Association Between Coronary Artery Disease and MicroRNA: Literature Review and Clinical Perspective

Background Until recently, circulating micro-RNAs (miRNAs) have attracted major interest as novel biomarkers for the early diagnosis of coronary artery disease (CAD). This review article summarizes the available evidence on the correlation of micro-RNAs with both the clinical and subclinical coronary artery disease and highlights the necessity for exploring miRNAs as a potential diagnostic and prognostic biomarker of early CAD in an adult population. Methods A systematic literature analysis and retrieval online systems Public/Publisher MEDLINE/ Excerpta Medica Database /Medical Literature Analysis and Retrieval System Online,(PUBMED/EMBASE/MEDLINE) search were conducted for relevant information. Search was limited to the articles published in English language and conducted on humans, January 2000 onwards. We excluded studies of heart surgery, coronary artery bypass grafting (CABG), angioplasty and heart transplant. Eighteen studies met the inclusion criteria. Results Seven out of 18 studies were multivariate, i.e. adjusted for age, gender, body mass index (BMI), smoking, hypertension, diabetes, and blood lipid profiles, while the remaining twelve studies were univariate analysis. Different sources of miRNAs were used, i.e. plasma/serum, microparticles, whole blood, platelets, blood mononuclear intimal and endothelial progenitor cells were investigated. Fourteen out of 18 studies showed up-regulation of different miRNA in CAD patients and in vulnerable plaque disease. Four out of 18 studies showed both the up-regulation and down-regulation of miRNA in the population, while only three studies showed down-regulation of miRNA. Various sources and types of miRNA were used in each study. Conclusion This review gives an extensive overview of up-regulation and down-regulation of miRNA in CAD and non-CAD patients. The pattern of miRNA regulation with respect to CAD/non-CAD study subjects varies across individual studies and different parameters, which could be the possible reason for this aberrancy. We suggest further trials be conducted in future for highlighting the role of miRNA in CAD, which may improve both the diagnostic and therapeutic approaches to stratifying CAD burden in the general population.


Introduction
Heart disease is the leading cause of death for both males and females with more than half of the deaths reported in 2009 in males [1]. Coronary heart disease is the most common type of heart disease with 370,000 annual deaths, i.e. each minute someone in the United States dies from a heart disease-related event [2]. Coronary heart disease alone each year costs the United States $108.9 billion, which includes the cost of health care services, medications, and lost productivity [3]. The total coronary artery disease (CAD) prevalence is 6.4% in US adults, which is expected to increase approximately 18% by 2030 [4]. Most individuals aged over 60 years have progressively enlarged deposits of calcium mineral in the plaques in their major arteries [5]. As atherosclerosis infiltrates the arterial wall long before it causes vessel obstruction and produces symptoms, earlier identification of this process should be part of risk prediction [6]. As such, there is a lack of cost-effective and specific biomarkers for the early clinical diagnosis and prognosis of CAD, and there is an immense clinical demand for specific and reliable noninvasive biomarkers for CAD.
With over 1900 MicroRNA (miRNAs) discovered in humans to date, many of them have already been implicated in common human disorders. However, the pattern among the miRNA-disease association remains largely unclear for most diseases. Until recently, circulating micro-RNAs (miRNAs) have attracted major interest as novel biomarkers for the early diagnosis of CAD [7]. MiRNAs are a class of small (~22 nucleotides long), highly specific, endogenous, singlestranded, non-coding RNAs that regulate the expression of target genes by binding to the 39 untranslated regions and degrading or inhibiting the translation of messenger ribonucleic acid (RNA) (mRNAs) [8]. Studies have shown miRNAs' involvement in the timing of cell death and cell proliferation, hematopoiesis, and other normal cellular homeostasis [9][10]. Various miRNAs are expressed in a tissue-specific manner and thus may regulate tissue-specific functions. This review article summarizes the available evidence correlating micro-RNA, clinical and subclinical CAD and further highlights the necessity for exploring the potential of micro-RNAs as useful diagnostic and prognostic biomarkers for early CAD in the adult population.

Materials And Methods
A computerized search of the Public/Publisher MEDLINE/ Excerpta Medica Database /Medical Literature Analysis and Retrieval System Online/Excerpta Medica Database (PubMed/Medline/EMBASE) database was done with the keywords and medical subject headings (MESH) terms such as "micro RNA," "coronary artery disease," "cardiovascular disease (CVD)," "Subclinical CVD," "coronary artery calcium and micro RNA," "miRNA and high sensitivity C-reactive protein (hs-CRP)," "miRNA and coronary intimal thickness," and "miRNA and pulse wave velocity." We included all the literature that was published from January 1, 2000, until January 1, 2017. The search was limited to articles published in the English language. Included studies were cross-sectional, case-control or prospective in design and conducted in adult populations ( Figure 1). CAD subjects diagnosed by symptoms, imaging, cardiac enzymes, electrocardiogram (EKG), diagnostic angiography or stress testing were included. We excluded studies with CAD patients who have had heart surgery, coronary artery bypass graft (CABG), angioplasty, and heart transplant. We also examined the references of all studies from the initial search for additional references. Demographic data was extracted from each study and results were collaborated into tables.

Results
A total of 18 clinical studies has been included in the review after a thorough analysis of the literature. Overall, there were 1720 subjects. The majority of the studies included were done in China, which was 11 in number, while two studies from Japan, one from the USA, one from Norway, one from Netherlands, one from the UK and one from Germany were included ( Table  1). All the studies had the same outcome, which was CAD. Studies were further divided into two groups of microRNA up-regulation and down-regulation. There were four studies that checked for both up-regulation and down-regulation of microRNA.

Up-regulation and microRNA
A total of 15 studies reported up-regulation of micro RNAs in patients with CAD. Ten studies out of the 15 used plasma as the source while two studies used peripheral blood mononuclear cells as the source, one study used microparticles (MP) from plasma, one study used endothelial progenitor cells (EPC) and one study used platelets. The majority of the studies employed quantitative reverse transcription polymerase chain reaction (QRT-PCR) for mircoRNA analysis while only one study employed Kyoto encyclopedia of genes and genomes (KEGG) method for microRNA analysis (Li, et al. 2013). All studies reported that there is up-regulation of specific miRNA in relationship to CAD. Univariate analysis was done in nine of the 15 studies, while remaining studies adjusted for age, sex, highdensity lipoprotein (HDL), low-density lipoprotein (LDL), aspartate aminotransferase (AST), alanine aminotransferase (ALT), hypertension HTN etc in their analysis (

Down-regulation and mircoRNA
A total of seven studies worked on the down-regulation of microRNA. All of the studies employed QRT-PCR as the method of analysis for detection of microRNA. Three studies used plasma as the source. Peripheral blood mononuclear cells were used by two studies. Two studies used whole blood as the source and one study used pericardial fluid and coronary arteries intima. All the studies reported down-regulation of microRNA in patients with CAD compared with non-CAD patients. The adjusted parameters were same as the up-regulation studies used.

Discussion
MiRNAs have been known to have an association with the physiological and pathological processes involved in the development of CADs such as endothelial dysfunction, inflammation, apoptosis, angiogenesis, atherosclerosis, and neointimal hyperplasia or restenosis [7,[29][30][31]. Our systematic review summarizes 18 articles comparing miRNAs in CAD patients. Ten studies found miRNAs that were up-regulated (21-3135b), five studies showed miRNAs that were both up-regulated (21-765) and down-regulated , and three studies showed downregulated miRNAs (19a-584). miRNAs derived from plasma/serum, microparticles, whole blood, platelets, blood mononuclear intimal and endothelial progenitor cells were investigated. Circulating miRNAs (plasma or serum) exhibit remarkable stability and are highly resistant to plasma ribonuclease (RNase) activity due to internalization in macro vesicles and the formation of protein-miRNA complexes and can be used as a reliable marker for both the early diagnosis and prognosis of CAD. Therefore, the levels of individual cardiac-enriched circulating miRNAs are related to the diagnosis and prognosis of heart diseases. Recent studies have shown the involvement of miRNAs in atherosclerosis, ranging from endothelial dysfunction to plaque rupture suggesting the use of miRNA as potential biomarkers in early diagnosis of CAD [32]. miRNA upregulation or downregulation is either related to the atherosclerotic disease process or the inflammatory compensation [21]. Ren, et al. stated that miRNA (21-5905p) that are involved in the pathogenesis of vulnerable plaque are mostly unregulated. In these patient populations, miRNA can be used as novel biomarkers in the diagnosis of disease. The severity of the disease can be potentially seen by aberrancy of miRNA in form of either up-regulation or down-regulation. Prior studies have shown that the levels of miRNA are linked with disease severity. The up-regulated miRNA leads to the evolution of plaque towards growth, instability, and rupture [33]. Fichtlscherer, et al. in a prospective case control trial studied miRNA derived from endothelial, cardiac, skeletal, and smooth muscle cells. Interestingly, in CAD patients endothelial and smooth muscle miRNAs were up-regulated and in cardiac and skeletal muscle cells miRNA 133a/208a/155 were downregulated in the same patient population. Prior studies have also documented the differential role of miRNA in skeletal, smooth and cardiac muscle [34]. The reason for this asymmetric regulation is an uptake of miRNAs in cardiac muscles by atherosclerotic lesions in apoptotic bodies and endothelial cells are exposed to inflammatory cells that can increase miRNA due to cellular stressors (Fichtlscherer, et al). miRNA can be specifically monitored in certain disease processes and used as prognostic markers as seen in CAD patients with diabetes, miRNA 145 being reduced compared to those without DM (Fichtlscherer, et al). For instance, Sondermeijer, et al. showed that the platelet-derived miRNA can potentially fine-tune the expression of specific gene products that may be involved in governing platelet reactivity. Therefore, a dysfunctional miRNA-based regulatory system could lead to the development of serious platelet-related cardiovascular diseases (Sondermeijer, et al Endothelial progenitor cells play an important level and in the regulation of miRNA after treatment with lipid therapy on lipometabolism related miRNA (Minami, et al.). Similarly, Takahashi

Limitations
Firstly, criteria for CAD participant enrolment across the studies were not standardized. Secondly, the study cohorts were not age and sex matched. Thirdly, the population size of different trials was very small. Fourthly, none of the included studies prospectively analyzed a specific miRNA regulation derived from one source or from different sources. Even specific miRNA regulation can be varied when compared across different studies and sources of origin. The pre and post CAD miRNA levels were not mentioned by any of the studies. Large randomized double-blinded studies are needed to eliminate population bias and increase the statistical power of each study. Prospective case-control trials with longer followup periods may play a role in deciphering the prognostic potential for the miRNA markers in CAD patients. Lastly, studies included did not mention specifically the number of patients on ACE/ARBs or statins.
Conclusions miRNA expression profile is associated with several human cardiovascular diseases, suggesting their role as a novel class of biomarkers as well as potential treatment targets for cardiovascular diseases. This systematic review provides a potential insight on miRNA regulations in subjects with and without CAD and highlights the different parameters which could be the reason for aberrant miRNA expression. Further understanding of miRNA expression may help to delineate their role in improving both the diagnostic and therapeutic approaches to stratifying CAD burden in the general population.