Treatment with anti-RANKL antibody reduces infarct size and attenuates dysfunction impacting on neutrophil-mediated injury.

Selective pharmacological treatments targeting reperfusion injury produced modest protective effects and might be associated with immunosuppression. In order to identify novel and better-tolerated approaches, we focused on the neutralization of receptor activator of nuclear factor kappa-B ligand [RANKL], a cytokine recently shown to activate inflammatory cells (i.e. neutrophils) orchestrating post-infarction injury and repair. Myocardial ischemia (60min) and reperfusion injury was surgically induced in C57Bl/6 mice. In hearts and serum, RANKL was early upregulated during reperfusion. A "one-shot" injection with neutralizing anti-RANKL IgG during ischemia ameliorated myocardial infarct size and function, but not adverse remodeling (determined by Magnetic Resonance Imaging [MRI]) as compared to Vehicle or control IgG. These beneficial effects were accompanied in vivo by reduction in cardiac neutrophil infiltration, reactive oxygen species (ROS) and MMP-9 release. Anti-RANKL IgG treatment suppressed sudden peak of neutrophil granule products in mouse serum early after reperfusion onset. In vitro, RANK mRNA expression was detected in isolated mouse neutrophils. Co-incubation with neutralizing anti-RANKL IgG abrogated RANKL-induced mouse neutrophil degranulation and migration, suggesting a critical role of RANKL in neutrophil-mediated injury. Conversely, anti-RANKL IgG did not affect salvage pathways in cardiac cells (i.e. ERK p42/p44, Akt and STAT-3) or macrophage cardiac infiltration. Finally, treatment with anti-RANKL IgG showed no effect on B and T lymphocyte polarization (in serum, spleen and infarcted myocardium) and circulating chemokines as compared with Vehicle or control IgG. In conclusion, acute treatment with anti-RANKL IgG improved cardiac infarct size and function by potentially impacting on neutrophil-mediated injury and repair.

Gastric bypass in morbid obese patients is associated with reduction in adipose tissue inflammation via N-oleoylethanolamide (OEA)-mediated pathways.

Paradoxically, morbid obesity was suggested to protect from cardiovascular co-morbidities as compared to overweight/obese patients. We hypothesise that this paradox could be inferred to modulation of the "endocannabinoid" system on systemic and subcutaneous adipose tissue (SAT) inflammation. We designed a translational project including clinical and in vitro studies at Geneva University Hospital. Morbid obese subjects (n=11) were submitted to gastric bypass surgery (GBS) and followed up for one year (post-GBS). Insulin resistance and circulating and SAT levels of endocannabinoids, adipocytokines and CC chemokines were assessed pre- and post-GBS and compared to a control group of normal and overweight subjects (CTL) (n=20). In vitro cultures with 3T3-L1 adipocytes were used to validate findings from clinical results. Morbid obese subjects had baseline lower insulin sensitivity and higher hs-CRP, leptin, CCL5 and anandamide (AEA) levels as compared to CTL. GBS induced a massive weight and fat mass loss, improved insulin sensitivity and lipid profile, decreased C-reactive protein, leptin, and CCL2 levels. In SAT, increased expression of resistin, CCL2, CCL5 and tumour necrosis factor and reduced MGLL were shown in morbid obese patients pre-GBS when compared to CTL. GBS increased all endocannabinoids and reduced adipocytokines and CC chemokines. In morbid obese SAT, inverse correlations independent of body mass index were shown between palmitoylethanolamide (PEA) and N-oleoylethanolamide (OEA) levels and inflammatory molecules. In vitro, OEA inhibited CCL2 secretion from adipocytes via ERK1/2 activation. In conclusion, GBS was associated with relevant clinical, metabolic and inflammatory improvements, increasing endocannabinoid levels in SAT. OEA directly reduced CCL2 secretion via ERK1/2 activation in adipocytes.

Statin treatment is associated with reduction in serum levels of receptor activator of NF-κB ligand and neutrophil activation in patients with severe carotid stenosis.

Systemic and intraplaque biomarkers have been widely investigated in clinical cohorts as promising surrogate parameters of cardiovascular vulnerability. In this pilot study, we investigated if systemic and intraplaque levels of calcification biomarkers were affected by treatment with a statin in a cohort of patients with severe carotid stenosis and being asymptomatic for ischemic stroke. Patients on statin therapy had reduced serum osteopontin (OPN), RANKL/osteoprotegerin (OPG) ratio, and MMP-9/pro-MMP-9 activity as compared to untreated patients. Statin-treated patients exhibited increased levels of collagen and reduced neutrophil infiltration in downstream portions of carotid plaques as compared to untreated controls. In upstream plaque portions, OPG content was increased in statin-treated patients as compared to controls. Other histological parameters (such as lipid, macrophage, smooth muscle cell, and MMP-9 content) as well as RANKL, RANK, and OPG mRNA levels did not differ between the two patient groups. Serum RANKL/OPG ratio positively correlated with serum levels of neutrophilic products, intraplaque neutrophil, and MMP-9 content within downstream portions of carotid plaques. In conclusion, statin treatment was associated with improvement in serum RANKL levels and reduced neutrophil activity both systemically and in atherosclerotic plaques.

Impact of obesity and bariatric surgery on metabolism and coronary circulatory function.

Increases in intra-abdominal visceral adipose tissue have been widely appreciated as a risk factor for metabolic disorders such as dyslipidemia, hypertension, insulin resistance, and type 2 diabetes, whereas this is not the case for peripheral or subcutaneous obesity. While the underlying mechanisms that contribute to these differences in adipose tissue activity remain uncertain, increases in visceral fat commonly induce metabolic dysregulation, in part because of increased venous effluent of fatty acids and/or adipokines/cytokines to the liver. Increased body weight, paralleled by an increase in plasma markers of the insulin-resistance syndrome and chronic inflammation, is independently associated with coronary circulatory dysfunction. Recent data suggest that plasma proteins originating from the adipose tissue, such as endocannabinoids (EC), leptin, and adiponectin (termed adipocytes) play a central role in the regulation and control of coronary circulatory function in obesity. Positron emission tomography (PET) in concert with tracer kinetic modeling is a well established technique for quantifying regional myocardial blood flow at rest and in response to various forms of vasomotor stress. Myocardial flow reserve assessed by PET provides a noninvasive surrogate of coronary circulatory function. PET also enables the monitoring and characterization of coronary circulatory function in response to gastric bypass-induced weight loss in initially morbidly obese individuals, to medication and/or behavioral interventions related to weight, diet, and physical activity. Whether the observed improvement in coronary circulatory dysfunction via weight loss may translate to diminution in cardiovascular events awaits clinical confirmation.

Nicotinamide phosphoribosyltransferase inhibition reduces intraplaque CXCL1 production and associated neutrophil infiltration in atherosclerotic mice.

Pharmacological treatments targeting CXC chemokines and the associated neutrophil activation and recruitment into atherosclerotic plaques hold promise for treating cardiovascular disorders. Therefore, we investigated whether FK866, a nicotinamide phosphoribosyltransferase (NAMPT) inhibitor with anti-inflammatory properties that we recently found to reduce neutrophil recruitment into the ischaemic myocardium, would exert beneficial effects in a mouse atherosclerosis model. Atherosclerotic plaque formation was induced by carotid cast implantation in ApoE-/- mice that were fed with a Western-type diet. FK866 or vehicle were administrated intraperitoneally from week 8 until week 11 of the diet. Treatment with FK866 reduced neutrophil infiltration and MMP-9 content and increased collagen levels in atherosclerotic plaques compared to vehicle. No effect on other histological parameters, including intraplaque lipids or macrophages, was observed. These findings were associated with a reduction in both systemic and intraplaque CXCL1 levels in FK866-treated mice. In vitro, FK866 did not affect MMP-9 release by neutrophils, but it strongly reduced CXCL1 production by endothelial cells which, in the in vivo model, were identified as a main CXCL1 source at the plaque level. CXCL1 synthesis inhibition by FK866 appears to reflect interference with nuclear factor-κB signalling as shown by reduced p65 nuclear levels in endothelial cells pre-treated with FK866. In conclusion, pharmacological inhibition of NAMPT activity mitigates inflammation in atherosclerotic plaques by reducing CXCL1-mediated activities on neutrophils. These results support further assessments of NAMPT inhibitors for the potential prevention of plaque vulnerability.

Update on the pathophysiological activities of the cardiac molecule cardiotrophin-1 in obesity.

Cardiotrophin-1 (CT-1) is a heart-targeting cytokine that has been reported to exert a variety of activities also in other organs such as the liver, adipose tissue, and atherosclerotic arteries. CT-1 has been shown to induce these effects via binding to a transmembrane receptor, comprising the leukaemia inhibitory factor receptor (LIFR ß ) subunit and the glycoprotein 130 (gp130, a common signal transducer). Both local and systemic concentrations of CT-1 have been shown to potentially play a critical role in obesity. For instance, CT-1 plasma concentrations have been shown to be increased in metabolic syndrome (a cluster disease including obesity) probably due to adipose tissue overexpression. Interestingly, treatment with exogenous CT-1 has been shown to improve lipid and glucose metabolism in animal models of obesity. These benefits might suggest a potential therapeutic role for CT-1. However, beyond its beneficial properties, CT-1 has been also shown to induce some adverse effects, such as cardiac hypertrophy and adipose tissue inflammation. Although scientific evidence is still needed, CT-1 might be considered as a potential example of damage/danger-associated molecular pattern (DAMP) in obesity-related cardiovascular diseases. In this narrative review, we aimed at discussing and updating evidence from basic research on the pathophysiological and potential therapeutic roles of CT-1 in obesity.

Receptor activator of NF- κB ligand (RANKL) increases the release of neutrophil products associated with coronary vulnerability.

The "blood vulnerability", resulting from the complex balance between serum molecules and inflammatory cell atherosclerotic activities, is a major determinant in the evaluation of the "global patient cardiovascular vulnerability". In the present study, we focused on the role of the soluble receptor activator of nuclear factor kappa-B (NF-κB) ligand (RANKL, a potential marker of coronary calcification and vulnerability) in the release of neutrophilic proteases. Then, the association between these mediators and the degree of coronary calcification (assessed by coronary calcium score [CCS]) was investigated in 20 subjects (aged ≥65 years) asymptomatic for cardiovascular disease. Results showed that RANKL dose-dependently induced matrix metalloprotease (MMP)-8 and MMP-9 release from human primary neutrophils cultured in Teflon dishes (suspension condition, mimicking cells circulating in the blood stream). Conversely, when adherent to polystyrene, neutrophils became unresponsive to RANKL. RANKL did not influence the release of other neutrophilic products in suspension and adherence cultures as well as neutrophil migration. RANKL-induced release of MMPs was dependent on the activation of defined intracellular signalling pathways (PI3K/Akt and ERK1/2). In asymptomatic subjects, serum levels of RANKL, MMP-8 and MMP-9 positively correlated with CCS, reflecting a potential relationship between circulating RANKL and coronary calcification. In conclusion, RANKL increased the release of neutrophilic products potentially related to the "blood" vulnerability via defined intracellular pathways. Serum levels of RANKL might represent a potential biomarker of coronary calcification and related cardiovascular risk.

Receptor activator of nuclear factor kappa B ligand/osteoprotegerin pathway is a promising target to reduce atherosclerotic plaque calcification.

Atherosclerotic plaque calcification represents a common pathophysiologic process in the advanced phases of the disease. Both inflammatory and vascular cells (such as osteoblast-like cells, osteoclast-like cells, dendritic cells, macrophages, smooth muscle cells, and endothelial cells) are active players in the balance between intraplaque bone deposition and resorption. Inflammatory processes underlying plaque calcification are regulated by soluble mediators that also contribute to plaque destabilization and increased vulnerability. Among different mediators, the receptor activator of nuclear factor-kappa B (NF-kappa B) ligand (RANKL)/osteoprogerin (OPG) system is a major determinant in inflammatory cell differentiation toward osteoclast-like cells. Thus, this system might be a promising parameter to be investigated as a marker of calcification-related cardiovascular risk and a therapeutic target. Despite some promising results, several limitations have been shown in the potential clinical use of serum RANKL/OPG to better assess the cardiovascular risk. At present, the potential relationship between RANKL/OPG and the content of calcium within the intima of the coronary arteries (CAC score, assessed by computed tomography) needs to be explored in large clinical studies. On the other hand, the antiatherosclerotic relevance of treatments antagonizing RANKL is still under investigation. Despite that clinical evidence is needed, this therapeutic approach might be of particular benefit in selective populations (such as rheumatoid arthritis patients) with an increased cardiovascular risk.

Coronary artery calcification and cardiovascular risk: the role of RANKL/OPG signalling.

BACKGROUND: Coronary artery disease (CAD) represents the most relevant cause of death and morbidity in the adult population of developed and developing countries. During the last decades, a strong research effort has been performed to identify more selective markers and better assess the cardiovascular risk in both primary and secondary prevention. MATERIALS AND METHODS: This review updates current knowledge regarding the pathophysiological relevance as possible markers of coronary calcification of the receptor activator of nuclear factor-kappa ligand (RANKL)/osteoprotegerin (OPG) system. Furthermore, the potential clinical use of both RANKL/OPG and coronary calcium score (CAC) to assess cardiovascular vulnerability has been discussed. RESULTS: Emerging evidence indicates that atherosclerotic plaque calcification is positively correlated with vulnerability. Several inflammatory mediators have been shown to modulate arterial calcification, thus increasing the risk of plaque rupture. Among these factors, RANKL/OPG axis might be of particular interest as a promising biomarker of plaque vulnerability in subjects with diffuse coronary calcification. CONCLUSION: Together with clinical parameters of coronary calcification (such as CAC), circulating RANKL/OPG levels could contribute to better assess and predict cardiac events.

Ezetimibe/simvastatin.

BACKGROUND: Treatments with ezetimibe/simvastatin (combined or alone) have been indicated as very promising approaches to strongly reduce cholesterol levels in hyperlipidemic patients. OBJECTIVE: We will discuss the efficacy and safety of ezetimibe/simvastatin and their potential to reduce atheroprogression. The molecular mechanisms underlying the possible benefits in atherosclerosis and its complications will also be described. RESULTS/CONCLUSIONS: Combined therapy with ezetimibe/simvastatin has been shown to improve lipid profile inducing a very rapid reduction of low-density lipoprotein cholesterol levels in clinical trials. In the near future, potential clinical benefits could be observed in the IMPROVE-IT or SHARP trials. Although clinical studies are needed to further confirm safety of ezetimibe/simvastatin therapy, the greater efficacy in lipid-lowering was not associated with the increase of adverse events. Also the possible association between ezetimibe and cancer, which was observed in the SEAS trial, was not confirmed by further studies and meta-analyses. At present, ezetimibe should be considered an effective lipid-lowering agent that can be used in conjunction with simvastatin at the beginning of therapy, or included in the treatment of patients who do not achieve their low-density lipoprotein cholesterol goal with statins alone.

Oxaprozin-induced apoptosis on CD40 ligand-treated human primary monocytes is associated with the modulation of defined intracellular pathways.

The modulation of CD40L activity might represent a promising therapeutic target to reduce monocyte inflammatory functions in chronic diseases, such as rheumatoid arthritis. In the present study, we investigated the possible influence of nonsteroidal anti-inflammatory drugs (NSAIDs) on CD40L-induced monocyte survival. Monocytes were isolated from buffy coats by using Ficoll-Percoll gradients. Monocyte apoptosis was evaluated by fluorescence microscopy on cytopreps stained with acridine orange or using flow cytometry analysis of Annexin-V and Propidium Iodide staining. Akt and NF-kappaB activation was assessed using western blot. Caspase 3 activity was determined spectrophotometrically. Among different NSAIDs, only oxaprozin dose-dependently increased apoptosis of CD40L-treated monocytes. Oxaprozin pro-apoptotic activity was associated with the inhibition of CD40L-triggered Akt and NF-kappaB phosphorylation and the activation of caspase 3. In conclusion, our data suggest that oxaprozin-induced apoptosis in CD40L-treated human monocytes is associated with previously unknown cyclooxygenase (COX)-independent pathways. These intracellular proteins might be promising pharmacological targets to increase apoptosis in CD40L-treated monocytes.

Intestinal radiation-induced stricture favours small bowel obstruction by phytobezoar: report of a case.

Bezoars represent the fifth most frequent cause of acute small bowel obstruction. Phytobezoar is the most common type of bezoar. It is a concretion of undigestible fibers derived from ingested vegetables and fruits. We report a case of a woman with a 1-year history of recurrent epigastric and periumbilical abdominal pain with intermittent vomiting caused by phytobezoar of the terminal ileum. After careful investigation of the case and review of literature, we identified the factor involved in bezoar formation as radiation-induced ileal stenosis due to previous treatment for a pelvic tumour. This report provides evidence to consider phytobezoar as a possible cause of small bowel obstruction in patients previously treated with abdominal radiotherapy.