Background Many 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).
Hypothesis Neutrophils influence plaque initiation and progression by releasing miRNA-containg microvesicles.
Aim To determine whether neutrophil microvesicles i) influence atherosclerotic plaque development and ii) contain miRNAs relevant to atherosclerosis.
Methods and results Mouse 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).
Conclusion Neutrophil 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.
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