterectomy for symptomatic carotid artery stenosis were analysed by polymerase chain reaction for the presence of DNA specific for H. pylori and M. pneumoniae. IgG antibody titres against H. pylori and M pneumoniae and plasma levels of soluble E-selectin, soluble intercellular adhesion molecule-1 and soluble vascular cell adhesion molecule-1 were determined. RESULTS: M. pneumoniae-specific DNA was detected in the atherosclerotic plaques of 13 of 36 (36.1%) patients, in the saphenous veins of 9 of 36 (25%) patients and in the leucocytes of 27 of 36 (75%) patients. No salient association was observed between the presence of M. pneumoniae-specific DNA in leucocytes and atherosclerotic plaques or veins. A marked correlation between the presence of M. pneumoniae in the respective specimens and the studied inflammatory markers or the presence of anti-M. pneumoniae antibodies was not observed. H. pylori-specific DNA could not be detected in the specimens tested. CONCLUSIONS: The absence of H. pylori and the random distribution of M. pneumoniae in tissue samples obtained from patients with symptomatic carotid artery stenosis do not support a role for these pathogens in the development of atherosclerosis due to a direct interaction of the bacteria with the vasculature.

Protease dependent activation of endothelial cells by peritoneal dialysis effluents.

Incubation of cultured human umbilical vein endothelial cells (HUVECs) with dilutions of peritoneal dialysis effluents (PDEs) from 11 individual patients undergoing continuous ambulatory peritoneal dialysis (CAPD) induced cellular procoagulant activity in a dose and time dependent manner. This procoagulant activity could be attributed to tissue factor (TF) expression since it was blocked by rabbit anti-TF IgG. These data was confirmed by FACS analysis yielding surface TF expression; In addition PDEs induced the expression of E-selectin in HUVECs. This TF and selectin inducing activity was heat labile and could be inhibited by protease inhibitors. Partial purification could be achieved using a benzamidine-Sepharose column. The TF inducing activity could not be attributed to LPS, IL-1, TNF-alpha, mast cell tryptase, active thrombin, or complement factor D. We therefore conclude that the peritoneal cavity contains a protease activity that induces a procoagulatory and proinflammatory phenotype in HUVECs.

Thrombin augments vascular cell-dependent migration of human mast cells: role of MGF.

Recent data suggest that auricular thrombosis is associated with accumulation of mast cells (MC) in the upper endocardium (where usually no MC reside) and local expression of MGF (mast cell growth factor) (25). In this study, the role of vascular cells, thrombin-activation and MGF, in MC-migration was analyzed. For this purpose, cultured human auricular endocardial cells (HAUEC), umbilical vein endothelial cells (HUVEC) and uterine- (HUTMEC) and skin-derived (HSMEC) microvascular endothelial cells were exposed to thrombin or control medium, and the migration of primary tissue MC (lung, n = 6) and HMC-1 cells (human MC-line) against vascular cells (supernatants) measured. Supernatants (24 h) of unstimulated vascular cells (monolayers of endocardium or endothelium) as well as recombinant (rh) MGF induced a significant migratory response in HMC-1 (control: 3025 +/- 344 cells [100 +/- 11.4%] vs. MGF, 100 ng/ml: 8806 +/- 1019 [291 +/- 34%] vs. HAUEC: 9703 +/- 1506 [320.8 +/- 49.8%] vs. HUTMEC: 8950 +/- 1857 [295.9 +/- 61.4%] vs. HSMEC: 9965 +/- 2018 [329.4 +/- 66.7%] vs. HUVEC: 9487 +/- 1402 [313.6 +/- 46.4%], p < 0.05) as well as in primary lung MC. Thrombin-activation (5 U/ml, 12 h) of vascular cells led to an augmentation of the directed migration of MC as well as to a hirudin-sensitive increase in MGF synthesis and release. Moreover, a blocking anti-MGF antibody was found to inhibit MC-migration induced by unstimulated or thrombin-activated vascular cells. Together, these data show that endocardial and other vascular cells can induce migration of human MC. This MC-chemotactic signal of the vasculature is associated with expression and release of MGF, augmentable by thrombin, and may play a role in the pathophysiology of (auricular) thrombosis.

Inhibitors of differentiation/DNA binding proteins Id1 and Id3 are regulated by statins in endothelial cells.

Id proteins (inhibitors of differentiation), which are involved in the control of cell cycle progression, can delay cellular differentiation and senescence and have been implicated in angiogenesis. The regulation of Id proteins in endothelial cells (ECs) by proangiogenic statins has not been investigated yet and remains unresolved. In this study, human dermal microvascular ECs (HDMECs) were stimulated with fluvastatin, vascular endothelial growth factor (VEGF), hepatocyte growth factor (HGF), and serum in vitro. The regulation of Id1, Id3, p21, p27, and p53 and the phosphorylation of AKT was investigated by Western blotting. Id1 was up-regulated by fluvastatin and serum, but not by VEGF and HGF. Fluvastatin did not regulate p21 and p27, but down-regulated Id3 and p53 slightly. In contrast to VEGF and HGF, fluvastatin did not result in AKT phosphorylation, indicating that this pathway is not involved in the control of endothelial Id1 expression. These experiments demonstrate for the first time that Id1 can be up-regulated and p53 down-regulated by a statin in HDMECs. Regulation of these proteins in ECs may account for the proangiogenic effect of statins.