TY - JOUR
AU - Girão, Henrique
AB - Abstract Aims MicroRNAs (miRNAs) have been implicated in the pathogenesis of pulmonary hypertension (PH), a multifactorial and progressive condition associated with an increased afterload of the right ventricle leading to heart failure and death. The main aim of this study was to correlate the levels of miR-424(322) with the severity and prognosis of PH and with right ventricle hypertrophy progression. Additionally, we intended to evaluate the mechanisms and signalling pathways whereby miR-424(322) secreted by pulmonary arterial endothelial cells (PAECs) impacts cardiomyocytes. Methods and results Using quantitative real-time PCR, we showed that the levels of circulating miR-424(322) are higher in PH patients when compared with healthy subjects. Moreover, we found that miR-424(322) levels correlated with more severe symptoms and haemodynamics. In the subgroup of Eisenmenger syndrome patients, miR-424(322) displayed independent prognostic value. Furthermore, we demonstrated that miR-424(322) targets SMURF1, through which it sustains bone morphogenetic protein receptor 2 signalling. Moreover, we showed that hypoxia induces the secretion of miR-424(322) by PAECs, which after being taken up by cardiomyocytes leads to down-regulation of SMURF1. In the monocrotaline rat model of PH, we found an association between circulating miR-424(322) levels and the stage of right ventricle hypertrophy, as well as an inverse correlation between miR-424(322) and SMURF1 levels in the hypertrophied right ventricle. Conclusions This study shows that miR-424(322) has diagnostic and prognostic value in PH patients, correlating with markers of disease severity. Additionally, miR-424(322) can target proteins with a direct effect on heart function, suggesting that this miRNA can act as a messenger linking pulmonary vascular disease and right ventricle hypertrophy. Pulmonary hypertension, Pulmonary arterial hypertension, Biomarkers, miR-424(322), SMURF1, Right ventricle hypertrophy 1. Introduction Pulmonary arterial hypertension (PAH) is a multifactorial pathophysiological condition1,2 characterized by the progressive increase of right ventricular (RV) afterload, which ultimately leads to heart failure and death. Despite important therapeutic advances, the prognosis of PAH remains poor,2 which is mainly related to the lack of effective diagnostic modalities that would enable an earlier detection.3 The heritable form of PAH is commonly associated with mutations in the bone morphogenetic protein receptor type 2 (BMPR2) gene.1 A finely tuned regulation of the BMPR2 pathway is needed for inhibiting the proliferation of pulmonary arterial smooth muscle cells (PASMCs) and to promote the survival of pulmonary arterial endothelial cells (PAECs).4 BMPR2 intracellular signalling is initiated when bone morphogenetic proteins (BMPs) bind to BMPR2, leading to phosphorylation of a set of cytoplasmic proteins known as r-SMAD1/5/8. R-SMADs bind to the cofactor SMAD4 and translocate to the nucleus, where they modulate the activity of genes involved in cell growth, proliferation, and differentiation.5 This pathway can be shut down by the proteasomal degradation of r-SMADs following their ubiquitination by the SMAD ubiquitination regulatory factor 1 (SMURF1).6 Unsurprisingly, impairment of the BMPR2 pathway results in PAECs dysfunction and PASMCs proliferation, which contribute to the obliteration of the pulmonary arteries and increased RV afterload.7 In an attempt to overcome this effect, the RV develops hypertrophy (RVH), a cardinal feature of PAH.8 RVH is initially adaptive (aRVH) but eventually progresses to a maladaptive phenotype (mRVH), clinically reflected by the impairment of functional capacity as measured by the World Health Organization (WHO) classification.8,9 In the last few years, microRNAs (miRNAs) have emerged as promising biomarkers and therapeutic targets for many diseases.10,11 In PAH, deregulation of circulating miRNAs is correlated with the onset and development of the disease.11 It was previously reported that the miR-424(322)12 is a key up-regulator of hypoxia inducible factor-1α (HIF-1α), the primary hypoxia-driven signalling pathway of the pulmonary vasculature.13,14 Hence, it is conceivable that an increase in miR-424(322) underlies HIF-1α up-regulation, a phenomenon observed in most forms of PAH.15 Moreover, overexpression of miR-424(322) in PAECs has been shown to promote a quiescent state and to inhibit the proliferation of PAMSCs, therefore ameliorating experimental PAH.16 Based on these studies, it might be suggested that miR-424(322) can constitute not only a biomarker for PAH but also a valuable therapeutic target. Since miRNAs can be detected extracellularly, its profile in biological fluids can reflect patterns of tissue expression.17 Moreover, miRNAs can circulate enclosed in extracellular vesicles (EVs), including exosomes (EXO),18 and EXO-mediated miRNA transfer between cells has been proposed as a mechanism for intercellular signalling. In the present study, we assessed the potential diagnostic and prognostic properties of circulating miR-424(322). Additionally, we investigated the mechanisms whereby miR-424(322) can modulate the BMPR2 pathway and contribute to RVH. 2. Methods Please see Supplementary material online for a detailed description of the methods. 2.1 Human plasma samples We conducted a multicentre, prospective cohort study including 88 patients and 34 healthy participants. Plasma samples were obtained according to the Declaration of Helsinki (2008), with local research ethical committee approval (#CE 035/2011) and written informed consent from all subjects. 2.2 PH rat model PH was induced in Wistar rats by a single intraperitoneal injection of monocrotaline (MCT).19 Animals were handled according to European Union guidelines (2010/63/EU) and were approved by ORBEA-IBILI (permit 09/2015). 2.3 Cell and organotypic cultures HEK293A cells, H9c2 rat cardiomyoblast cells,20 Human (H) PAECs, and HPASMCs were cultured according to the manufacturer’s recommendations. Primary cultures of neonatal rat ventricular myocytes21 (NRVMs) and organotypic cultures22 were obtained from Wistar neonatal rats following the protocol described in the Supplementary methods. 2.4 3′ UTR luciferase reporter assay HEK293A cells were co-transfected with the reporter vector (pMir-REPORT-SMURF1WT or pMir-REPORT-SMURF1MUT) and miR-424(322) or miR Control (miR-NC) using Lipofectamine 2000, according to the manufacturer‘s recommendations. 2.5 Statistical analysis The data were processed using GraphPad Prism 6 for Windows, version 6.01 (GraphPad Software, Fay Avenue, La Jolla, CA, USA) and STATA 12.0 (Lakeway Drive, College Station, TX, USA). 3. Results 3.1 Circulating miR-424(322) levels as a diagnostic marker for PH To evaluate the association between the levels of circulating miR-424(322) and PH, a total of 88 PH patients and 34 healthy subjects were enrolled (Table 1). The data presented in Figure 1A show that miR-424(322) levels were significantly elevated in the global cohort of PH patients and in the subgroup of PAH patients compared with healthy subjects (Figure 1B). As miR-424(322) is known to be positively correlated with hypoxia, we hypothesized that miR-424(322) levels would vary among the several PAH subgroups, characterized by different levels of oxygen saturation. Only congenital heart disease–associated PAH (PAH-CHD) patients had significantly higher levels of miR-424(322) compared with healthy subjects (Figure 1C). No association was detected between circulating miR levels and gender, age, and treatment (see Supplementary material online). A receiver operating characteristics curve (ROC) analysis was also performed to evaluate the potential of miR-424(322) as a diagnostic marker for PH or PAH, demonstrating high discriminative power for both conditions (Figure 1D and E). Table 1 Characteristics and clinical information of participants with pulmonary hypertension and controls   PAH*  IPAH HPAH DPAH  PAH-CTD  PAH-CHD  PoPH  CTEPH  Healthy controls  Patients, n  64  14  15  32  3  24  34  Age, years  47 (34–58)  54 (40–67)  59 (51–71)  37 (31–47)  52 (31–67)  60 (49–73)  69 (64–75)  Female sex, n (%)  46 (72)  12 (80)  12 (80)  19 (59)  3 (100)  14 (58)  19 (52)  WHO class, n (%)                 I–II  27 (41)  5 (36)  5 (33)  16 (50)  1 (33)  13 (53)     III–IV  37 (59)  9 (64)  10 (67)  16 (50)  2 (67)  11 (46)    6MWD, metres  426 (129)  433 (160)  361 (140)  450 (106)  425 (417–500)  419 (103)    Creatinine, mg/dL  0.93 (0.63)  0.84 (0.18)  1.32 (1.16)  0.83 (0.29)  0.6 (0.12)  0.89 (0.18)    Mean PAP, mm Hg  60 (20)  56 (16)  46 (15)  73 (18)  36 (3)  47 (17)    PCWP, mm Hg  11 (8–13)  10 (8–13)  9 (7–13)  12 (7–14)  13 (12–13)  11 (7–13)    mRAP, mm Hg  8 (5–10)  8 (7–10)  7 (3–9)  8 (6–12)  5 (4–8)  7 (5–10)    CO, L.min−1  4.1 (1.17)  3.7 (1.2)  3.6 (0.9)  4.4 (1.2)  5.2 (0.8)  3.91 (1.25)    PVR, WU  12.6 (6.9)  11.0 (5.8)  10.4 (6.4)  15.8 (6.9)  4.6 (1.2)  10.5 (5.9)    PAH therapy, n (%)                 None  6 (10)  0 (0)  2 (15)  5 (16)  0 (0)  9 (39)     PDE5 inhibitor  25 (43)  12 (86)  6 (46)  7 (22)  2 (67)  8 (35)     ETR antagonist  49 (84)  14 (100)  10 (77)  27 (84)  2 (67)  7 (30)     Prostacyclin analogue  16 (28)  9 (64)  3 (23)  5 (16)  0 (0)  2 (9)     Riociguat  2 (3)  1 (7)  0 (0)  0 (0)  1 (33)  1 (4)     Combo therapy  38 (55)  13 (93)  8 (54)  8 (25)  2 (67)  13 (52)      PAH*  IPAH HPAH DPAH  PAH-CTD  PAH-CHD  PoPH  CTEPH  Healthy controls  Patients, n  64  14  15  32  3  24  34  Age, years  47 (34–58)  54 (40–67)  59 (51–71)  37 (31–47)  52 (31–67)  60 (49–73)  69 (64–75)  Female sex, n (%)  46 (72)  12 (80)  12 (80)  19 (59)  3 (100)  14 (58)  19 (52)  WHO class, n (%)                 I–II  27 (41)  5 (36)  5 (33)  16 (50)  1 (33)  13 (53)     III–IV  37 (59)  9 (64)  10 (67)  16 (50)  2 (67)  11 (46)    6MWD, metres  426 (129)  433 (160)  361 (140)  450 (106)  425 (417–500)  419 (103)    Creatinine, mg/dL  0.93 (0.63)  0.84 (0.18)  1.32 (1.16)  0.83 (0.29)  0.6 (0.12)  0.89 (0.18)    Mean PAP, mm Hg  60 (20)  56 (16)  46 (15)  73 (18)  36 (3)  47 (17)    PCWP, mm Hg  11 (8–13)  10 (8–13)  9 (7–13)  12 (7–14)  13 (12–13)  11 (7–13)    mRAP, mm Hg  8 (5–10)  8 (7–10)  7 (3–9)  8 (6–12)  5 (4–8)  7 (5–10)    CO, L.min−1  4.1 (1.17)  3.7 (1.2)  3.6 (0.9)  4.4 (1.2)  5.2 (0.8)  3.91 (1.25)    PVR, WU  12.6 (6.9)  11.0 (5.8)  10.4 (6.4)  15.8 (6.9)  4.6 (1.2)  10.5 (5.9)    PAH therapy, n (%)                 None  6 (10)  0 (0)  2 (15)  5 (16)  0 (0)  9 (39)     PDE5 inhibitor  25 (43)  12 (86)  6 (46)  7 (22)  2 (67)  8 (35)     ETR antagonist  49 (84)  14 (100)  10 (77)  27 (84)  2 (67)  7 (30)     Prostacyclin analogue  16 (28)  9 (64)  3 (23)  5 (16)  0 (0)  2 (9)     Riociguat  2 (3)  1 (7)  0 (0)  0 (0)  1 (33)  1 (4)     Combo therapy  38 (55)  13 (93)  8 (54)  8 (25)  2 (67)  13 (52)    Data are presented as mean (SD) or median (interquartile range), unless otherwise stated. 6MWD denotes six-minute walking distance; BNP, B-type natriuretic peptide; PAH-CHD, congenital heart disease-PAH; CO, cardiac output, Combo, combination; PAH-CTD, connective tissue disorder associated-PAH; CTEPH, chronic thromboembolic pulmonary hypertension; DPAH, Drug induced-PAH; ETR, endothelin receptor; HPAH, heritable PAH; IPAH, idiopathic PAH; mRAP, mean right atrial pressure; PAP, pulmonary artery pressure; PCWP, pulmonary capillary wedge pressure; PDE5, phosphodiesterase-5; PoPH, portopulmonary hypertension; PVR, pulmonary vascular resistance; WHO, World Health Organization; WU, Wood units. *Includes IPAH, HPAH, PAH-CTD, CHD-PAH, and PoPH. Figure 1 View largeDownload slide Circulating levels of miR-424(322) are increased in PH patients. (A) Levels of miR-424(322) in PH patients were significantly higher compared with healthy subjects. (B) PAH patients displayed significantly higher levels of miR-424(322) than CTR, whereas CTEPH patients had no significant increase in miR-424(322) levels. (C) Among PAH patients, PAH-CHD patients had significantly higher levels of miR-424(322). (D) ROC curve analysis of miR-424(322) showed strong discrimination between the PH group and CTR. (E) PAH patients showed a better discrimination in this subgroup relative to CTR. MiR-424(322) levels were measured by qRT–PCR and expressed as logarithmic values of 2–ΔCt using a synthetic RNA spike as a normalizer in plasma samples from CTR and from patients with various forms of PH. Two-tailed Student‘s t-test. ***P < 0.001 relative to CTR. Healthy subjects: CTR (n = 34), PH: pulmonary hypertension (n = 88). IPAH: idiopathic pulmonary arterial hypertension; HPAH: heritable PAH; DPAH: drug-related PAH (n = 14); PAH-CTD: PAH associated with connective tissue disease (n = 15); PAH-CHD: PAH associated with congenital heart disease (n = 32); CTEPH: chronic thromboembolic pulmonary hypertension (n = 24), PoPH: portopulmonary hypertension (n = 3). Figure 1 View largeDownload slide Circulating levels of miR-424(322) are increased in PH patients. (A) Levels of miR-424(322) in PH patients were significantly higher compared with healthy subjects. (B) PAH patients displayed significantly higher levels of miR-424(322) than CTR, whereas CTEPH patients had no significant increase in miR-424(322) levels. (C) Among PAH patients, PAH-CHD patients had significantly higher levels of miR-424(322). (D) ROC curve analysis of miR-424(322) showed strong discrimination between the PH group and CTR. (E) PAH patients showed a better discrimination in this subgroup relative to CTR. MiR-424(322) levels were measured by qRT–PCR and expressed as logarithmic values of 2–ΔCt using a synthetic RNA spike as a normalizer in plasma samples from CTR and from patients with various forms of PH. Two-tailed Student‘s t-test. ***P < 0.001 relative to CTR. Healthy subjects: CTR (n = 34), PH: pulmonary hypertension (n = 88). IPAH: idiopathic pulmonary arterial hypertension; HPAH: heritable PAH; DPAH: drug-related PAH (n = 14); PAH-CTD: PAH associated with connective tissue disease (n = 15); PAH-CHD: PAH associated with congenital heart disease (n = 32); CTEPH: chronic thromboembolic pulmonary hypertension (n = 24), PoPH: portopulmonary hypertension (n = 3). 3.2 Circulating miR-424 levels as markers of prognosis and disease severity Since PH impairs RV function, elevated levels of miR-424(322) might plausibly be correlated with heart disease severity. To address this question, we correlated the levels of miR-424(322) with well-established prognostic markers, including the WHO functional class and cardiac output.1 This analysis demonstrated that better functional classes (WHO I–II) were significantly associated with lower levels of miR-424(322) compared with more advanced classes (WHO III–IV) (Figure 2A). Importantly, the levels of miR-424(322) could also be inversely correlated with cardiac output (Figure 2B). Given the long-term adaptation of the RV to the elevated afterload, the prognosis of patients with PH is particularly difficult to assess. Therefore, we investigated whether miR-424(322) could be used as a prognostic marker. Survival analysis showed no interaction in PH patients between miR levels and the primary endpoint incidence (Figure 2C). However, in the PAH-CHD subgroup, higher levels of miR-424(322) were predictive of event-free survival (Figure 2D). This association was independent after adjustment for potential confounders (Table 2), suggesting a unique prognostic value for miR-424(322) in PAH-CHD patients. Table 2 Cox survival analysis of the PAH-CHD cohort (primary endpoint: combined all-cause death, hospital admission for decompensated heart failure and emergency rooms visits for cardiovascular symptoms) Variable  Univariate model   Multivariate model   Hazard ratio (95% CI)  P-value  Hazard ratio (95% CI)  P-value  Age, per year  1.018 (0.944–1.097)  0.644      Female gender  0.093 (0.011–0.775)  0.028  0.201 (0.019–2.111)  0.182  WHO class, I–II vs. III–IV  1.451 (0.324–6.496)  0.627  1.957 (0.077–49.82)  0.684  mPAP, mm Hg  0.997 (0.947–1.051)  0.936      PCW, per mm Hg  0.986 (0.821–1.187)  0.894      mRAP, per mm Hg  0.909 (0.780–1.247)  0.909      CO, per L/min  0.899 (0.441–1.831)  0.899      PVR, per WU  1.023 (0.897–1.167)  0.734      6MWT, per m  0.998 (0.991–1.005)  0.686  1.007 (0.990–1.024)  0.447  Creatinine, per mg/mL  1.045 (1.011–1.079)  0.008  1.040 (0.999–1.102)  0.055  miR-424(322)−0.5, per unit  1.003 (0.999–1.007)  0.054  1.001 (1.001–1.008)  0.021  Variable  Univariate model   Multivariate model   Hazard ratio (95% CI)  P-value  Hazard ratio (95% CI)  P-value  Age, per year  1.018 (0.944–1.097)  0.644      Female gender  0.093 (0.011–0.775)  0.028  0.201 (0.019–2.111)  0.182  WHO class, I–II vs. III–IV  1.451 (0.324–6.496)  0.627  1.957 (0.077–49.82)  0.684  mPAP, mm Hg  0.997 (0.947–1.051)  0.936      PCW, per mm Hg  0.986 (0.821–1.187)  0.894      mRAP, per mm Hg  0.909 (0.780–1.247)  0.909      CO, per L/min  0.899 (0.441–1.831)  0.899      PVR, per WU  1.023 (0.897–1.167)  0.734      6MWT, per m  0.998 (0.991–1.005)  0.686  1.007 (0.990–1.024)  0.447  Creatinine, per mg/mL  1.045 (1.011–1.079)  0.008  1.040 (0.999–1.102)  0.055  miR-424(322)−0.5, per unit  1.003 (0.999–1.007)  0.054  1.001 (1.001–1.008)  0.021  6MWD denotes six-minute walking distance, a.u., arbitrary unit; CI, confidence interval; CO, cardiac output; mPAP, mean pulmonary artery pressure; mRAP, mean right atrial pressure; PAH-CHD, pulmonary arterial hypertension associated with congenital heart disease; PCWP, pulmonary capillary wedge pressure; PVR, pulmonary vascular resistance; WU, Wood units. Firstly, the prognostic potential of each individual variable was investigated by univariate Cox regression survival analysis. Secondly, gender, six-minute walking distance, creatinine levels, and transformed miR-424(322) values were analysed by multivariate Cox regression survival analysis to identify independent prognostic factors. Hazard ratios are for unit change per year. Figure 2 View largeDownload slide Circulating miR-424(322) levels have diagnostic and prognostic value. (A) Patients in WHO class III-IV (n = 48) had significantly higher levels of miR-424(322) compared with WHO class I-II (n = 40). Two-tailed Student‘s t-test. *P < 0.05 relative to WHO I-II. (B) Higher levels of miR-424(322) were significantly associated with lower cardiac output. (C) Kaplan–Meier survival curves showed no interaction between miR-424(322) levels and a combined endpoint of combined all-cause death, hospital admission for decompensated heart failure and emergency rooms visits. PH patients were stratified by the miR-424(322) best cutoff value derived from an ROC curve. Patients above the cutoff are indicated by the blue line, and patients below the cutoff by the red line. (D) In the PAH-CHD subgroup, patients with higher levels of miR-424(322) had better event-free survival. PH: pulmonary hypertension (n = 88); PAH-CHD: PAH associated with congenital heart disease (n = 32). Figure 2 View largeDownload slide Circulating miR-424(322) levels have diagnostic and prognostic value. (A) Patients in WHO class III-IV (n = 48) had significantly higher levels of miR-424(322) compared with WHO class I-II (n = 40). Two-tailed Student‘s t-test. *P < 0.05 relative to WHO I-II. (B) Higher levels of miR-424(322) were significantly associated with lower cardiac output. (C) Kaplan–Meier survival curves showed no interaction between miR-424(322) levels and a combined endpoint of combined all-cause death, hospital admission for decompensated heart failure and emergency rooms visits. PH patients were stratified by the miR-424(322) best cutoff value derived from an ROC curve. Patients above the cutoff are indicated by the blue line, and patients below the cutoff by the red line. (D) In the PAH-CHD subgroup, patients with higher levels of miR-424(322) had better event-free survival. PH: pulmonary hypertension (n = 88); PAH-CHD: PAH associated with congenital heart disease (n = 32). 3.3 SMURF1 as a miR-424(322) target Once we established the clinical relevance of miR-424(322), we used bioinformatic tools to identify potential targets of miR-424(322) that could mediate the effect of this miR upon PAH onset and progression. The in silico approach identified SMURF1, a key player in the maintenance of pulmonary vascular homeostasis,23 as a putative target of miR-424(322) (see Supplementary material online). To validate SMURF1 as a direct target of miR-424(322), we performed a luciferase reporter gene assay. After identifying miR-424(322) binding sites in the 3′ UTR of SMURF1 gene, wild-type and a mutant 3′ UTR were cloned into the pMIR-REPORTER vector after which the target gene transcripts were assessed by luciferase activity. Co-transfection of HEK293A cells with miR-424(322) and a luciferase construct containing the putative miR-424(322) binding sequences within the 3′ UTR of SMURF1 resulted in a significant decrease of luciferase activity (Figure 3B). On the other hand, mutation of the predicted miR-424(322) binding site abolished the effect of miR-424(322) on luciferase activity (Figure 3B). These results show that miR-424(322) binds to SMURF1 3′ UTR, thus leading to the down-regulation of the reporter gene. To further evaluate whether miR-424(322) mediates the down-regulation of SMURF1 in vivo, we assessed the levels of endogenous SMURF1 in HEK293A cells after transfection with miR-424(322). In agreement with the luciferase reporter assay, miR-424(322)-transfected cells showed a 50% reduction in SMURF1 levels, when compared with cells transfected with non-targeting miRNA (Figure 3C). Cells transfected with esiRNA directed against SMURF1 were used as positive controls for protein down-regulation (Figure 3C). Figure 3 View largeDownload slide MiR-424(322) directly targets SMURF1. (A) Diagram of the miR-424(322) binding sites in the 3′ UTR of the SMURF1 gene. The upper sequence shows the predicted miR-424(322) binding sequence within the SMURF1 wild-type (WT) 3′ UTR and the lower sequence shows the mutated form (MUT). (B) Luciferase activity significantly decreased in the presence of luciferase reporter constructs containing the WT 3′ UTR of the SMURF1 and miR-424(322). Two-way ANOVA, Tukey's multiple comparisons test. ***P < 0.001 relative to miR-NC; n = 6. (C) miR-424(322) reduced SMURF1 levels. HEK293A cells were transfected with miR-424(322) or esiRNA-SMURF1 for 48 h. Protein levels of SMURF1 were determined by western blotting and were normalized to GAPDH. One-way ANOVA, Tukey's multiple comparisons test. ***P < 0.001 relative to CTR; n = 6. (D) HIF-1α increased under hypoxia conditions. HPAECs or HPASMCs were subjected to hypoxia and normoxia during 72 h, and HIF-1α levels were evaluated by western blot (n = 6). (E) Hypoxia-conditioned medium (CMH) from HPAECs displayed significantly higher levels of miR-424(322) compared with normoxia-conditioned medium (CMN). CMH from HPASMCs displayed no significant differences from CMN. The CMH was collected from HPAECs or HPASMCs and the relative levels of miR-424(322) were assessed by qRT–PCR. Two-tailed Student‘s t-test. ***P < 0.001 relative to CMN; n = 6. (F) CMH led to a reduction of SMURF1 levels with CMN. SMURF1 levels were evaluated by western blot and were normalized to GAPDH. Two-way ANOVA, Tukey's multiple comparisons test. ***P < 0.001 relative to CMN, n = 6. (G) miR-424(322) leads to reduction of SMURF1 levels in organotypic cultures. Heart organotypic cultures was injected with either miR424(322) or miR-NC. SMURF1 levels were evaluated by western blot and was normalized to GAPDH. Two-tailed Student‘s t-test. ****P < 0.0001 relative to miR-NC, n = 6. Figure 3 View largeDownload slide MiR-424(322) directly targets SMURF1. (A) Diagram of the miR-424(322) binding sites in the 3′ UTR of the SMURF1 gene. The upper sequence shows the predicted miR-424(322) binding sequence within the SMURF1 wild-type (WT) 3′ UTR and the lower sequence shows the mutated form (MUT). (B) Luciferase activity significantly decreased in the presence of luciferase reporter constructs containing the WT 3′ UTR of the SMURF1 and miR-424(322). Two-way ANOVA, Tukey's multiple comparisons test. ***P < 0.001 relative to miR-NC; n = 6. (C) miR-424(322) reduced SMURF1 levels. HEK293A cells were transfected with miR-424(322) or esiRNA-SMURF1 for 48 h. Protein levels of SMURF1 were determined by western blotting and were normalized to GAPDH. One-way ANOVA, Tukey's multiple comparisons test. ***P < 0.001 relative to CTR; n = 6. (D) HIF-1α increased under hypoxia conditions. HPAECs or HPASMCs were subjected to hypoxia and normoxia during 72 h, and HIF-1α levels were evaluated by western blot (n = 6). (E) Hypoxia-conditioned medium (CMH) from HPAECs displayed significantly higher levels of miR-424(322) compared with normoxia-conditioned medium (CMN). CMH from HPASMCs displayed no significant differences from CMN. The CMH was collected from HPAECs or HPASMCs and the relative levels of miR-424(322) were assessed by qRT–PCR. Two-tailed Student‘s t-test. ***P < 0.001 relative to CMN; n = 6. (F) CMH led to a reduction of SMURF1 levels with CMN. SMURF1 levels were evaluated by western blot and were normalized to GAPDH. Two-way ANOVA, Tukey's multiple comparisons test. ***P < 0.001 relative to CMN, n = 6. (G) miR-424(322) leads to reduction of SMURF1 levels in organotypic cultures. Heart organotypic cultures was injected with either miR424(322) or miR-NC. SMURF1 levels were evaluated by western blot and was normalized to GAPDH. Two-tailed Student‘s t-test. ****P < 0.0001 relative to miR-NC, n = 6. Based on our data demonstrating that miR-424(322) displayed the most striking increase and prognostic impact in the subgroup of PAH-CHD patients (Figure 1C) and also on the fact that hypoxic endothelial cells up-regulate miR-424(322),23 we hypothesized that PAECs under hypoxia secrete miR-424(322) that is taken up by cardiomyocytes, where it induces a reduction in SMURF1 levels. To test this hypothesis, we first evaluated the effect of hypoxia on the amount of miR-424(322) secreted to the extracellular medium. The results obtained showed that after 72 h of hypoxia, the levels of miR-424(322) in the medium increased more than 50% (Figure 3E) compared with HPAECs maintained in normoxic conditions. However, the hypoxia-induced increased secretion of miR-424(322) was not observed in HPASMCs, suggesting that when arteries are subjected to hypoxia the endothelial cells are the main source of miR-424(322) (Figure 3E). The accumulation of HIF-1α was used as a positive control of hypoxia (Figure 3D). Afterwards, we evaluated the effect of HPAECs-derived hypoxia-conditioned medium on cardiomyocytes. In accordance to our model, conditioned medium from hypoxic HPAECs led to a down-regulation of SMURF1 protein levels both in NRVMs and H9c2 cells (Figure 3F). The reduction of SMURF1 levels was also observed in organotypic cultures of neonatal rat hearts injected with miR-424(322), demonstrating that miR-424(322) targets SMURF1 in heart tissue (Figure 3G). One of the putative mechanisms whereby miR-424(322) can be secreted is through EVs. To address this question, we isolated EXOs secreted by HPAECs cultured either in normoxic (EXON) or hypoxic conditions (EXOH), and in human plasma samples from PAH patients. The presence of proteins commonly used as EXO markers, vesicle size, and morphology analysis by transmission electron microscope (TEM) demonstrated that our vesicle extract was enriched in EVs (see Supplementary material online). The absence of calnexin revealed that the extract was devoid of cell debris (see Supplementary material online). We then evaluated the relative amount of miR-424(322) that is encapsulated in EXO compared with total miR-424(322). The results showed that about 50% of circulating miR-424(322) in the plasma was encapsulated in EXOs (see Supplementary material online), whereas in HPAECs almost 80% of miR-424(322) secreted into extracellular medium was detected in EXOs (see Supplementary material online). The presence of green puncta in the cytosol of H9c2 cells incubated with EXOs produced by HPAECs overexpressing CD63-GFP demonstrated that EXO were being taken up by cardiac cells (see Supplementary material online). Altogether, the data strongly suggest that EXOs produced by PAECs, likely carrying miR-424(322), can be internalized by cardiomyocytes. 3.4 MiR-424(322) modulates the BMPR2 pathway Since it has been reported that SMURF1-mediated impairment of BMPR2 signalling pathway is associated with pulmonary vascular disease,24 we proceeded to evaluate whether miR-424(322) could mediate the down-regulation of SMURF1, with the consequent increase in BMPR2 pathway activity. As expected, the presence of miR-424(322) led to a decrease in the amount of SMURF1, with a consequent increase in SMAD5 (Figure 4A). Furthermore, we assessed the effect of miR-424(322) on BMPR2 pathway activity by measuring the levels of phosphorylated SMAD5 (p-SMAD5) in cells incubated either in the presence or absence of BMP4, a BMPR2 agonist.5 The results showed that BMP4 induced the phosphorylation of SMAD5, this effect being exacerbated in the presence of miR-424(322) (Figure 4B). These results were further confirmed in cells transfected with esiRNA directed against SMURF1 (Figure 4B). Overall, these data support our hypothesis that miR-424(322) has a functional impact on the BMPR2 pathway, through the modulation of SMURF1 levels (Figure 4). Next, we evaluated the effect of hypoxia-conditioned medium from HPAECs on the BMPR2 pathway. We showed that incubation of NRVMs and H9c2 cells with conditioned medium from HPAECs subject to hypoxia when compared with conditioned medium from HPAECs in normoxia resulted in a reduction of SMURF1 (Figure 3F) with a concomitant increase in SMAD5 levels (Figure 4C). These observations demonstrate that miR-424(322) secreted by HPAECs can target SMURF1 in cardiomyocytes. Figure 4 View largeDownload slide MiR-424(322) modulates BMPR2 signalling. (A) miR-424(322) increased SMAD5 levels. HEK293A cells were transfected with miR-424(322) or esiRNA SMURF1 for 48 h. SMAD5 levels were determined by western blotting and were normalized to GAPDH. One-way ANOVA, Turkey's multiple comparisons test. **P < 0.01 relative to CTR (B) miR-424(322) increased p-SMAD5 levels. HEK293A cells were transfected with miR-424(322) or esiRNA SMURF1 for 48 h, and treated with BMP4. p-SMAD5 levels were determined by western blotting and was normalized to GAPDH. Two-way ANOVA, Tukey's multiple comparisons test. **P < 0.01 and ***P < 0.001 relative to CTR; n = 6. (C) CMH led to an increase of SMAD5 levels. H9c2 cells or NRVMs were treated 48 h with either CMH or CMN collected from HPAECs. SMAD5 levels were determined by western blotting and were normalized to GAPDH. Two-way ANOVA, Tukey's multiple comparisons test. ***P < 0.001 relative to CMN; n = 6. Figure 4 View largeDownload slide MiR-424(322) modulates BMPR2 signalling. (A) miR-424(322) increased SMAD5 levels. HEK293A cells were transfected with miR-424(322) or esiRNA SMURF1 for 48 h. SMAD5 levels were determined by western blotting and were normalized to GAPDH. One-way ANOVA, Turkey's multiple comparisons test. **P < 0.01 relative to CTR (B) miR-424(322) increased p-SMAD5 levels. HEK293A cells were transfected with miR-424(322) or esiRNA SMURF1 for 48 h, and treated with BMP4. p-SMAD5 levels were determined by western blotting and was normalized to GAPDH. Two-way ANOVA, Tukey's multiple comparisons test. **P < 0.01 and ***P < 0.001 relative to CTR; n = 6. (C) CMH led to an increase of SMAD5 levels. H9c2 cells or NRVMs were treated 48 h with either CMH or CMN collected from HPAECs. SMAD5 levels were determined by western blotting and were normalized to GAPDH. Two-way ANOVA, Tukey's multiple comparisons test. ***P < 0.001 relative to CMN; n = 6. 3.5 MiR-424(322) modulates SMURF1 in the hypertrophied RV of the MCT-treated rat MiR-424(322) has been previously associated with cardiac hypertrophy, either in engineered heart tissue from neonatal rats25 or in models of left ventricular (LV) hypertrophy.26 Although our results demonstrate that miR-424(322) levels correlate with disease severity in humans, the patients were heterogeneous regarding the stage of disease. It is conceivable that the levels of miR-424(322) vary during the course of the disease, a phenomenon difficult to capture in a human PH cohort. To investigate the association between miR-424(322) and RVH, we used the established MCT rat model of PH, which can partially reproduce the human phenotype of RVH progression, including the transition from an adaptive to a maladaptive phase.9 Our results show that MCT-treated animals presented cardinal features of mRVH, such as an increase in RV systolic pressure, an elevated RV/(LV + interventricular septum) ratio, and cavity dilation and decreased contractility, as shown by a reduced tricuspid annular plane systolic excursion on echocardiography (see Supplementary material online). These functional studies were corroborated with ultrastructural analysis, in which after 6 weeks of MCT treatment the animals showed marked structural changes in the RV, manifested by severe myofibrillar disarray, fibrosis, and mitochondrial degeneration (Figure 5A). To investigate the correlation between miR-424(322) levels and RVH progression, we began by evaluating the rat plasma levels of miR-424(322) along the progression of RVH. In Figure 5B, we show that miR-424(322) was increased two-fold 1 week after MCT injection and decreased after 2 weeks, after which it increased during the late stages of mRVH. Strikingly, and in agreement with the hypothesis that SMURF1 is a target of miR-424(322) in cardiomyocytes, we found an inverse correlation between the levels of circulating miR-424(322) and the amount of SMURF1 detected in the RV (Figure 5B and C). The levels of SMAD5 on RV inversely correlated with SMURF1 levels suggesting that circulating miR-424(322) levels can mirror disease-associated changes in RV (Figure 5D). Figure 5 View largeDownload slide SMURF1 and miR-424(322) levels are inversely modulated along with progression of RV hypertrophy. (A) Ultrastructural analysis showed marked structural changes in the RV, manifested by severe myofibrillar disarray, fibrosis (arrowhead) and mitochondrial degeneration (arrow) after 6 weeks of MCT treatment. (B) Plasmatic miR-424(322) levels increase with MCT treatment. MiR-424(322) levels were increased 1 week after MCT injection and decreased after 2 weeks, after which they increased again in the late stages of RVH. One-way ANOVA, Tukey's multiple comparisons test. **P < 0.01 relative to CTR; ##P < 0.01 relative to 6 weeks (n = 10). (C) SMURF1 levels in RV follow an inverse pattern of those of plasmatic miR-424(322) levels. One-way ANOVA, Tukey's multiple comparisons test. ***P < 0.001 relative to CTR; *P < 0.05 relative to 1 weeks; ##P < 0.01 relative to 2 weeks; (n = 8). (D) SMAD5 levels in RV follow a similar pattern of expression to as plasmatic miR-424(322) levels. One-way ANOVA, Tukey's multiple comparisons test. ****P < 0.0001 relative to CTR (n = 5). Figure 5 View largeDownload slide SMURF1 and miR-424(322) levels are inversely modulated along with progression of RV hypertrophy. (A) Ultrastructural analysis showed marked structural changes in the RV, manifested by severe myofibrillar disarray, fibrosis (arrowhead) and mitochondrial degeneration (arrow) after 6 weeks of MCT treatment. (B) Plasmatic miR-424(322) levels increase with MCT treatment. MiR-424(322) levels were increased 1 week after MCT injection and decreased after 2 weeks, after which they increased again in the late stages of RVH. One-way ANOVA, Tukey's multiple comparisons test. **P < 0.01 relative to CTR; ##P < 0.01 relative to 6 weeks (n = 10). (C) SMURF1 levels in RV follow an inverse pattern of those of plasmatic miR-424(322) levels. One-way ANOVA, Tukey's multiple comparisons test. ***P < 0.001 relative to CTR; *P < 0.05 relative to 1 weeks; ##P < 0.01 relative to 2 weeks; (n = 8). (D) SMAD5 levels in RV follow a similar pattern of expression to as plasmatic miR-424(322) levels. One-way ANOVA, Tukey's multiple comparisons test. ****P < 0.0001 relative to CTR (n = 5). 4. Discussion In this report, we demonstrate for the first time that miR-424(322) has diagnostic and prognostic value in PH patients, particularly in the PAH-CHD cohort. Additionally, we show that miR-424(322) targets SMURF1, leading to an increase in BMPR2 pathway activity. We also reveal that hypoxia induces PAECs to up-regulate and secrete miR-424(322), which are at least partially transported in EXOs, and can be taken up by cardiomyocytes, resulting in down-regulation of SMURF1. PAH-associated hypoxic environment in the lungs leads to secretion of miR-424(322) by PAECs which travel to the heart where it modulates the expression of SMURF1, contributing to RVH and heart failure. Our results support a model in which miR-424(322) acts as a signal to convey information from the lung to the heart. As the levels of circulating miRNAs are strongly correlated with their corresponding levels in the lungs and heart,17,27 it is conceivable that the peripheral miRNA profile mirrors the changes occurring in the tissues affected by PAH, a key aspect to consider a molecule as a biomarker.11 Our study establishes that plasmatic levels of miR-424(322) are elevated in PH patients, externally validating data from a pilot study previously carried out in 14 PH patients.17 miR-424(322) is an endothelial cell-specific miRNA that belongs to the hypoxamir family and is up-regulated during hypoxia. It was shown that not only does hypoxia induce an increase in miR-424(322), which prolongs the HIF-1α response,13,23 but also that increased expression of miR-424(322) in the lungs and PASMCs of rats subjected to hypoxia is mediated by HIF-1α, in a positive feedback loop fashion.14 Therefore, it is plausible that the miR-424(322) increase observed in PAH patients reflects the hypoxic component of the disease. Besides the association between miR-424(322) levels and PH, this study provides the first evidence that higher miR-424(322) levels correlate with disease severity, which might represent a compensatory response of the cardiovascular system to the increased RV afterload. Paradoxically, in PAH-CHD patients, characterized by a very well adapted but severely hypertrophied RV,28,29 higher levels of miR-424(322) were found to be associated with a better prognosis. It is conceivable that in these hypoxic patients, higher levels of miR-424(322) likely represent a truly adaptive and desirable compensatory mechanism to maintain higher levels of HIF-1α, vital to help tissues to cope with hypoxia and to maintain an adaptive RV phenotype.13,14,30,31 Patients with Chuvash polycythemia, caused by a mutation in the von Hippel–Lindau protein gene that impairs HIF-1α degradation, are also characterized by a very well adapted RV with no signs of heart failure.32 Although the antiproliferative properties of miR-424(322) in PASMCs16 and endothelial cells under normoxic conditions have been described,30,33 other studies have reported that miR-424(322) can induce proliferation and migration.13,14 Furthermore, studies carried out in animal models of PH showed that the administration of miR-424(322) led to phenotype rescue.16 These apparent contradictory results suggest a context-dependent mode of action30 of miR-424(322), which might be explained not only by differences in the intracellular levels of the miR but also by the different animal models and time points used to evaluate the association of miR-424(322) with PH. Nevertheless, most,17,16,34 but not all,35,36 studies that have used the well-established MCT rat model have found a down-regulation of miR-424(322) in the lungs. Conversely, in the chronic hypoxia mouse model, there was a consistent increase of miR-424(322),17,34 corroborating the human studies.17 Altogether, these observations demonstrate that PH aetiology is more complex than initially anticipated.17,16,35 It has been shown in different physiological and pathological contexts that miRNAs circulate enclosed in EVs, including EXO.18 Interestingly, the results obtained in this study show that about 50% of the amount of miR-424(322) detected in human plasma samples is packaged in EXOs, whereas in the case of PAECs most of the miR-424(322) secreted is carried in EXOs. This suggests that the source of miR-424(322) detected in circulation is not only from PAECs but can also originate from other cells and tissues. We also provide evidence that EXOs produced by PAECs, likely carrying miR-424(322), are internalized by cardiomyocytes. Since EXOs can constitute a strategy to carry biological material to specific acceptor cells, it is plausible that the encapsulation of miR-424(322) into EXOs is a means to target miR-424(322) produced by PAECs to cardiomyocytes. Genetic or functional abnormalities in the BMPR2 pathway are common in PAH.7 BMPR2 signalling down-regulation can be achieved by SMURF1-associated ubiquitination and degradation of SMAD1/5/8 proteins.6 SMURF1 levels have been reported to be elevated in the pulmonary arteries of animal models of PH and in the plasma of PAH patients, resulting in PASMC proliferation and vessel remodelling.24 Previous studies demonstrated that miR-424(322) regulates fibroblast differentiation during endothelial–mesenchymal transition, through a mechanism based on SMURF2.37 In our study, we demonstrated that miR-424(322) not only targets SMURF1 but also increases the activity of the BMPR2 pathway, a key event in the maintenance of vascular homeostasis.38 Moreover, it is conceivable that increased production and secretion of miR-424(322) by HPAECs under hypoxic conditions might modulate heart response. In agreement, our data demonstrated that conditioned medium from HPAECs had impact on several models of heart cells and tissues, namely a reduction in SMURF1 levels, suggesting that in PAH, miR-424(322) acts as a mediator of the crosstalk between the lungs and the heart, affecting both the HIF-1α and BMPR2 pathways.14 Importantly, in accordance with the literature,39 we show that similar experiments carried out using hypoxic HPASMCs does not lead to increased production of miR-424(322), which suggests that PAECs are the main source of miR-424(322) detected in the circulation of patients. Since prognosis is critically related to RV function,8 it is essential to determine whether the levels of miR-424(322) correlate with RVH progression. We performed a longitudinal analysis of the miRNA changes during the progression of RVH in the MCT rat model and observed that the levels of miR-424(322) increase about two-fold after 1 week of MCT treatment, followed by a drop to baseline after 2 weeks. In accordance, in another study, an increase in lung concentrations of miR-424(322) just 1 week after MCT treatment was found, with levels returning to baseline by 3 weeks.34 We speculate that this biphasic pattern likely reflects a dynamic process associated with the two stages of RVH (aRVH followed by mRVH). The abrupt increase in miR-424(322) during the first week may mimic the onset of the human disease and likely reflect the initial compensatory response to injury,8,40 a stage that most patients with Eisenmenger syndrome probably maintain throughout life.28 In agreement, proliferative insults such as mechanical vascular injury elicit a negative feedback loop that transiently increases the level of miR-424(322), promoting vascular quiescence through the targeting of cyclin D1.40 Therefore, the increase in miR-424(322) might result from an initial insult to the pulmonary vasculature, which can or cannot be maintained depending on each patient’s specific clinical condition. The levels of miR-424(322) are restored to baseline during the second week, perhaps reflecting a ‘honeymoon period’, which is clinically seen in chronic thromboembolic pulmonary hypertension patients.41 This period may correspond to a therapeutic window during which recovery is still possible. However, for sustained periods of afterload mismatch, the disease progresses towards a terminal stage, during which the levels of miR-424(322) progressively increase. This stage is likely to correspond to maladaptive RVH, in which the signalling pathways are severely deregulated and cell homeostasis and function are seriously compromised.9 Consistent with the hypothesis that miR-424(322) targets SMURF1, the amount of the protein in the RV and circulating miRNA levels varied inversely along the course of RVH. Our findings are concordant with those from an engineered model of heart tissue, where tension-induced hypertrophy resulted in up-regulated miR-424(322) levels, mimicking the increase seen in circulating miR-424(322) levels after the RV afterload challenge caused by PH.25 Conversely, pulmonary arterial banding induced SMURF1 up-regulation in the hypertrophied murine RV 1 week after surgery; however, no data are available for later time points.42 An active BMPR2 pathway is necessary for the development of pressure overload hypertrophy,43 and importantly, failure of BMP signalling can increase TGF-β/SMAD2/3 activity, which may be detrimental as it can cause maladaptive hypertrophy, myocardial dysfunction, and fibrosis.4 SMURF1 is a master regulator of both BMPR2 and TGF-β pathways, through the targeting of SMAD1/5/8 and SMAD2/3.38 In accordance, in this study, we demonstrate that in various models of cardiac tissue (H9c2 cells, NRVMs, and organotypic cultures), treatment with either miRNA-424(322)-enriched hypoxia conditioned medium or direct injection of miR-424(322) increased SMAD5.44 Therefore, it is likely that SMURF1 down-regulation in the heart may account for maladaptive hypertrophy and fibrosis, due to unopposed actions of both BMPR2 and TGF-β pathways.37,45 This study presents some limitations. The number of endpoints was small, limiting the statistical power for detecting small effects. Our study suffers from a potential mechanistic weakness in that we do not establish a direct causal relation between miR-424(322) levels, the SMURF1/BMPR2 axis, and RV failure, but rather provide ‘associative findings’. On the other hand, all the parameters follow an impressively similar temporal pattern, suggesting a direct and strong association. Other rat models of PH could have been used; however, the MCT model is the most widely utilized and accurately reproduces the natural history of RVH. In summary, plasmatic miR-424(322) levels are elevated in patients with PH and correlate with the prognosis and severity of disease. Moreover, miR-424(322) targets SMURF1 and can modulate the BMPR2 pathway activity in cardiomyocytes. Circulating miR-424(322) levels and SMURF1 levels in the hypertrophied RV of an animal model of PH are inversely correlated, suggesting that this miRNA can act as an exosome-carried messenger linking pulmonary vascular disease and RV hypertrophy. Supplementary material Supplementary material is available at Cardiovascular Research online. Authors’ contributions R.B. and C.M. performed the experiments, analysed the data, and contributed to writing the paper. S.C. performed the luciferase reporter assay and assisted in writing the paper. F.E. and M.C. performed the miRNA assays. P.Ma. and M.Z. performed the animal studies and TEM experiments, respectively. G.C. and A.R. conducted the clinical characterization of the patients. P.Mo. and M.P. contributed to the revision of the paper. P.P. designed and discussed the study. H.G. co-ordinated the study, designed the experiments, analysed the data, and contributed to the writing and revision of the paper. Acknowledgements The authors wish to thank the nursing and research staff of the Pulmonary Vascular Disease units at Hospitais da Universidade de Coimbra and Hospital Geral de Santo António (both in Portugal) for aiding in the collection of human plasma samples. Conflict of interest: none declared. Funding This work was supported by European Regional Development Fund (ERDF) through the Operational Program for Competitiveness Factors (COMPETE) under the projects HealthyAging2020 CENTRO-01-0145-FEDER-000012-N2323 to CNC.IBILI and national funds through the Portuguese Foundation for Science and Technology [SFRH/SINTD/60112/2009; PEST-C/SAU/UI3282/2011-2013; UID/NEU/04539/2013], PAC ‘NETDIAMOND’ POCI-01-0145-FEDER-016385 and by an research grant from Actelion Pharmaceuticals Portugal, Ltd. 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TI - MicroRNA-424(322) as a new marker of disease progression in pulmonary arterial hypertension and its role in right ventricular hypertrophy by targeting SMURF1
JF - Cardiovascular Research
DO - 10.1093/cvr/cvx187
DA - 2018-01-01
UR - https://www.deepdyve.com/lp/oxford-university-press/microrna-424-322-as-a-new-marker-of-disease-progression-in-pulmonary-40erw7JS1M
SP - 53
EP - 64
VL - 114
IS - 1
DP - DeepDyve
ER -