Gersina Rega

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.

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-κB activation

Summary.  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)‐α antibodies and tiron showed that the effect of C5a was not mediated by TNF‐α or oxidative burst. Furthermore C5a induced NF‐κB binding to the cis element in human macrophages and the C5a‐induced increase in PAI‐1 was completely abolished by an NF‐κB inhibitor. Conclusions: We conclude that C5a upregulates PAI‐1 in macrophages via NF‐κB 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.

Oncostatin M-enhanced vascular endothelial growth factor expression in human vascular smooth muscle cells involves PI3K-, p38 MAPK-, Erk1/2- and STAT1/STAT3-dependent pathways and is attenuated by interferon-γ

The pleiotropic cytokine oncostatin M (OSM), a member of the glycoprotein (gp)130 ligand family, plays a key role in inflammation and cardiovascular disease. As inflammation precedes and accompanies pathological angiogenesis, we investigated the effect of OSM and other gp130 ligands on vascular endothelial growth factor (VEGF) production in human vascular smooth muscle cells (SMC). Human coronary artery SMC (HCASMC) and human aortic SMC (HASMC) were treated with different gp130 ligands. VEGF protein was determined by ELISA. Specific mRNA was detected by RT-PCR. Western blotting was performed for signal transducers and activators of transcription1 (STAT1), STAT3, Akt and p38 mitogen-activated protein kinase (p38 MAPK). OSM mRNA and VEGF mRNA expression was analyzed in human carotid endaterectomy specimens from 15 patients. OSM increased VEGF production in both HCASMC and HASMC derived from different donors. OSM upregulated VEGF and OSM receptor-specific mRNA in these cells. STAT3 inhibitor WP1066, p38 MAPK inhibitors SB-202190 and BIRB 0796, extracellular signal-regulated kinase1/2 (Erk1/2) inhibitor U0126, and phosphatidylinositol 3-kinase (PI3K) inhibitors LY-294002 and PI-103 reduced OSM-induced VEGF synthesis. We found OSM expression in human atherosclerotic lesions where OSM mRNA correlated with VEGF mRNA expression. Interferon-γ (IFN-γ), but not IL-4 or IL-10, reduced OSM-induced VEGF production in vascular SMC. Our findings that OSM, which is present in human atherosclerotic lesions and correlates with VEGF expression, stimulates production of VEGF by human coronary artery and aortic SMC indicate that OSM could contribute to plaque angiogenesis and destabilization. IFN-γ reduced OSM-induced VEGF production by vascular SMC.

Thrombin induces the expression of oncostatin M via AP-1 activation in human macrophages: a link between coagulation and inflammation.

Macrophages as inflammatory cells are involved in the pathogenesis of atherosclerosis that today is recognized as an inflammatory disease. Activation of coagulation leads to the late complication of atherosclerosis, namely atherothrombosis with its clinical manifestations stroke, unstable angina, myocardial infarction, and sudden cardiac death. Thus inflammation and coagulation play fundamental roles in the pathogenesis of atherosclerosis. We show that the coagulation enzyme thrombin up-regulates oncostatin M (OSM), a pleiotropic cytokine implicated in the pathophysiology of vascular disease, in human monocyte-derived macrophages (MDMs) up to 16.8-fold. A similar effect was seen in human peripheral blood monocytes and human plaque macrophages. In MDMs, the effect of thrombin on OSM was abolished by PPACK and mimicked by a PAR-1-specific peptide. Thrombin induced phosphorylation of ERK1/2 and p38 in MDMs. The ERK1/2 inhibitor PD98059 blocked the effect of thrombin on OSM production in MDMs, whereas the p38 inhibitor SB202190 had no effect. Thrombin induced translocation of c-fos and c-jun to the nucleus of MDMs. Using OSM promoter-luciferase reporter constructs transfected into MDMs, we show that a functional AP-1 site is required for promoter activation by thrombin. We present another link between coagulation and inflammation, which could impact on the pathogenesis of atherosclerosis.

In Human Macrophages the Complement Component C5a Induces the Expression of Oncostatin M via AP-1 Activation

Objective—Macrophages produce the cytokine oncostatin M (OSM), which beside other functions is also involved in inflammation. The complement component C5a mobilizes and activates these cells at inflammatory sites. We examined the effect of C5a on OSM production in human monocytes and in human monocyte-derived macrophages. Methods and Results—For macrophage transformation peripheral blood monocytes were cultivated for 8 to 10 days in the presence of human serum. C5a significantly increased in these cells OSM antigen as determined by specific ELISA and mRNA as quantitated by real-time polymerase chain reaction in these cells as well as in plaque macrophages. This effect was blocked by antibodies against the receptor C5aR/CD88 and by pertussis toxin. The C5a-induced phosphorylation of p38 and JNK and the C5a-induced increase in OSM production in macrophages was abolished by 2 p38 inhibitors and by a JNK inhibitor. Furthermore C5a increased the nuclear translocation of c-fos and c-jun. Using different OSM promoter deletion mutant constructs we show that the putative AP-1 element is responsible for activation of OSM promoter activity by C5a. Conclusion—Our data establish a link between the complement system and the gp130 receptor cytokine family with possible implications for the pathology of inflammatory diseases.

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

Summary.  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.

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.— Hohensinner, P. J., Kaun, C., Rychli, K., Niessner, A., Pfaffenberger, S., Rega, G., Furnkranz, A., Uhrin, P., Zaujec, J., Afonyushkin, T., Bochkov, V. N., Maurer, G., Huber, K., Wojta, J. The inflammatory mediator oncostatin M induces stromal derived factor‐1 in human adult cardiac cells. FASEB J. 23, 774–782 (2009)

Monocyte chemoattractant protein (MCP-1) is expressed in human cardiac cells and is differentially regulated by inflammatory mediators and hypoxia

The chemokine MCP‐1 is thought to play a key role – among many other pathophysiological processes – in myocardial infarction. MCP‐1 is not only a key attractant for monocytes and macrophages and as such responsible for inflammation but might also be directly involved in the modulation of repair processes in the heart. We show that cultured human cardiac cells express MCP‐1 and that its expression is upregulated by inflammatory cytokines and downregulated by hypoxia. We hypothesize that inflammation but not hypoxia is the main trigger for monocyte recruitment in the human heart.

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

Summary.  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‐α (TNF‐α) 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 κB (NF‐κB) and activating protein 1 (AP‐1) in M‐CSF regulation, blockers to both pathways and an adenovirus overexpressing a dominant negative (dn) form of IκB kinase 2 (IKK2) were used. Only the NF‐κB blocker dimethylfumarate and the dn IKK2, but not januskinase inhibitor‐1 (JNK‐I), were able to block the TNF‐α‐induced increase in M‐CSF production in these cells, suggesting that the induction of M‐CSF through TNF‐α is mainly dependent on the activation of the NF‐κB 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‐α might contribute to monocyte and macrophage survival and differentiation.