Modulation of Endothelial Interleukin-1β Inflammation

Atherosclerosis, complex chronic inflammatory disease, has a heterogonous aetiology. Endothelium is critically involved in the pathogenesis of atherosclerosis by producing proinflammatory cytokines, including interleukin-1 beta (IL-1β). However, the mechanism by which IL-1β is released is unknown. Neutrophil elastase (NE; a potent serine protease) has been shown to cleave proIL-1β in vitro. Therefore, I hypothesised that NE induces IL-1β secretion from endothelial cells (ECs). I found that NE cleaves proIL-1β in ECs and causes significant secretion of mature IL-1β into supernatant. The release is via extracellular vesicles (EVs), associated with a transient increase in intracellular Ca2+. The released IL-1β is significantly attenuated by inhibition of NE, but not caspase-1. Intracellularly, IL-1β is detected within LAMP-1 positive organelles only after NE treatment. Two distinct populations of vesicles, containing IL-1β are found: at early time points, intracellular vesicles (100-200μm), associated with detection of MV shedding enriched of IL-1β; however, at later time points, IL-1β was detected inside ECs in (>200μm) multivesicular bodies (MVBs) containing exosomes. In a second study, in experimental atherosclerosis, I attempted to manipulate inflammation using omega-3 fatty acids (n3FAs). I hypothesised that docosahexaenoic acid (DHA), the main n3FAs in fish oil, would inhibit inflammation by an IL-1β driven mechanism. I found that DHA significantly decreased high blood pressure and left ventricular mass induced by high fat diet in ApoE-/- mice. Interestingly, this is associated with a reduction in distal vessel atheroma and plasma proinflammatory markers. Locally, DHA also significantly attenuates eNOS and endothelial IL-1β expressions. This study reveals a hitherto unexplained mechanistic link between NE expression in atherosclerotic plaques and concomitant bioactive IL-1β secretion from ECs, highlighting the possibility of targeting NE to control IL-1β-induced atherosclerosis. It also sheds a light, for the first time, on how DHA can act as an anti-atherogenic agent through its effects upon IL-1 system

Dietary Docosahexaenoic Acid Reduces Oscillatory Wall Shear Stress, Atherosclerosis, and Hypertension, Most Likely Mediated via an IL‐1–Mediated Mechanism

Background: Hypertension is a complex condition and a common cardiovascular risk factor. Dietary docosahexaenoic acid (DHA) modulates atherosclerosis and hypertension, possibly via an inflammatory mechanism. IL‐1 (interleukin 1) has an established role in atherosclerosis and inflammation, although whether IL‐1 inhibition modulates blood pressure is unclear. Methods and Results: Male apoE−/− (apolipoprotein E–null) mice were fed either a high fat diet or a high fat diet plus DHA (300 mg/kg per day) for 12 weeks. Blood pressure and cardiac function were assessed, and effects of DHA on wall shear stress and atherosclerosis were determined. DHA supplementation improved left ventricular function, reduced wall shear stress and oscillatory shear at ostia in the descending aorta, and significantly lowered blood pressure compared with controls (119.5±7 versus 159.7±3 mm Hg, P<0.001, n=4 per group). Analysis of atheroma following DHA feeding in mice demonstrated a 4‐fold reduction in lesion burden in distal aortas and in brachiocephalic arteries (P<0.001, n=12 per group). In addition, DHA treatment selectively decreased plaque endothelial IL‐1β (P<0.01). Conclusions: Our findings revealed that raised blood pressure can be reduced by inhibiting IL‐1 indirectly by administration of DHA in the diet through a mechanism that involves a reduction in wall shear stress and local expression of the proinflammatory cytokine IL‐1β

Selective improvement of pulmonary arterial hypertension with a dual ETA/ETB receptors antagonist in the apolipoprotein E−/− model of PAH and atherosclerosis

Idiopathic pulmonary arterial hypertension (IPAH) is increasingly diagnosed in elderly patients who also have an increased risk of comorbid atherosclerosis. Apolipoprotein E deficient (ApoE-/-) mice develop atherosclerosis with severe PAH when fed a high-fat diet (HFD), and have increased levels of endothelin (ET)-1. ET-1 receptor antagonists (ERAs) are used for the treatment of PAH but less is known about whether ERAs are beneficial in atherosclerosis. We therefore examined whether treatment of HFD-ApoE-/- mice with macitentan, a dual ETA/ETB receptor antagonist, would have any effect on both atherosclerosis and PAH. ApoE-/- mice were fed chow or HFD for 8 weeks. After 4 weeks of HFD, mice were randomised to a 4-week treatment of macitentan by food (30mg/kg/day dual ETA/ETB antagonist), or placebo groups. Echocardiography and closed-chest right heart catheterisation were used to determine PAH phenotype and serum samples were collected for cytokine analysis. Thoracic aortas were harvested to assess vascular reactivity using wire myography, and histological analyses were performed on the brachiocephalic artery and aortic root to assess atherosclerotic burden. Macitentan treatment of HFD-fed ApoE-/- mice was associated with a beneficial effect on the PAH phenotype and led to an increase in endothelial-dependent relaxation in thoracic aortae. Macitentan treatment was also associated with a significant reduction in interleukin 6 (IL-6) concentration but there was no significant effect on atherosclerotic burden. Dual blockade of ETA/ETB receptors improves endothelial function and improves experimental PAH but had no significant effect on atherosclerosis

Neutrophil Elastase Promotes Interleukin-1 beta Secretion from Human Coronary Endothelium

The endothelium is critically involved in the pathogenesis of atherosclerosis by producing pro-inflammatory mediators, including IL-1β. Coronary arteries from patients with ischemic heart disease express large amounts of IL-1β in the endothelium. However, the mechanism by which endothelial cells (ECs) release IL-1β remains to be elucidated. We investigated neutrophil elastase (NE), a potent serine protease detected in vulnerable areas of human carotid plaques, as a potential “trigger” for IL-1β processing and release. This study tested the hypothesis that NE potentiates the processing and release of IL-1β from human coronary endothelium. We found that NE cleaves the pro-isoform of IL-1β in ECs and causes significant secretion of bioactive IL-1β via extracellular vesicles. This release was attenuated significantly by inhibition of neutrophil elastase but not caspase-1. Transient increases in intracellular Ca2+ levels were observed prior to secretion. Inside ECs, and after NE treatment only, IL-1β was detected within LAMP-1-positive multivesicular bodies. The released vesicles contained bioactive IL-1β. In vivo, in experimental atherosclerosis, NE was detected in mature atherosclerotic plaques, predominantly in the endothelium, alongside IL-1β. This study reveals a novel mechanistic link between NE expression in atherosclerotic plaques and concomitant pro-inflammatory bioactive IL-1β secretion from ECs. This could reveal additional potential anti-IL-1β therapeutic targets and provide further insights into the inflammatory process by which vascular disease develops

“How to Release or Not Release, That Is the Question.” A Review of Interleukin‐1 Cellular Release Mechanisms in Vascular Inflammation

Cardiovascular disease remains the leading cause of death worldwide, characterized by atherosclerotic activity within large and medium‐sized arteries. Inflammation has been shown to be a primary driver of atherosclerotic plaque formation, with interleukin‐1 (IL‐1) having a principal role. This review focuses on the current state of knowledge of molecular mechanisms of IL‐1 release from cells in atherosclerotic plaques. A more in‐depth understanding of the process of IL‐1's release into the vascular environment is necessary for the treatment of inflammatory disease processes, as the current selection of medicines being used primarily target IL‐1 after it has been released. IL‐1 is secreted by several heterogenous mechanisms, some of which are cell type–specific and could provide further specialized targets for therapeutic intervention. A major unmet challenge is to understand the mechanism before and leading to IL‐1 release, especially by cells in atherosclerotic plaques, including endothelial cells, vascular smooth muscle cells, and macrophages. Data so far indicate a heterogeneity of IL‐1 release mechanisms that vary according to cell type and are stimulus‐dependent. Unraveling this complexity may reveal new targets to block excess vascular inflammation

192 dietary docosahexaenoic acid reduced experimental atherosclerosis by inducing protective haemodynamic conditions [abstract only]

Introduction Dietary omega-3 fatty acids have been associated with protection from atherosclerosis. However, the underlying mechanisms are incompletely understood. Blood flow generate a frictional force on endothelial cells called wall shear stress (WSS) that alters vascular wall function. The aim of this study was to determine whether docosahexaenoic acid (DHA), an omega-3 fatty acid, modulates vascular wall inflammation, blood flow velocity and WSS in experimental atherosclerosis.Methods ApoE–/– mice fed either high fat diet (control) or high fat diet plus DHA (300 mg/kg/day) for 12 weeks (n = 12/group). Blood flow velocity was assessed using pulsed wave doppler echocardiography and blood pressure was monitored using Visitech tail-cuff system. Atherosclerosis was measured in whole aorta using enface Oil red O stain, and in aortic roots and brachiocephalic sections stained with Alcian Blue & Elastic Van Gieson stain. Computational flow dynamics (CFD) was used to map WSS magnitude and oscillation in the aorta. Plasma cholesterol levels were quantified by gas chromatography.Results Plasma high density lipoprotein/total cholesterol ratio was significantly increased in DHA treated mice compared to controls (10.77 ± 1.86 vs. 6.63 ± 1.04, p < 0.05). DHA fed mice exhibited a 4–5 fold reduction in distal aortic and brachiocephalic atherosclerosis (p < 0.01) whereas lesion burden in the aortic arch was similar between groups. Dietary supplementation using DHA led to a reduction in blood pressure (119.5 ± 7.33 mmHg (+DHA) vs. 159.7 ± 2.482 mmHg (controls), p < 0.001 and a 12 decrease in aortic blood flow. CFD revealed that oscillatory shear in the descending aorta was reduced in DHA-fed mice compared to controls.Conclusions/Implications Our study suggests that dietary DHA can act systemically by enhancing levels of HDL. It can also act locally by reducing oscillatory shear stress in the descending aorta. Dietary DHA reduced lesion formation specifically in the descending aorta, an effect that can be explained by its dual effects on oscillatory shear and HDL. Therefore, the current study suggests novel and interacting protective mechanisms for DHA actions in atherogenesis with implications for the development of dietary interventions to prevent cardiovascular disease

Carbon monoxide releasing molecule A1 reduces myocardial damage after acute myocardial infarction in a porcine model

International audienceInfarct size is a major determinant of outcomes after acute myocardial infarction (AMI). Carbon monoxide releasing molecules (CORMs), which deliver nano-molar concentrations of carbon monoxide to tissues, have been shown to reduce infarct size in rodents. We evaluated efficacy and safety of CORM-A1 to reduce infarct size in a clinically relevant porcine model of AMI. We induced AMI in Yorkshire White pigs by inflating a coronary angioplasty balloon to completely occlude the left anterior descending artery for 60 minutes, followed by deflation of the balloon to mimic reperfusion. Fifteen minutes after balloon occlusion, animals were given an infusion of 4.27mM CORM-A1 (n=7) or sodium borate control (n=6) over 60 minutes. Infarct size, cardiac biomarkers, ejection fraction and hepatic and renal function were compared amongst the groups. Immunohistochemical analyses were performed to compare inflammation, cell proliferation and apoptosis between the groups. CORM-A1 treated animals had significant reduction in absolute infarct area (158+/-16 vs. 510+/-91 mm2, p<0.001) and infarct area corrected for area at risk (24.8+/-2.6% vs. 45.2+/-4.0%, p<0.0001). Biochemical markers of myocardial injury also tended to be lower and LV function tended to recover better in CORM-A1 treated group. There was no evidence of hepatic or renal toxicity with the doses used. The cardio-protective effects of CORM-A1 were associated with a significant reduction in cell proliferation and inflammation. CORM-A1 reduces infarct size and improves LV remodelling and function in a porcine model of reperfused MI via a reduction in inflammation. These potential cardio-protective effects of CORMs warrant further translational investigations

Table1_The interleukin-1 receptor type-1 in disturbed flow-induced endothelial mesenchymal activation.docx

IntroductionAtherosclerosis is a progressive disease that develops in areas of disturbed flow (d-flow). Progressive atherosclerosis is characterized by bulky plaques rich in mesenchymal cells and high-grade inflammation that can rupture leading to sudden cardiac death or acute myocardial infarction. In response to d-flow, endothelial cells acquire a mesenchymal phenotype through endothelial-to-mesenchymal transition (EndMT). However, the signaling intermediaries that link d-flow to EndMT are incompletely understood.Methods and ResultsIn this study we found that in human atherosclerosis, cells expressing SNAI1 (Snail 1, EndMT transcription factor) were highly expressed within the endothelial cell (EC) layer and in the pre-necrotic areas in unstable lesions, whereas stable lesions did not show any SNAI1 positive cells, suggesting a role for EndMT in lesion instability. The interleukin-1 (IL-1), which signals through the type-I IL-1 receptor (IL-1R1), has been implicated in plaque instability and linked to EndMT formation in vitro. Interestingly, we observed an association between SNAI1 and IL-1R1 within ECs in the unstable lesions. To establish the causal relationship between EndMT and IL-1R1 expression, we next examined IL-1R1 levels in our Cre-lox endothelial-specific lineage tracing mice. IL-1R1 and Snail1 were highly expressed in ECs under atheroprone compared to athero-protective areas, and oscillatory shear stress (OSS) increased IL-1R1 protein and mRNA levels in vitro. Exposure of ECs to OSS resulted in loss of their EC markers and higher induction of EndMT markers. By contrast, genetic silencing of IL-1R1 significantly reduced the expression of EndMT markers and Snail1 nuclear translocation, suggesting a direct role for IL-1R1 in d-flow-induced EndMT. In vivo, re-analysis of scRNA-seq datasets in carotid artery exposed to d-flow confirmed the IL-1R1 upregulation among EndMT population, and in our partial carotid ligation model of d-flow, endothelial cell specific IL-1R1 KO significantly reduced SNAI1 expression.DiscussionGlobal inhibition of IL-1 signaling in atherosclerosis as a therapeutic target has recently been tested in the completed CANTOS trial, with promising results. However, the data on IL-1R1 signaling in different vascular cell-types are inconsistent. Herein, we show endothelial IL-1R1 as a novel mechanosensitive receptor that couples d-flow to IL-1 signaling in EndMT.</p

Image2_The interleukin-1 receptor type-1 in disturbed flow-induced endothelial mesenchymal activation.jpeg

IntroductionAtherosclerosis is a progressive disease that develops in areas of disturbed flow (d-flow). Progressive atherosclerosis is characterized by bulky plaques rich in mesenchymal cells and high-grade inflammation that can rupture leading to sudden cardiac death or acute myocardial infarction. In response to d-flow, endothelial cells acquire a mesenchymal phenotype through endothelial-to-mesenchymal transition (EndMT). However, the signaling intermediaries that link d-flow to EndMT are incompletely understood.Methods and ResultsIn this study we found that in human atherosclerosis, cells expressing SNAI1 (Snail 1, EndMT transcription factor) were highly expressed within the endothelial cell (EC) layer and in the pre-necrotic areas in unstable lesions, whereas stable lesions did not show any SNAI1 positive cells, suggesting a role for EndMT in lesion instability. The interleukin-1 (IL-1), which signals through the type-I IL-1 receptor (IL-1R1), has been implicated in plaque instability and linked to EndMT formation in vitro. Interestingly, we observed an association between SNAI1 and IL-1R1 within ECs in the unstable lesions. To establish the causal relationship between EndMT and IL-1R1 expression, we next examined IL-1R1 levels in our Cre-lox endothelial-specific lineage tracing mice. IL-1R1 and Snail1 were highly expressed in ECs under atheroprone compared to athero-protective areas, and oscillatory shear stress (OSS) increased IL-1R1 protein and mRNA levels in vitro. Exposure of ECs to OSS resulted in loss of their EC markers and higher induction of EndMT markers. By contrast, genetic silencing of IL-1R1 significantly reduced the expression of EndMT markers and Snail1 nuclear translocation, suggesting a direct role for IL-1R1 in d-flow-induced EndMT. In vivo, re-analysis of scRNA-seq datasets in carotid artery exposed to d-flow confirmed the IL-1R1 upregulation among EndMT population, and in our partial carotid ligation model of d-flow, endothelial cell specific IL-1R1 KO significantly reduced SNAI1 expression.DiscussionGlobal inhibition of IL-1 signaling in atherosclerosis as a therapeutic target has recently been tested in the completed CANTOS trial, with promising results. However, the data on IL-1R1 signaling in different vascular cell-types are inconsistent. Herein, we show endothelial IL-1R1 as a novel mechanosensitive receptor that couples d-flow to IL-1 signaling in EndMT.</p