Walter S. Speidl

Simvastatin Blunts Endotoxin-Induced Tissue Factor In Vivo

Background—Beyond lipid lowering, various antiinflammatory properties have been ascribed to statins. Moreover, in vitro studies have suggested the presence of anticoagulant effects of 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitors, as lipopolysaccharide (LPS)-induced monocyte tissue factor (TF) was suppressed. In this study, we examined the role of statins in experimental endotoxemia on inflammatory and procoagulant responses in vivo. Methods and Results—In this double-blind, placebo-controlled, parallel-group study, 20 healthy, male subjects were randomized to receive either simvastatin (80 mg/d) or placebo for 4 days before intravenous administration of LPS (20 IU/kg IV). Plasma high-sensitive C-reactive protein (hsCRP), monocyte chemoattractant protein (MCP-1), sCD40L, sCD40, and prothrombin fragment F1+2 (F1.2) were determined by ELISAs at baseline and at 4 and 8 hours after LPS administration. Monocyte TF expression and monocyte-platelet aggregates were measured by whole-blood flow cytometry over the same time course. The increases in hsCRP and MCP-1, both known inducers of TF, were significantly suppressed by statin treatment after LPS challenge. Statin premedication blunted the increase of monocyte TF expression in response to LPS. In parallel, endotoxin-induced formation of F1.2 was significantly reduced by simvastatin after 4 and 8 hours. LPS infusion affected neither the formation and activation of monocyte-platelet aggregates nor plasma levels of sCD40 and sCD40L. Conclusions—Simvastatin suppresses the inflammatory response to endotoxin and blunts monocyte TF expression but does not affect platelet activation.

Early Metoprolol Administration Before Coronary Reperfusion Results in Increased Myocardial Salvage Analysis of Ischemic Myocardium at Risk Using Cardiac Magnetic Resonance

Background— &bgr;-Blockers improve clinical outcome when administered early after acute myocardial infarction. However, whether &bgr;-blockers actually reduce the myocardial infarction size is still in dispute. Cardiac magnetic resonance imaging can accurately depict the left ventricular (LV) ischemic myocardium at risk (T2-weighted hyperintense region) early after myocardial infarction, as well as the extent of necrosis (delayed gadolinium enhancement). The aim of this study was to determine whether early administration of metoprolol could increase myocardial salvage, measured as the difference between the extent of myocardium at risk and myocardial necrosis. Methods and Results— Twelve Yorkshire pigs underwent a 90-minute left anterior descending coronary occlusion, followed by reperfusion. They were randomized to metoprolol (7.5 mg during myocardial infarction) or placebo. Global and regional LV function, extent of myocardium at risk, and myocardial necrosis were quantified by cardiac magnetic resonance imaging studies performed 4 and 22 days after reperfusion in 10 survivors. Despite similar extent of myocardium at risk in metoprolol- and placebo-treated pigs (30.9% of LV versus 30.6%; P=NS), metoprolol resulted in 5-fold-larger salvaged myocardium (32.4% versus 6.2% of myocardium at risk; P=0.015). The LV ejection fraction significantly improved in metoprolol-treated pigs between days 4 and 22 (37.2% versus 43.0%; P=0.037), whereas it remained unchanged in pigs treated with placebo (35.1% versus 35.0%; P=NS). The extent of myocardial salvage was related directly to LV ejection fraction improvement (P=0.031) and regional LV wall motion recovery (P=0.039) at day 22. Conclusions— Early metoprolol administration during acute coronary occlusion increases myocardial salvage. The extent of myocardial salvage, measured as the difference between myocardium at risk and myocardial necrosis, was associated with regional and global LV motion improvement.

Rapid change in plaque size, composition, and molecular footprint after recombinant apolipoprotein A-I Milano (ETC-216) administration: magnetic resonance imaging study in an experimental model of atherosclerosis

OBJECTIVES This study sought to assess the effect of short-term apolipoprotein (apo) A-I(Milano) administration on plaque size and on suspected markers of plaque vulnerability. BACKGROUND Long-term lipid-lowering interventions can regress and stabilize atherosclerotic plaques. However, the majority of recurrent events occur early after the first episode. Interventions able to acutely induce plaque regression and stabilization are lacking. Regression of human coronary lesions after 5 weeks of treatment with apoA-I(Milano) administration has been shown. However, there are no data regarding its effect on plaque vulnerability. METHODS Advanced aortic lesions were induced in New Zealand White rabbits (n = 40). Plaque size was assessed by magnetic resonance imaging (MRI) at the end of atherosclerosis induction. Animals were randomized to placebo or apoA-I(Milano) phospholipids (ETC-216), 2 infusions 4 days apart. After the last dose, another MRI study was performed and aortas were processed for cellular composition and gene protein expression of markers associated with plaque instability. RESULTS Pre-treatment MRI showed similar plaque size in both groups, whereas post-treatment MRI showed 6% smaller plaques in apoA-I(Milano)-treated animals compared with placebo (p = 0.026). The apoA-I(Milano) treatment induced a 5% plaque regression (p = 0.003 vs. pre-treatment), whereas the placebo showed no significant effect. Plaque regression by apoA-I(Milano) was associated with a reduction in plaque macrophage density and a significant down-regulation in gene and protein expression of tissue factor, monocyte chemoattractant protein-1, and cyclooxygenase-2, as well as marked decrease in gelatinolytic activity. Conversely, cyclooxygenase-1 was significantly up-regulated. CONCLUSIONS Acute plaque regression observed after short-term apoA-I(Milano) administration was associated with a significant reduction in suspected makers of plaque vulnerability in an experimental model of atherosclerosis.

Complement in atherosclerosis: friend or foe?

Summary.  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.

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.

Catecholamines potentiate LPS-induced expression of MMP-1 and MMP-9 in human monocytes and in the human monocytic cell line U937: possible implications for peri-operative plaque instability

Plaque destabilization leading to myocardial infarction is observed after surgery even if the intervention is of noncardiovascular nature. Mediators of peri‐ or postoperative stress responsible for such events could include catecholamines and lipopolysaccharide (LPS). Monocytes may be involved in destabilization of atherosclerotic plaques by production of matrix metalloproteinases (MMP). We examined whether catecholamines could affect the expression of MMPs in human monocytes/macrophages and whether catecholamines could modulate LPS‐stimulated expression of particular MMPs in these cells. Epinephrine and norepinephrine up‐regulated MMP‐1 and potentiated LPS‐induced expression of MMP‐1 in peripheral blood monocytes and monocyte‐ derived macrophages. We further characterized this effect employing the monocytic cell line U937 and showed that catecholamines potentiate LPS‐induced effects on MMP‐1 and MMP‐9 antigen and activity. mRNA levels of the respective MMPs also increased. These effects did not result from higher mRNA stability but rather from increased transcription possibly induced by enhanced DNA binding of AP‐1 and were mediated by either β1‐ or β2‐receptors. If this mechanism is also effective in vivo, our findings might, at least in part, help to explain the observation that cardiac events are important causes of morbidity and mortality after noncardiac surgery and support the findings that peri‐operative β‐blockade has been shown to reduce postoperative mortality from cardiac events.

The complement component C5a is present in human coronary lesions in vivo and induces the expression of MMP-1 and MMP-9 in human macrophages in vitro

The complement component C5a is formed during activation of the complement cascade and exerts chemotactic and proinflammatory effects. Macrophages, which are localized in the rupture‐prone shoulder regions of coronary plaques, are thought to play a major role in plaque destabilization and rupture through the production of matrix metalloproteinases (MMPs). When human monocyte‐derived macrophages were stimulated in vitro with C5a, MMP‐1 and MMP‐9 mRNA levels were significantly increased. Furthermore, C5a up‐regulated MMP‐1 and MMP‐9 antigens and activity, as determined by ELISA and specific activity assays. These effects were blocked by antibodies against the receptor C5aR/CD88. In addition, blocking experiments revealed that MMP‐1 expression was mediated by activation of the transcription factor AP‐1, and MMP‐9 expression was induced by activation of NF‐κB and AP‐1. Immunohistochemical analysis of human coronary plaques demonstrated the colocalization of C5a, MMP‐1, and MMP‐9 in vivo. Together, these observations indicate that activation of the complement cascade and formation of C5a may play a role in the onset of acute coronary events by induction of MMPs in atherosclerotic lesions.—Speidl, W. S., Kastl, S. P., Hutter, R., Katsaros, K. M., Kaun, C., Bauriedel, G., Maurer, G., Huber, K., Badimon, J. J., Wojta, J. The complement component C5a is present in human coronary lesions in vivo and induces the expression of MMP‐1 and MMP‐9 in human macrophages in vitro. FASEB J. 25, 35–44 (2011). www.fasebj.org

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.

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.

Macrophages Transmit Potent Proangiogenic Effects of oxLDL In Vitro and In Vivo Involving HIF-1α Activation: a Novel Aspect of Angiogenesis in Atherosclerosis

Neovascularization has been linked to the progression and vulnerability of atherosclerotic lesions. Angiogenesis is increased in lipid-rich plaque. Hypoxia-inducible factor alpha (HIF-1α) is a key transcriptional regulator responding to hypoxia and activating genes, which promote angiogenesis, among them vascular endothelial growth factor (VEGF). Oxidized low-density lipoprotein (oxLDL) is generated in lipid-rich plaque by oxidative stress. It triggers an inflammatory response and was traditionally thought to inhibit endothelial cells. New data, however, suggest that oxLDL can activate HIF-1α in monocytes in a hypoxia-independent fashion. We hypothesized that HIF-1α activation in monocyte–macrophages could transmit proangiogenic effects of oxLDL linking hyperlipidemia, inflammation, and angiogenesis in atherosclerosis. First, we examined the effect of oxLDL on HIF-1α and VEGF expression in monocyte–macrophages and on their proangiogenic effect on endothelial cells in vitro in a monocyte–macrophage/endothelial co-culture model. OxLDL strongly induced HIF-1α and VEGF in monocyte–macrophages and significantly increased tube formation in co-cultured endothelial cells. HIF-1α inhibition reversed this effect. Second, we demonstrated a direct proangiogenic effect of oxLDL in an in vivo angiogenesis assay. Again, HIF-1α inhibition abrogated the proangiogenic effect of oxLDL. Third, in a rabbit atherosclerosis model, we studied the effect of dietary lipid lowering on arterial HIF-1α and VEGF expression. The administration of low-lipid diet significantly reduced the expression of both HIF-1α and VEGF, resulting in decreased plaque neovascularization. Our data point to oxLDL as a proangiogenic agent linking hyperlipidemia, inflammation, and angiogenesis in atherosclerosis. This effect is dependent on macrophages and, at least in part, on the induction of the HIF-1α pathway.