The regulatory role of microRNAs in angiogenesis‐related diseases

Abstract MicroRNAs (miRNAs) are small non‐coding RNAs that regulate gene expression at a post‐transcriptional level via either the degradation or translational repression of a target mRNA. They play an irreplaceable role in angiogenesis by regulating the proliferation, differentiation, apoptosis, migration and tube formation of angiogenesis‐related cells, which are indispensable for multitudinous physiological and pathological processes, especially for the occurrence and development of vascular diseases. Imbalance between the regulation of miRNAs and angiogenesis may cause many diseases such as cancer, cardiovascular disease, aneurysm, Kawasaki disease, aortic dissection, phlebothrombosis and diabetic microvascular complication. Therefore, it is important to explore the essential role of miRNAs in angiogenesis, which might help to uncover new and effective therapeutic strategies for vascular diseases. This review focuses on the interactions between miRNAs and angiogenesis, and miRNA‐based biomarkers in the diagnosis, treatment and prognosis of angiogenesis‐related diseases, providing an update on the understanding of the clinical value of miRNAs in targeting angiogenesis.


| INTRODUCTION
Vascular disease is a pathological process in clinical practice, including cardiovascular and peripheral vascular disease. 1 Cardiovascular disease (CVD) includes coronary artery disease (CAD), atherosclerosis, angina, coronary thrombosis, myocardial infarction (MI), congestive heart failure and stroke, 2,3 which is the most important cause of disability and premature death worldwide. 1 CVD and stroke produce a huge health and economic burden in the United States and the world. 1,3 Eighty per cent of the global burden of CVD occurs in developing countries where morbidity and mortality occur at younger ages. 2,4 In addition, the incidence of peripheral vascular disease is increasing, reducing the quality of life and exposing the risk of infection and thrombosis. 1 Peripheral artery disease (PAD) is characterized by severe ischaemic disease in the periphery that causes intermittent claudication and critical limb ischaemia (end stage), 5 leading to higher morbidity and mortality. 6 Furthermore, the greater prevalence of diabetes mellitus increases the risk of vascular disease, affecting the microvasculature, arteries and veins 7 and increasing amputation rates. 8 Therefore, vascular disease seriously affects the quality of life, increasing the psychological and economic burden. 1,9 Angiogenesis is the process of formation of new blood vessels from pre-existing vessels, involving cell proliferation, migration, differentiation, tube formation and regulation of angiogenic factors. It is responsible for a great variety of physiological and pathological processes, such as tumour, CVD, stroke, atherosclerosis, aneurysm, Kawasaki disease (KD), aortic dissection (AD), deep venous thrombosis (DVT), wound healing, diabetic microvascular complication, the formation of granulation tissue and other angiogenic disorders. [10][11][12][13] Therefore, regulation of angiogenesis is considered as an important therapeutic strategy for cancer and vascular disease. Emerging studies have demonstrated that dysregulation of microRNAs (miRNAs) expression may lead to abnormal angiogenesis, which has become a common feature of cancers and angiogenesis-related diseases. 14,15 Furthermore, strong supporting evidence has reported that miRNAs function as a class of oncogenes or tumour suppressor genes. 16 In this setting, the pro-angiogenic therapy with miRNAs may contribute to treating ischaemic diseases and the anti-angiogenic therapy with miRNAs in tumour may suppress the growth of cancer.
Lin-4 was the first miRNA to be identified in C. elegans in 1993, which began to reveal the importance of miRNAs. 17  of translation in miscellaneous biological processes. [18][19][20][21] The current findings indicate that compartmentalized stepwise processing of miRNAs takes place first in the nucleus and then in the cytoplasm.
Up to 40% of the miRNA genes are located in introns or even in exons of other genes and are generally transcribed into primary miRNA transcripts (pri-miRNAs) by RNA polymerase II (Pol II). 22 Pri-miRNAs are composed of one or more specific long hairpins with 5 0 cap and 3 0 poly (A) tail, which are further processed into 70-100 nt miRNA precursors (pri-miRNAs) by the microprocessor complex Drosha/DGCR8 in the nucleus. Pri-miRNAs are then exported from the nucleus into the cytoplasm by Exportin 5 and are sheared into approximately 22 nt mature miRNA duplexes by RNase III Dicer.
After Dicer processing, the mature double miRNA is incorporated into the RNA-induced silencing complex (RISC), where it is unwound into its mature, single-stranded form that binds to messenger ribonucleic acid (mRNA), the so-called miRNA targets, thus, down-regulating target mRNA levels, or by directly interfering with the translation mechanism to reduce protein levels. Expression levels of mRNA and protein can be regulated by the synthesis and silencing of miRNAs, thereby regulating the biological effects of cells and angiogenic factors, modulating angiogenesis and affecting the pathogenesis of angiogenesis-related diseases ( Figure 1). It is promising that miRNAs may be acted as a potential target to modulate angiogenesis for combating diseases characterized by either poor angiogenesis or F I G U R E 1 Biogenesis of miRNAs and the regulatory role of miRNAs in angiogenesis SUN ET AL. | 4569 abnormal vasculature. 23 Thus, miRNAs may provide novel and useful biomarkers, and new alternative treatment strategies for cancer and vascular disease detection, diagnosis and prognosis.
They are highly expressed in ECs, which is closely related to the regulation of angiogenesis. [24][25][26][27] Based on the studies regarding miRNA expression and function in angiogenesis, miRNAs fall into two major classes: (i) miRNAs that target genes involved in angiogenesis and (ii) miRNAs that can be regulated by pro-or anti-angiogenic stimuli. 28 The first group of miRNAs, including miR-34a, miR-124, miR-29, miR-126, miR-150, miR-221/222 and miR17-92 cluster, regulates angiogenesis mostly by targeting well-characterized target genes.

| miR-34a
Expression of miR-34a is significantly increased in CAD, 29 but is reduced within CD44-or CD133-positive prostate and breast cancer cells. 30 MiR-34a has the capability to impair angiogenesis and increase senescence via inhibiting silent information regulator 1 (SIRT1) and increasing the expression of Sirt1 effector-acetylated forkhead box O transcription factors 1 (FoxO1) and p53 in endothelial progenitor cells (EPCs) and WT human colon cancer cells. 29,31,32 In addition, the expression of miR-34a is down-regulated in ECs overexpressing Bcl-2. 33 Furthermore, the overexpression of miR-34a dramatically represses tumour angiogenesis, EC proliferation, migration and tube formation via the down-regulation of vascular endothelial growth factor (VEGF) and the upstream proteins of VEGF, such as E2F3, SIRT1, survivin and CDK4, in both head and neck squamous cell carcinoma (HNSCC) cell line and in cancer tissue samples. Moreover, the expression of VEGF significantly decreases overexpressing miR-34a in cell lines. 34 Thus, there is a feedback loop between miR-34a and VEGF. Consequently, it is interesting to develop miR-34a as a new biomarker and an innovative target for the treatment of cancers in future. 34 Taken together, miR-34a plays crucial roles that mainly involve SIRT1, according the recent studies.
The regulation of miR-34a activity provides an innovative therapeutic strategy for the treatment of HNSCC, prostate and breast cancer.
Recent studies found that miR-124-3p is significantly decreased in ECs from pulmonary arterial hypertension (PAH) patients and in various cancers tissues, associated with poor prognosis in patients, 35,36 whose up-regulation can attenuate glioma cell proliferation, migration and tumour angiogenesis in vitro and in vivo by NRP-1mediated PI3K/AKT/NFjB pathways and by targeting R-Ras and N-Ras. 35,37 Meanwhile, it also inhibits angiogenesis and proliferation by targeting Ets-1 and AKT2 in breast cancer cells. 38 Moreover, increased levels of miR-124-5p can inhibit angiogenesis and growth of glioma via suppressing LAMB in vitro and in vivo and may serve as a promising potential target of new therapeutic strategies for glioma. 39 These studies demonstrate that miR-124 may act as a promising and useful diagnostic/prognostic marker and new therapeutic target for tumour through inhibiting angiogenesis in future.

| MiR-29
MiR-29 includes miR-29a, miR-29b and miR-29c, and they show high sequence similarity and share a common seed sequence for target recognition. 40 MiR-29 is aberrantly increased in diabetic myocardial microvascular endothelial cells (MMEVCs), and inhibition of miR-29 can enhance angiogenesis in diabetic MMEVC by promoting cell proliferation and migration via increasing IGF1. 41 Moreover, serum miR-29c-3p in AAA patients is also significantly increased compared with controls and are correlated with aneurysm diameter, which inhibits VEGFA in ECs, 42 suggesting that it may inhibit angiogenesis in ECs. However, the levels of miR-29a/b/c are obviously downregulated in various cancers, including endometrial carcinoma, hepatocellular carcinoma (HCC), gastric cancer and breast cancer. 40,[43][44][45] MiR-29b can repress angiogenesis in endometrial carcinoma by targeting VEGFA via the MAPK/ERK and PI3K/AKT signalling pathways. 43 Parallelly, miR-29b is closely related to poor recurrence-free survival of HCC patients, and miR-29b overexpression can inhibit angiogenesis and tumourigenesis in vivo and weaken tube formation, and cell proliferation and migration in vitro via directly repressing MMP-2. 40 Importantly, therapeutic delivery of miR-29b can inhibit tumour angiogenesis and tumourigenesis with high efficiency by targeting AKT3 and inducing the expression of VEGF and C-myc. 44 Microvesicles (MVs) containing overexpressed miR-29a/c can efficiently repress VEGF in gastric cancer cells, inhibiting growth and tube formation of vascular cell and can also weaken angiogenesis and growth of tumour in vivo. 45 This indicates that therapy of miR-29a/b/c recovery has broad prospects in clinical applications. However, further studies are imperative to better understand the roles and mechanisms of miR-29 in different diseases.

| miR-17-92 cluster
The miR-17-92 gene cluster includes miR-17-5p, miR-17-3p, miR-18a, miR-19a, miR-20a, miR-19b and miR-92a, which exhibits miscellaneous biological functions in angiogenesis. 80,81 Studies have shown that miR-18 and miR-19 promote tumour angiogenesis by reducing connective tissue growth factor (CTGF) and thrombin sensitive protein 1 (Tsp1) and increasing VEGF. 51,80,81 However, based on this study, 82 miR-19b-1 suppresses cell migration and tube formation and obstructs the cell cycle from the S phase to the G2/M phase transition by inhibiting fibroblast growth factor receptor 2 (FGFR2) mRNA and the expression of cyclin D1 protein. Furthermore, antagonism of miR-19 improves arteriogenesis and blood flow recovery after ischaemia in aged mice by increasing FZD4/LRP6 signalling and b-catenin/TCF/LEF (T-cell factor/lymphoid enhancing factor)-dependent gene expression. 81 The contrary effects of miR-19 in vascular growth may be explained by the different mechanisms of miR-19 in angiogenesis in a tissue-specific way. That is, miR-19 targets specific mRNAs in a cell-type or cell context specific way and may serve as a valuable therapeutic agent in the specific context of angiogenesis.
Intriguingly, Panax notoginseng saponins (PNS) effectively suppresses tumour growth by reducing miR-18a and promotes myocardial ischaemia-induced angiogenesis by increasing miR-18a. 83 These results indicate that the expression of miR-18a can be altered by some medicines in a tissue-specific and bidirectional manner. Thus, it might be a riskless and practical therapeutic strategy for the treatment of cancers and vascular diseases by studying the drugs targeting miR-18a in future.
MiR-92a and miR-20a inhibit angiogenesis by targeting VEGFA and integrin subunit alpha5. 81 In the mouse models of limb ischaemia and MI, miR-92a inhibition leads to enhanced blood vessel growth and functional recovery of damaged tissue. 84 Recently, a novel study reported that irradiation effectively activates intradermally injected caged anti-miR-92a in the murine skin without substantially influencing miR-92a expression in other organs. 19 Furthermore, light activation of caged anti-miR-92a improved wound cell proliferation, wound healing and angiogenesis by derepressing the miR-92a targets Itga5 and SIRT1. 19 This interesting finding indicates that light-activatable anti-miRs may harbour great therapeutic potential for treating vascular diseases. Further studies are essential to implement deeper investigations and experiments for paving the way to examine anti-miRs as therapies for vascular diseases, which will contribute to inducting regeneration.

MiR-17 suppresses angiogenesis in ECs in vitro and in vivo by
significantly suppressing several targets, including the cell cycle inhibitor p21, the sphingosine 1-phosphate receptor 1 (S1P1/EDG1) and the protein kinase Janus kinase 1 (Jak1). 85 Interestingly, the inhibition of miR-17 and miR-20a selectively improves angiogenesis in ECs but does not influence tumour angiogenesis. 85 The results might The inhibition of miR-210 increases tumour cell apoptosis and autophagy and represses angiogenesis. 94 Thus, miR-210 might be a potential prognostic marker for judging tumour malignancy and be regarded as a valid target for the clinical auxiliary treatment of cancers and ischaemic diseases.

| miR-296
The expression of miR-296 induces angiogenesis in vascular disease and cancer. 95,96 A study showed that glioma or growth factormediated miR-296 in ECs leads to enhanced levels of pro-angiogenic growth factor receptors. Growth factor-induced miR-296 significantly enhances angiogenesis directly via reducing hepatocyte growth factor-regulated tyrosine kinase substrate (HGS), thereby increasing VEGFR2 and platelet-derived growth factor receptor-b (PDGFR-b) and inhibiting DLL4 and Notch1. 95,96 Additionally, the results have an effect on improving the expression of VEGF. Thus, these studies indicate an interesting feedback loop involving miR-296 and VEGF.
Intriguingly, epidermal growth factor (EGF) also induces miR-296, proposing a complex mechanism of miR-296 in angiogenesis. These studies suggest that miR-296 might enhance angiogenesis following a stroke in this setting. Furthermore, manipulation of miR-296 levels may demonstrate therapeutic effects in tumour growth and angiogenic disorders where angiogenesis is a pivotal component. 96

| miR-155
Growing evidences have shown that miR-155 is up-regulated in many types of human cancers and vascular diseases. [97][98][99] A recent study implied that miR-155 plays an important role in promoting tumour angiogenesis by inducing the down-regulation of von Hippel-Lindau (VHL), and the knockdown of miR-155 reduces the proliferation, migration and network formation of HUVECs. 97 In addition, the inhibition of miR-155 decreases the VEGF-induced tube formation abilities of human RMECs through the PI3K/AKT pathway and thereby inhibits retinal neovascularization. 100 Furthermore, VEGF induces the expression of miR-155. 101 Through studying the impact of surgery on the kinetics of miR-155, researchers found that surgery may up-regulate this angiogenesis-related microRNA. 102 Based on these findings, it might be possible that miR-155 will be a valuable prognostic marker and critical therapeutic target for angiogenesis-related diseases.

| miR-let-7
MiR-let-7f is down-regulated in the rat cortex in hypoxia and in diabetic BMACs. 103,104 Let-7f mimics enhance BMAC angiogenic function by reducing the expression of thrombospondin-2 (TSP-2). 103 However, further research is needed to clarify how let-7f and its regulatory pathways decrease the expression of TSP-2 in BMACs. In addition, let-7f mimics improve pro-angiogenic cell (PAC) number, proliferation, migration and network formation and promote angiogenesis in HUVECs exposed to cigarette smoke extracts (CSEs) by inhibiting the levels of TGF-bR1 (ALK5), SMAD2/3 and plasminogen activator inhibitor type 1 (PAI-1) both in vitro and in vivo. 105 Kong et al 106 found that miR-let-7e-5p is down-regulated in DVT patients and overexpression let-7e-5p enhances the ability of homing and thrombus revascularization in rat model of venous thrombosis (VT) via targeting Fas ligand (FASLG). This suggests that miR-let-7e-5p may be a novel therapeutic target in clinical treatment of DVT. In conclusion, using miRNA mimics might provide an innovative therapeutic strategy to improve angiogenesis in ischaemic diseases. 105

| miR-130a
miR-130a antagonizes anti-angiogenic homeobox proteins growth arrest homeobox (GAX) on EC proliferation and migration, and HoxA5 on tube formation. 107 MiR-130a inhibitor represses the growth and angiogenesis of haemangioma by targeting tissue factor pathway inhibitor 2 (TFPI2) via inhibiting the focal adhesion kinase (FAK)/phosphoinositide 3-kinase (PI3K)/Rac1/anti-mouse double minute (mdm2) signalling pathway. 108 In addition, the inhibition of miR-130a represses cell proliferation, migration and angiogenesis in gastric cancer by enhancing runt-related transcription factor 3 (RUNX3) protein expression. 109 Thus, miR-130a inhibitor might be regarded as a potential and useful therapeutic strategy for the treatment of cancers, and miR-130a mimic might be beneficial to ischaemia diseases. Nevertheless, it is worrying that the treatment of ischaemic diseases, using miR-130a mimic, might increase the risk of developing tumours, and the treatment of cancers using miR-130a inhibitor might lead to ischaemia diseases. Thus, further studies are required to better understand the specific target genes and signalling pathways of miR-130a in the regulation of angiogenesis.

| miR-483
MiR-483-5p is down-regulated under hypoxia condition and can inhibit the growth of HUVECs by targeting serum response factor (SRF), which recedes wound healing and tube formation. 110

| miR-206
The present research reported that miR-206 is regarded as a suppressor to modulate VEGF-mediated angiogenesis in triple negative breast cancer (TNBC) and NSCLS, which is significantly reduced under hypoxic condition. 112 In addition, the elevated levels of miR-206 can down-regulate expression of VEGF, MAPK3 and SOX9, thereby, particularly, inhibiting angiogenesis in TNBC tumours. 113,114 Furthermore, miR-206 represses proliferation, tube formation, growth and angiogenesis in NSCLC via targeting 14-33f and inhibiting the STAT3/HIF-1a/VEGF pathway. 114 A study suggested that miR-206 might be an innovative biomarker and suppress the progression of CAD by reducing VEGF. 115

| miR-26
MiR-26 consists of miR-26a and miR-26b. MiR-26a can be downregulated by pro-angiogenic stimuli such as VEGF or TNF, which can inhibit angiogenesis via targeting the SMAD1-Id1-p21 WAF/CIP1 /p27 signalling axis in ECs. 116 Importantly, a study showed that the inhibition of miR-26a can rapidly enhance angiogenesis and decrease AMI size with improved heart function in a mouse model of AMI, while overexpression miR-26a leads to the opposite results. 117 Mechanically, miR-26a represses VEGF signalling via directly targeting NgBR, and therefore inhibit angiogenesis by performing proliferation, migration and tube formation in HUVECs. 118 Furthermore, miR-26a/b is decreased in gastric cancer and HCC, 119

| miR-93
Based on the studies regarding hind-limb ischaemia and tumours, miR-93 promotes angiogenesis and/or inhibits angiogenesis in various molecular pathways. 124 Many studies support the roles of miR-93 in promoting angiogenesis and improving EC proliferation, migration, spreading and tube formation. 124,125 The overexpression of miR-93 increases perfusion recovery from hind-limb ischaemia 124 and improves angiogenesis in breast cancer via inhibiting homology 2 (LATS2). 125

| The miR-16 family
Members of the miR-16 family include miR-15a/b, miR-16, miR-195, miR-424 and miR-497. A study implied that hypoxia-induced reduction of miR-15b and miR-16 contributes to an increase in VEGF. 129 Thus, it is speculated that the overexpression of miR-15 and miR-16 may be an attractive anti-tumour strategy that reduces tumour cell proliferation and blocks VEGF-mediated angiogenesis.
Research results supported that miR-15b plays an important role of inhibiting brain tumour angiogenesis by neurofilament protein-2 (NRP-2) through the deactivation of the MEK/ERK pathway. 130 Ginsenoside-Rg1 plays a significant role in the wound healing pro- protein to inhibit angiogenesis in human dental pulp cells (hDPCs). 138 This finding indicates that the down-regulation of miR-424 might be an alternative strategy for the treatment of dental pulp diseases.
Moreover, miR-424 inhibits the proliferation, migration and tube formation of ECs by targeting VEGFR2, FGFR1 and the VEGF 3 0 UTRs, and the expression of VEGF and bFGF also up-regulates miR-424. 139 SUN ET AL.

| 4575
These results indicate that there exist regulatory circuits between miR-424 and the main vascular growth factors. Furthermore, miR-424 decreases angiogenesis by directly reducing VEGFA protein levels in endometrial and endometriotic cells. 140 In keeping with this, miR-424 reduces cell proliferation and angiogenesis in senile haemangioma by inhibiting the expression of MEK1 and cyclin E1. 141 Interestingly, MEK1 and cyclin E1 may remain with the same signalling pathway to manage the cell cycle, because MEK1 is the upstream molecule of ERK and cyclin E1 is a downstream target of ERK. 142,143 In summary, miR-424 has dual effects on angiogenesis, but it promotes angiogenesis in most cases. The contrary effects on angiogenesis may be explained by the assumption that miR-424 may activate the different signals to regulate angiogenesis in different conditions. However, it is a challenge for us to study the interactions between miR-424 and its specific target genes in complex situations.
Taken together, these findings regarding the members of the miR-16 family provide innovative insights into the complex regulation of angiogenesis. It will not be a surprise that targeting specific members of the miR-16 family may provide an interesting therapeutic perspective for cancers and vascular diseases.

| miR-27b
Recently, increasing studies demonstrated that the roles of miR-27b are completely different in different type cancers and vascular diseases. [144][145][146][147] A study showed that miR-27b improves angiogenesis in

| Other miRNAs in angiogenesis
There are other miRNAs without an in-depth study in the sense of function and mechanism of angiogenesis. Studies showed that miR-9, miR-135a, miR-181a, miR-181b, miR-199b and miR-204 may manage angiogenesis via targeting SIRT1. 150 MiR-200b, miR-361-5p, miR-874, miR-125-5p and miR-146 are involved in angiogenesis by regulating VEGF. [151][152][153] In addition, the miR-214/eNOS pathway is involved in Rg1-induced angiogenesis.   162 Moreover, plasma miR-26a is up-regulated in a model of AMI in mice and in human beings with ACS. 117 Interestingly, recent discoveries showed that circulating miR-214 was up-regulated in the early phase after AMI but then gradually decreased to near normal levels. 163 Aerobic exercise training can alter the cardiac miRNA expression in physiological cardiac remodelling, such as miR-1, miR-150, miR-21, miR-122, miR-126 and miR-208. 164 Mechanically, miR-1 is enriched in cardiomyocytes and modulates myogenesis, cardiac development and hypertrophy, and miR-208 is a cardiac-specific miRNA expressed by introns of myosin heavy chains and involved in stress-dependent cardiac growth and gene expression. 165 Furthermore, the up-regulated miR-26a in AMI can inhibit angiogenesis, and thereby aggravates MI. 117 However, further studies are necessary and important for exploring the role and mechanism of other specific miRNAs in the regulation of CVD pathogenesis.

| Arteriosclerosis
A study found that miR-21, miR-130a, miR-27b, miR-210, and let-7f in the intima of human atherosclerotic plaques are increased and in the accompanying serum miR-130a, miR-27b and miR-210 are also up-regulated. 166 In addition, miR-92a, miR-125a, miR-221, miR-34a, and miR-146a/b are up-regulated, while miR-10a is down-regulated in atherosclerotic portions of vessels. 166 Interestingly, some of them can promote endothelial activation and plaque formation, and miR-125a expression is significantly and negatively correlated with serum LDL-c levels of symptomatic patients. 160 Furthermore, miR-10a contributes to the inhibition of pro-inflammatory endothelial phenotypes in atherosusceptible regions in vivo, and miR-92a can impair endothelial functions during atherogenesis. 167 Consistently, miR-92a can promote atherogenesis and vascular inflammation the process of arteriosclerosis. 158 MiR-221 plays a major role in the regulation of Overall, present studies support differences in miRNA expression profiles in atherosclerosis vs controls and extend the knowledge base to miRNAs as potential biomarkers in patients with arteriosclerosis.

| Hypertension
Some recent studies reported that serum miR-510 is increased in hypertension patients 175 and serum miR-7-5p and miR-26b-5p are elevated in the left ventricular hypertrophy (LVH) hypertensive patients compared with healthy individuals. 176 Moreover, let-7 levels in ECs and plasma from hypertension patients are higher than those from healthy controls. 177 However, levels of miR-9 and miR-126 in peripheral blood mononuclear cells (PBMCs) of hypertensive patients are reduced compared with healthy controls. 178 Functionally, miR-7-5p can repress EC proliferation and angiogenesis by targeting RAF1, 176 and let-7 can induce oxidative stress and cell injury, 177 suggesting that the up-regulation of let-7 may aggravate atherosclerosis, thereby modulating hypertension. In addition, miR-9 can inhibit myocardin expression, and miR-9 mimic can reverse the hypertrophic response and improve cardiac structure and function. 179 Exhilaratingly, one study found for the first time that plasma miR-30a and miR-29 in white-coat hypertension (WCH) patients are significantly higher while plasma miR-133 in WCH patients is lower than hypertension patients and healthy controls, indicating that plasma miR-

| Pulmonary arterial hypertension
PAH eventually leads to heart failure and death, mainly because of the lack of effective diagnostic methods that can be detected earlier. Overall, miRNAs play an important role in the development of PAH and in diagnosis and treatment for PAH.

| Aneurysm
Aortic aneurysm (AA) is an increasingly common and ultimately fatal rupture with no effective drug treatment, 187  are decreased. 191,193 However, miR-29b was up-regulated in human tissues from thoracic AA patients. 187 Moreover, serum miR-29c-3p in AAA patients is significantly increased compared with controls and are correlated with aneurysm diameter, which inhibits VEGFA in ECs. 42 Additionally, the expression of miR-146a is different in different types of samples from AAA patients. It is increased in AAA tissues 188 but is decreased in plasma of AAA patients. 191 This may be because of different sources of sample and disease characteristics. Further, pre-miR-24, anti-miR-29b and anti-miR-33 treatment can attenuate aneurysm in mouse models. 187,192,194 These results strongly reveal that miRNA treatment may be a novel and useful therapeutic strategy for AA.
In addition, plasma levels of miR-16 and miR-25 are significantly increased in intracranial aneurysms (IA) patients. 195 Circulating miR-

| Kawasaki disease
KD is an acute, self-limited vasculitis that mainly affects mediumsized arteries, especially the coronary arteries. 197,198 Levels of miR-143, miR-199b-5p, miR-618, miR-223, miR-145-5p and miR- 145-3p in whole blood from acute KD are significantly up-regulated compared with convalescent KD. 199 Consistent with whole blood samples, plasma miR-145-5p levels are highly expressed in from patients with KD. 200 Plasma miR-125a-5p levels are also significantly elevated in both of acute and convalescent KD patients compared with controls, suggesting that miR-125a-5p may be potential diagnostic biomarkers for early KD. 201 In the recent study, miR-186 is confirmed to be significantly up-regulated in the serum of patients with KD and in HUVECs stimulated with KD serum, and its serum expression is down-regulated to normal levels in convalescent KD. 197

| Aortic dissection
AD is a catastrophe of the aorta, which is a rare but catastrophic disease. Given that, if left untreated, the mortality rate of acute aortic dissection (AAD) approaches 50% in the first 48 hours of onset. 203 Therefore, the early diagnosis and timely treatment of AD are essen- miR-4313, miR-933, miR-1281 and miR-1238 are up-regulated both in aortic tissue and in plasma from AAD patients. 207 In a word, some miRNAs may be acted as novel and useful biomarkers for diagnosis of AAD and more information on the role of miRNAs in the development and procession of AD needs to be studied.

| Phlebothrombosis
The first study has found that the serum levels of miR-582, miR-195 and miR-532 are higher in DVT patients, which suggests these miR-NAs present novel non-invasive biomarkers for detection of DVT. 156 Further study demonstrated that miR-195 inhibits proliferation, migration, angiogenesis and autophagy of hEPCs under hypoxia by targeting GABA Type A Receptor Associated Protein Like 1 (GABAR-APL1), 208 and miR-532 is associated with lipopolysaccharide (LPS)stimulated macrophage inflammatory response. 209 In plasma assays, one study found that miR-10b-5p, miR-320a, miR-320b, miR-424-5p and miR-423-5p are elevated, while miR-103a-3p, miR-191-5p, miR-301a-3p and miR-199b-3p are reduced in VTE patients. 210 Another study also found miR-424-5p is significantly up-regulated, whereas miR-136-5p is down-regulated in the plasma of DVT patients. 211 Both studies found that miR-424-5p is up-regulated in the plasma of VT patients at different stages, which suggests that miR-424-5p is highly correlated with VT and may be used as biomarkers for VT.

| Diabetic microvascular complication
Concerning diabetes, miR-503 and miR-377 are highly up-regulated in the plasma of diabetic patients with CLI and in the model of diabetic nephropathy (DN), respectively. 213 In addition, miR-200b is increased in the model of diabetic retinopathy (DR), which plays a protective role in DR by modulating neuronal sensitivity to oxidative stress via inhibiting oxidation resistance (Oxr-1) protein. 214 Serum miR-320a and miR-27b are elevated, which is related to T1DR. 215 Moreover, miR-21, miR-181c and miR-1179 are significantly upregulated in patients with proliferative DR (PDR) compared with non-proliferative DR (NPDR), 216 suggesting that miRNAs may serve as potential biomarkers for detecting the progression of PDR from NPDR. 213 Functionally, miR-21 is highly related to angiogenesis with the microenvironment of high glucose by protecting EC against high glucose-induced endothelial cytotoxicity, 217 and miR-181c may be linked with vascular proliferation in high glucose because of its high level in vein ECs treated in a diabetic-like environment. 218 Besides, elevated serum levels of miR-217 are associated with the occurrence of proteinuria in T2DN patients. 215 215 Studies reported that the three decreased miR-NAs are related to increased levels of IFTA and tubulointerstitial inflammation, suggesting the role of these miRNAs in fibrosis formation in DN. 219 Notably, studies reported that miR-192 is increased in the early stage of DN patients but it is decreased in the late stage, 220 Among others, the detection of miRNAs in urine may act as a fresh non-invasive approach for diagnosis and dynamic monitoring of diabetic microvascular complication to especially improve the prediction, treatment and prognosis without the need for invasive diagnostic or radiological procedures.

ANGIOGENESIS-RELATED DISEASES
Currently, miRNA therapy in the clinical studies mainly uses miRNA inhibitors (anti-miRs)/miRNA mimics or miRNA transfected cells. It is generally considered that inhibiting an endogenous miRNA is less risky than overexpressing a miRNA. 222 The common miRNA-based therapies include intravenous, intraperitoneal or intramuscular injections. The blood flow in the ischaemic hindlimb is significantly improved via intravenous injection of miR-126-loaded Bubble liposomes. 223 Nevertheless, systemic injections may result in promoting tumour growth and platelet activation. Interestingly, local intramuscular injection can reduce systemic side-effects. Furthermore, altering specific miRNA expression using exercise training or drugs appears to be a new and powerful therapy to treat angiogenesis-related diseases. Long non-coding RNAs (lncRNAs) may act as miRNA sponges to decrease miRNA levels. Studies have shown that lncRNA CHRF binds and sequesters miR-489, thus, preventing the miRNA from acting on its target genes that activate hypertrophic responses, 164 revealing that the participation of the lncRNA-miRNA-mRNA axis provides an intriguing approach for tackling angiogenesis-related diseases. Thus, the clinical application of miRNAs may contribute to the diagnosis and treatment of angiogenesis-related diseases.
However, there are some limitations because of the use of miR-NAs that are not cell-type or organ specific, which may cause potential side-effects. A recent review article has shown that the challenges of miRNA-based therapy are tissue-specific delivery, minimizing off-target effects and dose optimization. 51