Pre-diagnostic role of platelet miRNA in coronary heart disease of healthy overweight subjects via platelet leptin receptor activation

Obesity and overweight have become a global problem that development of various coronary artery diseases (CAD), such as myocardial infarction, atherosclerosis and congestive heart failure. Effective diagnosis is needed for effective treatment and prevention, particularly in healthy overweight subject. Platelets is an important component of hemostatic balance, that maintains the coagulation physiology. Platelets are involved in different pathological events such as thrombosis and CAD in various inflammatory conditions. There are evidences that highlight an important role of miRNA in regulation of gene expression profiling in platelets. Current hypothesis has shown the likelihood of using miRNAs as diagnostic markers in the event of CAD. This review article describes the association between overweight/obesity and platelets activation in elucidating the gene expression profiling in platelet miRNAs in CAD patient. The application of platelet miRNAs as predictive markers in overweight/obese individuals may become a marginal milestone in the history of this diet-related disorder treatment.


INTRODUCTION
Overweight is a major public health issue, which affects conspicuous portion of the world population. It constitutes a risk factor for metabolic events that predispose an individual to diabetes, hypertensions and atherosclerosis. Overweight has been recently linked to low grade chronic inflammatory diseases, like type 2 diabetes, cardiovascular disease and cancer. However, acute inflammatory responses are important for defensive and homeostatic mechanisms of healing, repair and tissue regeneration 1 . Chronic inflammatory conditions include hypertension, diabetes, cardiovascular disorders (CVDs) and cancer 2 . A report by World Health Organization (WHO) in 2016 showed that over 1.9 billion adults above 18 were overweight worldwide, and 650 million adults were obese. These figures show, that 39 % of adults ≥ 18 years of age (representing 40 % women and 39 % men) were overweight, while 13 % of world adult population (representing 15 % women and 11 % men) were obese. Thus, from 1975 to 2016 3 , the global prevalence of overweight and obesity cases has nearly tripled. Epidemiological evidence and clinical studies have clearly demonstrated, that overweight predisposed an individual to an increased incidence of thrombotic occlusion, or atherosclerosis that corre-lated with epicardial fat thickness 4 and visceral obesity 5 . Endothelial dysfunction, monocytes recruitment, inflammation and platelet activation are associated with dyslipidemia. Studies showed that leptin resistance could be a possible candidate that links obesity with cardiovascular diseases [6][7][8] . Meta-analysis data by Coronary Heart Prevention of West Scotland Studies suggested, that leptin could induce CHD independently 9 . Although, leptin receptor (ObRb) on platelet membrane can signal angiogenesis, regulate bone formation, accelerate vascular endothelial injuries and further enhance platelet aggregation via platelet leptin receptor 10,11 (Figure 1). Activated platelet releases cytokines, chemokines, hemostatic factor, adhesion protein, antigen receptor, mitogens and platelet miRNA 12 . Platelet miRNA gene expression pattern has been demonstrated to be more accurate and precise, compared to mRNA expression pattern in differentiation and characterizing various diseases (such as atherosclerosis, myocardial infarction and cancers 13 ). Thus, this review article describes the relationship between overweight, leptin resistance, platelet activation and platelets miRNAs expression profiling, as well as to advocates the usefulness of miRNAs as predictive biomarkers of CAD in healthy overweight subjects.  Plateletfree calcium concentration may be involved in platelet response to leptin stimulation via ObRb on platelet membrane. The stimulation of phospholipase C (PLC) in phospholipids membrane is an early step in cellular activation, including platelets that resulted in the hydrolysis of phosphatidylinositol 4,5 diphosphate. This, in turn, leads to the formation of 1,2-diacylglycerol (DAG) and inositol 1,4,5 triphosphate (IP3). DAG is known to stimulate protein kinase C (PKC) and IP3 is able to mobilize intracellular calcium for platelet activation.

THE ASSOCIATION BETWEEN PLATELET ACTIVATION AND ATHEROSCLEROSIS IN OVERWEIGHT
Overweight can be referred as a leptin resistance condition. Leptin is a product of adiponectin from ob gene 14 , that signals the energy stores level via receptor in central nervous system. Leptin regulatesactivity of the hypothalamic nuclei, responsible for appetite and energy homeostasis. This hormone is antioverweight, with its level decreasing during fasting and increasing after overfeeding in order to maintain energy status. Thus, leptin resistance in overweight subjects may signify the relationship between leptin concentration and overweight 15 . Multiple evidences have demonstrated that this hormone could engage in many pathophysiological pathways that could result in leptin resistance in different cells, arterial thrombosis formation 16 , platelet activation and aggregation 17 , arterial hypertension 18 and vascular response to in-flammation 19,20 . Other studies have shown that leptin is a prognosticator of atherosclerosis, myocardial infarction, stroke and coronary artery episode which is independent of body fat 21 . The overall ability of this hormone to increase human platelet activation has been suggested in OB/OB mice and healthy control subjects 11,21 . However, Ozata et al. did not observe leptin on epinephrine, collagen and ADP platelet activation in both obese and overweight healthy subjects, and in leptin-deficient individuals 22 . Therefore, the notion of presence of leptin receptor on platelet membrane, signaling the prothrombotic episode of leptin resistance is still controversial. However, the association between platelet activation in overweight/obesity with dyslipidemia and endothelial dysfunction, triggers human platelet activation and aggregation, thus further enhancing the risk of atherosclerosis events 23 . Furthermore, studies have shown that obesity triggers platelet activation via release of an inflammatory mediator that activates multiple internal signaling networks, includ-ing platelet miRNA 24 . The role of miRNA (miR) on leptin resistance has been demonstrated in several gene expression profiles. MiRNA plays a modulation role in different metabolites. Thus, the exact effect of platelet miRNA on leptin in hypothalamic center remains elusive. The up-regulation of miR-200b and miR-200a were observed in hypothalamus center of obese-deficient ob/ob mice, and down-regulation of miR-200b and miR-200a were reported after leptin treatment in the hypothalamus 25 . Reduced level of miR-200b and miR-200a in the hypothalamic center induced the expression of both insulin and leptin receptors that signal the reduction of fat deposit and further restored insulin function in the liver 26 . These miRNAs were present in activated platelets, suggesting that up regulation of miR-200a and miR-200b from activated platelets in overweight/obesity was altered ( Table 1). Such alteration influenced leptin and insulin receptor signaling pathways in the hypothalamic nuclei 25 which could serve as a target for therapeutic intervention in obesity/overweight individuals.

REGULATORY AND B IOGENESIS OF PLATELET MIRNA
MiRNAs are a short class of non-coding endogenous RNAs, that post-regulate gene expression transcriptionally via either translational mRNA degradation, or repression. MiRNAs play crucial roles in controlling biochemical functions and mechanisms of different types of cells, including cell differentiation, developmental timing, tumorigenesis, proliferation, apoptosis as well as thrombosis and platelet functions. Similarly, miRNA itself as a regulatory element, is coordinatively modulated by multifarious effectors when carrying out basic functions, such as miRNA editing, single-nucleotide polymorphism, circadian clock and methylation. Platelet miRNAs act together in order to fine-tune and regulate a wide variety of this molecules in form of mRNA in different cellular functions. Those include aggregation, cell adhesion, proliferation, activation, chemotaxis, coagulation, proteolysis and cell survival. During these events, the mRNA is synthesized from DNA transcription and further translated into protein. Certain specific regions of these mRNA molecules are not normally translated into proteins. These regions include the 5 ′ untranslated region (5' UTR), 5 ′ cap, poly-A tail and 3 ′ untranslated region (3' UTR). 3 ′ UTR often contains regulatory site that influences and regulates the posttranscriptional gene expression profiling 29 . Britton and Davidson, (1969) 30 postulated that "activator" mRNA transcript may work to turn on and off genes as predicted by Watson-Crick base pairing to region, located within genes 31 . Three major forms of sR-NAs regulation in plants and animals were identified, which include small interfering RNAs (siRNA), miR-NAs (miRNA) and piwi-interacting RNAs (piRNA). Landry et al., 2009 32 found, that the platelet miRNA was not usually translated into protein, but it regulated mRNA by annealing to the recognition site on 3' UTR of mRNA, coding gene sequence of 2-8 nucleotide, complementary to miRNA seed region 33 . This pairing was based on Watson-Crick nucleotide base pairing, such as miRNA-mRNA. The hybridization of these complexes does not require full complementary homology 34 and complete homolog usually leads to degradation of a target mRNA, while partial pairing homology is a translational repression. Therefore, miRNA influences degradation to regulate mRNA by switching the functional gene transcript on and off, while translational repression of miRNA regulates the function of the protein expression 34,35 . This functional role of miRNA presumes a wide range of miRNA-mRNA interactions, following either convergent (many miRNA-Single targets), or divergent (single miRNA-many target mRNA) interaction. This results in robust pathways that regulate the synthesis of protein in a cell 35 . Currently, the nature and extent of the involvement of platelet miRNAs in a non-(protein)-coding ribonucleic acids (RNAs) in physiology and pathological processes (in both plant and animals) has become clearer 35 . More than 280 miRNAs have been recognized and characterized from human nucleated platelets 31,35 . The miRNAs biosynthesis in biological systems is strictly regulated. The biogenesis of platelet miRNAs began with transcription of the gene in non-coding region of miRNA using RNAs poly-III from the genome of megakaryocytes, harboring miRNA gene in either intergenic or intronic at promoter site. The first primary miRNA as a non-coding transcript is known as pri-miRNA, which acts upon by RNA poly III (called "Drosha"), that bounds to the microprocessor subunits DGCR-8 (di-george syndrome critical region-8) to produce a small length hairpin called pre-miRNA stemloop with 80-110 nucleotides base in length. Exporting-5 assists the nuclear export of pre-miRNA into the cytoplasm 36 . The presence of pre-miRNAs in the cellular cytoplasm is identified by ribonuclease (RNase) or Dicer enzymes which is a part of transactivation response binding protein (TRBP) complex. The complex Dicer/TRBP cleaves the pre-miRNAs loop to release a short length of double stranded miRNA, such as miRNA/miRNA, * or duplex 37 . The duplex (such as miRNA/miRNA) are transported into the RNA Induced Silencing Complex (RISC) 38,39 and Helicase enzymes are used to unwind the double stranded miRNA into a single 22 nucleotides base, which is released as matured miRNA. The 2 nd strand miRNA, known as passenger strand is thought to be digested by the RISC complex substrate. Argonauts (Ago) protein family will assemble in RISC and provide stability and protection to the mature miRNA strand against RNase enzymes activities and will further guide the matured miRNA to its target 3 ′ -untranslated region (UTR) of mRNA transcripts 40 (Figure 3 ). However, the inverse modulation of platelet miRNA profiling during adipose tissue development in overweight and obesity is important for understanding miRNA dysregulation in adipose tissue of both obese humans and mice. Such modulation enhances chronic inflammation observed in obesity subject with insulin resistance 41 . Thus, a single nucleotide base change of mature and pre-miRNA may drive the emerging of new miRNA by influencing the biological function of these cells 40 .

PATHOLOGICAL ROLE OF PLATELET MIRNA IN CORONARY ARTERY DISEASE
The mutations or single-nucleotide polymorphism (SNP) that occur in the biogenesis of both mRNA and miRNA can be classified as follows: (1) Mutations, that affect biogenesis enzymes of miRNA, or the promoter region of mRNA.
(2) Mutations, that affect the 3' UTR region of the mRNA.

Mutation or SNP that affects miRNA biogenesis enzymes
SNP in the processing regions can be categorized as pri-miRNA, pre-miRNA, mature miRNA sequences and mRNA biogenesis machinery (promoter region). In fact, gene variations may influence either miRNA hairpin and biogenesis proteins, or enzymes in the processing accuracy 43 .

Mutation in the miRNA seed region
SNP in the seed region of miRNA may reduce the binding strength between miRNA and mRNA target sites, which will influence hundreds of gene expressions 44 .

Mutation in the mRNA 3' UTR-region
Approximately 180,000 mutations or SNP found within the 3' UTR region mRNA were demonstrated with its corresponding mature miRNA 2,600 sequences. These were found in database sequence 45 . Mutations in the seed region of miRNA and 3' UTR region of mRNA are almost identical in terms of variations and regulation of miRNA-mRNA* complex, affecting functions and disease vulnerability 42 . However, further molecular mechanism underlying disease-associated with 3' UTR SNPs in mRNA still require further investigation 46 . For example, SNP meta-analysis of 8,120 patients and 8,364 controls identified four different SNPs in the following miR-NAs (miRNA-149, miRNA-196a2, miRNA-499 and miRNA-146a), which increased patients CAD vulnerability 47 . Other articles have demonstrated that miR-146a was upregulated in CAD 47,48 due to the change of G to C in pre-miRNA. That affected the expression of mature miRNA-146a in CAD patients 49 . That finding was confirmed by Wang et al. and showed, that C allele was greater than G allele in gene that predisposed individuals to CAD 50,51 . However, in type 2 diabetes patients with ischemic stroke, the miR-146a was downregulated ( Table 1) 52 . Other abundant miRNAs found in platelets miRNA were miR-223 and miR-126. In addition, miR-223, miR-107, miR-495, miR-96 and miR-200b were actively involved in various platelet functions, such as platelet release, adhesion and aggregation, 53 as summarized in Table 2.

MIRNA AS BIOMARKERS
The fact of discovery of miRNA as a stable molecule in plasma and serum is surprising despite the level of RNase enzymes activities 54 . The expression of miRNA in tissue, or organ-specific, and its release into the plasma in response to tissue injury is considered to be a potential biomarker for different diseases 55 . The hypothesis that miRNA in blood cells could be used as diagnostic parameter in different diseases was first postulated by Mitchell et al.. He showed, that miRNA remained highly stable in both plasma and serum even after prolonged storage at room temperature, or repeated cycles of freezing and thawing. For example, tumor-derived miRNA could easily be identified in plasma sample from cancer patient with standard laboratory procedure 54,56,57 . The diagnostic role of circulating miRNA has been evaluated not only in cancer, but also in other clinical disorders, such as hepatic disease 58 , heart failure 59 and diabetes 60 . Few studies have claimed platelets in obesity and overweight healthy individuals as the culprit factor, responsible for coronary artery disease 61 . The application of specific platelet miRNAs as markers for platelet activation will be a marginal milestone in the history of this diet-related disorder.

PLATELET-MIRNA IN ENDOTHELIAL AND VASCULAR SMOOTH MUSCLE (VSCM) CELLS IN CORONARY ARTERY DISEASE (CAD)
Large scale studies, presented by Nagalla et al. and Landry et al. found, that thirty different platelet miRNAs modulate platelet and endothelial angiogenesis (angio-miRs) 32,35 . These miRNAs, (miR-21, miR-200, miR-210 and miR-126), are well known Production pri-miRNA* from miRNA genes is processed by RNase-II/III and the pri-miRNA form is cleaved by Drosha-DGCR8 complex to produce pre-miRNA* in the nucleus. Pre-miRNA* is exported by exportin-5 from the nucleolus to the cytoplasm. The pre-miRNA* in the cytoplasm is further digested by another enzme called RNase-Dicer complex with TRBP that catalyzed the pre-miRNA* hairpin to mature miRNA duplex. Matured miRNA strand is transported into RISC in assemble of argonaute-2 (Ago2) proteins to guide the silencing of target mRNA to fully complementary matched for degradation and partially complementary matched for repression. This figure is adopted and modified DOI: 10.5772/ intechopen.81847.
to play critical roles in the vessel and capillary formation. The regulation of angiogenesis by miRNA in the endothelial cell was confirmed, using Dicerknockdown mice experiment, demonstrating the essential role of Dicer in miRNA biogenesis 62,63 . Furthermore, Dicer gene deletion was shown to cause early death of mice during the embryonic stage, due to impaired angiogenesis 64 . In addition, Dicer knockdown of vascular smooth muscle cells VSMC specific caused the late embryonic death due to internal bleeding 65 , suggesting that platelet miRNA was an integral part of vascular development. Therefore, clusters of miR-221 and miR-222 are the most abundant and well distributed miRNAs across these three cells(platelet, endothelial and VSMC) 66,67 . Both miR-221 and miR-222 function as pro-inflammatory inhibitor of angiotensin II and reversed leucocytes adhesion (in vivo and in vitro) 68

ROLE OF PLATELET MIRNA AS A MARKER IN CORONARY ARTERY DISEASE (CAD)
Previous reports showed that more than 80 different diseases were associated with dysregulation, or mutation in miRNAs 70 . The mechanisms of platelet activation in various thrombotic diseases were well established 71 . The accurate etiology of miRNA in activated platelet in overweight and obesity is not clear, but obesity is always a risk factor for atherothrombotic episodes 71 . Landry et al. 2009 demonstrated that the platelets and megakaryocyte miRNAs had 219 different types of miRNA in platelet expression patterns and profiles 72 . The authors observed three most abundant miRNAs in human platelets; miR-19a, let-7c, and miR-223. Binding of miRNA-223 to 3' UTR region of P2Y12 mRNA receptor in HEK293 cell line repressed P2Y12 gene and decreased the activities of both platelet and megakaryocyte. However, dysregulation of miRNA-233 was observed in hyperreactive platelet, leading to overexpression of miRNA-223 73 . Thus, miR-223, miR-197 and miR-126 were involved in platelet hyperreactive and endovascular inflammation, stretching the application of these miR-NAs as predictive biomarkers for diagnosis of CAD ( Table 1) 73 .

CAD, CORONARY ARTERY DISEASE; QRT-PCR, REAL-TIME QUANTITATIVE REVERSE TRANSCRIPTION PCR
In 2013, Osman and Fälker discovered 281 transcripts, of which 228 were mature miRNAs and 53 were pre-miRNAs. Six miRNAs: miR-339-3 p, miR-15 a, miR-365, miR-495, miR-361-3 p and miR-98, were upregulated, or downregulated in hyperreactive platelets 28 . The level of expression pattern, or characteristic of miRNAs in platelets were associated with a procoagulant (such as thrombin stimulation). Nagalla et al., 2011 demonstrated that there were 284 miRNA transcripts in human platelets, by which 74 were differentially expressed, based on the platelet reactivity 35 . Seven miRNA expression profiles (miR-320b, miR-190, miR-320d, miR-19b, miR-34b, miR-320c and miR-320a) were shown to have a strong relationship with the degree of platelet response to adrenaline 35 . In fact, the most abundant expressed platelets miRNA is miR-223, followed by miR-126 57 . Others were identified as miR-200b, miR-107, miR-96 and miR-495 53 . All of these miRNAs were involved in platelet hyperreactivity (such as platelet activation, adhesion and aggregation, as summarized in Table 2).  Continued on next page  86 . This finding suggested a possible relationship between the platelet activation and the miRNA modification, which may an induce agonist-specific platelet function. Jeanine et al., 2014 also reported the expression profile of platelet miRNA in patients with acute coronary syndrome after a series of diagnostic investigations. In blood sample of STEMI patients, miR185-5p and miR186-5p were downregulated, while miR221-3p and miR127-3p were upregulated in platelets. On the other hand, in blood samples of NSTEMI patients, miR942 and miR20a-5p were downregulated, whereas miR146a-5p and miR483-5p were upregulated in platelets ( Table 2) 74 . Thus, in body circulation, the pathophysiology of platelet miRNA can exhibit clear characteristic differences of expression profiling in various platelet disorders. This condition however is dangerous if urgent measure is not taken, since the risk of heart failure (HF) in such case is eminent. Patients with systolic heart failure were found to have different platelet miRNA expression profiles, as compared to control subjects 56 . Platelets miRNA-150 expression level decreased more than three-fold in blood platelets of patients with heart failure, secondary to atrial fibrillation ( Table 2) 75 .
In addition, the study, conducted by Duan et al., 2014 showed, that the expression of platelet miRNA-223 and miRNA-146a in patients with ischemic stroke and diabetes mellitus was significantly higher, compared to healthy donors. The expression level of these two platelet miRNAs was suggested to correlate with platelet activation ( Table 2) 52 .

CONCLUSION
The gene expression and bioinformatic analysis procedures have become the state-of-the-art methods for diagnosing a number of human diseases. Several studies have reported different miRNA expression profiles in platelets. The application of these miRNAs for diagnostic purpose in overweight healthy subjects will be a milestone in the history of treatment of this diet-related disorder. Six notable candidates of platelet miRNAs have been discovered and proposed for diagnostic accuracy in CAD upregulation: ↑ miR483-5p* ↑ miR146a-5p* ↑miR340* ↑miR624*, ↑miR451* and ↑miR454*. Current modern diagnostic laboratory method for identification of coronary obstruction is still in its early stage. Nevertheless, current molecular medicine and diagnostic methods are capable to identify if an individual is predisposed to the illness. Platelet miRNA expression profile detection is associated with platelet hyperactivity and may serve as an important marker for prevention of coronary artery disease (CAD) in human overweight/obesity.