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.

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.

Statin treatment reduces matrix degradation capacity of proinflammatory polarized macrophages.

BACKGROUND AND AIMS: Macrophages are versatile immune cells involved in tissue degradation and remodeling. Proinflammatory macrophages have the highest capacity of matrix degradation and proteolysis. Within atherosclerotic lesions, proinflammatory macrophages are associated with unstable plaques. Statins have been demonstrated to increase plaque stability. Possible changes of polarized macrophage tissue degradation behavior under statin treatment are currently unknown. METHODS: Polarized macrophages were tested in vitro for matrix degradation capacity with or without statin treatment. RESULTS: Proinflammatory macrophages show high matrix degradation capacity, which is lost after statin treatment. Statin concentrations were within a physiological range and did not influence overall macrophage polarization. Proinflammatory macrophages showed however a loss of filopodia where activators of MMPs are located. Loss of matrix degradation in proinflammatory macrophages was associated with changes of MMP14 activation and loss of uPAR localization at filopodia. Supplementation of mevalonate restored localization of uPAR to cellular protrusions and matrix degradation capacity. CONCLUSION: Statins reduce the matrix degradation potential of proinflammatory macrophages by reducing uPAR localization to cellular filopodia and reducing intracellular MMP14 activation.

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.

Differential in vivo activation of monocyte subsets during low-grade inflammation through experimental endotoxemia in humans.

Human monocytes are a heterogeneous cell population, which can be divided into a classical (CD14++CD16-), a non-classical (CD14+CD16+), and an intermediate (CD14++CD16+) subset. We hypothesized that low-grade inflammation may differentially affect monocyte subsets. We used a human lipopolysaccharide (LPS) infusion model to mimic low-grade inflammation to identify, which monocyte subsets are preferentially activated under these conditions. Monocyte subsets were identified by staining for CD14 and CD16, activation status of monocytes was analyzed by staining for CD11b and a novel in situ mRNA hybridization approach to detect IL-6 and IL-8 specific mRNA at the single-cell level by flow cytometry. After LPS challenge, cell numbers of monocyte subsets dropped after 2 h with cell numbers recovering after 6 h. Distribution of monocyte subsets was skewed dramatically towards the intermediate subset after 24 h. Furthermore, intermediate monocytes displayed the largest increase of CD11b expression after 2 h. Finally, IL-6 and IL-8 mRNA levels increased in intermediate and non-classical monocytes after 6 h whereas these mRNA levels in classical monocytes changed only marginally. In conclusion, our data indicates that the main responding subset of monocytes to standardized low-grade inflammation induced by LPS in humans is the CD14++CD16+ intermediate subset followed by the CD14+CD16+ non-classical monocyte subset. Circulating classical monocytes showed comparably less reaction to LPS challenge in vivo.

Macrophage-modulating cytokines predict adverse outcome in heart failure.

Cytokines regulating the mobilisation, recruitment and survival of mononuclear cells may play an important role in progression of heart failure. Therefore, we investigated the role of granulocyte colony stimulating factor (G-CSF), monocyte chemoattractant protein 1 (MCP-1) and macrophage colony stimulating factor (M-CSF) in patients with advanced heart failure. G-CSF, MCP-1 and M-CSF were determined in plasma of 351 patients with advanced heart failure by specific ELISAs. During a median follow up period of 16 months (95% confidence interval [CI]: 15-17 months) 175 patients (50%) experienced the composite endpoint rehospitalisation and all-cause mortality. M-CSF tertiles were associated with a gradually increasing risk with hazard ratios (HR) of 2.2 (95% CI: 1.5-3.2; for trend, p<0.001) for the composite endpoint and 2.6 (95% CI: 1.5-4.6; for trend, p=0.002) for all-cause mortality comparing third and first tertile. These associations remained significant in a multivariable Cox regression model after adjustment for BNP and other known risk factors (p=0.043 and p=0.024). High MCP-1 concentrations were associated with an increased risk of all-cause mortality with an adjusted HR of 1.9 (third vs. first tertile, 95% CI: 1.1-3.3; for trend, p=0.034). In contrast, G-CSF tertiles were not significantly associated with the composite endpoint or all-cause mortality in multivariable Cox regression. In conclusion, the independent and concentration-dependent association of macrophage-modulating cytokines and in particular of M-CSF with adverse outcome in advanced HF patients suggests that these cytokines may play an important pathophysiological role in progression of cardiomyopathy.

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

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.