Inhibition of NET Release Fails to Reduce Adipose Tissue Inflammation in Mice

Obesity-associated diseases such as Type 2 diabetes, liver disease and cardiovascular diseases are profoundly mediated by low-grade chronic inflammation of the adipose tissue. Recently, the importance of neutrophils and neutrophil-derived myeloperoxidase and neutrophil elastase on the induction of insulin resistance has been established. Since neutrophil elastase and myeloperoxidase are critically involved in the release of neutrophil extracellular traps (NETs), we here hypothesized that NETs may be relevant to early adipose tissue inflammation. Thus, we tested the effect of the Peptidyl Arginine Deiminase 4 inhibitor Cl-amidine, a compound preventing histone citrullination and subsequent NET release, in a mouse model of adipose tissue inflammation. C57BL6 mice received a 60% high fat diet for 10 weeks and were treated with either Cl-amidine or vehicle. Flow cytometry of adipose tissue and liver, immunohistological analysis and glucose and insulin tolerance tests were performed to determine the effect of the treatment and diet. Although high fat diet feeding induced insulin resistance no significant effect was observed between the treatment groups. In addition no effect was found in leukocyte infiltration and activation in the adipose tissue and liver. Therefore we concluded that inhibition of neutrophil extracellular trap formation may have no clinical relevance for early obesity-mediated pathogenesis of the adipose tissue and liver

Atherosclerotic plaque destabilization in Mice: A comparative study

Atherosclerosis-Associated diseases are the main cause ofmortality and morbidity in western societies. The progression of atherosclerosis is a dynamic process evolving from early to advanced lesions thatmay become rupture-prone vulnerable plaques. Acute coronary syndromes are the clinical manifestation of life-Threatening thrombotic events associated with high-risk vulnerable plaques. Hyperlipidemic mouse models have been extensively used in studying the mechanisms controlling initiation and progression of atherosclerosis. However, the understanding of mechanisms leading to atherosclerotic plaque destabilization has been hampered by the lack of proper animalmodelsmimicking this process. Although various mouse models generate atherosclerotic plaques with histological features of human advanced lesions, a consensus model to study atherosclerotic plaque destabilization is still lacking. Hence, we studied the degree and features of plaque vulnerability in different mouse models of atherosclerotic plaque destabilization and find that the model based on the placement of a shear stress modifier in combination with hypercholesterolemia represent with high incidence the most human like lesions compared to the other models

Histone Deacetylase 9 Activates IKK to Regulate Atherosclerotic Plaque Vulnerability

Rationale: Arterial inflammation manifested as atherosclerosis is the leading cause of mortality worldwide. Genome-wide association studies have identified a prominent role of histone deacetylase 9 (HDAC9) in atherosclerosis and its clinical complications including stroke and myocardial infarction. Objective: To determine the mechanisms linking HDAC9 to these vascular pathologies and explore its therapeutic potential for atheroprotection. Methods and Results: We studied the effects of Hdac9 on features of plaque vulnerability using bone marrow reconstitution experiments and pharmacological targeting with a small molecule inhibitor in hyperlipidemic mice. We further employed two-photon and intravital microscopy to study endothelial activation and leukocyte-endothelial interactions. We show that hematopoietic Hdac9 deficiency reduces lesional macrophage content whilst increasing fibrous cap thickness thus conferring plaque stability. We demonstrate that HDAC9 binds to IKKα and β resulting in their deacetylation and subsequent activation, which drives inflammatory responses in both macrophages and endothelial cells. Pharmacological inhibition of HDAC9 with the class IIa HDAC inhibitor TMP195 attenuates lesion formation by reducing endothelial activation and leukocyte recruitment along with limiting pro-inflammatory responses in macrophages. Transcriptional profiling using RNA-Seq revealed that TMP195 downregulates key inflammatory pathways consistent with inhibitory effects on IKKβ. TMP195 mitigates the progression of established lesions and inhibits the infiltration of inflammatory cells. Moreover, TMP195 diminishes features of plaque vulnerability and thereby enhances plaque stability in advanced lesions. Ex vivo treatment of monocytes from patients with established atherosclerosis reduced the production of inflammatory cytokines including IL-1β and IL-6. Conclusions: Our findings identify HDAC9 as a regulator of atherosclerotic plaque stability and IKK activation thus providing a mechanistic explanation for the prominence of HDAC9 as a vascular risk locus in genome-wide association studies. Its therapeutic inhibition may provide a potent lever to alleviate vascular inflammation

Acute mental stress drives vascular inflammation and promotes plaque destabilization in mouse atherosclerosis

Publisher Copyright: © 2021 The Author(s). Published by Oxford University Press on behalf of the European Society of Cardiology.Aims: Mental stress substantially contributes to the initiation and progression of human disease, including cardiovascular conditions. We aim to investigate the underlying mechanisms of these contributions since they remain largely unclear. Methods and results: Here, we show in humans and mice that leucocytes deplete rapidly from the blood after a single episode of acute mental stress. Using cell-tracking experiments in animal models of acute mental stress, we found that stress exposure leads to prompt uptake of inflammatory leucocytes from the blood to distinct tissues including heart, lung, skin, and, if present, atherosclerotic plaques. Mechanistically, we found that acute stress enhances leucocyte influx into mouse atherosclerotic plaques by modulating endothelial cells. Specifically, acute stress increases adhesion molecule expression and chemokine release through locally derived norepinephrine. Either chemical or surgical disruption of norepinephrine signalling diminished stress-induced leucocyte migration into mouse atherosclerotic plaques. Conclusion: Our data show that acute mental stress rapidly amplifies inflammatory leucocyte expansion inside mouse atherosclerotic lesions and promotes plaque vulnerability.publishersversionPeer reviewe

Neutrophils as regulators of cardiovascular inflammation

Neutrophils have traditionally been viewed as bystanders or biomarkers of cardiovascular disease. However, studies in the past decade have demonstrated the important functions of neutrophils during cardiovascular inflammation and repair. In this Review, we discuss the influence of traditional and novel cardiovascular risk factors on neutrophil production and function. We then appraise the current knowledge of the contribution of neutrophils to the different stages of atherosclerosis, including atherogenesis, plaque destabilization and plaque erosion. In the context of cardiovascular complications of atherosclerosis, we highlight the dichotomous role of neutrophils in pathogenic and repair processes in stroke, heart failure, myocardial infarction and neointima formation. Finally, we emphasize how detailed knowledge of neutrophil functions in cardiovascular homeostasis and disease can be used to generate therapeutic strategies to target neutrophil numbers, functional status and effector mechanisms

Inhibition of NET Release Fails to Reduce Adipose Tissue Inflammation in Mice.

Obesity-associated diseases such as Type 2 diabetes, liver disease and cardiovascular diseases are profoundly mediated by low-grade chronic inflammation of the adipose tissue. Recently, the importance of neutrophils and neutrophil-derived myeloperoxidase and neutrophil elastase on the induction of insulin resistance has been established. Since neutrophil elastase and myeloperoxidase are critically involved in the release of neutrophil extracellular traps (NETs), we here hypothesized that NETs may be relevant to early adipose tissue inflammation. Thus, we tested the effect of the Peptidyl Arginine Deiminase 4 inhibitor Cl-amidine, a compound preventing histone citrullination and subsequent NET release, in a mouse model of adipose tissue inflammation. C57BL6 mice received a 60% high fat diet for 10 weeks and were treated with either Cl-amidine or vehicle. Flow cytometry of adipose tissue and liver, immunohistological analysis and glucose and insulin tolerance tests were performed to determine the effect of the treatment and diet. Although high fat diet feeding induced insulin resistance no significant effect was observed between the treatment groups. In addition no effect was found in leukocyte infiltration and activation in the adipose tissue and liver. Therefore we concluded that inhibition of neutrophil extracellular trap formation may have no clinical relevance for early obesity-mediated pathogenesis of the adipose tissue and liver

Inhibition of NET Release Fails to Reduce Adipose Tissue Inflammation in Mice.

Obesity-associated diseases such as Type 2 diabetes, liver disease and cardiovascular diseases are profoundly mediated by low-grade chronic inflammation of the adipose tissue. Recently, the importance of neutrophils and neutrophil-derived myeloperoxidase and neutrophil elastase on the induction of insulin resistance has been established. Since neutrophil elastase and myeloperoxidase are critically involved in the release of neutrophil extracellular traps (NETs), we here hypothesized that NETs may be relevant to early adipose tissue inflammation. Thus, we tested the effect of the Peptidyl Arginine Deiminase 4 inhibitor Cl-amidine, a compound preventing histone citrullination and subsequent NET release, in a mouse model of adipose tissue inflammation. C57BL6 mice received a 60% high fat diet for 10 weeks and were treated with either Cl-amidine or vehicle. Flow cytometry of adipose tissue and liver, immunohistological analysis and glucose and insulin tolerance tests were performed to determine the effect of the treatment and diet. Although high fat diet feeding induced insulin resistance no significant effect was observed between the treatment groups. In addition no effect was found in leukocyte infiltration and activation in the adipose tissue and liver. Therefore we concluded that inhibition of neutrophil extracellular trap formation may have no clinical relevance for early obesity-mediated pathogenesis of the adipose tissue and liver

Cl-amidine treatment does not affect metabolic changes associated with high fat diet feeding.

<p><b>A)</b> 3D reconstruction of a NET in obese adipose tissue of HFD fed C57Bl6 mice imaged by immunofluorescence confocal microscopy. Co-localisation of the markers Ly6G (green), cathelicidin (red), and dapi (blue) is shown. Dotted circle shows a NET-releasing neutrophil between the dotted lines. <b>B)</b> Isolated neutrophils were incubated with medium, 100 nM PMA, or 100 nM PMA and 1 mM Cl-amidine (Cl-ami). NETosis was measured with the extracellular DNA dye Sytox green over a period of 4 hours (n = 7). <b>C-G)</b> Mice receiving a 60% HFD for ten weeks were treated with either Cl-amidine (n = 10; 10 mg/kg; s.c.; daily) or vehicle (PBS) (n = 10; s.c.; daily). <b>C)</b> The mice were weighted weekly. <b>D)</b> Prior (pre-HFD) and after 10 weeks of the HFD (Post-HFD) a glucose tolerance test was performed. The mice were fasted for 12 hours after which they received a subcutaneous injection of 1 mg glucose per g mice. Blood glucose levels were measured at different time points. <b>E)</b> Area under the curve was calculated from the GTT graphs Fig 1D. <b>F)</b> Plasma levels of cholesterol were measured by CHOD-PAP (Roche). <b>G)</b> Haematoxylin/eosin staining was performed on paraffin sections of EpAT and the diameter of 100 adipocytes per mice was calculated. Data is represented as mean ± SEM, *p = 0.05, **p = 0.01, ***p = 0.001, ****p = 0.0001, n = 10 for each group unless stated otherwise.</p