Ovariectomy increases vascular calcification via the OPG/RANKL cytokine signalling pathway.

BACKGROUND: Observational studies suggest a strong relationship between menopause and vascular calcification. Receptor activator of nuclear factor-kappaBeta ligand (RANKL) and osteoprotegerin (OPG) are critical regulators of bone remodelling and modulate vascular calcification. We assessed the hypothesis that ovariectomy increases vascular calcification via the OPG/RANKL axis. MATERIALS AND METHODS: Age-matched sexually mature rabbits were randomized to ovariectomy (OVX, n = 12) or sham procedure (SHAM, n = 12). One month post-procedure, atherosclerosis was induced by 15 months 0.2%-cholesterol diet and endothelial balloon denudations (at months 1 and 3). Aortic atherosclerosis was assessed in vivo by magnetic resonance imaging (MRI) at months 9 and 15. At sacrifice, aortas were harvested for ex vivo microcomputed tomography (microCT) and molecular analysis of the vascular tissue. RESULTS: Vascular calcification density and calcific particle number were significantly greater in OVX than SHAM (8.4 +/- 2.8 vs. 1.9 +/- 0.6 mg cm(-3), P = 0.042, and 94 +/- 26 vs. 33 +/- 7 particles cm(-3), P = 0.046, respectively). Calcification morphology, as assessed by the arc angle subtended by the largest calcific particle, showed no difference between groups (OVX 33 +/- 7 degrees vs. SHAM 33 +/- 5 degrees , P = 0.99). By Western blot analysis, OVX increased the vascular OPG:RANKL ratio by 66%, P = 0.029, primarily by decreasing RANKL (P = 0.019). At month 9, MRI demonstrated no difference in atheroma volume between OVX and SHAM, and no significant change was seen by the end of the study. CONCLUSIONS: In contrast to bone, vascular OPG:RANKL ratio increased in response to ovariectomy with a corresponding fourfold increase in arterial calcification. This diametrical organ-specific response may explain the comorbid association of osteoporosis with calcifying atherosclerosis in post-menopausal women.

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.

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.

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.

Inflammation and coagulation in atherosclerosis.

Cardiovascular diseases remain to be the leading cause of death in Western societies. Despite major findings in vascular biology that lead to a better understanding of the pathomechanisms involved in atherosclerosis, treatment of the disease has only changed slightly within the last years. A big body of evidence suggests that atherosclerosis is a chronic inflammatory disease of the vessel wall. Accumulation and peroxidation of LDL-particles within the vessel wall trigger a strong inflammatory response, causing macrophage and T-cell accumulation within the vessel wall. Additionally, B-cells and specific antibodies against LDL-particles, as well as the complement system are implicated in atherogenesis. Besides data from clinical trials and autopsy studies it was the implementation of mouse models of atherosclerosis and the emerging field of direct gen-modification that lead to a thorough description of the pathophysiological mechanisms involved in the disease and created overwhelming evidence for a participation of the immune system. Recently, the cross-talk between coagulation and inflammation in atherogenesis has gained attention. Serious limitations and disparities in the pathophysiology of atherosclerosis in mice and men complicated the translation of experimental data into clinical practice. Despite these limitations, new anti-inflammatory medical therapies in cardiovascular disease are currently being tested in clinical trials.

Complement in atherosclerosis: friend or foe?

Atherosclerosis is a chronic inflammatory disease and the complement system plays a central role in innate immunity. Increasing evidence exists that the complement system is activated within atherosclerotic plaques. However, the role of complement in atherogenesis is not fully understood. Whereas complement activation by the classic and lectin pathway may be protective by removing apoptotic cells and cell debris from atherosclerotic plaques, activation of the complement cascade by the alternative pathway and beyond the C3 convertase with formation of anaphylatoxins and the terminal complement complex may be proatherogenic and may play a role in plaque destabilization leading to its rupture and the onset of acute cardiovascular events. In this review article we present evidence for complement activation within atherosclerotic plaques and we discuss recent data derived from experimental animal models that suggest a dual role of complement in the development of the disease. In addition, we summarize the role of complement components as biomarkers for cardiovascular disease.

High soluble Fas and soluble Fas Ligand serum levels before stent implantation are protective against restenosis.

Percutaneous coronary intervention (PCI) represents the most important treatment of coronary artery stenosis today. But instent restenosis (ISR) is a limitation for the outcome. Fas and Fas Ligand have been implicated in apoptosis and vessel wall inflammation. Their role in ISR is not known so far. In this prospective study we studied 137 patients with stable coronary artery disease who underwent elective PCI. Blood samples were taken directly before and 24 hours after PCI. Soluble (s)Fas and sFas Ligand serum levels were measured by ELISA. Restenosis was evaluated six to eight months later either by coronary angiography or by exercise testing. During the follow-up period, 18 patients (13%) developed ISR. At baseline, patients with ISR had significantly lower median sFas, as well as sFas Ligand levels compared to patients without ISR (sFAS: ISR 492 pg/ml, no ISR 967 pg/ml, p=0.014; sFAS Ligand: ISR: 26 pg/ml, no ISR: 42 pg/ml, p=0.001). After PCI median sFas levels significantly decreased in patients with ISR compared to patients without ISR [ISR: -152 pg/ml (IQR -36 to -227), no ISR: -38 pg/ml (IQR -173 to +150 pg/ml), p=0.03]. sFas Ligand levels after PCI significantly increased in ISR patients compared to patients without ISR [ISR: 14 pg/ml (IQR -3 to +26 pg/ml), no ISR -6 pg/ml (IQR -22 to +21 pg/ml), p=0.014]. In conclusion, sFas and sFas Ligand seem to be associated with the development of ISR. Determination of serum levels before and after PCI might help identifying patients at higher risk of ISR.

Plasminogen activator inhibitor-1 predicts coronary in-stent restenosis of drug-eluting stents.

BACKGROUND: We tested the hypothesis that plasma levels of plasminogen activator inhibitor-1 (PAI-1) are influenced by percutaneous coronary intervention (PCI) with the implantation of drug eluting stents (DES) and are able to predict the occurrence of in-stent restenosis (ISR). METHODS AND RESULTS: PAI-1 active antigen plasma levels were determined in 75 patients before and 24 h after PCI with DES implantation. Patients with ISR after six to eight months (16%) showed significantly lower PAI-1 plasma levels before PCI (ISR, 11.7 +/- 8.1 ng mL(-1); non-ISR, 22.8 +/- 18.8 ng mL(-1); P <0.05). PAI-1 levels in the lowest tertile were associated with a 9.5-fold increased risk of ISR, independent of clinical risk factors, angiographic or procedural characteristics, compared to the highest tertile (P < 0.05). The induced change of PAI-1 active antigen 24 h after PCI was significantly higher in patients with ISR (ISR, +5.6 +/- 8.0 ng mL(-1); non-ISR, -3.2 +/- 12.1 ng mL(-1); P < 0.05) with positive correlation to late lumen loss (r = 0.30; P < 0.05). CONCLUSIONS: ISR after DES implantation is significantly related to plasma levels of PAI-1 active antigen before and after PCI. If confirmed by larger multicenter studies, the determination of PAI-1 plasma levels might be clinically helpful in the identification of patients at high risk of developing of ISR, even after DES implantation.

The gp130 ligand oncostatin M regulates tissue inhibitor of metalloproteinases-1 through ERK1/2 and p38 in human adult cardiac myocytes and in human adult cardiac fibroblasts: a possible role for the gp130/gp130 ligand system in the modulation of extracellular matrix degradation in the human heart.

There is ample evidence supporting the view that alterations in the balance between matrix deposition and matrix degradation brought about by changes in the respective activities of matrix metalloproteinases (MMPs) and tissue inhibitors of matrix metalloproteinases (TIMPs) contribute significantly to cardiac dysfunction and disease. Here we show that TIMP-1 was upregulated up to threefold after treatment with the inflammatory mediator and gp130 ligand oncostatin M (OSM) in human adult cardiac myocytes and fibroblasts. The Erk1/2 inhibitor PD98059 and the p38 inhibitor SD202190 abolished the effect of OSM on TIMP-1 production in both cell types. Human cardiac myocytes and human cardiac fibroblasts also express MMP-1, 2, 3 and 9, and TIMP-2 constitutively. OSM, however, did not affect the expression of these proteins. In addition also the other gp130 ligands tested, cardiotrophin-1 (CT-1), interleukin-6 (IL-6) and leukemia inhibitory factor (LIF) had no effect on the expression of TIMPs and MMPs studied. We speculate that OSM by inducing TIMP-1 expression counteracts excessive proteolysis and unrestricted matrix degradation during inflammatory processes in the heart. The notion that OSM favors matrix stabilization in the human heart is further supported by our earlier observation that OSM also upregulates PAI-1, the physiological inhibitor of the protease urokinase-type PA (u-PA), which in turn is essential for extracellular proteolysis. Therefore we propose a role for the gp130 ligand OSM in the modulation of cardiac remodeling and repair processes.