Background Atherosclerotic plaque rupture is accompanied by an acute decrease in the carotid plaque expression of microRNAs (miRs) -221 and -222. Circular RNA (circR)-284 is a potential inhibitor of miR-221/-222 activity. We aimed to determine whether changes in the serum levels of these non-coding RNAs (ncRNAs) are observed in patients with asymptomatic high-grade carotid disease versus patients with acutely symptomatic carotid disease and recent ischemic stroke. Additionally, we tested the use of functionally related ncRNA pairs to enhance the discriminatory power of ncRNAs as circulating biomarkers. Methods and Results Serum levels of miR -221,-222, -145 and circR-284 were measured in 24 asymptomatic (Asymptomatic) and 17 acutely symptomatic patients ([Urgent] ischemic cerebrovascular event within the previous 5 days) undergoing carotid endarterectomy (CEA). miR-221 was significantly lower while circR-284 was elevated in the serum of the Urgent compared to the Asymptomatic group. The ratio of serum circR-284:miR-221, was significantly elevated in the Urgent group (p = 0.0002) and exhibited favorable characteristics as a biomarker indicative of carotid plaque rupture and stroke. A validation study in 112 patients (47 Asymptomatic, 41 Urgent, and 24 patients with a cerebrovascular event between 5 to 180 days of the CEA [Symptomatic]) confirmed elevation of serum circR-284:miR-221 uniquely in the Urgent group (p < 0.001) and favorable sensitivity and specificity for detecting plaque rupture and stroke. Conclusions Serum circR-284:miR-221 has potential as a diagnostic biomarker of carotid plaque rupture and stroke. Moreover, we demonstrate use of functionally related pairs of circulating ncRNAs as biomarkers in cardiovascular disease.

Background and Purpose Atherosclerotic plaque vulnerability is accompanied by changes in the molecular and cellular function in the plaque shoulder, including a decrease in vascular smooth muscle cell (VSMC) proliferation. We aimed to determine if the expression of three miRNAs that regulate VSMC proliferation (miR-145, miR-221, and miR-222) are altered with plaque rupture, suggesting a role in regulating plaque stability. Methods miRNAs were measured in the plaque shoulder of carotid plaques obtained from patients undergoing carotid endarterectomy (CEA) for three distinct clinical scenarios: 1) patients without prior neurologic events but high-grade carotid stenosis (asymptomatic), 2) patients with an acute neurologic event within 5 days of the CEA (urgent), and 3) patients undergoing CEA > 5 days after a neurologic event (symptomatic). Results Mean time from plaque rupture event to CEA was 2.4 days in the urgent group. The urgent group exhibited a significant decrease in miR-221 and miR-222 expression in the plaque shoulder while no significant differences were seen in miR-145 across the three groups. Regression analysis demonstrated a significant correlation between time from the neurologic event to CEA and increasing miR-221 and miR-222, but not miR-145. mRNA encoding p27Kip1, a target of miR-221 and -222 that inhibits VSMC proliferation, was increased in the urgent group. Conclusions Atherosclerotic plaque rupture is accompanied by a loss of miR-221 and -222 and an increase in p27Kip1 mRNA expression in the plaque shoulder, suggesting an association between these miRNAs and atherosclerotic plaque stability.

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