Potential of circulating pro‐angiogenic microRNA expressions as biomarkers for rapid angiographic stenotic progression and restenosis risks in coronary artery disease patients underwent percutaneous coronary intervention

Abstract Background This study aimed to investigate the correlation of pro‐angiogenic microRNA (miRNA) expressions with rapid angiographic stenotic progression (RASP) and restenosis risks in coronary artery disease (CAD) patients underwent percutaneous coronary intervention (PCI) with drug‐eluting stents (DES). Methods A total of 286 CAD patients underwent PCI with DES were consecutively recruited in this study. Plasma samples were collected before PCI operation, and 14 pro‐angiogenic miRNAs were measured by real‐time quantitative reverse transcription‐polymerase chain reaction. Rapid angiographic stenotic progression at nontarget lesions and restenosis at stented lesions were evaluated by quantitative coronary angiography at 12 months after PCI operation. Results The occurrence rates of RASP and restenosis were 39.5% and 22.4%, respectively. Let‐7f, miR‐19a, miR‐19b‐1, miR‐92a, miR‐126, miR‐210, and miR‐296 were decreased in RASP patients than non‐RASP patients, among which let‐7f, miR‐19a, miR‐126, miR‐210, and miR‐296 independently correlated with lower RASP occurrence by multivariate analysis, followed by receiver‐operating characteristic (ROC) curve exhibited that these five miRNAs showed great value in predicting RASP risk with area under curve (AUC) 0.879 (95% CI: 0.841‐0.917). Besides, let‐7f, miR‐19a, miR‐92a, miR‐126, miR‐130a, and miR‐210 were reduced in restenosis patients than non‐restenosis patients, among them miR‐19a, miR‐126, miR‐210, and miR‐378 independently correlated with lower restenosis occurrence by multivariate analysis, followed by ROC curve disclosed that these four miRNAs had good value in predicting restenosis risk with AUC 0.776 (95% CI: 0.722‐0.831). Conclusions Circulating let‐7f, miR‐19a, miR‐126, miR‐210, and miR‐296 independently correlate with reduced RASP risk, while miR‐19a, miR‐126, miR‐210, and miR‐378 independently correlate with decreased restenosis risk in CAD patients underwent PCI with DES.


| INTRODUC TI ON
Coronary artery disease (CAD) is the leading cause of deaths in both developing and developed countries with the annual deaths more than 17.5 million worldwide, accounting for approximately one third of all-cause deaths. [1][2][3] In clinical practice, there are several revascularization treatments for CAD, such as percutaneous coronary intervention (PCI), coronary artery bypass graft surgery (CABG), and drug therapy, among which PCI represents the most common therapy for CAD patients, and PCI with drug-eluting stents (DES) achieves significant relief of clinical symptoms and reduction in mortality in CAD patients compared with previous baremetal stents (BMS). [4][5][6][7][8][9] However, in clinical practice, CAD patients underwent PCI with DES may also present with rapid angiographic stenotic progression (RASP) at nontarget lesion and restenosis at stented lesion in CAD patients. Although antiproliferative drugs loaded in stents are designed to reduce the occurrence of RASP as well as restenosis which have made some progresses, especially restenosis rate could decrease to 3%-20%, while the incidences of RASP and restenosis are still not negligible due to a very large CAD population treated by PCI with DES. [10][11][12][13] Hence, it is meaningful to explore better strategies for predicting RASP and restenosis risks in CAD patients underwent PCI with DES, thereby guiding optimization of clinical treatment strategies and taking preventive measures as soon as possible.
Angiogenesis is of great importance in the development and progression of CAD by expanding microvascular network and assisting vascular repair to improve vascular functions. 14 Accordingly, proangiogenic microRNA (miRNA) is a class of approximately 20-25 nt, single-stranded noncoding RNAs that regulate vascular functions through post-transcriptional modulation of anti-angiogenic genes expressions, and a few pro-angiogenic miRNAs have been identified until now. [15][16][17] For instance, miR-130a induces angiogenesis through targeting C-MYB in vascular endothelial cells 15 ; miR-126 and miR-210 improve angiogenesis and then present with cardioprotective effect in rat. 17 Since angiogenesis is benefiting for the microvascular network rebuilding and vascular repair, we hypothesized that pro-angiogenic miRNA expressions might correlate with the risks of RASP and restenosis in CAD patients underwent PCI with DES, while no related data have been reported. Therefore, we selected 14 pro-angiogenic miRNAs referring to a previous comprehensive review on angiogenesis-related miRNAs, 18 and this study aimed to explore the correlation of these 14 pro-angiogenic miRNA expressions with RASP and restenosis risks in CAD patients underwent PCI with DES.

| Participants
During the period from January 2012 to December 2016, 286 CAD patients who underwent PCI with DES implantation at the Central Hospital of Wuhan were consecutively recruited in this study. The inclusion criteria included (a) confirmed diagnosis of severe CAD based on the coronary angiography, which was defined as the luminal stenosis ≥70% in at least one major coronary vessel, (b) age ≥18 years old, (c) scheduled to undergo PCI with DES implantation, and (d) able to be regularly followed up. Patients were excluded if they (a) had contraindications to the PCI, (b) were allergic to the medications: clopidogrel, rapamycin, paclitaxel, stainless steel, or cobalt alloy, (c) previously underwent major surgery, PCI, or coronary artery bypass grafting, (d) received treatment with immunosuppressive drugs within 3 months, (e) complicated with inflammatory diseases, autoimmune diseases, (f) with a history of severe infection or malignancies, and (g) were pregnant women or lactating women. The present study was approved by the Institutional Review Board of the Central Hospital of Wuhan and conducted in accordance with the Declaration of Helsinki. All patients provided the written informed consents before enrollment.

| Data collection
After the eligibility was confirmed and the informed consents were provided, patients' baseline features were recorded, which included age, gender, body mass index (BMI), smoke, hypertension, diabetes mellitus, hypercholesteremia, hyperuricemia, and family history of CAD and left ventricular ejection fraction (LVEF), and the laboratory detections were also recorded including mean arterial pressure (MAP), fasting blood glucose (FBG), serum creatinine (Scr), serum uric acid (SUA), cardiac troponin I (cTnI), N-terminal pro-brain natriu-

K E Y W O R D S
coronary artery disease, percutaneous coronary intervention, pro-angiogenesis microRNA, rapid angiographic stenotic progression, restenosis balloon dilation pre-stent) as well as drugs used after surgery (such as aspirin, nitrates, statins, β receptor blockers, angiotensin-converting enzyme inhibitors/angiotensin receptor blockers (ACEIs/ARBs), and calcium channel blockers) were recorded accordingly.

| Measurement of candidate miRNAs
Blood samples were collected from all patients before PCI and centrifuged at 880 g for 10 minutes to separate plasma. Total RNA was extracted from the plasma using TRIzol solution (Invitrogen) according to the manufacturer's protocol. The extracted RNA in solution was stored at −80°C until use. RNA concentration and purity from all samples were measured using NanoDrop ND-1000 Spectrophotometer (Thermo Fisher Scientific). The integrity of RNA was determined by 1.2% agarose electrophoresis (Sigma). Before real-time quantitative reverse transcription-polymerase chain reaction (RT-qPCR) was performed, the complementary DNA (cDNA) was synthesized from total RNA using a RevertAid RT Reverse Transcription Kit (Thermo Fisher Scientific) following the manufacturer's instructions. RT-qPCR was carried out on an Applied Biosystems 7900 HT thermocycler (Applied Biosystems) with TB Green™ Fast qPCR Mix (Takara), and the reactions were incubated at 95°C for 3 minutes, followed by 40 cycles of 95°C for 5 seconds and 61°C for 30 seconds. All reactions were run in triplicate. Relative quantitation of gene expression was analyzed by the 2 −ΔΔCq method, and the U6 was utilized as an internal reference for normalizing miRNA quantity. The primers used in the study are listed in Table S1.

| Quantitative coronary angiography (QCA) analysis
The procedures of PCI and DES implantation and the perioperative managements were performed in accordance with the recommendations of the guidelines of PCI, and the rapamycin-eluting stent and paclitaxel-eluting stent were used as DES (the choice of stent depended on the patients' willingness). Quantitative coronary angiography was performed before PCI (baseline), post-operation, and at 12th month after PCI or earlier if indicated by clinical symptoms or evidence of myocardial ischemia. Coronary angiograms were obtained after intracoronary nitroglycerin administration. Baseline, postoperative, and follow-up coronary angiograms were digitally recorded, and the QCA analyses were performed with an automated edge-detection system (Medis Medical Imaging Systems) by experienced specialists.

| Definitions
Rapid angiographic stenotic progression of nontarget lesion and restenosis of stented lesions were evaluated by QCA. Rapid angiographic stenotic progression of nontarget lesion was defined as the occurrence of any of the following conditions 19 : (a) the increase in stenosis ≥10% at 12th month if the original stenosis was ≥50% before PCI; (b) the increase in stenosis ≥30% at 12th month if the original stenosis was <50% before PCI; (c) newly developed stenosis ≥30% at 12th month if no original stenosis existed before PCI; (d) and the stenosis aggravated and turned to complete occlusion lesion at 12th month. Restenosis of stented lesion was defined as the lumen stenosis at stent-implanted segment was ≥50% at 12th month (compared with postoperative).

| Statistics analysis
Statistical analysis was performed with the use of SPSS 22.0 statistical software (SPSS Inc), and figures were made using GraphPad Prism 7.00 software (GraphPad Software Inc). Data were expressed as count (percentage), mean ± standard deviation, or median (25th-75th quantiles). Comparison was determined by the Wilcoxon F I G U R E 1 Study flow rank-sum test. Univariate and multivariate logistic regression model analyses with the forward stepwise (conditional) method were used to determine miRNAs related to the RASP or restenosis occurrence.
Receiver-operating characteristic (ROC) curves and the area under the ROC curve (AUC) were used to assess the predictive value of miRNAs for the RASP or restenosis occurrence. P value < .05 was considered significant.

| Study flow
Initially, 581 CAD patients who were about to undergo PCI were in-

| The occurrence rates of RASP and restenosis
There were 113 cases (39.5%) and 64 cases (22.4%) occurred RASP and restenosis, respectively, in CAD patients underwent PCI with DES implantation (Figure 2).

| Predictive value of pro-angiogenic miRNAs for RASP risk determined by ROC curves
Since let-7f, miR-19a, miR-126, miR-210, and miR-296 independently correlated with decreased RASP occurrence, ROC curve analysis was further carried out to evaluate their predictive value for RASP risk, which disclosed that let-7f (

| Correlation of pro-angiogenic miRNA expressions with restenosis risk
To assess the correlation of pro-angiogenic expressions with restenosis risk, univariate and multivariate logistic regression analyses were carried out. Univariate logistic regression analysis uncovered

| D ISCUSS I ON
In the present study, we observed that in CAD patients underwent Rapid angiographic stenotic progression at non-stented sites and restenosis at stented sites are two common major risks in CAD F I G U R E 4 ROC curve analysis for RASP risk. ROC curve disclosed that let-7f, miR-19a, miR-126, miR-210, and miR-296 could predict RASP risk, and combination of all these five miRNAs presented even better predictive value for RASP occurrence (red color line). The analysis was determined by ROC curve analysis. RASP, rapid angiographic stenotic progression; ROC, receiveroperating characteristic F I G U R E 5 Pro-angiogenic miRNA expression levels in restenosis and non-restenosis patients. Decreased baseline expression levels of let-7f (B), miR-19a (F), miR-92a (I), miR-126 (J), miR-130a (K), and miR-210 (L) were observed in restenosis patients compared with nonrestenosis patients, while no difference in the other eight miRNA expression levels (including let-7b (A), miR-17-5p (C), miR-17-3p (D), miR-18a (E), miR-19b-1 (G), miR-20a (H), miR-296 (M), and miR-378 (N)) at baseline was discovered between restenosis patients and nonrestenosis patients. Comparison was determined by the Wilcoxon rank-sum test. P < .05 was considered as significant. miRNA, microRNA patients underwent PCI with DES. Rapid angiographic stenotic progression is characterized by a rapid plaque progression and a consequent luminal obstruction at nontarget locations, which possibly arises from plaques disruption and endothelial mechanical injury caused by stent implantation lead to accretion of exfoliated plaques, endothelial dysfunction, and subsequent responsive processes including inflammatory reaction, ectopic proliferation, and migration of vascular smooth muscle cells (VSMCs) at nontarget sites. [20][21][22] As to restenosis, it occurs at stented sites probability due to the vulnerability of stented arteries and the effect of regeneration of endothelium, which manifests as distinct neointimal hyperplasia at stented sites. 21,22 Briefly, RASP and restenosis derive from several adverse effects caused by stent implantation, including endothelial cell (EC) dysfunction, ectopic proliferation, and migration of VSMCs as well as inflammatory reaction. 3,22 Recently, accumulating studies disclose that pro-angiogenic miR-NAs play critical roles in vascular remodeling and pathophysiology of cardiovascular diseases. [23][24][25][26][27][28][29][30][31][32][33][34][35] For instance, miR-126, as a common  25 ; meanwhile, it reduces ectopic VSMC proliferation and limits neointima formation by inhibiting low-density lipoprotein receptor-related protein 6 (LRP6). 28 In addition, miR-126 is also a negative regulator of inflammation, which is a key contributor to cardiovascular pathology, including endothelial dysfunction and ectopic proliferation and migration of VSMCs. [26][27][28] As to another common pro-angiogenic miRNA, miR-92a facilitates angiogenesis via upregulating vascular endothelial growth factor A (VEGFA) and re-endothelialization, preventing neointima formation following vascular injury, 30,31 and it protects VSMCs against apoptosis via suppressing of both mitogen-activated protein kinase 4 (MKK4) and c-Jun N-terminal kinase (JNK) pathways in vascular remodeling of several vascular diseases, such as aneurysms, post-angioplasty restenosis, and atherosclerosis, which leads to inhibition of plaque instability and rupture. 32 Furthermore, another pro-angiogenic miR-210 is also observed to accelerate angiogenesis via activating notch signaling pathway and able to decrease inflammatory cells density as well as reduce the apoptosis and dysfunction of ECs through inhibiting the receptor tyrosine kinase ligand Ephrin-A3, protecting from vascular ischemic damage. [33][34][35] Considering the functions of pro-angiogenesis miRNAs in vascular remodeling and the pathological processes of RASP and restenosis in blood vessels, we hypothesized that pro-angiogenic  There were several limitations in this study. Firstly, angiographic surveillance of RASP and restenosis might elevate the restenosis rate; hence, other alternatives of assessment could be utilized, such as intravascular ultrasonography, optical coherence tomography, and fractional flow reserve. 43 Secondly, we only assessed the shortterm incidence of RASP or restenosis in CAD patients underwent PCI with DES; thereby, further study would be performed with longer follow-up time. Thirdly, this was a single-center study with relatively low patients which might lead to patient selection bias and reduced statistical efficiency; thus, further study with more medical centers and patients should be conducted in the future.

ACK N OWLED G EM ENTS
This study was supported by National Natural Science Foundation of China (No. 81300214).

CO N FLI C T O F I NTE R E S T
The authors declare that they have no conflicts of interest.