Let-7a-transfected mesenchymal stem cells ameliorate monocrotaline-induced pulmonary hypertension by suppressing pulmonary artery smooth muscle cell growth through STAT3-BMPR2 signaling
© The Author(s). 2017
Received: 1 December 2016
Accepted: 12 January 2017
Published: 10 February 2017
Cell-based gene therapy has become a subject of interest for the treatment of pulmonary arterial hypertension (PAH), a devastating disease characterized by pulmonary artery smooth muscle cell (PASMC) hyperplasia. Mesenchymal stem cells (MSCs) have been recently acknowledged as a potential cell vector for gene therapy. Here, we investigated the effect of MSC-based let-7a for PAH.
After isolation and identification of MSCs from rat bone marrow, cells were infected with recombinant adenovirus vector Ad-let-7a. Lewis rats were subcutaneously injected with monocrotaline (MCT) to induce PAH, followed by the administration of MSCs, MSCs-NC (miR-control), or MSC-let-7a, respectively. Then, right ventricular systolic pressure (RVSP), right ventricular hypertrophy, and pulmonary vascular remodeling were evaluated. Rat pulmonary artery smooth muscle cells (rPASMCs) under hypoxia were co-cultured with MSCs or MSC-let-7a. Cell proliferation and apoptosis were separately determined by 3H thymidine incorporation and flow cytometry analysis. The underlying mechanism was also investigated.
MSC transplantation enhanced let-7a levels in MCT-induced PAH rats. After injection with MSC-let-7a, RVSP, right ventricular hypertrophy, and pulmonary vascular remodeling were notably ameliorated, indicating a protective effect of MSC-let-7a against PAH. When co-cultured with MSC-let-7a, hypoxia-triggered PASMC proliferation was obviously attenuated, concomitant with the decrease in cell proliferation-associated proteins. Simultaneously, the resistance of PASMCs to apoptosis was remarkably abrogated by MSC-let-7a administration. A mechanism assay revealed that MSC-let-7a restrained the activation of signal transducers and activators of transcription 3 (STAT3) and increased its downstream bone morphogenetic protein receptor 2 (BMPR2) expression. Importantly, preconditioning with BMPR2 siRNA dramatically abated the suppressive effects of MSC-let-7a on PASMC proliferation and apoptosis resistance.
Collectively, this study suggests that MSCs modified with let-7a may ameliorate the progression of PAH by inhibiting PASMC growth through the STAT3-BMPR2 signaling, supporting a promising therapeutic strategy for PAH patients.
KeywordsPulmonary arterial hypertension mesenchymal stem cells let-7a pulmonary artery smooth muscle cell STAT3 pathway BMPR2
Pulmonary arterial hypertension (PAH) is a well-recognized, devastating, and life-threatening disease. As a common progressive disease, it is characterized by a sustained increase in pulmonary arterial pressure, right heart dysfunction, and vascular remodeling, leading to the high morbidity and mortality of patients with lung and heart diseases [1–3]. Despite therapeutic advances in PAH treatment, the 3-year survival rate is still less than 60% . Therefore, an understanding of the molecular mechanism underlying PAH development is urgently required for improving the therapeutic strategies.
Recently, cell-based gene therapies have garnered increasing interest due to their beneficial roles in ameliorating the progression of PAH [5–7]. Mesenchymal stem cells (MSCs) are the preferred cell model for cell-based therapy because of their high expansion in culture, reproducible potential, and capacity to differentiate into various lineages. They can be mobilized and migrate into injury sites to orchestrate the repair response . Application of naïve MSCs attenuates monocrotaline (MCT)-induced PAH, indicating a beneficial effect of stem cell therapy for PAH disorders [9, 10]. More interestingly, administration of MSCs modified with prostacyclin synthase or endothelial nitric oxide synthase not only abrogated right ventricular (RV) hypertrophy and cardiovascular remodeling, but also improved survival time in PAH rats [5, 6]. Additionally, MSCs expressing heme oxygenase-1 enhanced the protective effect of MSCs on hypoxia-triggered PAH by attenuating lung inflammation and smooth muscle cell proliferation, suggesting a promising strategy against PAH based on MSC-based gene therapy .
MicroRNAs (miRNAs) orchestrate various pathophysiological processes including lipid metabolism, vascular remodeling, and tissue repair [12–15]. Abnormal expression of miRNAs has been corroborated in several lung-related diseases, such as PAH . The presence of some miRNAs that are correlated with PAH severity can retard the pathological progression of PAH, indicating a potential innovative therapy for the treatment of PAH . Animals with PAH show a dramatic downregulation of let-7a in the lungs . An analogous decrease in let-7a was also validated when cells were exposed to chronic hypoxia, suggesting a potential role of let-7a in the development of PAH . Moreover, let-7 expression also negatively correlated with the severity of PAH in patients with systemic scleroderma . Unfortunately, its function in PAH remains poorly defined. In this study, we aimed to explore the function of MSCs transfected with let-7a in PAH development. Furthermore, the effects of MSC-let-7a on pulmonary artery smooth muscle cell (PASMC) growth, as well as the underlying mechanism, were also explored.
Antibodies and reagents
The signal transducer and activator of transcription 3 (STAT3) inhibitor, S31-201, was acquired from Sigma (St. Louis, MO, USA). The primary antibodies against STAT3, phosphorylated STAT3-Tyr-705 (p-STAT3), proliferating cell nuclear antigen (PCNA), and bone morphogenetic protein receptor 2 (BMPR2) were purchased from Abcam (Cambridge, UK). Rabbit antibodies against rat p21 and α-smooth muscle actin (SMA) were obtained from Sigma. Anti-rat antibodies of Ki67 and cyclin D1 were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA).
For experiments using animals, 6-week-old male Lewis rats were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. (Beijing, China). All animal care and experimental procedures were undertaken with approval from the Institutional Animal Care and Use Committee of the First Affiliated Hospital of Xi’an Jiaotong University (SCXK-2012-003). Rats were acclimatized for 1 week prior to the subsequent treatments.
Isolation and expansion of rat MSCs
Preparation of rat MSCs (rMSCs) was conducted following previously published methods . Briefly, bone marrow cells from Lewis rats were mechanically dissociated by flushing the femur and tibia cavities under sterile conditions. The isolated cells were then suspended in α-MEM medium containing 10% fetal bovine serum (FBS), 100 U/mL penicillin and 100 μg/mL streptomycin on 75-cm2 tissue culture flasks. Three days later, non-adherent cells were removed, and the adherent cells that had grown to 90% confluence were considered as passage (P)0 MSCs. For expansion, cells were diluted in the ratio of 1:2 per passage and continuously cultured in rMSC medium. Passages 3–4 were used for the subsequent experiments.
Characterization of MSCs in vitro
The surface marker proteins of MSCs were identified by incubation with fluorescein isothiocyanate (FITC)-conjugated monoclonal antibodies against CD34, CD45, CD44, and CD90 (all from Abcam, Cambridge, MA, USA). Then, the labeled cells were analyzed by a FACScan flow cytometer (Becton Dickinson, NJ, USA). To evaluate osteogenic ability of MSCs, MSCs of P3 were incubated with osteogenic medium for 4 weeks and subsequently stained with 2% Alizarin Red S (Sigma). Cells were stained with 0.3% Oil Red O (Sigma) to assess adipogenic ability. All specimens were analyzed microscopically (IX70, Olympus, Tokyo, Japan).
Adenovirus production for let-7a in rMSCs
Adenoviral vectors of AdmiRa-rno-let-7a expressing green fluorescent protein (GFP) and miR-control (normal control; NC) were obtained from Applied Biological Materials Inc. (Richmond, BC, Canada). The above adenovirus was packaged according to the protocol provided by the manufacturers. After amplification and purification, virus titers were measured by the p24 ELISA kit (Cell Biolabs, Inc., San Diego, CA, USA). Then, the obtained rMSCs at P3–P4 were infected with the above adenovirus to generate the let-7a-overexpressed MSCs. The infection efficiency was evaluated by GFP and quantitative RT-PCR. Cell that overexpressed let-7a were then harvested with phosphate-buffered saline (PBS) buffer supplement with 0.25% trypsin and 1 mmol/L EDTA before use.
To construct the PAH model, Lewis rats received a subcutaneous injection of monocrotaline (MCT; Sigma, St. Louis, MO, USA; 60 mg/kg) as previously reported . Rats injected with 0.9% saline were defined as the negative control.
About 3 weeks later, rats treated with MCT were randomized to four groups (n = 10/group, n = 40 in total): rats treated with PBS (MCT + PBS group); rats received MSCs (MCT + MSC group); rats injected with MSC-let-7a (MCT + MSC-let-7a group); rats received MSC-NC (MCT + MSC-NC group). For cell implantation, approximately 3 × 106 cells in PBS were slowly injected into rats via the tail vein. Rats without MCT administration were defined as the control group.
Collection of blood specimens
EDTA-anticoagulated whole blood samples were collected at 7, 14, and 21 day after the injection of MSC-let-7a, MSCs, or PBS. The bloods were centrifuged at 1000 g for 10 min. The obtained specimens were then stored at –70 °C until analysis.
Hemodynamic evaluation and right ventricular hypertrophy
Three weeks after MSC administration, rats were anesthetized with sodium pentobarbital (30 mg/kg) to perform the hemodynamic study. The 3-F Miller catheters were inserted into the right ventricle through the right jugular vein to assess the right ventricular systolic pressure (RVSP). After euthanizing with an overdose of pentobarbital, the lungs and hearts were collected. The ratios of right ventricle weight to body weight (RVW/BW) and right ventricle/left ventricle + septum weight (RV/LV + Sep) were detected and defined as the indexes of right ventricular hypertrophy [5, 6]. For vascular remodeling evaluation, lung tissues were embedded in paraffin and then were cut into serial 4-μm thick sections. After staining with hematoxylin and eosin, the wall thickness (n = 5 measurements/rat in 10 rats) was calculated as follows: wall thickness (%) = (external diameter – internal diameter)/ external diameter × 100 .
Rat pulmonary artery smooth muscle cells (rPASMCs) were purchased from Pricells Company (Wuhan, China) and cultured in DMEM medium containing 5% FBS, 100 U/ml penicillin, and 100 μg/ml streptomycin. Cells from P5–P10 were used for the subsequent experiments. All cells were maintained in 5% CO2 at 37 °C.
Knockdown of BMPR2 by siRNA
To knockdown BMPR2 expression in MSCs, three specific pairs of siRNA sequences were designed according to rat BMPR2 mRNA (GenBank accession number: NM_080407.1). Their sequences were as follows: siRNA-BMPR2-1500 (sense, 5′-GGUUGGCACAAUCCGCUAU-3′; antisense, 5′-AUAGCGGAUUGUGCCAACC-3′), siRNA-BMPR2-1947 (sense, 5′-GCAGAACGAACGCAACCUA-3′; antisense, 5′-UAGGUUGCGUUCGUUCUGC-3′), and siRNA-BMPR2-2173 (sense, 5′-CCAGAAACAAGCGUCACAA-3′; antisense, 5′- UUGUGACGCUUGUUUCUGG-3′). The scramble siRNA (siRNA-NC) were used as negative control, and the sequences were as follow: forward 5′-GGUCACGCUAACGCGUUAU-3′, reverse 5′-AUAACGCGUUAGCGUGACC-3′. For transfection, cells were grown to approximately 50–60% confluence. Then, cells were transfected with siRNAs at 4 nM using Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA) according to the manufacturer's directions. Approximately 24 h later, the efficiency of BMPR2 siRNA was analyzed by Western blotting.
Co-culture of MSCs and rPASMCs
MSCs and PASMCs were co-cultured using the transwell system as previously described . Briefly, rPASMCs were exposed to hypoxia with 3% oxygen concentration or normoxia for 24 h. Cells were then seeded in the transwell insert membrane with 0.4-mm diameter pores. Then, the modified MSCs (MSC-NC or MSC-let-7a) were cultured in the lower chamber. Twenty-four hours later, the PASMCs were collected for cell proliferation and apoptosis assay.
RNA extraction and quantitative RT-PCR
Total RNA from blood samples was obtained by the RNAprep Pure Hi-Blood Kit (TIANGEN, Beijing, China) according to the manufacturer’s directions. Total RNA from lung tissues and cells was extracted using TRIzol reagent (Invitrogen Life Technologies, Carlsbad, CA, USA). For mRNA detection, reverse transcription was performed to synthesize the cDNA with RevertAidTM First Strand cDNA Synthesis Kits (Fermentas, St. Leon-Rot, Germany). Then, quantitative RT-PCR was carried out to evaluate the mRNA levels of let-7a using SYBR premix Ex Taq (TaKaRa) according to the manufacturer’s protocol. The specific primers for let-7a, Ki67, cyclinD1, PCNA, and fibronectin (Fn) were used as described previously [21–23]. The expression of let-7a was normalized to U6, and β-actin was used as a quality control to normalize other gene expression. All data were calculated with the 2–ΔΔCt equation.
The collected tissues and cells were lysed with RIPA lysis buffer (Thermo scientific, Rockford, IL, USA) followed by the detection of protein concentration using the BCA assay kit (Pierce, Rockford, IL, USA). Then, equal amounts of samples were subjected to 12% SDS–PAGE, and the separated proteins were electro-blotted onto PVDF membranes (Sigma), which were blocked with 5% nonfat dry milk in TBST for 2 h. The membranes were then incubated at room temperature with the primary antibodies against Ki67, cyclin D1, p21, PCNA, and α-SMA. The HRP-conjugated secondary antibodies were then added for further incubation for 1 h. The binding proteins were visualized using chemiluminescence (Santa Cruz, CA, USA) and quantified by a Gel DocTM XR imaging system (Bio-Rad Laboratories, Hercules, CA, USA).
Cell proliferation assay
PASMC proliferation was assessed using 3H thymidine (3H-TdR; Atomic Nucleus Research Institute, Shanghai, China) incorporation in accordance with the manufacturer’s instructions. Briefly, 37 kBq/ml of 3H-TdR was added to the co-culture system. Twenty-four hours later, cell viability was analyzed by detecting radioactivity with a liquid scintillation counter (Beckmen, USA). At least three independent experiments were performed to evaluate cell viability.
Flow cytometry analysis
Following co-incubation with MSCs for 24 h, the PASMCs were collected. Then, cells were trypsinized and suspended with binding buffer. About 10 μl Annexin V-FITC and 5 μl PI were added for further incubation at room temperature. Fifteen minutes later, all specimens were subjected to a FACScan flow cytometer (Becton Dickinson, NJ) to analyze the relative apoptosis via at least three independent repeats.
Caspase-3 activity was performed using the Caspase-Glo 3/7 Assay kit (Promega, Madison, WI, USA). Following the co-culture with MSC-NC or MSC-let-7a, caspase-3 activity was detected according to the manufacturer’s instructions. The results were obtained from three independent experiments.
All experiments were conducted independently at least three times and all results are shown as mean ± SD. The unpaired Student’s t test was used to analyze the statistical significance of differences between two groups. Comparisons among three or more groups were made with ANOVA followed by the post-hoc test, and p < 0.05 was considered statistically significant.
Characterization of MSCs isolated from bone marrow
Expression levels of let-7a in MSCs
Transplantation of MSC-let-7a ameliorates MCT-triggered right ventricular impairment
Treatment with let-7a/MSCs attenuates pulmonary vascular remodeling induced by MCT
Pulmonary vascular remodeling is the major characterization of PAH with hyperplasia of PASMCs and deposition of extracellular matrix (ECM) [1, 7]. To further clarify the effect of MSC-let-7a on MCT-induced pulmonary vascular remodeling, the expression of ECM protein fibronectin (Fn) was determined. As shown in Fig. 3f, MCT induced an approximately 4.57-fold increase in Fn mRNA levels in the lung, which was notably attenuated following MSC treatment. Moreover, a more obvious decrease in Fn expression was seen in the MSC-let-7a groups than that in the MSC groups. Specifically, the elevated expression of α-SMA, a marker for SMCs that is used to evaluate muscularization of pulmonary arteries, was obviously restrained following injection with MSC-let-7a (Fig. 3g). Importantly, the notable increase in wall thickness of pulmonary arterioles was markedly restrained when rats received MSC-let-7a administration (Fig. 3h). In brief, these data suggested that MSC-let-7a might participate in MCT-induced pulmonary vascular remodeling.
Overexpression of let-7a in MSCs regulates hypoxia-induced PASMC proliferation
MSC-let-7a administration abrogates apoptosis resistance of PASMC
As shown in Fig. 4d, MSC treatment deteriorated PASMC cell apoptosis upon hypoxia. A higher apoptotic rate of PASMCs was validated after co-culture with let-7a-elevated MSCs and reached 31.2 ± 1.04% (Fig. 4e). The increase in caspase 3 activity further identified the enhanced impact of MSC-let-7a on PASMC apoptotic response, in contrast to the MSC-treated group (Fig. 4f). Thus, MSC-let-7a could suppress PASMC growth by dampening cell proliferation and antagonizing cell resistance to apoptosis.
STAT3-BMPR2 signaling is involved in the growth inhibition of PASMCs triggered with MSC-let-7a
Recently, let-7a has generated broad interest as a promising therapeutic agent for several diseases associated with proliferative and metabolic abnormalities, including cancer . A previous study corroborated the downregulation of let-7a in lung injury . Interestingly, its decrease has also been validated in PAH animal models and patients [16, 19]. In this study, depression of let-7a was observed in PAH rats. Moreover, MSC treatment enhanced let-7a expression in the lungs of PAH rats, indicating a potential role of let-7a in MSC-mediated therapy for PAH. Concomitantly, we also simulated the condition of PAH by hypoxia exposure in vitro and observed a high expression of let-7a in hypoxia-treated MSCs, suggesting that MSCs could induce the expression of let-7a in the progression of PAH. Next, we explored the effect of let-7a-modified MSCs on PAH. As expected, we successfully detected the retention of MSCs in lung tissues. Importantly, let-7a-MSC treatment obviously suppressed RVSP and RVH, which is the final pathway leading to the death of patients . Furthermore, application of MSC-let-7a also attenuated the wall thickness of pulmonary arterioles and restrained the expression of ECM protein Fn and α-SMA, indicating an inhibitory effect of MSC-let-7a on vascular remodeling. Importantly, the MSCs modified with let-7a exhibited more favorable effects in ameliorating MCT-induced PAH compared to the MSC-only groups, indicating a promising potential therapeutic role of MSC-let-7a in PAH.
Excessive proliferation and apoptosis resistance of PASMCs are major pathological features of PAH and rank as key events in the pathogenesis of PAH . Furthermore, PASMC proliferation and migration also facilitate the remodeling of the pulmonary artery, which is a vital element of current PAH therapies . Hypoxia is widely accepted as a trigger of pulmonary vascular remodeling and the subsequent pulmonary hypertension. To elucidate the underlying mechanism involved in the MSC-let-7a-mediated protective effect against PAH, the proliferation and apoptosis of PASMCs were investigated. Here, hypoxia exposure significantly induced the proliferation of PASMCs, as supported by previous reports . Nevertheless, this increase was mitigated when cells were incubated with MSC-let-7a. Moreover, MSC-let-7a treatment remarkably attenuated cell resistance to apoptosis. Therefore, these results confirmed that MSC-let-7a suppressed the growth of PASMCs by suppressing cell proliferation and cell resistance to apoptosis, which may ameliorate the progression of PAH.
Signal transducers and activators of transcription 3 (STAT3) signaling exerts important roles in various physiopathological processes, such as angiogenesis, immune response, and cellular functions [28, 29]. Recent research corroborated the abnormal activation of STAT3 signaling in PAH patients . Moreover, the increase in the pro-proliferative phenotype of PASMCs has been shown to be associated with the activation of the STAT3-BMPR2 pathway [17, 30–32]. Emerging studies suggest that let-7a may restrain tumor cell proliferation by blocking the STAT3 pathway [28, 33]. Accordingly, to elucidate the mechanism involved in the inhibitory effect of MSC-let-7a on PASMC growth, STAT3 signaling was evaluated. Consistent with our previous hypothesis, MSC-let-7a significantly antagonized p-STAT3 levels, concomitant with the upregulation in its downstream BMPR2. When blocking the STAT3 signaling with S31-201, an antagonist of the STAT3 pathway, BMPR2 expression was increased. As a surface protein receptor of BMPs, BMPR2 can be expressed on vascular SMCs and identified as a well-established inhibitor of vascular SMC proliferation, but it provokes cell death [31, 34]. Interestingly, suppression of PASMC proliferation and enhancement of cell apoptosis triggered by MSC-let-7a were obviously abated when silencing BMPR2 expression. Therefore, these data suggest that STAT3-BMPR2 signaling might account for MSC-let-7a-regulated PASMC survival. However, blocking BMPR2 levels could not absolutely abolish the effect of MSC-let-7a on PASMC growth. Whether other molecules or pathways are involved in this process will be explored in our future research.
This study indicated that MSC-let-7a could ameliorate the development of MCT-induced PAH by suppressing the growth of PASMCs. Furthermore, the STAT3-BMPR2 pathway was involved in MSC-let-7a-mediated PASMC proliferation. As a whole, our research may illustrate the protective role of MSC-let-7a in the progression of PAH. Therefore, this study provides the groundwork for the further study of MSC-let-7a as a therapeutic strategy against PAH. Thus, it may provide a promising therapeutic approach for PAH. However, further studies will explore the above mechanism in vivo. This study only clarifies the effect of MSC-let-7a on PAH in an animal model. There is still a long way to go to investigate the application of MSC-let-7a in human therapy, which will be the focus of our future research. Futhermore, recent research has also corroborated the critical implication of lipids, especially the oxidized lipids, in the pathophysiological hallmarks of PAH, such as SMC proliferation . Interestingly, a previous study also corroborated the protective effect of let-7a on oxidized low-density lipoprotein-induced endothelial cell injury . Whether MSC-let-7a can attenuate the development of PAH by regulating lipid metabolism will provide a new direction for further research and will be explored in our future research.
Bone morphogenetic protein receptor 2
Fetal bovine serum
Green fluorescent protein
Mesenchymal stem cell
Pulmonary arterial hypertension
Pulmonary artery smooth muscle cell
Proliferating cell nuclear antigen
Rat mesenchymal stem cell
Rat pulmonary artery smooth muscle cell
Right ventricular hypertrophy
Right ventricular systolic pressure
Right ventricle weight
Small interfering RNA
Smooth muscle actin
Smooth muscle cell
Signal transducers and activators of transcription 3
No financial support was used for this study.
Availability of data and materials
The raw data cannot be shared for now since the data may be used in our subsequent research.
Both GC and XW participated in the design of this research. GC performed cell culture and construction. XW and YL carried out the animal experiment assay. GC and LH conducted the assessment of PASMC growth and mechanism analysis. XW wrote the manuscript. All authors read and approved the final manuscript.
The authors declare that they have no competing interests.
Consent for publication
All animals care and experimental procedures received approval from the Institutional Animal Care and Use Committee of the First Affiliated Hospital of Xi’an Jiaotong University.
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