Background
Pulmonary arterial hypertension (PAH) is a rare but life-threatening disease characterized by pulmonary vasoconstriction, endothelial cell proliferation, smooth muscle cell proliferation, and
in situ thrombosis, leading to progressive pulmonary hypertension and ultimately causing right ventricular (RV) failure and death [
1‐
3]. Current therapy with prostacyclin analogs, endothelin-1 receptor blockers, and phosphodiesterase inhibitors improves symptoms and exercise tolerance, but persistent morbidity and mortality indicate that important pathogenic mechanisms are minimally affected. Thus more effective therapeutic approaches are urgently needed [
4,
5]. Recently, transfer of the human prostacyclin synthase (PGIS) gene has been shown to ameliorate monocrotaline (MCT)-induced PAH progression in rats [
6]. However, the molecular networks regulating prostacyclin are largely unknown. Coupling factor 6 (CF6), a novel essential vascular constrictor, is a known endogenous inhibitor of prostacyclin (PGI
2) [
7].
CF6 is a subunit of the stalk that joins the membrane domains, F1 and Fo (F
1F
o complex) of mitochondrial adenosine triphosphate (ATP) synthase. CF6 is released from the surface of vascular endothelial cells by mechanical forces such as the tumor necrosis factor-alpha (TNF-α), shear stress, and high glucose levels [
8,
9]. More recently, studies showed that plasma membrane-bound ATP synthase in the vascular endothelial cells functions as a receptor for CF6 and may play an important role in affecting vascular function by increasing the concentration of intracellular proton, acidosis [
10]. CF6 suppresses PGI
2 synthesis by inhibiting cytosolic phospholipase A [
7] as well as the synthesis of nitric oxide (NO) generation via upregulation of asymmetric dimethylarginine [
10], thereby profoundly affecting vascular function. Specifically, the CF6 injection consistently induces an elevation in arterial blood pressure [
9]. In CF6-overexpressing transgenic mice, endothelial dysfunction increases, and wall thickness and inflammation are increased in small vessels [
11]. These data suggests that CF6, which directly inhibits PGI
2 synthase, may be present in pulmonary arteries and function as a novel target in the treatment of PAH.
Therefore, we aimed to determine the involvement of CF6 in PAH development using MCT-induced PAH rat models. CF6 RNA interference (RNAi) was used to further evaluate the effects of CF6 inhibition on PAH progression. Here, we have demonstrated for the first time that CF6 is released into the extracellular space in MCT-injected rats and that it contributes to PAH development. Our findings provide novel insights into the molecular basis of PAH and present a potential therapeutic target for this disease.
Methods
Preparation of AAV2-CF6-shRNA
A U6 promoter-driven shRNA expression system was established in an AAV2 vector. Green fluorescent protein (GFP) expression was separately controlled by a CMV promoter as a marker for transduction efficiency. CF6 shRNA was designed based on the siRNA sequence (GenBank Acc. NM_053602.2) using an siRNA design tool (Biomiao, Beijing, China) and was screened according to the guidelines reported by Elbashir SM et al. [
12]. Four selected siRNA target sequences were inserted between the KpnI and EcoRI sites in a U6-CMV-EGFP/AAV vector, and an optimal CF6 target (sequence: 5′-CAGGACTTAAAGGCTCTTAAT-3′) was selected. A recombinant adenovirus carrying an siRNA sequence targeting the eGFP reporter gene (sequence: 5′-CACCGTTCTCCGAACGTGTCACGTCAAGAGATTACGTGACACGTTCGGAGAATTTTTTG-3′) was included as a control. Both the adenovirus-CF6-shRNA and negative control vectors contained the sequence encoding GFP. All constructs were verified by DNA sequencing, all viral vectors were generated by triple-plasmid cotransfection of human 293 cells, and recombinant virions were column purified as previously described [
13]. Next, viral titers were determined using qPCR [
14]. The resulting AAV2-CF6-shRNA titer was determined to be 2.5 × 10
11 vector genomes (vg)/ml, and the AAV2-GFP titer was 1 × 10
12 vg/ml.
Animal models
Sprague–Dawley rats (6–7 weeks old, weighing 200–250 g, obtained from the Laboratory Animal Center, Chinese Academy of Science, Beijing) were used in this experiment. The rats were housed at 20 ± 3 °C under a 12-h light/12-h dark cycle with free access to food and water. All procedures were carried out according to approved protocols and guidelines established by the Shandong University Institutional Animal Care and Use Committee.
CF6 expression study was performed in 2 models of PAH: I: animals received a single subcutaneous injection of MCT (Sigma, St. Louis, MO) (60 mg/kg), which is a pyrrolizidine alkaloid that has been previously reported to induce PAH at 2 to 3 weeks after injection [
15]. The animals in the sham group received subcutaneous injection of saline. The 24 survivors of the 40 enrolled rats (mean survival rate = 60 %) were randomly assigned to the following 4 groups: 1) the MCT-vehicle-1 week group (
n = 6); 2) MCT-vehicle-2 week group (
n = 6); 3) MCT-vehicle-3 week group (
n = 6); and 4) MCT-vehicle-4 week group (
n = 6). II: animals intubated via tracheotomy, and ventilated with a small-animal ventilator (HX-300S, TME, Chengdu, China), with an adjusted rate of 60 breaths/min and a tidal volume set to 1.1–1.3 ml/100 g body weight, then received left unilateral pneumonectomy as described previously [
16]. Seven days after pneumonectomy rats received MCT or saline injection. As a result, 24 survivors of the 56 enrolled rats (mean survival rate = 43 %) were randomly assigned to the 4 groups as reported in I (
n = 6 per group).
CF6 knockdown experiment was performed to evaluate the prevention and reversal effect of CF6 inhibition, the rats were randomly assigned to the following groups: the sham group (
n = 20), MCT-vehicle group (
n = 30), MCT-control group (
n = 60) and MCT-shRNA group (
n = 40). The MCT-control group received intratracheally injection of AAV-GFP-shRNA, while the MCT-shRNA group received intratracheally of AAV-shRNA-CF6 (80 μl; 2.5 × 10
11 vg/ml) at 2 weeks prior or 2 weeks after MCT injection [
6]. The MCT-control and MCT-shRNA groups were further divided into two subgroups by virus injection time-point: i.e. -2w-MCT group (
n = 30), 2w-MCT group (
n = 30), −2w-shRNA group (
n = 20), and 2w-shRNA group (
n = 20). All animals were sacrificed at 4 weeks after MCT injection. The rats were monitored daily until they developed pulmonary hypertension symptoms such as weight loss and tachypnea until 28 days after MCT administration. Hemodynamic, morphologic, and biochemical assessments were performed.
Echocardiography and hemodynamic measurements
The rats in the experimental groups were anesthetized by intraperitoneal injection of sodium pentobarbital (30 mg/kg). The room temperature was maintained at approximately 25 °C to avoid hypothermia. A Visual Sonics Vevo 770 echocardiographic machine (Visual Sonics, Toronto, Canada) equipped with a 14-MHz linear transducer was used to assess cardiac function. The measurements were performed in a blinded manner by an echocardiographic expert. Short- and long-axis B-dimensional parasternal views of both ventricles at the level of the papillary muscles were acquired to visualize the areas of the left ventricle (LV) and the right ventricle (RV). Cardiac output and stroke volume were obtained from the B-mode long axis according to Simpson's method, while the pulmonary artery diameter and RV wall thickness were obtained in M-mode. Doppler was applied to the pulmonary artery to obtain the pulmonary artery acceleration time [
17].
Blood pressure was evaluated by the tail-cuff method, using a non-invasive automatic blood pressure recorder (BP-98A; Softron, Tokyo, Japan). Each value was the average of at least three consecutive data [
18]. Prior to sacrifice of the animals, RV systolic pressure (RVSP) was transduced from the right jugular vein into the vena cava, into the right atrium and then into the right ventricle using a 1.4 F Millar Mikro-Tip catheter transducer (Millar Instruments Inc., Houston, TX). The position of the catheter into the right ventricular was vadilated by acutely increased pressure wave accompanied by the loss of resistance, then RVSP was measured with Power Lab monitoring equipment (Millar Instruments). Hemodynamic values were automatically calculated using a LabChart 7.0 physiological data acquisition system (AD Instruments, Sydney, Australia). The animals were then euthanized before being sacrificed.
Tissue processing and histology
After completion of the above measurements, cardiac arrest was induced by injection of 2 mmol KCl through the catheter. Blood from the RV was stored at room temperature for 1 h and was then centrifuged at 3000 rpm at 4 °C for 15 min. Blood serum was collected and stored at −80 °C, and the rats were euthanized by exsanguination. Next, the lungs were weighed, and the L/BW ratio was calculated. The right lung was removed and frozen in liquid nitrogen for Western blot analysis. The left lung was inflated with 0.5 % low-melting agarose at a constant pressure of 25 cm H
2O, fixed in 10 % formalin for 24 h and used for small pulmonary artery and IHC analyses. Then, the heart was excised and the weight ratio of the right ventricle to the left ventricle plus the septum (RV/LV + S) was determined using Fulton’s index [
19].
Immunohistochemistry
The left lung lobes were longitudinally cut and processed as described previously [
17] by preparing standard formalin-fixed, paraffin-embedded tissues for HE or regular immunohistochemistry staining. Tissue samples were sectioned at a thickness of 5 μm [
20]. Sections stained with hematoxylin and eosin (HE) were examined at 400x magnification to determine the severity of PAH in vessels with diameters of either < 50 or 50 to 100 μm, including the percentage with medial hyperplasia and the percentage occluded [
21]. Anti-CF6 (1:150, Abcam), α-SMA (1:500; Abcam), PCNA (1:1000; Millipore) and anti-CD68 (1:400; Santa Cruz) antibodies were used as primary antibodies. Subsequently, slides were incubated with an ABC Elite Kit (Vector Laboratories) and DAB substrate (Vector Laboratories) and counterstained with hematoxylin.
Lung samples from 10 patients with NCSCL at the time of a lobectomy or pneumonectomy for localized lung cancer in the Department of Thoracic Surgery (Qianfoshan Hospital of Shandong University) from 2012 to 2016. Human lung specimens was fixed in formalin for at least 72 h, and then paraffinembedded and cut into 5-mm slices for histology processing. Immunohistochemistry was performed as described above, except that the anti-CF6 polyclonal antibody was used at a dilution of 1:300.
A pathologist blinded to the study reviewed 10 sections per lung. All images were obtained using an Olympus LCX100 Imaging System and analyzed with ImageJ software (version 1.38x; National Institutes of Health). The studies were approved by the Institutional Review Board of Shandong University.
Western blotting
For immunoblot analyses, RIPA buffer (Beyotime Institute of Biotechnology, Jiangsu, China) was used to extract total protein from frozen lung tissues. The quantity of protein extracted from the tissues was measured using a BCA protein assay reagent kit (Pierce). An equal amount of total protein (80 μg of lane) from each sample was resolved on a 5–8 % SDS-PAGE gel and transferred onto a polyvinylidene difluoride (PVDF) membrane. The membranes were blocked with 5 % nonfat dry milk in PBST containing 0.05 % Tween 20 and incubated overnight at 4 °C with a CF6 primary antibody (1:1500; Abcam). The blots were developed using an enhanced chemiluminescence (ECL) detection kit (Millipore) and visualized using a FluroChem E Imager (Protein-Simple, Santa Clara, CA, USA). Measurements to determine the relative densities were normalized to that of a standard protein (GAPDH) (Proteintech, Wuhan, China) using NIH Image J software.
qRT-PCR
Total RNA was extracted from lung tissues with Trizol reagent (Invitrogen). cDNA was synthesized from 2 μg RNA using a Prime Script RT Reagent Kit (TaKaRa, Dalian, China) according to the manufacturer's instructions. CF6 mRNA expression was determined using gene-specific primers and SYBR Green 1 with a Bio-Rad iQ5 Multicolor Real-Time PCR machine (Bio-Rad Laboratories). For each sample, both GAPDH and the target gene were amplified in triplicate in separate tubes. Relative gene expression was calculated by the 2
-ΔΔCT method [
22] and normalized to GAPDH expression. The primers used in this study were as follows:
-
CF6: forward: 5′-TGTCCTTCGGTCAGCAGTCTC-3′, reverse, 5′-AACTTATCCATCTCTCCTTTA-3′;
-
GAPDH: forward, 5′-AGATCCACAACGGATACATT-3′, reverse, 5′-TCCCTCAAGATTGTCAGCAA-3′.
Determination of endothelial dysfunction in the pulmonary artery
After the rats received MCT for 28 days, the branch of the pulmonary artery next to the heart was removed, and the pulmonary arteries from the individual rats were rapidly separated and placed in cold oxygenated Krebs’ solution composed of the following (mmol/L): 118.3 NaCl, 4.7 KCl, 2.5 CaCl2, 1.2 KH2PO4, 1.2 MgSO4-7H2O, 25 NaHCO3 and 11.1 glucose. The tissues were then oxygenated with carbogen gas at 37.0 ± 0.5 °C. The right and left pulmonary arteries were carefully dissected to remove fat and connective tissue and cut into 3 mm-wide rings. After an equilibration period of 2 h at 1.5 g resting tension, the preparations were exposed to increasing concentrations of Phe (10−10 to 10−5 mol/L). After a contraction plateau was reached, the pulmonary artery rings were exposed to increasing concentrations of ACh (10−10 to 10−5 mol/L) to determine the vasodilation capacity and endothelial dysfunction.
ELISA
The blood samples obtained from the RV were stored at room temperature for 1 h and were then centrifuged at 3000 rpm at 4 °C for 15 min. Serum samples were collected and stored at −80 °C. The level of 6-keto PGF1a, a stable metabolite of prostacyclin, was measured to assess prostacyclin production. The serum 6-keto PGF1a and CF6 levels were measured using a commercial enzyme immunoassay kit (EIA Assay Design, Inc.; Ray Biotech, Norcross, GA, USA) according to the manufacturer's instructions. Rat organs were harvested and homogenized to measure the 6-keto PGF1a levels in the tissues. The results were expressed as picograms of 6-keto-PGF1a per milligram of protein.
Statistics
The data are presented as the mean ± standard deviation (SD). The unpaired t-test was used to compare values between two groups. ANOVA was used to compare differences between more than two groups, followed by a Newman–Keuls test. Analyses were performed using SPSS 17.0 software (SPSS Inc. Chicago, IL, USA). A p-value of < 0.05 was considered statistically significant.
Discussion
To our knowledge, the current study is the first to elucidate the role of CF6 in the pathology of PAH. A single injection of MCT caused persistent severe PAH, which shares important features with human diseases, such as pulmonary arterial endothelial cell (EC) injury and subsequent pulmonary artery smooth muscle cell (PASMC) hypertrophy [
26]. Based on the model, we demonstrated that (1) CF6 was significantly upregulated in lungs and serum of PAH rats, specifically, in the lung tissues; (2) CF6 gene silencing using AAV2-CF6-shRNA induced down-regulation of CF6 protein in the lung tissues of the PAH rats, along with reversed prostacyclin synthesis; and (3) transfer of CF6 shRNA ameliorated MCT-induced pulmonary vascular remodeling and PAH progression. Finally, we demonstrated that (4) CF6 knockdown as a therapeutic approach in the MCT rats without causing definite adverse effects.
CF6, a component of the peripheral stalk in ATP synthase under physiological conditions, is known to elicit its novel function outside the cells after being released from the plasma-membrane of ATP synthase in a pathological state [
27]. In clinical settings, we and others have previously shown that the plasma CF6 level increases in various cardiovascular disorders, where a cause–result connection between increased plasma CF6 and decreased PGI2 has been indicated [
8,
28‐
30]. However, the CF6 level in PAH has not been explored. There exhibited significant overproduction of CF6 in the lung tissues and elevation in circulating CF6 among PAH rats of MCT models and MCT plus pneumonectomy models, most likely due to mechanical stress secondary to the elevated pulmonary pressure and the inflammation activation induced by MCT, such as NF-kB activation and TNF-α releasing [
7,
31,
32]. ECs represents a main source of circulating CF6 [
9]. In hypertension study, intravenous injection of recombinant CF6 mimetics increased blood pressure transiently, apparently by suppressing PGI
2 synthesis, whereas a specific antibody against CF6 decreased systemic blood pressure with a concomitant increase in plasma PGI
2 in hypertension rats. Except for endocrine manner, our data showed that the limited distributed CF6 observed in ECs in normal rats was released into surrounding tissues and may interact with the interact with plasma membrane ATP synthase, the receptor of CF6 presented on vascular cells such as ECs and vascular smooth muscle cells (VSMCs) function in a paracrine manner, supported by a previous study identified that the high local CF6 levels in tissues such as resistance arterioles could directly enhancing Ca
2+ signaling in VSMCs and vasoconstriction in the mesenteric arteriolar network in a paracrine manner [
10]. In addition, the CF6 level did not affect systematic blood pressure in PAH rats. Therefore, it appears that different from the direct role in pathological hypertensive as a systemic hormone and vasoconstrictor, CF6 may function on PAECs and PASMCs in a paracrine manner in PAH, which is consistent with the fact that pulmonary circulation is selectively inhibited in humans with PAH.
We chose the RNAi strategy over CF6 antibodies to further determine whether the high CF6 level is a cause or merely an effect of PAH given that a therapeutic antibody should decrease the circulating levels of free CF6 to zero and have a limited duration of effect [
33], and especially considering that CF6 signaling proteins have short half-lives. Various “RNA interference (RNAi) therapeutics” have already entered clinic trials [
34]. Investigators showed that AAV-2 is an efficient vector for delivering genes to pulmonary vessels and lungs to treat PAH [
35]. We adopted the intratracheal route for AAV2 delivery of CF6 shRNA because the majority of drugs targeting the vasculature will, if administered systemically, affect normal circulation as well; in contrast, gene transfer of specific peptides to bronchial epithelial cells and alveolar cells selectively affects the pulmonary artery in animal models [
36,
37]. As a result, CF6 RNAi reversed ECs dysfunction and PA remodeling, indicating that through the secretion of CF6, ECs regulates the proliferation of SMCs in their vicinity. The data described above revealed that, in addition to its vasoconstrictive properties, CF6 is also involved in the proliferation of PASMCs. The downstream signaling of CF6 is mediated via plasma membrane ATP synthase receptor. After combination, the molecular rotary motor, F1-ATPase in ECs and VSMCs, forcefully hydrolyzes ATP in a mode that is in the reverse direction of the mode used in the mitochondria. Then, this hydrolysis inversely rotates the Fo motor against the original clockwise direction, resulting in proton import. CF6 and the plasma membrane-F1Fo complex mediated VSMC remodeling is well established and that the downstream the intracellular pH (pHi) changes are associated with cell proliferation and migration in VSMCs [
28]. Additional methods of CF6 intervening in vitro are needed to define the underlying post receptor signaling mechanisms and the direct role of CF6 on ECs and PASMCs.
Suppressed PGI
2 is a well-known target of the increasing intracellular acidosis of CF6. In PAH, the primary effects of IP receptor activation (e.g. by prostacyclin or its analogues) are the induction of pulmonary artery dilation and the inhibition of vascular smooth muscle cell proliferation. Treatments that target the prostacyclin pathway are crucial for the effective management of patients with PAH [
25,
28,
38,
39]. Our results indicated that the impaired PGI
2 generation was enhanced in PAH by the overproduction of CF6 and restored by CF6 silencing. Therefore, we speculate that the impaired pulmonary vascular PGI
2 generation may be the target of CF6 and contributes to pulmonary vasoconstriction and the excessive medial hypertrophy observed in this PAH model. In addition, the CF6 peptide may influence PAH by a more direct mechanism that may be independent of the complex effects of PGI
2. After binding to the β-subunit of the ATP synthase at the cell surface, CF6 could further activate c-Src or RAC1 [
7,
10,
40], which potentially enhances Ca
2+ influx and sensitization [
10,
28,
41,
42]. Aberrant expression of these downstream signaling proteins cause a mitochondrial metabolic abnormality that plays a pathophysiological role in PAH. Moreover, RNAi of CF6 effectively prevented macrophage infiltration, which is involved in the development of PAH-associated vascular lesions and has been well documented in humans [
43,
44]. This suggests that CF6 plays a critical role in the inflammation process of PAH; however, the complex mechanism remains poorly understood. These theories provide a strong impetus for ongoing efforts to define the mechanisms of CF6.
Prospective
Although many vasodilators have antiproliferative effects on VSMCs, there is no definitive evidence that pulmonary vascular remodeling in human PH is reversible. Therefore, novel approaches that directly target pulmonary vessel wall pathology are needed to reverse the established pulmonary vascular pathology in PH patients. We found that CF6 levels increased in MCT model, MCT plus left pneumonectomy model and human lung cancer tissue, while the AAV2-based RNAi targeting CF6 reversed the PA remodeling and PH in MCT-induced PAH. The above makes CF6 a target molecule for novel therapeutic strategies in the treatment of clinical PH regardless of the different clinical forms. Future experiments on hypertrophied pulmonary arterioles and lung tissues from human samples with PAH are needed to confirm CF6 expression. Besides, despite of the fact that CF6 is implicated in multiple clinical settings and experimental models, no pharmacologically active small molecule inhibitors of CF6 has been developed. Therefore, the specific plans aiming at blocking CF6 pathway that can be applied in PAH treatment is warranted to be explored in the future.
Limitations
First, the MCT-induced PAH model does not accurately reflect the clinical picture of PAH in humans and the high death rate could cause potential selective bias. Further investigations of other animal models of PAH (Sugen-hypoxia) are needed to determine the clinical efficacy of CF6 inhibition. Second, our study was performed within a limited window of time and involved only short-term animal research; therefore, the results only provide an impetus for further clinical investigation. Third, the AAV vectors used were capable of inserting their genome at random positions in the host chromosomes, which eventually restricts gene function and may cause mutations. Moreover, the interpretation of RNAi data is complicated by unintended interactions between silencing molecules and cellular components, and additional shRNA sequences should be designed to decrease the risk of unwanted artifactual off-target effects [
45]. Despite these limitations, our data demonstrate a potential role of airway delivery of shRNA-CF6 in paracrine inhibition of CF6 secretion into the pulmonary vasculature in PAH human clinical trials.
Acknowledgements of grant support
The work was supported by the following grants: the National Natural Science Foundation of China (NSFC, 81370157), the Doctoral Fund of the Ministry of Education of China (20130131110069), Science and Technology Development Planning of Shandong Province (2013GGB14056), the Independent Innovation Foundation for Jinan Science and Technology Development Planning (201311020) and the Shandong Taishan Scholarship (Suhua Yan).