Neutrophils are implicated in the pathogenesis of atherosclerosis but are seldom detected in atherosclerotic plaques. We investigated whether neutrophil-derived microvesicles may influence arterial pathophysiology. Here we report that levels of circulating neutrophil microvesicles are enhanced by exposure to a high fat diet, a known risk factor for atherosclerosis. Neutrophil microvesicles accumulate at disease-prone regions of arteries exposed to disturbed flow patterns, and promote vascular inflammation and atherosclerosis in a murine model. Using cultured endothelial cells exposed to disturbed flow, we demonstrate that neutrophil microvesicles promote inflammatory gene expression by delivering miR-155, enhancing NF-κB activation. Similarly, neutrophil microvesicles increase miR-155 and enhance NF-κB at disease-prone sites of disturbed flow in vivo. Enhancement of atherosclerotic plaque formation and increase in macrophage content by neutrophil microvesicles is dependent on miR-155. We conclude that neutrophils contribute to vascular inflammation and atherogenesis through delivery of microvesicles carrying miR-155 to disease-prone regions.

Neutrophils have been implicated in the pathogenesis of atherosclerosis, a lipid-driven disease of arteries, but they are seldom found in atherosclerotic plaques. To resolve this longstanding paradox, we investigated whether neutrophil-derived microvesicles may influence arterial pathophysiology. Clinical and pre-clinical studies revealed that levels of circulating neutrophil microvesicles were enhanced by exposure to a high fat diet, a known risk factor for atherosclerosis. Neutrophil microvesicles accumulated at disease-prone regions of arteries that are exposed to complex flow patterns, and they promoted vascular inflammation and atherosclerosis in a murine model. Using cultured endothelial cells exposed to disturbed flow, it was demonstrated that neutrophil microvesicles promoted inflammatory gene expression by delivering a microRNA (miR-155) that enhanced NF-κB activation. Similary, neutrophil microvesicles increased miR-155 and enhanced NF-κB at disease-prone sites of disturbed flow in arteries of mice. We conclude that delivery of microvesicles carrying miR-155 to disease-prone regions of arteries provides a novel mechanism by which neutrophils contribute to vascular inflammation and atherogenesis.Key words: atherosclerosis, vascular inflammation, microvesicle, microRNA All rights reserved. No reuse allowed without permission.(which was not peer-reviewed) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity.The copyright holder for this preprint . http://dx.doi.org/10.1101/319392 doi: bioRxiv preprint first posted online May. 11, 2018; (which was not peer-reviewed) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity.The copyright holder for this preprint . http://dx.doi.org/10.1101/319392 doi: bioRxiv preprint first posted online May. 11, 2018; Gomez et al. Neutrophil microvesicles and atherogenesis 4 A causal role for neutrophils in atherosclerosis is now evident and these abundant leukocytes have been shown to play a part both in plaque development 1,2 and plaque erosion 3 , as well as being implicated in plaque rupture 4,5 . Increased levels of circulating neutrophils exacerbate atherosclerotic plaque formation in mice 2 and indirect evidence also links increased circulating leukocyte counts and infection with an increased risk of cardiovascular disease 6,7 . Nevertheless, detection of neutrophils in lesions is rare, possibly due to their short life span, the rapid removal of senescent neutrophils by macrophages within the developing plaque, and/or the lack of a highly specific detection method (see 8 for review). In order to address this paradox, we have investigated whether neutrophils exacerbate vascular inflammation through the release of pro-inflammatory microvesicles (MVs), thus influencing atherosclerotic plaque formation without entering the vessel wall.MVs are 0.1-1 µm vesicles released from the cell membrane in response to various stimuli or during apoptosis. Depending on the cell source, MVs var...

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BackgroundMany cell types are involved in the initiation and progression of atherosclerosis but neutrophils are rarely considered to be among them. However, despite their infrequent detection in plaques, neutrophils have been shown to facilitate the movement of monocytes into the vessel wall and increase plaque growth. One possible mechanism for this is through release of microvesicles that contain cargo such as microRNAs (miRNAs).HypothesisNeutrophils influence plaque initiation and progression by releasing miRNA-containg microvesicles.AimTo determine whether neutrophil microvesicles i) influence atherosclerotic plaque development and ii) contain miRNAs relevant to atherosclerosis.Methods and resultsMouse blood neutrophils were stimulated for 1h with fMLP (3 × 105 M) and supernatants subjected to differential centrifugation (twice at 300g for 6 min followed by 20,000g for 20 mins). Microvesicles were quantified using calibrated flow cytometry. ApoE–/– mice were fed Western diet for 6 weeks and injected twice a week with sterile PBS or microvesicles (4 × 106) isolated from wild type mice. After 6 weeks aortae were dissected and stained with Oil Red-O to identify areas of plaque formation. To detect miRNA, human neutrophils were isolated and stimulated with fMLP (10–5 M), AcLDL (20 µg/ml) or PBS for 2 h and microvesicles pelleted by differential centrifugation. MiRNA extraction and quantitative RT-PCR were performed in microvesicles, HUVEC and HUVEC incubated with microvesicles for 2 h.The number of neutrophils isolated from mice injected with microvesicles was increased (PBS = 67.3 ± 3.9/μl; microvesicles = 88.25 ± 4.0/μl, P = 0.031; n = 3 in each group). In addition neutrophil reactivity with respect to microvesicle formation was also increased (PBS = 4.5 ± 0.8 microvesicles per neutrophil; microvesicle = 8.3 ± 0.7 microvesicles per neutrophil, P = 0.0249; n = 3). Consequently plaque formation was more advanced in mice injected with microvesicles (% lesion area in aortic arch: PBS = 0.4563 ± 0.09452; microvesicles = 1.643 ± 0.3891, P = 0.0118; n = 7 in each group).MiR-223 and miR-150, found to be constitutively expressed in microvesicles and not in HUVEC, are involved in inflammation and atherosclerosis. These miRNAs were increased in after fMLP and AcLDL stimulation compared to PBS and were found to be significantly increased in HUVEC after 2h incubation with microvesicles (miR-223: 1.74 ± 0.44, P = 0.0305; 2.68 ± 0.50, P = 0.0034 and 6.79 ± 0.99, P = 0.0012 fold increase with microvesicles from PBS, fMLP and AcLDL stimulated neutrophils respectively and for miR-150: 3.79 ± 0.21, P = 0.0131 and 3.89 ± 0.78, P = 0.0109 fold increase with microvesicles from fMLP and AcLDL stimulated neutrophils respectively; n = 5 for each group).ConclusionNeutrophil microvesicles induce increased plaque formation and contain miRNA that may be transferred to endothelial cells suggesting neutrophils may be involved in atherogenesis through the release of microvesicles.

Background: Atherosclerosis is the most common underlying cause of cardiovascular disease. Many cells types are involved in the initiation and progression of atherosclerotic plaques but, until recently, neutrophils were not considered to be among them. Despite their rare detection in plaques, neutrophil depletion has been shown to reduce plaque formation and monocyte recruitment to the vessel wall. Hypothesis: Neutrophils may influence plaque initiation and progression through the formation of microvesicles. Aim: To determine the role of neutrophil-derived-microvesicles in the early stage of atherosclerosis. Methods and Results: Mouse peripheral blood neutrophils were isolated by negative magnetic separation. Following stimulation for 1h with fMLP (3x105M), supernatants were subjected to differential centrifugation (twice at 300g for 6 min followed by 20,000g for 20 mins) to isolate the microvesicle population. Microvesicles were quantified using flow cytometry calibrated with MegaMix and Spherocount beads. Apo E mice were fed a Western diet for 6 weeks and injected twice a week with PBS or neutrophil microvesicles (4.52x106 ± 3.05x105) isolated from control mice. After 6 weeks, blood was collected for neutrophil and microvesicle quantification. The aorta was dissected and stained by conventional procedures using Oil Red. Areas of plaque formation was determined using computer based imaging and semi-quantitative scoring. Injection with microvesicles increased circulating neutrophil number (PBS treated = 67.3 ± 3.9 neutrophils per μl; microvesicle treated = 88.25 ± 4.0 neutrophils per μl, P = 0.031; n = 3) and neutrophil reactivity with respect to microvesicle formation (PBS treated = 4.5 ± 0.8 microvesicles per neutrophil; microvesicle treated = 8.3±0.7 microvesicles per neutrophil, P = 0.0249; n = 3). After 6 weeks, the plaque formation was more advanced in the MVs group (% lesion area in the arch in mice injected with PBS = 0.4563 ± 0.09452, with MVs = 1.643 ± 0.3891, P = 0.0118, n=7). Conclusion: Neutrophil microvesicles induce increased plaque formation and increase circulating neutrophil levels in a mouse model of atherosclerosis. This suggests neutrophils may be involved in atherogenesis through the release of microvesicles.