Plasma MicroRNA-100 Is Associated With Coronary Plaque Vulnerability

Abstract

Background: Although numerous studies have reported altered plasma levels of various microRNAs (miRNAs) in patients with cardiovascular disease, there are no data on the relationship between plasma miRNAs and vulnerable coronary plaque. In this study, we investigated whether plasma miRNAs might be a sensitive marker of coronary plaque vulnerability.

Methods and Results: Integrated backscatter intravascular ultrasound (IB-IVUS) was performed in 32 consecutive patients with angina pectoris who underwent percutaneous coronary intervention. Three-dimensional analysis of IB-IVUS was performed to determine the percentage of lipid volume (%LV) and fibrous volume (%FV). Circulating miRNAs were measured in EDTA-plasma simultaneously obtained from the aorta and the coronary sinus (CS). Muscle-enriched (miR-133a, miR-208a, miR-499), vascular-enriched (miR-92a, miR-100, miR-126, miR-127, miR-145), and myeloid cell-enriched miRNAs (miR-155, miR-223) were measured. Plasma miR-100 was higher in the CS than in the aorta, but there were no significant differences in the levels of other miRNAs between the aorta and CS. Plasma miR-100 in the aorta was positively correlated with %LV (r=0.48, P<0.01) and negatively correlated with %FV (r=–0.41, P<0.05). Importantly, transcoronary concentration gradient of circulating miR-100 was more strongly correlated with %LV (r=0.53, P<0.01) and %FV (r=–0.56, P<0.01).

Conclusions: miR-100 might be released into the coronary circulation from vulnerable coronary plaques. This study provides insights into the role of miRNAs in coronary atherosclerotic disease. (Circ J 2015; 79: 413–418)

MicroRNAs (miRNAs) are short non-coding RNA sequences consisting of 19–23 nucleotides that regulate gene expression on the post-transcriptional level by binding to target mRNA, which leads to either degradation or translational repression.¹ Recent studies have demonstrated that miRNAs, which can be detected in circulating blood in a remarkably stable form, may be useful as biomarkers for disease.^2,3 In the nucleus, miRNA is transcribed from DNA. After being transported into the cytoplasm, pre-miRNA can be cleaved into mature miRNA duplexes. Cytoplasmic miRNA can be released by microvesicles, which are then released from the cell through blebbing of the plasma membrane.⁴

Editorial p 303

Atherosclerotic plaque rupture is considered the most important mechanism underlying most acute ischemic syndromes, including acute coronary syndrome (ACS) and stroke. Although the pathophysiology of plaque rupture is not completely understood, it is now well accepted that lesion vulnerability is more closely correlated to plaque composition than size.⁵ Integrated backscatter intravascular ultrasound (IB-IVUS) has recently been developed, allowing for analysis of the tissue components of coronary plaques in vivo.^6–9 Apart from its diagnostic utility, IB-IVUS has also proved useful in assessing prognosis in patients with coronary atherosclerosis, and the risk of experiencing a coronary event.^10,11

Although numerous studies have reported altered plasma and serum levels of various miRNAs in patients with cardiovascular disease, there are no data on the relationship between plasma miRNAs and vulnerable coronary plaque. Therefore, in the present study, we investigated whether plasma miRNA level might constitute a sensitive marker of coronary plaque vulnerability.

Methods

Subjects

A total of 32 patients with angina pectoris with ≥50% stenosis of at least 1 coronary artery who underwent percutaneous coronary intervention (PCI) were analyzed. Patients were excluded from the study if they had active myocardial infarction, previous coronary revascularization surgery, history of malignancy, significant renal or hepatic dysfunction, active collagen vascular disease, or apparent heart failure. Written informed consent was obtained from each patient, and the study protocol was approved by the Institutional Review Board of Tokushima University Hospital.

IB-IVUS and Tissue Characterization

For IVUS, the Atlantis ultrasound catheter (40 MHz; Boston Scientific/Cardiovascular System, San Jose, CA, USA) was used. Data were collected with auto-pullback at 0.5 mm/s and analyzed using an IVUS console (Galaxy, Boston Scientific/Cardiovascular System). To prevent coronary spasms, optimal dose of i.c. isosorbide dinitrate was given prior to measurement. In the non-culprit moderate stenotic lesion in the proximal or mid-portion on angiography, the most clearly visible plaque with IB-IVUS was selected as the target area. A total of 10 IB-IVUS images (5 mm in length) were captured at intervals of 0.5 mm using a motorized pullback system in each plaque, as previously described.¹²

IB-IVUS parameters were measured immediately before PCI. As previously described,¹⁰ plaque properties were classified into 4 types by combining spectral parameters of IVUS posterior scattering signals: lipid pool; fibrosis; dense fibrosis; or calcification. The percent area of each component was automatically measured for each plaque. The percent volume was calculated using integration. IVUS measurements were performed independently by 2 physicians who were blinded to patient clinical characteristics.

Plasma miRNA Measurement

Blood samples were simultaneously obtained from the coronary sinus (CS) and the aortic bulb during cardiac catheterization, before heparin or any contrast agents were administered and before any interventional procedures were initiated. After rapid centrifugation in EDTA-containing tubes, samples were transferred to RNase/DNase-free tubes and stored at –80℃.

Total plasma RNA was isolated using the TRIzol LS RNA isolation kit (Invitrogen, Tokyo, Japan). Reverse transcription was performed using the TaqMan microRNA Reverse transcription Kit (Applied Biosystems, CA, USA) with 10ng of total RNA. Subsequently, TaqMan miRNA assay kits (Applied Biosystems) were used to detect miRNA expression on quantitative polymerase chain reaction (PCR) for each miRNA. The relative cycle threshold (Ct) for U6 small nuclear RNA was used as an endogenous control for normalizing the respective miRNA Ct as previously described.^13,14

Ten miRNAs were chosen for this study. miR-133a, miR-208a, and miR-499 were chosen as “myocardial muscle”-enriched miRNAs because it has been suggested that these miRNAs are released from the heart into the coronary circulation during myocardial injury in patients with ACS.¹⁵ miR-92a, miR-100, miR-126, miR-127, and miR-145 were chosen as vascular-enriched miRNAs because some studies have suggested that they are associated with atherosclerotic plaque development, progression, and disruption.^16,17 miR-155 and miR-223 were chosen as “myeloid cell”-enriched miRNAs because it has been suggested that they are involved in the regulation of immune cell function in atherosclerotic lesions.^18,19

Statistical Analysis

Data are given as mean±SD. Comparison between 2 groups was performed using unpaired t-test or one-way analysis of variance followed by Bonferroni correction. Correlations between 2 parameters were assessed using simple linear regression. Multivariate logistic regression was used to determine the effects of clinical atherosclerotic risk factors on the impact of miRNA level on IB-IVUS parameters. Differences were considered significant for P<0.05.

Results

miRNA in Aortic and CS Blood

The clinical characteristics are listed in Table 1, and target lesion characteristics for coronary angiography and IVUS in patients with angina pectoris who underwent PCI are given in Table 2. Among the 10 miRNAs measured in the present study, miR-208a and miR-499 were not detected with the present PCR protocol, so we analyzed the profile of the remaining 8 miRNAs.

Table 1. Patient Characteristics

Characteristic
Age (years)	69±7
M/F	21/11
Hypertension	26 (81)
Diabetes	12 (38)
LDL cholesterol (mg/dl)	140±176
HDL cholesterol (mg/dl)	52±15
Triglycerides (mg/dl)	137±75
BMI (kg/m2)	25.6±2.8

Data given as mean±SD, n (%) or n/n. BMI, body mass index; HDL, high-density lipoprotein; LDL, low-density lipoprotein.

Table 2. Target Lesion Characteristics

Target plaque location
LAD/LCX/RCA	12 (38)/8 (25)/12 (38)
Proximal/Mid	23 (72)/9 (28)
IVUS
Mean vessel area (mm2)	15.5±4.4
Mean lumen are (mm2)	8.7±3.1
Plaque volume (mm3)	19.2±7.5
Lipid volume (mm3)	7.3±4.4
Fibrous volume (mm3)	10.6±4.7
Lipid volume (%)	37.2±13.3
Fibrous volume (%)	55.3±10.2

Data given as mean±SD or n (%). IVUS, intravascular ultrasound; LAD, left anterior descending coronary artery; LCX, left circumflex coronary artery; RCA, right coronary artery.

First, we compared the levels of miRNAs in the aorta and CS. As shown in Figure 1, one of the vascular-enriched miRNAs, miR-100, was significantly higher in the CS than in the aorta. miR-126 was lower in the CS than in the aorta, but it did not reach statistical significance. There were no significant differences, however, in miR-133, miR-92a, miR-127, miR-145, miR-155, and miR-223 between the aorta and the CS.

Figure 1.

Circulating microRNA (miRNA) level in plasma from the aorta and coronary sinus (CS). miR-100 was significantly higher in the CS than in the aorta, and miR-126 was lower in the CS than in the aorta, but this difference did not reach statistical significance. There were no significant differences, however, in the levels of other miRNAs between the aorta and CS.

Plasma miR-100 and Coronary Plaque Characteristics

Plasma miR-100 in the aorta was positively correlated with percentage of lipid volume (%LV; r=0.48, P<0.01) and negatively correlated with percentage of fibrous volume (%FV; r=–0.41, P<0.05; Figure 2). Importantly, transcoronary concentration gradient of circulating miR-100 was more strongly correlated with %LV (r=0.53, P<0.01) and %FV (r=–0.56, P<0.01) compared with plasma miR-100 level in the aorta (Figure 3). In contrast, there were no significant correlations observed between %LV or %FV and plasma level of other miRNAs. There were also no significant correlations between mean vessel area or mean lumen area or plaque volume and plasma level of all miRNAs.

Figure 2.

Plasma miR-100 aorta level and coronary plaque characteristics. Plasma miR-100 aorta level was positively correlated with the percentage of lipid volume (%LV) and negatively correlated with the percentage of fibrous volume (%FV).

Figure 3.

Transcoronary concentration gradient of circulating miR-100 (∆miR-100) level and coronary plaque characteristics. ∆miR-100 was more strongly correlated with percentage of lipid volume (%LV) and percentage of fibrous volume (%FV) than miR-100 aorta level.

On multiple regression analysis transcoronary concentration gradient of circulating miR-100 was positively correlated with %LV independent of age, body mass index, hypertension, diabetes mellitus, smoking, lipid profile, and renal function (Table 3). In addition, transcoronary concentration gradient of circulating miR-100 was negatively correlated with %FV independent of age, body mass index, hypertension, diabetes mellitus, smoking, lipid profile, and renal function (Table 4).

Table 3. Multivariate Indicators of %LV

	Standard regression coefficient	t statistic	P-value
ΔmiR-100 (CS−aorta)	0.591	2.514	0.021
Age	−0.146	−0.538	0.596
BMI	0.026	0.095	0.925
Hypertension	−0.012	−0.058	0.954
Diabetes mellitus	−0.146	−0.693	0.496
Smoking	−0.134	−0.632	0.534
LDL cholesterol	0.052	0.254	0.802
HDL cholesterol	−0.153	−0.625	0.539
Triglycerides	0.224	0.741	0.467
eGFR	0.045	0.218	0.829

ΔmiR-100, transcoronary concentration gradient of circulating microRNA-100; %LV, percentage of lipid volume; CS, coronary sinus; eGFR, estimated glomerular filtration rate. Other abbreviations as in Table 1.

Table 4. Multivariate Indicators of %FV

	Standard regression coefficient	t statistic	P-value
ΔmiR-100 (CS−aorta)	−0.555	−2.560	0.019
Age	−0.006	−0.024	0.980
BMI	−0.215	−0.842	0.410
Hypertension	−0.048	−0.246	0.808
Diabetes mellitus	0.115	0.590	0.562
Smoking	0.131	0.668	0.512
LDL cholesterol	−0.159	−0.834	0.414
HDL cholesterol	0.111	0.491	0.628
Triglycerides	−0.082	−0.294	0.772
eGFR	0.175	0.911	0.374

%FV, percentage of fibrous volume. Other abbreviations as in Tables 1,3.

Transcoronary concentration gradient of circulating miR-100 was higher in patients with 3-vessel disease than in patients with disease in 1 or 2 vessels, but this difference did not reach statistical significance (Figure 4).

Figure 4.

Transcoronary concentration gradient of circulating miR-100 (∆miR-100) according to the number of vessels with ≥50% stenosis on coronary angiography. ∆miR-100 was higher in patients with 3-vessel disease than in those with disease in 1 or 2 vessels, but this difference did not reach statistical significance.

miRNA and Cardiac Function or Chronic Inflammation

Transcoronary concentration gradient of circulating miRNAs, including miR-100, was not correlated with brain natriuretic peptide, left ventricular end-diastolic diameter, left ventricular ejection fraction, or C-reactive protein (CRP; data not shown).

Discussion

In the present study, we have demonstrated for the first time that plasma miR-100 is positively correlated with %LV and negatively correlated with %FV as determined on IB-IVUS. In addition, we found that plasma miR-100 was higher in blood samples from the CS compared with the aorta, and that transcoronary concentration gradient of circulating miR-100 is more strongly correlated with %LV and %FV compared with plasma miR-100 level in the aorta. This suggests that miR-100 may be released into the coronary circulation from vulnerable coronary plaques and therefore may be useful as a biomarker of plaque vulnerability.

These findings are compatible with a previous study showing that the expression of miR-100 was higher in symptomatic plaques than in asymptomatic plaques collected in carotid endarterectomy.²⁰ In accordance with this finding, a recent study showed that miR-100 modulates proliferation, tube formation, and sprouting activity of endothelial cells and migration of vascular smooth muscle cells, and it functions as an endogenous repressor of mammalian target of rapamycin (mTOR) in mice with hind-limb ischemia.²¹ A recent study showed that knockdown of mTOR ameliorated dysregulated blood lipid metabolism and stabilized aortic atherosclerotic plaques by decreasing plaque area and increasing the size of fibrous cap and cap-to-core ratio via a decrease in the number of macrophages by autophagy in apolipoprotein E-deficient mice.²² Another recent study showed that increased mTOR activity upregulated sterol regulatory element-binding protein-2-mediated cholesterol uptake through the phosphorylated retinoblastoma tumor suppressor protein.²³ In accordance with these studies, a recent clinical study also showed that mRNA expression of mTOR and other pro-inflammatory mediators, including tumor necrosis factor-α and monocyte chemotactic protein-1, was significantly higher in mononuclear cells extracted from carotid endarterectomy specimens compared with peripheral mononuclear cells.²⁴ Along with these prior studies, the present study suggests that the expression of miR-100 is enhanced in unstable coronary atherosclerotic plaques, and that it might stabilize plaque at least in part by suppression of the mTOR signaling pathway. Another recent study showed that decreased expression of miR-100 in human nasopharynx carcinoma cells resulted in disease progression, and correlated with higher levels of the mitotic regulator polo-like kinase 1, suggesting that miR-100 may be involved in the regulation of cell proliferation.²⁵

In the present study, transcoronary concentration gradient of circulating miR-100 was not correlated with CRP level, which may indicate that miR-100 is released into the coronary circulation from increasing foam cells before apoptosis or necrosis, rather than from vulnerable coronary plaques. Some recent studies, however, reported that CRP is less specific than local inflammatory markers such as pentraxin 3 as an indicator of coronary plaque vulnerability.^26,27 In which case, we need to clarify the role of local inflammation in the production of miR-100 by using not only CRP but more specific inflammatory markers such as pentraxin 3 in further studies.

In the present study, miR-126 tended to be lower in the CS than in the aorta. Vascular miR-126 has been posited to play a role in inflammation control and leukocyte adherence. Vascular cell adhesion molecule-1 (VCAM-1)-mediated adhesion can be inhibited by miR-126 in human umbilical vein endothelial cells. Decreased expression of miR-126 upregulates VCAM-1 expression, which in turn enhances leukocyte adherence to the endothelium.²⁸ In addition, miR-126 was found to be a component of microparticles produced by apoptotic endothelial cells during atherosclerosis. These microparticles convey a survival signal to neighboring endothelial cells via miR-126 and its target, regulator of G-protein signaling 16, which is a G protein-coupled receptor inhibitor. This allows for the production of CXCL12 and thereby antagonizes apoptosis.²⁹ Furthermore, a previous clinical study showed that circulating endothelial-enriched miRNAs including miR-126 were downregulated in patients with coronary artery disease.³⁰ The present findings and these previous studies suggest that miR-126 is a paracrine mediator of the survival signal in endothelial cells within atherosclerotic lesions, and that endothelial dysfunction associated with some risk factors such as diabetes leads to decreased local expression of miR-126, which might further aggravate atherosclerosis in coronary plaques.

With regards to muscle cell-enriched miRNAs, miR-208a and miR-499 were not detected using the present PCR protocol and there were no significant differences in miR-133 level between the aorta and the CS. Systemic plasma concentration of muscle cell-enriched miRNAs, including these specific miRNAs, was elevated in patients with ACS.^31–33 Furthermore, miR-499 and miR-133a were elevated across the coronary circulation in patients with ACS. Transcoronary concentration gradient of these miRNAs was significantly correlated with the extent of myocardial damage as measured with high-sensitivity troponin T.¹⁵ These data suggest that myocardial injury is associated with a significant release of myocyte-specific miRNAs into the circulation, which is compatible with the results of the present study that excluded patients with ACS.

The present study had some limitations. First, the sample size was small. We measured plasma level of miRNAs in only a limited number of patients; therefore, we were unable to definitively exclude the predictive value of miRNAs other than miR-100 with respect to vulnerable plaques. Second, we could not determine whether the increased miR-100 level in the CS was a consequence or a cause of vulnerable plaque. Although we hypothesized that increased miR-100 might be a compensatory reaction to stabilize the plaque as mentioned here, we could not rule out the contrary hypothesis that elevated miR-100 might unstabilize the plaque. Therefore, further studies are needed to clarify the causation between serial changes in miRNA level and the coronary plaque characteristics.

Conclusions

Plasma miR-100 was higher in the CS than in the aorta, but there were no significant differences in plasma levels of other miRNAs. In addition, transcoronary concentration gradient of circulating miR-100 was significantly correlated with %LV and %FV. This suggests that miR-100 might be released into the coronary circulation from vulnerable coronary plaque. In addition, the present findings raise the interesting possibility, which needs to be confirmed in future studies, that the regulation of miR-100 might provide a novel form of therapy for plaque stabilization.

Acknowledgments

This work was partially supported by JSPS Kakenhi Grants (Number 25670390 and 25293184 to M. Sata).

Disclosures

None.

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