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Role for miR-204 in human pulmonary arterial hypertension

Role for miR-204 in human pulmonary arterial hypertension

+ Author Affiliations

Department of Medicine, Medical School, 3Institut universitaire de cardiologie et de pneumologie de Québec, Université Laval, Québec, Québec G1V 0A6, Canada; 2Centre de research en cancérologie de l’Université Laval, Hôtel-Dieu of Québec;

CORRESPONDENCE Sébastien Bonnet: sebastien.bonnet@crhdq.ulaval.ca

Abstract

Pulmonary arterial hypertension (PAH) is characterized by increased proliferation and decreased death of pulmonary artery smooth muscle cells (PASMCs). We proposed that microRNAs, which have just been associated with the control of cell proliferation and demise, may also be involved in the etiology of PAH. In this study, we show that miR-204 expression in PASMCs is downregulated by PAH in both rodents and humans. MiR-204 down-regulation, which is linked with the severity of PAH, accounts for the proliferative and antiapoptotic traits of PAH-PASMCs. SHP2 expression is directly targeted by miR-204, and miR-204 expression is inhibited by STAT3 activity. MiR-204 down-regulation causes SHP2 to be up-regulated, which in turn causes activated T cells to release Src kinase and nuclear factor (NFAT). Furthermore, STAT3 directly encourages the expression of NFATc2. NFAT and SHP2 were necessary to maintain PAH-PASMC proliferation and resistance to apoptosis. Finally, synthesized miR-204 was administered to the lungs of PAH-affected rats, significantly reducing the severity of the illness. The results of this study demonstrate a novel regulatory pathway involving miR-204, which is crucial to the etiology of PAH. They also indicate that miR-204 re-expression should be looked into as a potential therapeutic treatment for this condition.

Pulmonary arterial hypertension (PAH), a vascular disease, mostly impacts the small pulmonary arteries (PAs). Despite the possibility of uncommon familial and idiopathic variants, congenital heart disease, HIV, connective tissue disorders, and anorexigen use are all associated with a syndrome that commonly includes PAH as a component. Numerous illnesses can contribute to this state of congested, constrained small PAs. Serotonin, IL-6, platelet-derived growth factor (PDGF), and endothelin are among the neurotransmitters and cytokines whose blood levels vary as a result. (Stewart et al., 1991; Christman et al., 1992; Steudel et al., 1997; Perros et al., 2008). The nuclear factor of activated T cells (NFAT) is also more active in the media, which results in increased [Ca2+]i-mediated PA smooth muscle cell (PASMC) proliferation and decreased mitochondrial-dependent apoptosis. (Bonnet et al., 2006, 2007b). In addition, metalloprotease activity is present and inflammatory cells have invaded the adventitia.(Humbert et al., 2004). Despite recent medical developments like endothelin-1 receptor blockers (like Bosentan),; Dupuis and Hoeper, 2008), type 5 phosphodiesterase inhibitors (e.g., sildenafil; Li et al., 2007), or PDGF receptor blockers (e.g., imatinib; Ghofrani et al., 2005), mortality rates remain high (Archer and Rich, 2000). Furthermore, the preservation of the PAH phenotype in cultured PASMCs isolated from PAH patients suggests that genetic remodeling processes rather than circulating growth factors or agonists are necessary for maintaining the PAH phenotype. (Yildiz, 2009; Dumas de la Roque et al., 2010). The BMPR2 gene (bone morphogenetic receptor-2) has been found to be mutated in at least 50% of familial PAH patients over the past ten years, and its down-regulation is now recognized as a hallmark of PAH. (Tada et al., 2007; Zakrzewicz et al., 2007). Recent research has connected the activation of both the tyrosine kinase Src and BMPR2 in human PASMCs. (Wong et al., 2005) and a STAT3/miR-17-92 microRNA (miRNA) secondary to IL-6 exposure, suggesting the implication of miRNAs in the etiology of PAH (Brock et al., 2009).

Small noncoding RNAs called miRNAs (21-23 nt) are now understood to play a significant role in controlling how genes are expressed. They create imperfect RNA-RNA duplexes and interact with messenger RNAs (mRNAs), primarily in the 3′ untranslated region, using their seed region. (UTR; Khan et al., 2009). The relevant mRNAs are negatively posttranscribedally regulated as a result of this interaction. Recently, pulmonary hypertension and other cardiovascular disorders have been linked to miRNA misexpression. (Latronico and Condorelli, 2009; Mishra et al., 2009; Zhang, 2009; Caruso et al., 2010), However, their molecular function in these pathologies has not yet been determined.

RESULTS

miR-204 is aberrantly expressed in human PAH-PASMCs

PASMCs were isolated from distal PAs of two nonfamilial PAH patients (two idiopathic PAH [iPAH] patients A and B; based on the World Health Organization [WHO] classification) and two control patients (A and B), and cultured as previously described, to ascertain whether miRNAs are aberrantly expressed in human PAH. (passage 3 and less; McMurtry et al., 2005). 377 distinct miRNAs’ expression levels were evaluated. When compared to control PASMCs, seven miRNAs (miR-204, -450a, -145, -302b, -27b, -367, and -138) had aberrant expression; Fig. S1 A). Only miR-204’s level was downregulated among them (Fig. S1 A). Quantitative RT-PCR (qRT-PCR) was used to determine if miR-204 was down-regulated in PASMCs extracted from three PAH (all from group 1 according to WHO classification patients A-C) as opposed to five control patients (A–E). Notably, there were no discernible variations in the expression of miR-204 between the control patients and the PAH patients (Fig. S1 B). All subsequent cell-based experiments in the study used the three PAH-PASMC cell lines in addition to the five control PASMC cell lines.

Interestingly, miR-204 down-regulation has been linked to increased cell proliferation and membrane potential depolarization in retinal epithelial cells and a number of cancer cells. (Lee et al., 2010; Wang et al., 2010), both of which are present in PAH-PASMCs. (Bonnet et al., 2006, 2007b). Recently, we demonstrated in a number of cancer cells and PAH-PASMCs (Bonnet et al., 2007a,b) that the Src-STAT3 pathway was partially responsible for this pro-proliferative phenotype (BMPR2 down-regulation),; Wong et al., 2005) and NFAT pathways (Bonnet et al., 2007b). This shows a potential connection between the down-regulation of miR-204, NFAT activation, and cell growth. As a result, miR-204 is probably involved in PAH and may play a part in maintaining the pro-proliferative and anti-apoptotic phenotype of PAH-PASMC. As a result, the current study will concentrate on miR-204’s function in the genesis of PAH. Interestingly, we found that only 165 of the 461 identified targets of miR-204 (TargetScan 5.1) were elevated by artificial miR-204 suppression in control human PASMCs (n = 2 patients; Fig. S1 C) using in silico and microarray gene expression studies. Several Src-STAT3- and NFAT-related genes were discovered in agreement with the pro-proliferative and anti-apoptotic phenotypes reported in PAH (Fig. S1 C).

miR-204 expression is decreased in human PAH and correlates with PAH severity

We compared the expression levels of miR-204 in the lungs of 8 people with non-familial PAH to the expression levels in the lungs of 8 people without pulmonary hypertension, in the lungs of 6 mice with hypoxia-induced pulmonary hypertension compared to the lungs of 5 control littermates, and in the lungs of 5 rats with monocrotaline (MCT)-induced pulmonary hypertension compared to the expression levels in the lungs of 10 control rats. (Fig. 1 A). In contrast to normotensive lung samples, we discovered lower levels of miR-204 in the pulmonary hypertension lung tissues of both humans and rodents. We investigated organ-specific levels of miR-204 between normal and pulmonary hypertensive rats in order to determine if down-regulated miR-204 levels were unique to the lung in rat models of pulmonary hypertension. (Fig. 1 B). MiR-204 levels in rats 3 weeks after MCT injection (pulmonary hypertensive rats) were only decreased in the lung and PAs as opposed to the aorta, liver, heart, and kidney, even though we were only able to detect tiny amounts of the miRNA in most organs. (Fig. 1 B).

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Figure 1.

expression of miR-204 and the severity of PAH are correlated. (A) PAH-induced lung damage reduces miR-204 in humans, mice, and rats. In comparison to human (n = 8), mouse (n = 10) and rat (n = 5) control (Ctrl) lungs, qRT-PCR analysis of miR-204 expression was performed in human lungs with PAH (n = 8), mouse lungs with hypoxia-induced pulmonary hypertension (n = 6), and rat lungs with MCT-induced pulmonary hypertension (n = 5). (B) The distal PAs are where miR-204 is primarily expressed. miR-204 expression in several rat organs with MCT-induced pulmonary hypertension (n = 5) was analyzed using qRT-PCR in comparison to control rats (n = 5). (C) The degree of PAH is correlated with miR-204 downregulation. qRT-PCR analysis of miR-204 expression in the lungs of healthy individuals (n = 8) and patients with varying degrees of pulmonary arterial hypertension (n = 3), in the lungs of mice with varying degrees of hypoxia-induced pulmonary hypertension (n = 3), and in the lungs of rats with varying degrees of MCT-induced pulmonary hypertension (n = 3), in comparison with control animals (n = 5 for both rats and mice; n = 3 experiments per patient or Every experiment compares the amount of miR-204 to the control RNA U6. Means and SEM are used to express the data (*, P 0.05; **, P 0.01; ***, P 0.001).

We looked at humans, mice, and rats with various levels of PAH to see if miR-204 down-regulation was associated with disease progression. MiR-204 lung levels were directly associated with the degree of PAH in both human subjects and rats, as determined by pulmonary vascular resistance in humans and mean PA pressure in mice. (Fig. 1 C). According to our findings, miR-204 levels are correlated with the seriousness of PAH in people and experimental pulmonary hypertension.

miR-204 is confined to PASMCs in the lung

Rat bronchi, veins, and PAs were used to evaluate miR-204 expression by qRT-PCR in order to determine its lung distribution. According to our findings, the bulk of miR-204 expression is seen in PAs but not in veins or bronchial tissue. (Fig. S2 A). Both cultured (passage 3 and fewer) human PASMCs and PA endothelium cells had miR-204 expression levels evaluated in order to determine the cell type distribution of miR-204 within PAs (PAECs). Our findings show that miR-204 is primarily restricted to the PASMCs within PAs since control-cultured PASMCs expressed seven times more miR-204 than control PAECs (Fig. S2 B).

miR-204 expression level is a potent biomarker for PAH

The fact that miRNAs are currently employed in humans as a biomarker for cancer is the final point. (Ferracin et al., 2010), We measured the expression of miR-204 in human buffy coats isolated from patients with PAH and non-pulmonary hypertensive patients to further support the role of miR-204 in PAH.(Table S1). We have previously demonstrated that there are many similarities between human PAH-PASMCs and cells from PAH patients’ buffy coats in terms of the pathways that are activated. For instance, both have turned on NFAT. (Bonnet et al., 2007b); Consequently, miR-204 expression in PASMCs and the buffy coat may be similar. In fact, patients with PAH had considerably lower miR-204 expression than those with PAH-PASMCs (Fig. S2 C). This finding verifies the role of miR-204 in PAH and raises the possibility that it could serve as an accurate biomarker for the disease. It is therefore of tremendous clinical interest.

Diminution of miR-204 level promotes PASMC proliferation and resistance to apoptosis

Cultured human PAH-PASMCs were either exposed to 10% FBS to promote proliferation or 0.1% FBS to promote apoptosis in order to study the impact of miR-204 on PASMC proliferation and apoptosis in vitro. (Bonnet et al., 2007b). PAH-PASMCs showed a higher rate of cell proliferation and resistance to induced apoptosis when compared to control PASMCs that had a high level of miR-204. (Fig. 2 A). In control PASMCs, where miR-204 inhibition boosted proliferation and resistance to apoptosis to levels similar to those reported in PAH-PASMCs, the role of miR-204 in controlling PASMC proliferation and apoptosis was verified. (Fig. 2 A).

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Figure 2.

The Src-STAT3-NFATc2 pathway is altered by miR-204 in PASMCs from a patient with PAH (PAH-PASMCs). (A) Human PASMC proliferation and apoptosis are regulated by miR-204. Analysis of serum starvation-induced apoptosis (TUNEL staining) and PASMC proliferation (PCNA nuclear localization) in PAH patients (n = 3 patients) and healthy controls (n = 5 people). As indicated, control (Ctrl) or miR-204 antagonists (Inh) were introduced. (B and C) Src and STAT3 activation is elevated in PAH-PASMCs with decreased miR-204 expression. The Western blots of PASMCs from three PAH and five control patients show total and phosphorylated Src (B) and total and phosphorylated STAT3 (C). When necessary, control or miR-204 antagomirs or mimics were introduced. As a loading control, smooth muscle actin (SM-actin) was utilized. There are examples of Western blots displayed. (D) The expression and activation of NFAT is increased in human PASMCs when miR-204 is downregulated in PAH-PASMCs. By using qRT-PCR (left) and a luciferase assay (right), NFATc2 mRNA expression and activity were assessed in PASMCs from control or PAH patients who had either received miR-204 antagomir or control. Error bars show the mean value and standard error of the mean (SEM) (*, P 0.05; **, P 0.01; ***, P 0.001).

Altering miR-204 level promotes the activation of the pro-proliferative and antiapoptotic Src–STAT3–NFAT pathway in PAH-PASMCs

The Src-STAT3-BMPR2 pathway has been linked to the rise in PASMC proliferation and resistance to apoptosis seen in PAH. (Wong et al., 2005; Brock et al., 2009) and NFAT pathways (Bonnet et al., 2007b). So, it was looked into how miR-204 might be involved in these pathways. As anticipated, we noticed a rise in Src activity (an increase in the phosphorylated Src [p-Src]/Src ratio).; Fig. 2 B), STAT3 (upregulated p) ratio of STAT3/STAT3 and p nuclear translocation of STAT3; Fig. 2 C and Fig. S3 A), and NFATc2 (increased expression, nuclear translocation, and luciferase activity; Fig. 2 D and Fig. S3 A) inside PAH-PASMCs. Src, STAT3, and NFATc2 activation were altered in PAH-PASMCs by an increase in miR-204 level, whereas they were stimulated in control PASMCs by a decrease in miR-204. (Fig. 2 and Fig. S3). These results show that the Src-STAT3-NFAT pathway is activated in PAH-PASMCs when miR-204 is downregulated. While our team has previously demonstrated that NFATc2 activation occurs in the lungs of PAH patients, STAT3 activation was also verified in lung biopsies from PAH patients (Fig. S3 B).(Bonnet et al., 2007b).

Next, we looked for the mechanism that was causing PAH-PASMCs to downregulate miR-204. Through stimulation studies, we discovered that PDGF, endothelin-1, and angiotensin II, all of which are well established to have a role in the etiology of pulmonary hypertension, decrease the expression of miR-204. (Zhao et al., 1996; Archer and Rich, 2000). Because STAT3 is primarily involved in the signaling of PDGF, endothelin-1, and angiotensin II (Yellaturu and Rao, 2003; Banes-Berceli et al., 2007), Additionally, the impact of miR-204 on STAT3 inhibition by small interfering RNA (siRNA) was examined. We found that the down-regulation of miR-204 seen in PAH-PASMCs was eliminated by siSTAT3. (Fig. 3 A). Finally, we discovered that the expression of miR-204 was negatively correlated with the activation of STAT3, meaning that the more STAT3 was activated, the more strongly miR-204 was downregulated. (Fig. 3 B). The human TRPM3 gene’s intron 6 contains the miR-204 coding sequence (transient receptor potential melastatin 3; Wang et al., 2010). An earlier study found that miR-204 and TRPM3 originate from the same transcription unit and share the same regulatory motif for transcription. (Wang et al., 2010). This was confirmed in PAH and in control PASMCs exposed to pro-PAH factors (Fig. 3 C). We performed promoter region analysis of TRPM3 using ENCODE (encyclopedia of DNA elements) chromatin immunoprecipitation (ChIP) sequencing (seq; ChIP-seq) data for the STAT family of transcription factors and discovered three putative STAT-binding sites close to the promoter region of TRPM3 to further illustrate the role of STAT3 in the regulation of miR-204 expression. So, we conducted a test to see if STAT3 could bind to these regulatory sites directly. PCR and ChIP using p-STAT3 antibody provided direct evidence of a STAT3-TRPM3 interaction. (Fig. 3 D). These findings imply that STAT3 may decrease the TRPM3/miR-204 gene locus, activating Src and NFAT in the process.

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Figure 3.

The down-regulation of miR-204 in PAH-PASMCs is attributed to a primary STAT3 activation by circulating pro-PAH factors. (A) In PAH-PASMCs, siSTAT3 elevates miR-204 expression. In PAH treated with control siRNA (siRNA ctrl) or siSTAT3 as specified (n = 3), the amount of miR-204 was determined by qRT-PCR. (B) In PASMCs, the expression of miR-204 and STAT3 activity are inversely associated. Analysis of the relationship between miR-204 expression (measured by qRT-PCR; n = 2 experiments/patient in three PAH and five control patients) and STAT3 activation (measured by the pY705-STAT3/STAT3 ratio monitored by Western blot). (C) In control PASMCs, pro-PAH factors reduce the expression of miR-204 and TRPM3 in a manner that is STAT3-dependent. On control cells treated with the pro-PAH factors PDGF, endothelin-1 (ET-1), or angiotensin II (AII) as indicated, the expression of miR-204 (top) and TRPM3 (middle) was assessed by qRT-PCR (n = 3 experiments/patient/in three PAH and five controls). (Bottom) Comparison of the miR-204 and TRPM3 expression patterns as determined by qRT-PCR in control, PAH, and PAH treated as indicated with siSTAT3. (D) The TRPM3 gene is located downstream of p-STAT3-binding sites. ChIP-PCR analyses of the TRPM3 gene’s upstream (Up1) and downstream (Dw1 and Dw2) STAT3-binding sites. A positive control was the VEGF gene, whereas a negative control was the OR8J1 gene. The means and SEM are represented by graphs (*, P 0.05; **, P 0.01; ***, P 0.001).

The down-regulation of TRPM3 in human vascular smooth muscle cells stimulates IL-6 production through an unidentified mechanism. (Naylor et al., 2010). As TRPM3 is down-regulated in PAH (Fig. 3 C) and IL-6 has been reported to be increased in PAH (Humbert et al., 1995), The etiology of PAH may be related to TRPM3, which would explain the phenotype of PAH. TRPM3 inhibition, however, did not imitate the PAH phenotype in control PASMCs (no changes in [Ca2+]i, DYm, and PASMC proliferation and apoptosis).; Fig. S4). MiR-204 was not related to these effects. In fact, because miR-204 is confined to a section of TRPM3’s intronic region, siTRPM3 had no effect on miR-204 levels (Fig. S4). Additionally, we show that TRPM3 is not a mediator of miR-204 effects because within 48 hours, ectopic increases in miR-204 inhibit the Src-STAT3 axis, reducing PAH-PASMC proliferation, resistance to apoptosis, and IL-6 secretion without restoring TRPM3 expression, whereas miR-204 down-regulation in control PASMCs mimics PAH without lowering TRPM3 expression.(Fig. S4, A and B; Wang et al., 1999). MiR-204’s control of IL-6 secretion (Fig. S4 B) and enhancement of Src activity (Fig. 2) in PAH-PASMCs (which had been associated with a number of pathophysiological PAH processes, such as cell proliferation [Steiner et al., 2009] and migration and K+ channel inhibition [Wong et al., 2005], as well as BMPR2 down-regulation [Wong et al., 2005]) suggests that BMPR2 may be indirectly downregulated by miR-204 downregulation. We found that both human PAH-PASMCs and PAs from PAH-rats treated with miR-204 mimics significantly up-regulated the gene for BMPR2. (Fig. S5, A and B). This might be the case because the rise in miR-204 inhibits STAT3, preventing the previously described STAT3-dependent BMPR2 down-regulation. (Brock et al., 2009) due to the fact that siSTAT3 also causes human PAH-PASMCs to express more BMPR2 (Fig. S5, A and B).

Src activation by miR-204 promotes STAT3 and NFAT activation in PAH-PASMCs

TargetScan 5.1 in silico analysis revealed that STAT3 and the three NFAT isoforms that are activated in PAH-PASMCs, NFATc1, -c2, and -c3, were not present.; Bonnet et al., 2007b) is anticipated to be miR-204’s target. However, because miR-204 expression is downregulated, STAT3 and NFAT activation are upregulated. (Fig. 2, B and C), It’s possible that miR-204 indirectly contributes to the activation of STAT3 and NFAT. JAK2 or SRC pathway activation is the primary cause of STAT3 activation. (Gharavi et al., 2007; Cheranov et al., 2008; Li et al., 2008). It’s interesting to note that JAK2 and two Src activators are among miR-204’s predicted targets. (SHP2 [Wu et al., 2006] and SHC [Src homology 2 domain containing; Sato et al., 2002]) were identified. Among JAK2, SHP2, and SHC, only SHP2 was up-regulated in PAH-PASMCs (Fig. 4 A and Fig. S5 C). In addition, raising the level of miR-204 in PAH-PASMCs reduced SHP2, whereas miR-204 inhibition in control PASMCs cells increased SHP2 expression.(Fig. 4 A). These findings imply that SHP2 might be miR-204’s main target in PAH. We carried out a reporter assay in which the luciferase reporter gene was under the control of the SHP2 3′ UTR to test this theory. We found that miR-204 is involved in the regulation of SHP2 through its 3′ UTR and that both point mutations that disrupt the miR-204 binding site in the SHP2 3′ UTR and the sequestration of miR-204 with a particular inhibitor increase the expression of the luciferase reporter. (Fig. 4 B). Finally, as predicted, SHP2 knockdown by siRNA or the Src inhibitor PP2 (4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine) significantly reduce STAT3 activation in PAH-PASMCs. This suggests that SHP2-dependent activation of Src is responsible for the increase in STAT3 activation in PAH-PASMCs. (Fig. S6 A). As a result, the level of miR-204 is reduced, which encourages SHP2 expression, Src activation, and enhanced STAT3 activation.

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Figure 4.

In PAH-PASMCs, SHP2 up-regulation by miR-204 encourages activation of the Src-STAT3-NFAT axis. (A) In PAH-PASMCs, miR-204 induces the expression of SHP2. Western blot was used to assess the levels of total and phosphorylated JAK2, SHC1, and SHP2 proteins in PASMCs from three patients with PAH and five control individuals. (right) MiR-204 mimic and antagomir (Inh) were introduced as well as the necessary controls (Ctrl). Smooth muscle actin is SM-actin. (B) The SHP2 3′ UTR is directly targeted by miR-204. (left) MiR-204 binding sites were discovered in SHP2’s 3′ UTR. The luciferase reporter mutations are displayed in red. (right) After transfection in control PASMCs (n = 5), relative firefly luciferase activity produced from the SHP2 3′ UTR and SHP2 3′ UTR mutant reporter constructs was observed. As indicated, a control and a miR-204 inhibitor (n = 3) were introduced. Luciferase unit, or L.U. (C) NFATc2 expression is controlled by STAT3. In PASMCs from control and PAH patients treated as needed with control or STAT3 siRNA, NFATc2 mRNA level (left) relative to 18S was evaluated by qRT-PCR (n = 3 qRT-PCR/patient in three PAH and five control patients). Studies of STAT3 binding on the genes encoding the indicated NFAT isoforms using ChIP-PCR (right) (NFATc1, -c2, and -c3). A positive control was the VEGF gene, whereas a negative control was the OR8J1 gene. Means and SEM are depicted in the graphs (*, P 0.05; **, P 0.01).

The activation of either calcineurin or NFAT is the primary cause of NFAT activation. (Macian, 2005) or Pim-1 (Glazova et al., 2005), however, none of them are miR-204 predicted targets. Luciferase assay served as confirmation of this The ChIP-seq data showed that several STAT-binding sites were present near NFAT genes. (Fig. S6 C; Chen et al., 2008; Bourillot et al., 2009). Therefore, we concluded that STAT3 activation follows SHP2-Src activation, and STAT3 activation, in turn, activates NFAT in PAH-PASMCs. The impact of STAT3 knockdown on NFATc2 expression was assessed in PAH-PASMCs and control PASMCs to show the role of STAT3 in the regulation of NFATc2 expression in PAH. When PAH-PASMCs were exposed to siRNA targeting STAT3, their expression of NFATc2 was significantly reduced in comparison to PAH-PASMCs treated with control siRNA. (Fig. 4 C), indicating that PAH-PASMCs express NFATc2 as a result of STAT3 activation. Additionally, real-time PCR (ChIP-PCR) analysis of ChIP demonstrated that STAT3 binds to the NFATc2 gene. (Fig. 4 C). NFAT activation by SHP2–Src has been described in skeletal muscle (Fornaro et al., 2006), and our findings not only support this earlier discovery but also suggest a new mechanism that involves STAT3.

Increasing miR-204 level in PAH-PASMCs reverses the pro-proliferation and antiapoptotic phenotype of PAH-PASMCs

In PAH-PASMCs, Src–STAT3–NFAT-mediated proliferation (Wong et al., 2005; Bonnet et al., 2007b) has been linked to the down-regulation of K+ channels (Platoshyn et al., 2000; Bonnet and Archer, 2007), resulting in membrane depolarization (Yuan, 1995; Platoshyn et al., 2000), opening the voltage-dependent Ca channels, thereby increasing intracellular Ca concentration ([Ca2+]i; Yuan, 1995; Wong et al., 2005; Bonnet et al., 2007b). We evaluated the impact of miR-204 modification on [Ca2+]i and PASMC proliferation using Fluo-3AM and proliferating cell nuclear antigen (PCNA). When miR-204 is inhibited in control PASMCs, [Ca2+]i and PASMC proliferation reach levels similar to those of PAH-PASMCs, whereas when miR-204 is increased in PAH-PASMCs, [Ca2+]i and proliferation decline to levels similar to those of control PASMCs. (Fig. 5, A and C). Using the Src inhibitor PP2, STAT3 siRNA, and the NFAT inhibitor VIVIT, we treated cells in order to further demonstrate that these effects were mediated by the Src-STAT3 and NFAT pathway. (Bonnet et al., 2007b). In PAH-PASMCs where the miR-204 level has been restored, VIVIT treatment does not further reduce [Ca2+]i. (Fig. 5, A and C), PP2 and siSTAT3, in contrast, reduce [Ca2+]i in PAH-PASMCs to a level comparable to that in control PASMCs. (Fig. 5 B). The decrease in [Ca2+]i brought on by raising the miR-204 level in PAH-PASMCs suppresses cell proliferation similarly to SHP2 inhibition (siSHP2).(PCNA; Fig. 5 C). In addition to the STAT3-NFAT axis, the inhibition of the RhoA-ROCK (Rho-associated, coiled-coil-containing protein kinase) pathway by siRNA or miR-204 mimics may also be responsible for the antiproliferative and proapoptotic effects of SHP2 inhibition.(Fig. S6 D; Lee and Chang, 2008; Kimura and Eguchi, 2009), which is increased and implicated in PAH-PASMC proliferation (Barman et al., 2009).

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Figure 5.

Restoring miR-204 lowers [Ca2+]i and depolarizes the membrane potential of the mitochondria. (A–C) Analysis of cell proliferation (PCNA nuclear localization) and [Ca2+]i (Fluo3-AM) in PASMCs from PAH and control (Ctrl) patients. (n = 50–150 cells/patient in three PAH and five control patients) miR-204 antagonists (Inh) and mimics, VIVIT (NFAT competitor peptide), PP2 (Src inhibitor) compared with PP3, its negative control, STAT3 siRNA, SHP2 siRNA, and appropriate controls for each therapy were added as indicated. (D–F) Analysis of serum starvation-induced apoptosis (TUNEL staining) and mitochondrial membrane potential (m; TMRM) in PASMCs from PAH and control patients. PP2 (Src inhibitor), STAT3 siRNA, SHP2 siRNA, VIVIT (NFAT competitor peptide), miR-204 antagomir (Inh) and mimics, as well as the necessary controls for each treatment were added (n = 50–150 cells/patient in three PAH and five control patients). Fluorescence unit, abbreviated F.U. The means and SEM are represented by graphs (*, P 0.05; **, P 0.01; ***, P 0.001).

Considering that miR-204 is encoded by TRPM3 (Wang et al., 2010) Since TRPM3 expression modification may be responsible for variations in [Ca2+]i, we investigated whether TRPM3 was responsible for the miR-204-dependent modulation of [Ca2+]i. Because miR-204 is situated inside an intronic region, siRNA suppression of TRPM3 had no effect on miR-204 expression, and PASMC proliferation and [Ca2+]i were unaffected. This showed that miR-204 restoration is the major therapeutic target (Fig. S4).

Hyperpolarization of the mitochondrial membrane potential (m), which would prevent the release of proapoptotic mediators like cytochrome c, has been linked to PAH-PASMCs’ resistance to apoptosis. c (Bonnet et al., 2007a, b, 2009). To determine whether miR-204 modulation can influence mitochondrial hyperpolarization, we measured the effects using tetramethylrhodamine methyl ester (TMRM). We found that inhibiting miR-204 causes m to hyperpolarize to a level similar to that seen in PAH-PASMCs in control PASMCs. (Fig. 5 D), whereas depolarizing m in PAH-PASMCs to a level comparable to that observed in control PASMCs requires either raising the level of miR-204 or using SHP2 siRNA, Src inhibitor PP2, or STAT3 siRNA. (Fig. 5 E). Last but not least, serum starvation-induced apoptosis (terminal deoxynucleotidyl transferase dUTP nick end labeling) is increased by mitochondrial depolarization caused by up-regulating miR-204 expression in PAH-PASMCs. [TUNEL]; Fig. 5 F). TRPM3 inhibition had no effect on [Ca2+]i, and m.

The STAT3–miR-204–Src–STAT3–NFAT axis is activated in the PAH animal model

We next tested the contribution of miR-204 in the MCT-injected rat model of PAH (Frasch et al., 1999; Bonnet et al., 2007b). We found that miR-204 down-regulation occurs concurrently with the onset of PAH, confirming that the severity and progression of PAH are correlated with the level of miR-204. (Fig. 6, A and E). Rats were sacrificed at different intervals following the injection of MCT in order to more thoroughly investigate the timing of the STAT3-miR-204-Src-STAT3-NFAT axis’ activation in the development of PAH. Right heart catheterization was used to directly assess pulmonary arterial pressure in closed chest mice prior to sacrifice. A rise in STAT3 activation was seen. (≤1 wk; Fig. 6 C) prior to the down-regulation of miR-204 SHP2 is elevated and STAT3 activation is pushed up to reach and maintain a maximal level from weeks 2 to 4 after miR-204 is down-regulated (2 wk). (Fig. 6, B and C). Once STAT3 activation becomes maximal, NFAT gets activated (≥3 wk; Fig. 6 D), increasing pulmonary arterial pressure (Fig. 6 E). Therefore, the time course analysis supports our in vitro data that show STAT3 activation happens before miR-204 levels drop, amplifying the activation of STAT3 and NFAT. These findings supported the hypothesis that STAT3 was involved in the attenuation of miR-204 in PAH. Upon miR-204 downregulation, STAT3 activation is subsequently increased and sustained over an extended period of time, enabling NFAT-dependent PASMC proliferation and resistance to apoptosis as well as rising PA remodeling and pressures.

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Image 6.

During the fourth week of PAH development, rats receiving MCT injections show a drop in the amount of miR-204 in their distal PAs. Rat distal PAs were used to evaluate the expression of miR-204 in relation to U6 using qRT-PCR. (B) Immunofluorescence (F.U., fluorescent unit) on lung slices was used to quantify SHP2 protein expression in distal PAs (n = 5 measurements per rat in five rats per time point). (C and D) Rats’ distal PAs were examined for STAT3 and NFAT activation using the proportion of cells displaying p-STAT3 and NFAT nuclear localization, respectively (n = 5 measurements per rat in 10 rats per time point). (E) Mean PA pressure in rats with closed chests as determined by right catheterization (n = 5 rats per group). The means and SEM are represented by graphs (*, P 0.05; **, P 0.01; ***, P 0.001).

MiR-204 mimic nebulization prevents MCT-induced PAH

Synthetic miR-204 RNA molecules were specifically delivered to the lung of MCT-induced PAH (MCT-PAH) rats by intratracheal nebulization 10-15 d after MCT injection (when endogenous miR-204 down-regulation reached its peak and PAH was established) in order to test whether restoration of miR-204 level can reverse symptoms of PAH in the rat model. We assessed the mRNA levels of nebulized miR-204 in numerous tissues using qRT-PCR, and we examined the distribution of the mimic DY547 tagged (control of transfection) using immunofluorescence.; Fig. S7 A).According to our findings, nebulized synthetic miR-204 primarily targets intraparenchymal resistance PAs and has little to no negative effects.

Using noninvasive measurements, a 2-week longitudinal study was carried out to evaluate the effectiveness of our treatment. In MCT-PAH rats, we saw that local delivery of synthetic miR-204 decreased pulmonary arterial pressure. (Fig. 7 A), as determined by the PAAT, a Doppler parameter associated with PA pressure In addition, when compared to MCT-PAH rats treated with nonspecific synthetic RNA molecules, synthetic miR-204 reduced the thickness of the right ventricle wall (Fig. S7 B). On the other hand, nebulizing miR-204 antagonist caused PAH development within 3 weeks in control rats. (Fig. 7 A and Fig. S7 B), Contrarily, nebulized antagomir negative control had no impact on the two animals (not depicted). We assessed medial wall thickness to ascertain if synthetic miR-204 administration can lessen PA remodeling in MCT-PAH rats. We noticed that mice given synthetic miR-204 showed a notable decrease in the medial thickness of small (300 m) and medium (600 m) PAs. (Fig. 7 B). Rats given synthetic miR-204 also showed a significant reduction in SHP2, p-STAT3, NFATc2 activation, PASMC proliferation (measured by PCNA distribution), and resistance to apoptosis (TUNEL). (Fig. 7 C and Fig. S8).

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Figure 7.

MCT-PAH is reversed by nebulizing to raise the amount of miR-204. (A) Restoring miR-204 lowers mean PA pressure. The mean PA pressure (n = 5 rats per group) was determined via right catheterization in closed chest rats. After two weeks of MCT injection, miR-204 antagomir (Inh), mimic, and the appropriate controls (Ctrl) were intratracheally nebulized. (B) Restoring miR-204 reduces PA wall thickness. The percentage of medium wall thickness on lung sections stained with hematoxylin and eosin was used to quantify PA remodeling (n = 5 measurements/rat in 10 rats). The graph displays mean values, and the images show representative distal arteries. (C) MiR-204 restoration promotes apoptosis and lowers proliferation in MCT-rat PASMCs via lowering SHP2, STAT3, and NFAT activation. Immunofluorescence (F.U., fluorescent unit) on lung sections was used to measure SHP2 protein expression in the distal PAs; STAT3 and NFAT activation (middle) was determined by the percentage of cells exhibiting p-STAT3 and NFAT nuclear localization, respectively, in the distal PAs of rats; and apoptosis and proliferation were determined by the percentage of cells exhibiting TUNEL and PCNA nuclear localization, respectively, in the After two weeks of MCT injection, control and miR-204 mimic rats were intratracheally nebulized (n = 5 measures per rat in five rats per group). The means and SEM are represented by graphs (*, P 0.05; **, P 0.01; ***, P 0.001).

DISCUSSION

Despite the fact that a prior study claimed that a number of miRNAs were expressed in PAH in an abnormal manner (Caruso et al., 2010), The first study to offer a mechanistic perspective on their role in the etiology of human PAH is ours. We concentrated on miR-204 because it was the only miRNA down-regulated in PAH-PASMCs, and because putative mRNA targets predicted in silico (TargetScan 5.1) were members of pathways associated with cell proliferation and resistance to apoptosis, such as Src and p53. (Wong et al., 2005), STAT3 (Shibata et al., 2003), and NFAT (Fig. S1; Bonnet et al., 2009). We concentrated our research on the role of miR-204 in PAH-PASMC proliferation and resistance to apoptosis because PASMCs express miR-204 seven times more than PAECs do. However, we cannot rule out a role for miR-204 in PAH-PAECs, which are also implicated in the etiology of PAH. (Jurasz et al., 2010).

Although other miRNAs have been linked to vascular disorders, their significance is yet unclear. For instance, despite the finding from a recent study showing miR-21 up-regulation was related to vascular neointimal lesions, (Ji et al., 2007), According to studies, miR-21 is down-regulated in the lung tissue of MCT rats but remains unaffected by chronic hypoxia. (Caruso et al., 2010). Interestingly, miR-21 is unaltered in our human PAH-PASMCs, indicating that miR-21 may not necessarily be a key player in the etiology of PAH. Curiously, in the Caruso et al. (2010) According to our findings in PASMCs and human lungs, miR-204 is down-regulated by 45% and 40% in chronic hypoxic and MCT rat lungs, respectively.

Unknown is miR-204’s function in vascular tissues. The down-regulation of miR-204 has been linked to increased PDGFb expression, cell proliferation, and the down-regulation of K+ channels, which in turn depolarizes the membrane potential of epithelial cells, according to a recent study conducted in retinal epithelial cells and several cancer cells. (Wang et al., 2010). These results confirm the significance of miR-204 in the genesis of PAH and are compatible with our findings and the previously described pathophysiological mechanisms of PAH. In fact, PAH has been associated with elevated PDGFb. (Barst, 2005), and this is consistent with STAT3 activation (PDGFb being an activator of STAT3; Yu et al., 2003), PASMC proliferation, and resistance to apoptosis (Bonnet et al., 2009).

The TRPM3 gene contains the miR-204 coding region. However, PAH has been linked to transient receptor potential cation channels. (Yu et al., 2004), TRPM3’s function is still unknown. In this research, we show that TRPM3 is not necessary for miR-204 effects to occur. First, ectopic increases in miR-204 suppress the SHP2-Src-STAT3 axis, reducing the proliferation of PAH-PASMCs, their susceptibility to apoptosis, and their ability to secrete IL-6 without increasing TRPM3 expression (Fig. S4, A and B). Second, miR-204 downregulation mimics PAH in control PASMCs without lowering TRPM3 levels (Fig. S4 A). Third, in control PASMCs, TRPM3 inhibition (without changing miR-204 levels) did not result in a PAH phenotype (Fig. S4 C).

A recent study using human PASMCs connected the activation of an IL-6-STAT3-miR-17-92 axis to the down-regulation of BMPR2. (Brock et al., 2009). In their model, IL-6 activates STAT3, which boosts the expression of the miR-17-92 cluster and inhibits BMPR2. Even though the miR-17-92 cluster expression is unaltered in our PAH-PASMCs, our research sheds new light on the process by which BMPR2 is down-regulated in PAH. MiR-204’s control of IL-6 secretion (Fig. S4 B) and enhancement of Src activity (Fig. 2) suggests that miR-204 downregulation may indirectly downregulate BMPR2 in PAH-PASMCs. The BMPR2 gene was significantly up-regulated in both human PAH-PASMCs and PAs from PAH-rats treated with miR-204 mimics, according to preliminary results (Fig. S5).

The fact that we provide a clear proof of a mechanism for the origin of the miRNA deregulation in around 10 PAH patients, which is a first in the field of vascular disorders, is one of the study’s primary strengths. We demonstrate that circulating pro-PAH factors such endothelin-1, PDGF, and angiotensin II (which all rise with the onset of PAH) can activate STAT3 in a main manner.; Archer and Rich, 2000) explains why miR-204 is downregulated in control PASMCs. This discovery was supported by promoter analysis and ChIP-PCR results, which demonstrate direct STAT3 binding to the miR-204 gene (within TRPM3).; Fig. 3 D). Additionally, we demonstrated that miR-204 down-regulation comes before STAT3 activation in vivo in the PAs of rats given MCT injections. (Fig. 6 C). The Src activator SHP2 is immediately up-regulated after miR-204 is down-regulated, which leads to the production of IL-6 (Fig. S4 B) and PDGF. (Wang et al., 2010) increased, enabling NFAT activation and supporting STAT3 activation via Src. This could explain why cultured PAH-PASMCs have a persistent pro-proliferative and antiapoptotic phenotype. According to a recently published RNA profiling study conducted on 18 iPAH patients, this mechanism is activated.(Rajkumar et al., 2010). Reanalyzing the data from this study confirms that TRPM3 was downregulated while SHP2 (also known as PTPN11) and NFATc2 were both upregulated. We show that STAT3 binds to the NFAT gene promoter region and increases NFAT expression; however, STAT3 can only activate NFAT if the Ca-calcineurin pathway or other NFAT activators, such as Pim-1, are also activated. In our model, [Ca2+]i is indeed elevated, which may help to activate calcineurin. Additionally, Pim-1 is a protooncogene whose expression rises in vascular diseases and is controlled by STAT3.(Katakami et al., 2004). Therefore, it’s plausible that STAT3 activation not only causes NFAT expression but also encourages it by increasing Pim-1 expression. (Rainio et al., 2002). Finally, we examined the expression of miR-204 in buffy coats from 13 PAH patients and 7 control donors, finding that it is down-regulated just as it is in PAH lungs, indicating that miR-204 may be a useful PAH biomarker.

Finally, we present the first proof that aberrantly expressed miRNAs are a significant factor in the etiology of human PAH. We show that miR-204 can be therapeutically targeted both in vitro and in vivo, which results in a reduction in proliferation, vascular remodeling, and PA blood pressure and thus represents a new therapeutic strategy for PAH. Additionally, there is preliminary evidence that suggests miR-204 may control the RhoA-ROCK pathway in PAH-PASMCs, another significant PAH component (Fig. S6 D; Doggrell, 2005). Although additional research is needed to pinpoint the precise mechanism, Bregeon et al. [2009] and Kimura and Eguchi [2009] have proven that SHP2 plays a part in the RhoA-ROCK pathway in the past. This serves as the foundation for an additional investigation. In order to achieve clinical benefit, our study hypothesizes that therapeutic modulation of a single miRNA (miR-204) may simultaneously affect numerous pathways linked to PAH. The regulation of hundreds of targets in multiple pathways by miRNAs may lessen the emergence of drug resistance as is currently seen in PAH compared to currently used therapies that target a single protein (ET-1 receptor blockers, PDE5 inhibitor, etc.). This is because it would take numerous simultaneous mutations to reverse the effects of miRNA expression. MiRNA-based therapies will nevertheless need in-depth preclinical validation because these widespread effects may occasionally have negative effects on health. However, in our rats, this was not noted.

In summary, we provide a comprehensive model (Fig. S8 E) linking miRNA abnormal expression to already known pathophysiological processes in PAH, including NFAT activation, BMPR2 down-regulation, IL-6 production, the Rho pathway, PASMC proliferation, and resistance to apoptosis (Cowan et al., 2000; Sakao et al., 2005; Bonnet and Archer, 2007; Bonnet et al., 2007b; Tuder et al., 2007). Thus, our study does not only demonstrate the importance of miRNAs in PAH but also suggests that reestablishing the miR-204 level might represent a novel therapeutic approach for human PAH.

MATERIALS AND METHODS

Ethics.

All experiments were performed in accordance with the Université Laval’s Ethics and Biosafety Committee (protocol number 20142) and the Centre Hospitalier Universitaire de Québec’s Ethics Committee. The investigation conforms to the Guide for the Care and Use of Laboratory Animals, published by the National Institutes of Health (publication no. 85–23, revised 1996) and with the principles outlined in the Declaration of Helsinki.

Human tissue samples.

See Table S1. All patients gave informed consent before the study. Normal lung tissues (controls) were obtained during lung resection for benign (n = 3) or malignant (n = 5) tumors. Only the healthy parts of the lungs were used in this study. All the PAH tissues were from open lung explants from transplant or autopsy.

Cell culture.

We used cells in the first to third passage. PAH-PASMCs were obtained as described previously (McMurtry et al., 2005) from ∼1,500-µm-diameter small PAs from two males with iPAH (31- and 48-yr-old patients A and B) and one female with PAH group 1 (lupus; 54-yr-old patient C) from lung explants. All patients had right catheterization that confirmed pulmonary hypertension (mean pulmonary arterial pressure >25 mmHg). Age- and sex-matched control PASMCs (three males A, B, and C 45, 21, and 64 yr old; and two females D and E 17 and 35 yr old), and PAECs were purchased from Cell Application USA. PASMCs were grown in high-glucose DME supplemented with 10% FBS (Invitrogen) and 1% antibiotic/antimitotic (Invitrogen; Bonnet et al., 2007a). STAT3 and SHP2 were inhibited by a specific siRNA (20 nM for 48 h; Applied Biosystems) as previously described (Bonnet et al., 2007a). NFAT was inhibited by 4 µM VIVIT as previously described (Bonnet et al., 2007b).30 ng/ml PDGF, 10 nM endothelin-1, 200 nM angiotensin II, or 100 ng/ml TNF were given to control PASMCs. The effects of the Src inhibitor PP2 and its antagonist PP3 (4-amino-7-phenylpyrazol [3,4-d] pyrimidine; 10 M for 48 h) were compared. MiR-204 antagomirs (200 nM for 48 h) or miRIDAN miR-204 mimics (400 nM for 48 h) were transfected using the Ca phosphate transfection technique. We used a suitable control for every experiment (the hairpin inhibitor negative control #1 from Thermo Fisher Scientific or mimics). Fig. S8 C displays the dose response, transfection effectiveness, and siRNA effectiveness.

Low density TaqMan arrays

Four patients had TLDA, two for each ailment, in accordance with the manufacturer’s procedure (Applied Biosystems). Each sample was examined twice. Then, two distinct normalization techniques were used to adjust the raw CT data, one normalizing in relation to U6 small nuclear RNA and the other in relation to the median CT. The significantly modulated miRNAs were found using an empirical Bayesian approach in the Bioconductor program limma. Both normalizations needed a large modulation of miRNAs. The GEO DataSets under Accession contain TLDA data that have been deposited.no. GSE21284.

DNA microarrays.

The Whole Human Genome microarray kit was used for DNA microarray studies (Agilent Technologies). The data were retrieved from the images using the Feature Extraction program after the arrays were scanned using an Agilent Technologies dual-laser DNA microarray scanner. RNAs were taken from two control patients and hybridized on Cy3, whereas RNAs were taken from two PAH patients and hybridized on Cy5. This was done to compare the results between the control and PAH patient groups. In the miR-204 inhibition experiment, RNA was isolated from control PASMCs that had been given a 48-hour treatment with 200 nM miR-204 antagomir from Thermo Fisher Scientific and hybridized with Cy3. Before limma in Bioconductor’s Empirical Bayes method-based significant modulation assessment, data were background-subtracted and standardized inside the array using the LOESS normalization. In our model, miR-204 targets were defined as genes that were identified as miR-204 targets in TargetScan 5.1, had an expression level 100 in log2 base, and were up-regulated following miR-204 suppression. Microarray data are accessible under the accession number and have been deposited in GEO DataSets. no. GSE21284.

qRT-PCR.

Applied Biosystems’ mirVana kit was used to extract total RNA from control or PAH-PASMCs in order to measure the expression of miR-204. On a real-time PCR apparatus, stem-loop qRT-PCR for mature miRNAs was carried out (AB 7900; Applied Biosystems). Normal qRT-PCR was carried out as previously mentioned. (Bonnet et al., 2007b).

ChIP-PCR.

In a nutshell, endothelin was used to control PASMC cells that were developing asynchronously at 10 nM. Chromatin was extracted using lysis buffer, which contains 50 mM Tris-HCl, pH 8, 10 mM EDTA, 0.2% SDS, and 5 mM Na-butyrate, and cross-links were created using 1% formaldehyde. After that, chromatin was sonicated (Bioruptor; Diagenode) on ice until it reached a mean length of 750 bp. 80 g of chromatin was used for immunoprecipitation with the appropriate antibodies (10 ml p-Sat3 [Tyr705; 9131; Cell Signaling Technology] and 10 mg regular rabbit IgG [I-1000; Vector Laboratories]) in a total volume of 300 ml after preclearing with a mixture of protein A/G-Sepharose beads (4°C for 1 h). 25 l of protein A Dynabeads (Invitrogen) were added and incubated for more than an hour after an overnight incubation at 4°C. The immunoprecipitated complexes were eluted in buffer E (50 mM Na bicarbonate and 1% SDS), after the beads had undergone a thorough washing procedure. At 65°C, cross-links were reversed overnight. Proteinase K was used to treat the samples, and phenol-chloroform was used to extract the DNA. Using SYBR green I, quantitative real-time PCR was carried out (LightCycler 480; Roche). The comparative Ct approach was used to calculate enrichment for a particular DNA sequence. The data is based on two biological repeats (cells/chromatin/immunoprecipitation) and is supplied with standard errors. PCR primers used in reactions (Table S2) were analyzed for specificity, linearity range, and efficiency to accurately evaluate occupancy (percentage of immunoprecipitation/input). Vascular endothelial growth factor (VEGF) primers were used as positive control, whereas OR8J1 primers were used as negative control.

Confocal microscopy.

NFATc1 and -c2 and STAT3 nuclear translocation assays were performed using antibodies (1:250; Abcam) as previously described (Bonnet et al., 2007b). TMRM, TUNEL, PCNA, and Fluo-3 were measured as previously described (Bonnet et al., 2009; Bonnet et al., 2007b).

Transfection and luciferase assay for different 3′ UTR constructions.

Every gene of interest had its 3′ UTR cloned and introduced into the psiCHECK2 plasmid right after firefly luciferase’s stop codon. When everything was prepared, cells were transfected with the reporter plasmid along with 200 nM of unrelated small RNA duplex (mimic control; Invitrogen), miR-204 mimic (Thermo Fisher Scientific), miR-204 inhibitor (Thermo Fisher Scientific), or Caenorhabditis elegans miR-67 inhibitor used as a control (Thermo Fisher Scientific). A dual-luciferase assay was used to evaluate the simultaneous activity of Firefly and Renilla luciferases 48 hours after transfection. The firefly luciferase activity would be decreased if miR-204 and the target mRNA interacted (normalized to Renilla luciferase activity expressed from the psiCHECK2 plasmid). Point mutations were introduced into the 3′ UTR sequence, which corresponds to the miR-204 binding site, at positions 2, 4, and 6 from the 5′ end of miR-204 in order to abolish the binding of miR-204 to the SHP2 3′ UTR, as shown in Fig. 4 B.

In vivo model rats.

Male rats were injected s.c. with a 60-mg/kg MCT solution (Todorovich-Hunter et al., 1988). Hemodynamic measurements (with Swan-Gan catheters) and echocardiography (with Vevo 2100; VisualSonics), which were carried out as previously described, were used to evaluate PAH. (Bonnet et al., 2007b). In vivo, miR-204 mimic (mature sequence, 3′-UUCCCUUUGUCAUCCUAUGCCU-5′) or mimic negative (20 M once a week for two weeks) were nebulized into rats with developed PAH (measured by Echo-Doppler). In accordance with the recommendations of the manufacturer, Invivofectamine (Invitrogen) was employed as the transfected agent. qRT-PCR was used to evaluate tissue distribution and transfection effectiveness. Utilizing the fluorescence distribution of the commercially available, DY547-labeled mimic control, tissue distribution was evaluated (Thermo Fisher Scientific).

Chronic hypoxic mice model.

Mice were kept in normobaric hypoxic chambers with 5.5 liters per minute of hypoxic air (10% O2 and 90% N2) for two to three weeks. Twice a week, doors to the chambers were opened for cleaning and food and water replenishment. Blood gas analyzers were used to continuously measure oxygen levels. To reduce the carbon dioxide concentration, soda lime was utilized.

Statistical analysis.

Fold change, mean, and SEM are used to express values. Unpaired The Student’s t test was employed to compare two means. We used one-way analysis of variance followed by a Dunn’s test for comparisons involving more than two means. Statistical significance was defined as p 0.05. (and indicated with asterisks). Data from TLDA and microarrays were examined inside R (https://www.r-project.org) using the limma package in Bioconductor.

Online supplemental material.

Seven miRNAs are abnormally expressed in human PAH-PASMCs when compared to control PASMCs, as shown in Fig. S1. The assessments of miR-204 levels in the buffy coat and pulmonary vasculature are shown in Fig. S2. According to Fig. S3, miR-204 down-regulation in PAH-PASMCs encourages STAT3 and NFAT activation. The effect of miR-204 is not dependent on TRPM3 expression, as seen in Fig. S4. The miR-204 mimic molecule restores BMPR2 expression in PAH, as shown in Fig. S5. According to Fig. S6, a reduction in miR-204 levels stimulates the Src-STAT3 axis and encourages the production of NFAT. According to Fig. S7, miR-204 mimic intratracheal nebulization enhances the PAAT and lessens right ventricular hypertrophy by restoring miR-204 expression in distal PAs. The effects of siRNA and miR-204 mimic/antagomir transfection on PASMCs are validated in Fig. S8. Patients who donate tissue are listed in Table S1. Primer sets for ChIP-real-time PCR are listed in Table S2. There is further content online. at https://www.jem.org/cgi/content/full/jem.20101812/DC1.

Acknowledgments

Bonnet, M.J. Simard, and J. Côté received grants from the Canadian Institutes of Health Research (CIHR) to fund this research. M.J. Simard is a new investigator at the CIHR. J. Côté and S. Bonnet both hold Canada Research Chairs.

There are no competing financial interests of the writers.

Footnotes

Utilized abbreviations:

ChIP

Immunoprecipitation of chromatin

iPAH

PAH idiopathic

MCT

monocrotaline

miRNA

microRNA

mRNA

RNA messenger

NFAT

nuclear factor of T-cell activation

Pneumatic artery

PAAT

Acceleration time for PA

PAEC

Endothelial cells in PA

PAH

hypertension in the pulmonary arteries

PASMC

Smooth muscle cell, PA

PCNA

nuclear antigen for proliferating cells

PDGF

growth factor derived from platelets

qRT-PCR

statistical RT-PCR

ROCK

Coiled-coil-containing, Rho-associated protein kinase

seq

sequencing

siRNA

interfering RNA small

TLDA

Low-density TaqMan array

TMRM

methyl ester of tetramethylrhodamine

TUNEL

nick end labeling with terminal deoxynucleotidyl transferase

UTR

region not yet translated

VEGF

Blood vessel endothelial growth factor Date submitted: August 31, 2010

Acceded: January 19, 2011

For the first six months following the date of publication, this article is made available under the terms of an Attribution-Noncommercial-Share Alike-No Mirror Sites license. (see https://www.rupress.org/terms). After six months it is available under a Creative Commons License (Attribution–Noncommercial–Share Alike 3.0 Unported license, as described at https://creativecommons.org/licenses/by-nc-sa/3.0/).

↵
1. Archer, S.,
2. S. Rich

. 2000. “Work in progress” vascular biology and translational research on primary pulmonary hypertension. 102:2781–2791 of circulation.

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