Micro-RNA Analysis in Pulmonary Arterial Hypertension: Current Knowledge and Challenges

STATE-OF-THE-ART REVIEW Micro-RNA Analysis in Pulmonary Arterial Hypertension Current Knowledge and Challenges Cátia A. Santos-Ferreira, MD, a,d Mónica T. Abreu, PHD, b,c,d Carla I. Marques, PHD, b,c,d Lino M. Gonçalves, MD, PHD, a,b,c,d Rui Baptista, MD, PHD, a,b,c,d,e Henrique M. Girão, PHD b,c,d HIGHLIGHTS The role of miRNAs in PAH is fast expanding, and it is increasingly difficult to identify which molecules have the highest translational potential. This review discusses the challenges in miRNA analysis and interpretation in PAH and highlights 4 promising miRNAs in this field. Additional pre-clinical studies and clinical trials are urgently needed to bring miRNAs from the bench to the bedside soon. SUMMARY Pulmonary arterial hypertension (PAH) is a rare, chronic disease of the pulmonary vasculature that is associated with poor outcomes. Its pathogenesis is multifactorial and includes micro-RNA (miRNA) deregulation. The understanding of the role of miRNAs in PAH is expanding quickly, and it is increasingly difficult to identify which miRNAs have the highest translational potential. This review summarizes the current knowledge of miRNA expression in PAH, discusses the challenges in miRNA analysis and interpretation, and highlights 4 promising miRNAs in this field (miR-29, miR-124, miR-140, and miR-204). (J Am Coll Cardiol Basic Trans Science 2020;5:1149–62) © 2020 The Authors. Published by Elsevier on behalf of the American College of Cardiology Foundation. This is an open access article under the CC BY-NCND license (http://creativecommons.org/licenses/by-nc-nd/4.0/). Pulmonary arterial hypertension (PAH) is a rare multifactorial condition, hemodynamically definedasanincreaseinthemeanpulmonary arterial pressure (>20 mm Hg, according to the recently revised 6th World Symposium on Pulmonary Hypertension definition), with normal left ventricular filling pressures and elevated pulmonary vascular resistance (1,2). It is characterized by structural and functional changes in the pulmonary arterial vasculature that evolve from isolated medial hypertrophy to end-stage plexiform fibrosis, originating an increase in pulmonary vascular resistance (3). The progressive increase in right ventricular (RV) afterload leads to RV hypertrophy and, ultimately, RV failure and death (4). Despite considerable progress in the understanding of the epidemiology, pathophysiology, and management of PAH, the prognosis remains poor, especially in patients with severe disease (i.e., World Health Organization [WHO] functional class IV) (1,5). ISSN 2452-302X https://doi.org/10.1016/j.jacbts.2020.07.008 From the a Cardiology Department, Centro Hospitalar e Universitário de Coimbra, Coimbra, Portugal; b University of Coimbra, Coimbra Institute for Clinical and Biomedical Research, Faculty of Medicine, Coimbra, Portugal; c University of Coimbra, Center for Innovative Biomedicine and Biotechnology, Coimbra, Portugal; d Clinical Academic Centre of Coimbra, Coimbra, Portugal; and the e Cardiology Department, Centro Hospitalar Entre Douro e Vouga, Santa Maria de Feira, Portugal. The authors attest they are in compliance with human studies committees and animal welfare regulations of the authors’ institutions and Food and Drug Administration guidelines, including patient consent where appropriate. For more information, visit the JACC: Basic to Translational Science author instructions page. Manuscript received June 3, 2020; revised manuscript received July 23, 2020, accepted July 24, 2020. JACC: BASIC TO TRANSLATIONAL SCIENCE VOL. 5, NO. 11, 2020 ª2020 THE AUTHORS. PUBLISHED BY ELSEVIER ON BEHALF OF THE AMERICAN COLLEGE OF CARDIOLOGY FOUNDATION. THIS IS AN OPEN ACCESS ARTICLE UNDER THE CC BY-NC-ND LICENSE (http://creativecommons.org/licenses/by-nc-nd/4.0/). Patients in WHO class IV have higher right atrial pressure and pulmonary vascular resistance, lower exercise capacity and peak oxygen consumption, and a 3-year survival rate of 38%, compared with >60% observed for patients in WHO classes I to III (5–7). Current PAH therapies primarily target 3 vasomotor pathways: the nitric oxide, the endothelin-1, and the prostacyclin pathways (1). However, these interventions might not accurately target the intricate molecular primum movens of PAH, because genetic susceptibility, hypoxia, inflammation, DNA damage, viral infection, and shear stress are all involved in various amounts within the pathogenic events that contribute to the patient-specific phenotype of disease manifestation and progression (8). PAH involves a single organ, the lungs, but multiple pulmonary arterial cell types—pulmonary artery endothelial cells (PAECs), pulmonary artery smooth muscle cells (PASMCs), and pulmonary arterial adventitial fibroblasts (PAAFs) (9). The inability to fully understand this complex interplay of cells and soluble mediators has hampered the development of new drugs. In the last 15 years, no new drug aimed at novel pathological pathways has been approved. One of the players of this complex network of stimuliaremicro-RNAs(miRNAs).Mostofour genome is not directly implicated in protein synthesis but, rather, in the production of noncoding RNAs (ncRNAs) with regulatory functions (10). Among the several RNA species, 2 varieties of ncRNAs, loosely defined as long and small RNA, have been described recently. Among the small RNAs, miRNAs are short length, noncoding endogenous RNA molecules that consist of approximately 22-nucleotide-long sequences profusely expressed in all human cells and are believed to control approximately 50% of all protein-coding messenger RNA (mRNA) (11). ThebiogenesisofmiRNAsisclassified into 2 pathways: the canonical (the dominant pathway by which miRNAs are processed), and non-canonical pathways. In the canonical pathway, transcription of miRNAs genes produces primary miRNAs that are hairpin-like and can be >1,000 nucleotides long; this transcription is dependent on polymerase II and is regulated by transcription factors (12). Primary miRNAs are cleaved in the nucleus to form precursor miRNAs, which are a shorter hairpin approximately 70 nucleotides long, by the Microprocessor complex, which consists of an RNA-binding protein DiGeorge Syndrome Critical Region 8 and a ribonuclease III enzyme, Drosha. Precursor miRNAs are then exported from the nucleus to the cytoplasm by exportin-5. In thecytoplasm,precursormiRNAsareprocessedinto short, double-stranded, immature miRNA by another ribonuclease, Dicer. In the following step, the 2 strands are separated, and 2 of these strands (mature miRNA) are recruited by Argonaute proteins to incorporate a multiprotein complex known as the RNA-induced silencing complex (13). In addition to the canonical miRNA biogenesis pathway, multiple non-canonical pathways have been revealed by using different combinations of the proteins involved in the canonical pathway. In general, they can be classified as Droshaand Dicer-independent pathways (13). MiRNAs are able to modify protein expression by binding to complementary sequences on mRNA, mainly in the 30-untranslated region of the target mRNA transcripts, thereby promoting translational inhibition and degradation by altering the stability of target mRNAs (14).ThematuremiRNAisintegrated into the RNA-induced silencing complex and then binds to its mRNA target. Once bound to an mRNA, the miRNARNA-induced silencing complex promotes the downregulation of the protein that the mRNA encodes, mainly through direct mRNA degradation (15). A specificmiRNAcantargetmultiple mRNAs (the so-called divergent pathway), and a mRNAcanhavemultiplebindingsitesforseveral miRNAs. In addition, various related miRNAs can affect a pathway at different levels, termed a convergent pathway (14). The interactome of miRNAs with mRNA is a multidimensional network and plays a crucial role in regulating cellular pathways and biological functions in health and disease (16). During pathological processes, such as pulmonary vascular processes involved in PAH, the dysregulation of specific and distinct miRNAs has profound consequences in cell function due to changes in protein synthesis (14). Therefore, miRNAs have been recognized as promising biomarkers and therapeutic options for many cardiovascular diseases (14,17). However, one of the issues regarding the study of miRNA in PAH resides in the large heterogeneity of models and cells analyzed (18,19). miRNA regulation is not conserved between the various pulmonary hypertension (PH) animal models. These differences may have important implications in how preclinical miRNA variations should be interpreted as potential biomarkers and therapeutic targets and consequently translated to patients with PAH (19). Among the various techniques available to study miRNAs regulation in cell lines, animal models, and patients, microarray analysisisofgreatuse,because ABBREVIATIONS AND ACRONYMS BMPR2 =bone morphogenetic protein receptor type 2 EPC =endothelial progenitor cell HIF =hypoxia-inducible factor HPAH =hereditary pulmonary arterial hypertension lncRNA =long noncoding RNA MCT =monocrotaline miRNA =micro-RNA mRNA =messenger RNA ncRNAs =noncoding RNAs PAAF =pulmonary arterial adventitial fibroblast PAEC =pulmonary artery endothelial cell PAH =pulmonary arterial hypertension PASMC =pulmonary artery smooth muscle cells PH =pulmonary hypertension RV =right ventricle SU/Hx/Nx =association of Sugen 5416 with chronic hypoxia followed by normoxia WHO =World Health Organization Santos-Ferreira et al.JACC: BASIC TO TRANSLATIONAL SCIENCE VOL. 5, NO. 11, 2020 MicroRNA in Pulmonary Arterial Hypertension NOVEMBER 2020:1149–62 1150 it allows the detection of genome-wide gene expression (20). The development of these powerful, effective, and high-throughput data methodologies enables the unbiased screening of mechanisms altered in a specific dataset and the search for differentially expressed genes, according to the experimental design (e.g., healthy control subjects vs. patients with disease). They have been broadly applied to investigate the pathophysiology of diverse cardiovascular diseases (14,17). Recently, the role of long non-coding RNAs (lncRNA) in the pathobiology of PAH has also been explored. LncRNAs are longer than 200 nucleotides and are abundantly present in the genome (21). Based on their subcellular localization, lncRNAs are able to regulate gene expression through different mechanisms. In contrast to miRNA, lncRNA can yield their regulatory effects, both at the transcriptional and post-transcriptional levels, through interactions with DNA, chromatin, and other species of RNA. In addition, they can act as epigenetic modulators (22). Emerging evidence suggests that these molecules play an important role in PAH pathogenesis as fundamental drivers and gatekeepers in the regulation of key cellular and molecular trafficking in PASMC and PAEC dysregulation (21). In this review, we summarize the current knowledge on miRNA expression in experimental models of PH and patients with PAH, focusing our analysis on thosemiRNAsthathavebeenfoundtohavea conserved regulation across animal models and human PAH samples using microarray analysis (Central Illustration). Finally, we highlight future perspectives and potential challenges concerning miRNA application in the clinical arena, namely, in the development of novel diagnostic and therapeutic tools for PAH. CHALLENGES IN MICRO-RNA ANALYSIS AND INTERPRETATION IN PAH The first evidence that suggested that miRNAs contribute to the pathogenesis of PAH resulted from the observation that bone morphogenetic protein receptor type 2 (BMPR2) protein, but not mRNA levels, was reduced in animal models of PH (23). Since then, miRNAs have been implicated in a wide range of pulmonary vascular processes involved in PAH (14). However, many challenges limit the validation of miRNAs discovered in experimental models. LACK OF CONCORDANCE AMONG ANIMAL PH MODELS. First, although all PH animal models are characterized by an increase in RV afterload and progression to RV failure, the trigger is substantially different between them (19,24). Therefore, there is a lack of concordance in the pattern of miRNA expression among different experimental models of PAH (19,24). For example, administration of monocrotaline (MCT) to rats mostly targets the pulmonary vascular endothelium and elicits a strong pulmonary inflammation process, especially monocyte recruitment, which plays an important role in human idiopathic PAH (24). Conversely, in the chronic hypoxia murine model, persistent hypoxia causes vascular structural remodeling in all 3 layers of the pulmonary arteriolar wall, with hypertrophy of PASMCs being a major histological finding and having less inflammation compared with the MCT model (19,25). The model induced by the association of vascular endothelial growth factor receptor antagonist, Sugen 5416, with chronic hypoxia followed by normoxia (SU/Hx/ Nx) causes profound and sustained PH, along with inflammation and angio-obliteration, culminating in RV remodeling and failure, in both rats and mice (24). The subjacent mechanism is believed to involve death of PAECs, with subsequent proliferation of an apoptosis-resistant cell type. The restitution of normoxia results in the development of neointimal lesions extremely similar to human plexogenic arteriopathy (24). Although the SU/Hx/Nx model has been suggested as a more relevant model of human PAH (19,26), miRNAs have predominantly been studied in the MCT or chronic hypoxia models. Despite reproducing important characteristics of human PAH, these models generally fail to reproduce the severe pulmonary arteriopathy usually present in patients with PAH (19). Other models have been developed to better reproduce the natural history of human PAH. Recently, a rat model with a monoallelic deletion of 71 bp in exon 1 in the Bmpr2 gene was developed. This model showed not only some of the pivotal cellular and molecular dysfunctions described in human PAH, but also the gradual phenotype seen in humans. It also showed myocardial abnormalities, supporting the hypothesis that the RV might also be affected in PAH besides the increased afterload imposed by pulmonary vascular disease (27). Considering these pathophysiological differences, it is not surprising there is divergence in miRNA regulation among the different animal models reported in the literature (18,19). Schlosser et al. (19) explored these disparities in the pattern of expression of a set of miRNAs causally implicated in PAH in the plasma, lung, and RV of different animal models and in plasma from patients with PAH (19). They found that most of the miRNA investigated had discordant patterns across the different tissues and models (19). Moreover, after analyzing the mRNA targets of JACC: BASIC TO TRANSLATIONAL SCIENCE VOL. 5, NO. 11, 2020 Santos-Ferreira et al. NOVEMBER 2020:1149–62 MicroRNA in Pulmonary Arterial Hypertension 1151 miRNAs, they also found discordant regulation in the different models, which suggested that in these scenarios miRNA might not be the only or the most significant regulation mechanism (19). HETEROGENEITY IN REGULATION AMONG DIFFERENT HUMAN PAH ETIOLOGIES. Second, phenotypic variability between predominantly hypoxic and nonhypoxic human forms of PAH regarding miRNA levels might therefore be recapitulatedbywhatisfoundintheanimalmodels,which suggests different pathological mechanisms of disease.Asinanimalmodels,thedifferentformsof human PAH are unlikely to be equivalent, and it remains unclear whether the various types of PAH share the same pathophysiology (26). Although they have CENTRAL ILLUSTRATION Key Micro-RNAs Associated With Cardinal Features of Pulmonary Arterial Hypertension Santos-Ferreira, C.A. et al. J Am Coll Cardiol Basic Trans Science. 2020;5(11):1149–62. The miRNA dysregulation plays an important role in the hyperproliferative and apoptosis-resistant phenotype of pulmonary vascular cells in PAH, including PAECs, PASMCs, and PAAFs. This figure illustrates the lack of concordance in the pattern of expression of most miRNAs between human PAH and different animal PH models and highlights 4 miRNAs that might ultimately represent a greater potential of translating into the clinical arena. miRNA ¼microRNA; PAAF ¼pulmonary arterial adventitial fibroblasts; PAEC ¼pulmonary artery endothelial cell; PAH ¼pulmonary arterial hypertension; PASMC ¼pulmonary artery smooth muscle cell; PH ¼ pulmonary hypertension; SU/Hx/Nx ¼association of Sugen 5416 with chronic hypoxia followed by normoxia. Santos-Ferreira et al.JACC: BASIC TO TRANSLATIONAL SCIENCE VOL. 5, NO. 11, 2020 MicroRNA in Pulmonary Arterial Hypertension NOVEMBER 2020:1149–62 1152 similar histopathological reflections in the pulmonary artery, there are different forms of PAH within the same classification; therefore, differential miRNA profiles may reflect different mechanisms of disease (18). We have previously reported that in patients in whom the hypoxic phenotype is predominant (e.g., Eisenmenger syndrome, lung-disease related PH, or advanced right heart failure, in which low cardiac output leads to hypoxia at a peripheral tissue level), the upregulation of miR-424(322) promotes hypoxiainducible factor (HIF)-1 a accumulation,which,in turn, contributes to increased miR-424(322) levels (28). Although in this hypoxic phenotype—exposed to high HIF-1 a levels—miR-424(322) likely promotes some amount of pulmonary vascular proliferation, it is also necessary to maintain the life-saving compensatory mechanisms that mitigate the pathophysiological manifestations of chronic hypoxia (e.g., erythrocytosis). This might explain why lower levels of miR-424(322) were associated with a poorer prognosis in patients with Eisenmenger syndrome in our cohort, because they might be a cause (or a consequence) of a detrimental lower global activation of HIF-1 a compensatory pathways (28). For example, it is well recognized that relative anemia due to iron deficiency is a marker of poor prognosis in Eisenmenger syndrome (29,30). Conversely, in other patients with PAH, such as those with idiopathic PAH, hereditary PAH, drug-induced PAH, or connective tissue diseaseassociated PAH, the hypoxic component is likely not as relevant to the mechanism of disease. For instance, in patients with hereditary PAH, the BMPR2 pathway can be downregulated by mutations in several genes (BMPR2, ACVRL1, ENG, SMAD1, SMAD4, SMAD9)(31). In addition, even if there are no identified mutations, the pathway can be downregulated in patients with idiopathic PAH (32). PAECs subjected to hypoxia conditions show downregulation of miR-424(322) levels, whereas, in PASMCs, no differences have been found compared with control cells (28). Paradoxically, we (28)and others (19) found that circulating miR-424(322)/503 levels were elevated in patients with PAH and patients with chronic thromboembolic PH. However, those patients were themselves in various phases of thediseaseatthetimeofanalysis.Onceagain,the timepoint of miR-424(322) assessment and organspecific differences (lung, heart) might have an important role in these apparently contradictory results (19,28). Together, these findings suggest that the balance of hypoxic and inflammatory and/or genetic stimuli in each patient will determine the specific patient-level deregulation of miRNAs, again adding complexity to translational approaches. DIFFERENT miRNA EXPRESSION PROFILE BETWEEN RV HYPERTROPHY AND RV FAILURE. Finally, there aredifferencesinthemiRNAexpressionprofile between RV hypertrophy and RV failure, which suggests specific changes in the signature of miRNAs during PAH progression (33,34). The comparison of the available data on miRNA expression in the RV reveals few overlaps among the studies (35–37). The different approaches to induce RV hypertrophy and failure (pulmonary artery bandingvs.chronichypoxia), different surgical interventions leading to different banding gradients, and the use of different species (mouse,rat,ovine)arelikelyreasons(34). The mechanisms underlying the transition from TABLE 1 Human Microarray Studies in PAH Patient Population Tissue Microarray Platform Analysis Method Observations Dataset 6 patients with PAH-SSc, 20 SSc patients without PAH and 9 healthy control subjects EPCs Affymetrix Human Exon 1.0 ST Array A linear model was fitted to the log-transformed expression values using the limma package 2,175 genes were found differentially expressed between control and SSc PAH GSE73674 15 patients with PAH (6 IPAH, 4 PAH CTD-PAH, 4 PAH secondary to CHD and 1 CTEPH) and 11 normal subjects Lung Affymetrix Human Gene 1.0 ST Array A linear model was fitted to the log-transformed expression values using the limma package 12,834 genes were found differentially expressed in the PAH cohort compared with control subjects GSE113439 6 patients with PAH and 3 healthy control subjects PASMCs Affymetrix Multispecies miRNA-4 Array A linear model was fitted to the log-transformed expression values using the limma package —GSE108707 14 patients with PAH and 14 sex-/ agematching control subjects PBMCs Affymetrix GeneChip Human Gene 1.0 ST Array A linear model was fitted to the log-transformed expression values using the limma package 2,624 genes were found differentially expressed between control subjects and patients with PAH GSE131793 CHD ¼congenital heart disease; CTD-PAH ¼connective tissue disease-associated pulmonary arterial hypertension; CTEPH¼chronic thromboembolic pulmonary hypertension; EPC ¼endothelial cells derived from circulating endothelial progenitor; IPAH ¼idiopathic pulmonary arterial hypertension; miRNA ¼micro-RNA; PASMC ¼pulmonary artery smooth muscle cell; PAH ¼pulmonary arterial hypertension; PBMC ¼peripheral blood mononuclear cell; SSc ¼systemic sclerosis. JACC: BASIC TO TRANSLATIONAL SCIENCE VOL. 5, NO. 11, 2020 Santos-Ferreira et al. NOVEMBER 2020:1149–62 MicroRNA in Pulmonary Arterial Hypertension 1153 compensated RV hypertrophy to RV failure remain unclear, and further investigations are needed to gain a better understanding of miRNA expression patterns in RV pathological remodeling (34,36). MICROARRAY TECHNOLOGY AND DATA ANALYSIS Genome-wide studies, as microarrays and RNA sequencing, deliver screening technologies that can rapidly identify genes, clusters of co-regulated genes, or pathways that are engaged in pathological processes. Microarray technology has been extensively applied in the study of complex disorders, including cancer, diabetes, and cardiovascular diseases (38). The intricate pathogenesis ofPAHisasuitabletarget for microarray technology, which provides an additional tool to further explore the expression of key genes and regulatory networks (18). In addition, highthroughput screening is useful in the detection of novel molecular targets for diagnosis, prognosis, and treatment. Therefore, in addition to the identification of coding RNAs, the expression of miRNAs can also be analyzed by microarray technologies. These technologies rely on nucleic acid hybridization target miRNAs and their corresponding complementary probes (39). First, complementary DNA is synthesized from isolated miRNAs by reverse transcription, labeled with a fluorescent dye, and then hybridized to the microarray. After eliminating any unbound complementary DNAs with a series of washing steps, the microarray is scanned to measure the fluorescence intensity on each probe spot, which is translated into the relative amount of each target miRNA from the original sample (39). MiRNA arrays have experienced substantial technological progress in recent years, with innovations in probe design, immobilization techniques, sample labeling, and signal detection methods (40). Although genome-wide miRNA microarray platforms convey an inexpensive way to analyze a large number of simultaneous measurements, they are unable to identify new miRNAs (41), which is only possible with RNA sequencing (42). Additional disadvantages of these platforms are related to miRNA intrinsic characteristics, such as a small margin to optimize the hybridization conditions secondary to their short length. Another disadvantage is the limited specificity for miRNAs that have similar sequences and maydifferbyaslittleasasinglenucleotide,which decreases the sensitivity and specificity of microarrays. Another pitfall of the microarray is the inability to quantify absolute miRNA abundance (39). Despite all the limitations, microarrays are still the best available tool for comparing the relative abundance of pre-defined miRNAs between 2 conditions (e.g., patients with disease and healthy control subjects) (39). Regarding PAH, the information on expression, function, and role of miRNA in human tissues is still limited. Overall, the array studies provide a solid characterization of end-stage damage in PAH (18). Although the current goal of microarray analysis in PAH has been to explore patterns of expression that identify novel biomarkers or help to understand the pathophysiology behind the disease andtheeffectsofdrugs,theyarealsouseful at confirming the relevance of possible etiologies unveiled in experimental models in humans (18). Considering this, we performed an analysis of human multitissue microarray datasets (Table 1). These datasets included endothelial cells derived from circulating endothelial progenitor (EPCs) (GSE73674), lung (GSE113439), PASMCs (GSE108707), and peripheral blood mononuclear cell (GSE131793) samples of both patients with PAH and healthy control subjects. We used GEOquery library to extract GEO datasets. Differentially expressed genes were determined by TABLE 2 Dysregulated miRNAs in Pulmonary Hypertension Organized by Their Mechanistic Role Mechanism miRNA Proliferation miR-210; miR-204; miR-424; miR-503; miR-130/301; miR-193; miR-17w92; miR-145; miR-21; miR-124; miR-140; miR-500b-3p; miR-206; miR-200; miR-338-3p; miR-141-3p; miR-133a-3p: miR-29a; miR-214; miR-223; miR-100; miR-222; miR-25; miR-138; miR-1; miR-497, miR-1268, miR-665; miR-98; miR-191; miR-30a-5p; miR-593-5p; miR-203; miR-429; miR-371b-5p; miR-760; miR-1181; miR-143; miR-92b-3p; miR-135a-5p; miR-23a; miR-1281; miR-361-5p; miR-195-5p; miR-150; miR-4632; miR-221-3p; miR-125; miR-34; miR-103/107 miR-322: miR-199b-5p; let-7a-5p; miR-20a; miR-328 Vasoconstriction miR-130/301; miR-328; miR-190; miR-29b; miR-1; miR-543; miR-27b; mR-328 DNA damage miR-223; miR-204 Estrogen signaling and sex-specific miR-96; miR-29 Angiogenesis miR-208; miR-126; miR-495 miR-206 Inflammation miR-181a/b-5p; miR-146b; miR-135; miR-124 Abbreviation as in Table 1. Santos-Ferreira et al.JACC: BASIC TO TRANSLATIONAL SCIENCE VOL. 5, NO. 11, 2020 MicroRNA in Pulmonary Arterial Hypertension NOVEMBER 2020:1149–62 1154 using the limma package (43). A design matrix was created for the 2 groups of samples (control subjects and patients with PAH), and a linear model was fitted to the log-transformed expression values. The differential expression was achieved after an empirical Bayes adjustment. MiRNA WITH SIMILAR PATTERNS OF EXPRESSION IN ANIMAL PH MODELS AND HUMAN PAH. Over the years, mounting evidence has explored the role of dysregulated miRNAs in the hyperproliferative and apoptosis-resistant phenotype of pulmonary vascular cells, including PAECs, PASMCs, and PAAFs, in the TABLE 3 A List of Well-Established Dysregulated miRNAs in PAH miRNA Changes in PH Sample type Targets Function (Ref. #) miR-29 YHypoxic PH rat PAAFs a -SMA, extracellular matrix collagen Fibrosis (44) MCT PH rat PASMCs Collagen I (45) (in adaptative RV hypertrophy) [ Fetally-implanted aortopulmonary shunt ovine Collagen A1, Collagen 3A1 (37) miR-124 YHuman PAH PASMCs NFAT PASMC proliferation (46) Human PAH PAAFs and human PAH PAECs PTPB1, MCP-1 Proliferation (47-49) miR-140 Y [ MCT PH rat PASMCs TNFa PASMC proliferation (50) SU/Hx/Nx and MCT PH rat PASMCs SMURF1 PASMC proliferation (51) Human hypoxia PASMCs SOD2 PASMC proliferation (52) SU/Hx/Nx PH rat RV MFN1 Apoptosis of cardiac myocytes (53) miR-204 YHuman PAH PASMCs HIF-1 a PASMC proliferation (54) Human PAH PASMCs RUNX2 PASMC proliferation (55) Human PAH and MCT PH rats PASMCs SHP2, Src kinase, NFAT PASMC proliferation (56) Human PAH and hypoxia rat PAECs ATG7 Autophagy (57) miR-210 [Hypoxic PH rat PASMCs ISCU PASMC proliferation (58) E2F3 PASMC proliferation (59) MKP-1 PASMC proliferation (60) miR-1 [ Y Plasma human PAH and Su/Hx/Nx PH rat lungs SOD1, Cx43, CAV2, KLF4 Endothelial dysfunction (61) SU/Hx/Nx PH rat lungs KCNA5 PASMC hypertrophy (62) Human PAH and hypoxic PH rat PASMCs SphK1 PASMC proliferation (63) MCT PH rat RV TGF b RV hypertrophy (64) miR-130/301 [SU/Hx/Nx mouse model EDN1 Vasoconstriction (65) MCT PH mouse PAECs BMPR2 Lung vascular remodeling (66) Pulmonary fibrosis mouse lung PPAR g -APOE-LRP8 Fibrosis (67) miR-138 [Hypoxic PH rat PASMCs Mst1 PASMC proliferation (68) Human PAH PASMCs MCUC PASMC proliferation (69) Human PAH PASMCs TASK-1 PASMC proliferation (70) miR-17w92 [Human PAH PAECs BMPR2 PAEC survival (71) Hypoxic PH mouse and MCT PH rat lungs p21 PASMC proliferation (72) Human PAH PASMCs PDLIM5 PASMC dedifferentiation (73) Hypoxic PH mouse PASMCs PHD2 PASMC proliferation (74) miR-21 [Human PAH PAECs BMPR2, RhoB Decrease angiogenesis and vasodilation (75) Human PAH PASMCs PTEN PASMC proliferation (76,77) Human PAH lung tissue DDAH1 PASMC proliferation (78) Hypoxia PH mouse lung BMPR2 PASMC proliferation (79) miR-214 [Human PAH PASMCs PTEN, CCNL2 PASMC proliferation (80,81) Hypoxia PASMCs ARHGEF12 PASMC proliferation (82) Human PAH PASMCs MEF2C, MYOCD, SMC PASMC proliferation (83) miR-223 YHuman PAH PASMCs PARP-1 PASMC proliferation (84) Hypoxia PH mouse and rat lung and hypoxia PASMCs RhoB, MYPT1, MLC2 PASMC proliferation (85) MCT PH rat PASMCs ITGB3 PASMC proliferation (86) miR-424 [Human plasma and MCT PH rat PAECs SMURF1, BMPR2 RV hypertrophy (28) YSU/Hx/Nx, MCT PH rat and human PAH PAECs FGF2, FGFR1 PASMC proliferation (87) MCT ¼monocrotaline; PAAF ¼pulmonary arterial adventitial fibroblasts; PAEC ¼pulmonary artery endothelial cell; PASMC ¼pulmonary artery smooth muscle cell; PH ¼pulmonary hypertension; RV ¼right ventricle; SU/Hx/Nx ¼association of Sugen 5416 with chronic hypoxia followed by normoxia; other abbreviation as in Table 1. JACC: BASIC TO TRANSLATIONAL SCIENCE VOL. 5, NO. 11, 2020 Santos-Ferreira et al. NOVEMBER 2020:1149–62 MicroRNA in Pulmonary Arterial Hypertension 1155 different PH rodent models and human PAH tissues. Because of the exhaustive list of such molecules (Table 2), we initially focused on key miRNAs with a well-established role in PAH pathogenesis (Table 3, Supplemental Figure 1). We then constructed a heatmap that showed the log-fold change expression of these miRNAs between patients with PAH and healthy control subjects in different tissues (Figure 1). As depicted in the heatmap, miRNAs could be distinctly expressed between the different tissues. Although miR-29, miR-17, and miR-223 were highly expressed in human PAH lung samples, miR-1, miR-124, miR-130, miR-138, miR-140, and miR-210 were downregulated. Regarding PASMCs, miR-204 and miR-29 were downregulated; conversely, miR-424 was upregulated. In EPCs, miR-1, miR-140, and miR-21 were upregulated, and miR-301 showed lower levels of expression compared with those of healthy control subjects. Finally, miR-130, the only miRNA with differential expression in patients with PAH versus healthy control subjects in peripheral blood mononuclear cells, was downregulated. In addition, in line with previous reports in the literature (19), we observed a lack of concordance in the pattern of expression of most miRNAs between human PAH (Figure 1) and different animal PH models (Table 3). This suggested that different miRNA-dependent mechanisms might contribute to experimental PH and human PAH, impairing potential diagnostic and therapeutic applications. Among all this heterogeneity, we highlighted 4 miRNAs, that due to their similar expression pattern among the different models, might ultimately represent a greater potential of translating into the clinical arena, namely, miR-29, miR-124, miR140, and miR-204 (Figure 2). MiR-29. The hypoxamiR miR-29 (88)isaproapoptotic miRNA family that targets myeloid cell leukemia 1 (MCL1), which is a B-cell lymphoma 2 (BCL2) family apoptosis regulator that can be suppressed by peroxisome proliferator-activated receptor gamma (Pparg) agonists in rat hearts (89). Likewise, the miR-29 family directly targets >16 extracellular matrix genes, providing solid evidence for antifibrotic effects in different organs, including the lungs and heart (90). In human PAH, miR-29 is significantly upregulated in the lung, but downregulated in PASMCs and has shown a trend to lower levels in EPCs and peripheral blood mononuclear cells FIGURE 1 Heatmap Showing logFC Expression of the miRNAs Between Patients With PAH and Healthy Control Subjects in Different Tissues The highlighted boxes represent p values <0.05. EPC ¼endothelial cells derived from circulating endothelial progenitor; logFC ¼log-fold change; miR ¼micro-RNA; PAH ¼pulmonary arterial hypertension; PASMC ¼pulmonary artery smooth muscle cell; PBMC ¼peripheral blood mononuclear cell. Santos-Ferreira et al.JACC: BASIC TO TRANSLATIONAL SCIENCE VOL. 5, NO. 11, 2020 MicroRNA in Pulmonary Arterial Hypertension NOVEMBER 2020:1149–62 1156 (Figure 1). Chen et al. (91) found an elevation of miR29 in lung tissue among patients with hereditary PAH, with a sex-specific interaction of miR-29 with estrogen metabolism. Estrogen exposure led to significant reductions in PPARgand CD36 via the upregulation of miR-29 in Bmpr2 transgenic mice lungs (91), which suggested a correlation between energy metabolism and female hormone signaling, with a predisposition to PAH. Regarding PASMCs, TGFb1/SMAD3signaling negatively regulated the expression of miR-29b and promoted collagen synthesis in an MCT rat model (45). MiR-29b treatment suppressed collagen synthesis by directly targeting collagen I and blocking PI3K/AKT signaling (45). Activation of PAAFs also played a role in the pulmonary vascular remodeling of a chronic hypoxia rat model through a drastic decrease of miR-29a, and it induced the expression of a -smooth muscle actin ( a -SMA) and extracellular matrix collagen. In contrast, a miR-29a mimic repressed the proliferation, migration, and secretion of PAAFs induced by hypoxia and ameliorated pulmonary vascular remodeling (44). miR-29 seemed to be FIGURE 2 Networks Representing Known Interactions Networks representing known interactions involving (A) miR-29, (B) miR-124, (C) miR-140, and (D) miR-204 related to PAH. The green arrows depict activation, whereas the red lines show inhibition. PAAF ¼pulmonary arterial adventitial fibroblasts; PAEC ¼pulmonary artery endothelial cell; other abbreviations as in Figure 1. JACC: BASIC TO TRANSLATIONAL SCIENCE VOL. 5, NO. 11, 2020 Santos-Ferreira et al. NOVEMBER 2020:1149–62 MicroRNA in Pulmonary Arterial Hypertension 1157