Experimental murine model with MCT-induced cardiopulmonary disease (pulmonary hypertension, and associated perivascular/pulmonary fibrosis and right ventricular hypertrophy) injected or not with siRNA AP-1
A total of thirty-nine male Golden Syrian hamsters, approx. 3 months of age, with a body weight of 100-130g, were randomly assigned into three experimental groups, including: normal healthy animals for control group (C) (n = 12); MCT group (n = 14) that received a single subcutaneous injection of 60mg/kg body weight MCT at the beginning of the experiment (day 0), in order to induce pulmonary arterial hypertension (PAH) with subsequent right ventricular hypertrophy; MCT-siRNA AP-1 group (n = 13), which 2 weeks after the first injection with MCT, received treatment with siRNA AP1 (five doses with a concentration of 100 nM each in a volume of 300 µl of phosphate-buffered saline (PBS)), administered subcutaneously every two weeks until the end of the 3-month experimental period (Fig. 1).
Previous animal studies (performed on mice or rats) indicated that a high dosage of 80 mg/kg of MCT was fatal in the first 3 weeks [26] so we chose to administer a dose of 60 mg/kg MCT to our animal model. This selected concentration was sufficient to generate the animal model with pulmonary arterial hypertension developed over an extended period of time, reflecting the chronic pathological processes present in sick human subjects as well. The aqueous solution of MCT was prepared according to the protocol described in 1967 by Hayashi et al., [27]: MCT (170mg, Sigma-Aldrich) was dissolved in 1.2 ml of 1 N HCl solution followed by addition of 3 ml distilled water, pH was adjusted to 7.4 using 1 M NaOH solution, and later distilled water was added up to a final volume of 12 ml.
During the entire experimental period of 3 months, all three experimental groups of hamsters were kept in the same housing conditions with a 12/12 h light/dark cycle, a temperature of 25°C, humidity of 55%, were fed with standard chow containing basal 1%NaCl and received free access to tap water. Body weight was measured at the beginning of the experiment to adjust the treatment doses accordingly and at the end of the 3 months (12 weeks) of experiment, when the hamsters were lightly anesthetized with 2% isoflurane for blood collection. Subsequently, they were sacrificed under intraperitoneal anaesthesia (a solution containing 80mg ketamine, 10mg xylazine, 2mg acepromazine/kg body weight in a sterile isotonic saline (0.9% saline)), perfused with PBS containing 1mM CaCl2 for tissues blood removal in order to collect the organs of interest (pulmonary artery, lung, right ventricle) for biochemical, structural and functional assays. The experimental protocols were approved by the Ethics Committee from Institute of Cellular Biology and Pathology „Nicolae Simionescu” according to Decision no.11/08.08.2017 and National Sanitary Veterinary and Food Safety Authority (Bucharest, Romania) in compliance with Project Authorization no. 575/13.11.2020. Also, all the animal procedures were carried out in strict accordance with the Guide of the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH publication no. 85 − 23, revised 1996) and were conducted in accordance with National, European and International legislation on the use of experimental animals in biomedical research. All surgical procedures were performed under anaesthesia (mild or total) without causing the animals suffering.
Biochemical analysis of plasmatic parameters
Blood samples were taken from all experimental animal groups by collecting approximately 1ml of venous blood from the retro-orbital plexus in vacutainers containing ethylenediaminetetraacetic acid (EDTA) solution and centrifuged at 2500 x g for 10 min at 4°C to obtain plasma. For this, animals were lightly sedated via inhalation with 2% isoflurane (ISOFLUTEK 1000mg/g) mixed with oxygen under fasting conditions. The procedure was performed at 4 weeks and at the end of the 3-month experimental period. Plasma levels assessing the lipid profile (total cholesterol, HDL-cholesterol, LDL-cholesterol, triglyceride), glucose and hepatic transaminases (ALT-Alanine Aminotransferase, AST-Aspartate Aminotransferase), were determined by a colorimetric/kinetic method using commercially available kits from DIALAB GmbH, Vienna, Austria. The samples were measured in duplicate using a spectrophotometric assay at a wavelength between 340–500/600 nm (Tecan Infinite M200 PRO).
Non-invasive measurement of haemodynamic parameters
Blood pressure in hamsters was measured with the ADInstruments ML125NIBP (Non-Invasive Blood Pressure) Controller connected to a PowerLab 4/26 system, using specialized tail cuffs. The advantage of this technique is that it provides an accurate recording of pulse rate and blood pressure in the caudal artery in real time. The LabChart software allows the opening of two channels, one for the pulse signal (BPM) and the other for pressure (mmHg), for a typical recording of these parameters. As the cuff inflates, the blood flow in the tail is obstructed and the measurement of the systolic arterial pressure is performed at the moment of the appearance of the first noise determined by the pressure of the cuff. The last noise picked up by the transducer before total deflation at the cuff corresponds to the diastolic blood pressure. Before starting the measurements, the animals were immobilized with the help of a special cage and kept in the dark for 30 minutes. Every measurement was repeated 8–10 times for the reproducibility of the results. Five animals from each group were evaluated.
Echocardiographic assessments
Transthoracic echocardiography was performed at the end of the 12 experimental weeks. The high-resolution ultrasonic imaging system for small animals Vevo 2100 equipment (VisualSonics Inc., Toronto, ON, Canada) with MS 250 transducer (12–24 MHz) was used for this diagnostic procedure. The animals have been prepared in advance by removing the fur from the chest with a hair clipper so as not to interfere with the signal of the device. Throughout the procedure, hamsters were lightly anesthetized with 2% inhaled isoflurane in combination with oxygen and placed on a heated platform to maintain a constant body temperature. Vital signs (heart rate, pulse) were constantly monitored. Through this analysis, parasternal sections were obtained both on the long and the short axis to determine the structure and function of the right ventricle and the pulmonary artery. B mode (two-dimensional), M mode were used to assess anatomical and functional characteristics and Pulsed Wave (PW) Doppler mode was used to measure the hemodynamic characteristics of blood flow. The right side of the heart was investigated in: (1) parasternal long axis view, in order to measure pulmonary artery (PA) diameter, right ventricle wall thickness (RVWT) at the level of proximal right ventricle (PRRV), proximal ventricle outflow chamber during systole and diastole (PRVOFd, PRVOFs) and pulsed waved doppler (PW) for PA peak flow velocity (PFV) and velocity time integral (VTI); (2) parasternal short axis view for PA PFV and VTI measurement; and (3) in apical four chamber were measured RV systolic and diastolic area and tricuspid annular plane systolic excursion (TAPSE). VevoLab300 software was used to process and analyse images obtained from all groups of animals. Five animals from each group were followed.
Hamster echocardiography
Echocardiographic assessment was performed focusing the right ventricular (RV) function at the
end of the study (week 12). Hamsters were anesthetized using isoflurane and subjected for
transthoracic ultrasound examination using Vevo 2100 equipment (VisualSonics Inc., Toronto,
ON, Canada) with MS 250 transducer (12–24 MHz). We investigated the right side of the heart
in: parasternal long axis view, in order to measure pulmonary artery (PA) diameter, right
ventricle wall thickness (RVWT) at the level of proximal right ventricle (PRRV), proximal
ventricle outflow chamber during systole and diastole (PRVOFd, PRVOFs) and pulsed waved
doppler (PW) for PA peak flow velocity (PFV) and velocity time integral (VTI); parasternal
short axis view for PA PFV and VTI measurement; and in apical four chamber view we
measured RV systolic and diastolic area and tricuspid annular plane systolic excursion (TAPSE)
Hamster echocardiography
Echocardiographic assessment was performed focusing the right ventricular (RV) function at the
end of the study (week 12). Hamsters were anesthetized using isoflurane and subjected for
transthoracic ultrasound examination using Vevo 2100 equipment (VisualSonics Inc., Toronto,
ON, Canada) with MS 250 transducer (12–24 MHz). We investigated the right side of the heart
in: parasternal long axis view, in order to measure pulmonary artery (PA) diameter, right
ventricle wall thickness (RVWT) at the level of proximal right ventricle (PRRV), proximal
ventricle outflow chamber during systole and diastole (PRVOFd, PRVOFs) and pulsed waved
doppler (PW) for PA peak flow velocity (PFV) and velocity time integral (VTI); parasternal
short axis view for PA PFV and VTI measurement; and in apical four chamber view we
measured RV systolic and diastolic area and tricuspid annular plane systolic excursion (TAPSE)
Hamster echocardiography
Echocardiographic assessment was performed focusing the right ventricular (RV) function at the
end of the study (week 12). Hamsters were anesthetized using isoflurane and subjected for
transthoracic ultrasound examination using Vevo 2100 equipment (VisualSonics Inc., Toronto,
ON, Canada) with MS 250 transducer (12–24 MHz). We investigated the right side of the heart
in: parasternal long axis view, in order to measure pulmonary artery (PA) diameter, right
ventricle wall thickness (RVWT) at the level of proximal right ventricle (PRRV), proximal
ventricle outflow chamber during systole and diastole (PRVOFd, PRVOFs) and pulsed waved
doppler (PW) for PA peak flow velocity (PFV) and velocity time integral (VTI); parasternal
short axis view for PA PFV and VTI measurement; and in apical four chamber view we
measured RV systolic and diastolic area and tricuspid annular plane systolic excursion (TAPSE)
Myographic analysis of functional responses of isolated pulmonary arteries
In order to see the response capacity of the pulmonary artery to contract and relax after MCT injection or siRNA AP-1 treatment and to analyse vascular dysfunction, the myographic system (Multi Myograph System-model 620M in combination with the Automatic Buffer Filler System-625FS - Danish Myo Technology, DMT), a device that investigates the vascular reactivity, was used. The isolated arterial fragments (150 ÷ 200 µm in diameter) were placed on an elastomer to remove any lipid residue and cut under a microscope to a length of 2 mm. They were then mounted in the wire-myograph chamber and immersed in HEPES sodium salt buffer, pH = 7.45 at 37°C. Subsequently, ACh (acetylcholine) was used to induce vessel relaxation and NA (noradrenaline) to induce vessel contraction. Before recordings starts, the calibration of the optimal operating diameter was performed and the standard start procedure was checked for the integrity of the mounted blood vessel and the presence of the intact endothelium by stimulation with 3x10− 7 M NA. Oxygen bubbling in the myograph’s organ chamber was constant throughout the experiment to maintain optimal blood vessel function. The responses of pulmonary artery in the presence of curves of increasing concentrations of NA or ACh were recorded in real time. The concentration curve used was as follows: 10− 8 M, 3x10− 8 M, 10− 7 M, 3x10− 7 M, 10− 6 M, 3x10− 6 M, 10− 5 M, 3x10− 5 M, 10− 4 M for both NA and ACh. Following the presence of the vasodilator agent ACh or vasoconstrictor agonist NA, the forces that developed in the vascular wall were measured every 2 minutes. Finally, the wire tension (mN/mm) developed by the blood vessels in the presence of the vasoconstrictor NA was evaluated and the relaxation capacity of the arteries in the presence of the vasodilator ACh was calculated as a percentage (%) of the maximum value of precontraction to NA. PowerLab 4/26 hardware (ADInstruments) was used for data acquisition and LabChart7 (multi-channel chart recorder) software was used for image recording.
Measurements of serum inflammatory markers
Blood samples were collected on anticoagulant from the retro-orbital venous plexus from all groups of animals and analysed after centrifugation at 2500 x g for 10 min at 4°C. Transforming growth factor beta 1 (TGF-β1), Endothelin-1 (ET-1), Interleukin-1 beta (IL-1β) and Tumor Necrosis Factor alpha (TNF-α) were evaluated at 4 weeks and at the end of the 3-month experimental period. The tests were performed using commercially available enzyme-linked immunosorbent assay (ELISA) colorimetric kits: Human / Mouse TGF-beta 1 Uncoated ELISA Kit (Invitrogen), ET-1 Kit (R&D Systems), Mouse IL-1β and TNF-α DuoSet ELISA (R&D Systems) with a lower detection limit of 7.8pg/ml, 0.39pg/ml, 15.6pg/ml, respectively 31.3pg/ml according to the manufacturer's protocol. To determine the optical density of the samples, a microplate reader set to a wavelength at 450 nm with a correction set to 540nm was used, as suggested by the manufacturer.
Analysis of different cell types in bronchoalveolar lavage fluid
To isolate bronchoalveolar lavage (BAL) fluid, the hamsters were anesthetized with a solution administered intraperitoneally containing 80mg ketamine, 10mg xylazine, 2mg acepromazine/kg body weight and the neck area was disinfected with 70% ethanol. With the help of a surgical scissors, a longitudinal incision was made in the neck to remove the muscle and salivary glands and to expose the trachea without injuring or cutting other blood vessels, keeping the area clean. With an 18G needle, a small semi-excision was made in the trachea under the laryngeal cartilage between two cartilaginous rings and with a cotton thread, the catheter (consisting of a 20G needle covered by a transparent plastic polypropylene tube) was fixed very well. The insertion of the lavage tube should not be made too low to avoid structural damage to the lung. A syringe was loaded with 1ml of sterile Hanks′ Balanced Salt Solution (HBSS buffer from Gibco) containing 100µM EDTA and gently 500µl of salt/EDTA solution were injected into the lung. While the lavage was being aspirated, a chest massage was performed, and then the syringe was detached from the needle and transferred to a clean collection tube, the process being repeated 8–10 times. A volume of 10 ml salt/EDTA solution was used for each animal. The collected sample was kept on ice. The whole procedure was performed under a stereomicroscope. The tube with the pulmonary aspirate was centrifuged at 800 x g for 10 min at 4°C. This protocol was adapted after previously described by [28, 29]. The resulting pellet, containing the cellular influx in the lung, was washed with filtered PBS, centrifuged at 800 x g for 10 min at 4°C and the supernatant was removed. Subsequently, the cell pellet was resuspended in 200 µl Ammonium-Chloride-Potassium (ACK) Lysis Buffer (Gibco) and incubated for 2 min at room temperature (RT), over which 1 ml of PBS was added and again centrifuged. The pelleted cells were resuspended in 500 µl of PBS, counted on the hemocytometer chamber and then labelled with specific antibodies for different cell types: T cells (CD3e+, Invitrogen), T helper cells (CD4+, Abcam), alveolar macrophages (Singlec-1+, SantaCruz Biotechnology), dendritic cells (CD11c+, SantaCruz Biotechnology) and proinflammatory macrophages (MHC-II+, Invitrogen). After a 40-minute incubation at RT in the dark, the cells thus marked were analysed by flow cytometry. The analysis of the different cell types in the BAL fluid was done according to the protocol described by [30]. For all these markers, the gates were set using the unmarked sample. A total of 10,000 events were counted for each sample.
Western Blot analysis
Protein extraction. Snap-frozen pieces of the pulmonary artery were processed for protein extraction. For this procedure, the samples were finely ground with a pair of scissors, then immersed in RIPA buffer (Thermo Scientific) containing 100mM PMSF and a cocktail of phosphatase B inhibitors and protease inhibitors, and finally they were homogenized using 1mm diameter glass beads and Minilys Personal High Power Tissue Homogenizer (Bertin Technologies) at 5000 rpm x 10 rounds for 2 min each with a 2 minute break on ice. After processing, the samples were kept for 3 hours at -80°C, then allowed to thaw at RT and centrifuged at 15600 rpm, for 5 min, at 4°C. The supernatant containing the cytosolic protein fractions (protein lysate) was collected in new tubes and the protein concentration was quantified using the BCA Protein Assay Kit (ThermoScientific). Sample replicates with a working range of 20-2000µg/ml were measured at a wavelength of 562nm on a reading plate and reported on a standard curve of bovine serum albumin (BSA).
Gel electrophoresis and immunoblotting. Equal amounts of protein (100µg/lane) were separated by 8%-12% SDS-PAGE gel electrophoresis under denaturing conditions and transferred onto nitrocellulose membrane. For each set of experiments, a wide range molecular weight marker (6.5 ÷ 200 kDa) (Sigma-Aldrich) was loaded into one or two lanes serving as standard. Blockade of nitrocellulose membranes was performed prior to antibody labelling in a non-specific site blocking solution (TBST solution with 3% BSA (AppliChem) or TBST solution with 5% Blotto, non-fat dry milk (Santa Cruz Biotechnology)) for 1hour at RT. The membranes were incubated at 4°C overnight with the following primary monoclonal antibodies: AP-1 28kDa (1:500, Sigma-Aldrich), pFAK 125kDa (1:200, SantaCruz Biotechnology), FAK 125kDa (1:200, SantaCruz Biotechnology), pERK 42/44kDa (1:1000, R&D Systems), ERK 42/44kDa (1:200, Abcam), and incubated for 1 hour with β-actin 42kDa or GAPDH 36kDa – used as loading control (1:200, SantaCruz Biotechnology/ 1:1000, Abcam 37168) which recognize the proteins of interest. The membranes were washed thoroughly three times with TBST (Tris Buffered Saline and 0.05% Tween 20) and subsequently incubated with horseradish peroxidase-conjugated secondary antibodies: anti-mouse antibodies (1:5000, ThermoFisher Scientific) or anti-rabbit antibodies (1:5000, ThermoFisher Scientific), which specifically recognize primary antibodies, for 1 hour at RT. Detection was performed with enhanced chemiluminescence reagents (ECL) (AppliChem) and the protein bands were quantified with the TotalLab program. The intensity of the bands was normalized to β-actin/GAPDH levels, a housekeeping reference protein or to the total form of the protein.
Transmission Electron Microscopy
Under deep anesthesia, the animals were perfused through the left ventricle with modified Karnovsky's fixative consisting of 2.5% glutaraldehyde and 1.5% paraformaldehyde in 0.1M sodium cacodylate buffer (pH 7.2). Small fragments of pulmonary artery, lung and right ventricle free wall were harvested and processed for routine Transmission Electron Microscopy (TEM). Briefly, approximately 1 cubic mm tissue blocks were postfixed with 1% osmium tetroxide for 90 min, and stained en bloc with 0.5% uranyl acetate for 30 min. Then, the tissue samples were dehydrated in a graded series of ethanol, cleared in propylene oxide, embedded in Epon 812 epoxy resin, and sectioned for TEM. Ultrathin sections (70nm) were mounted on copper grids, double-stained with uranyl acetate and lead citrate, and examined in a FEI Tecnai G2 Spirit BioTwin TEM (Eindhoven, The Netherlands) at 100kV. Digital images were recorded with a bottom-mounted FEI Eagle 4k CCD camera and its TIA software (Eindhoven, The Netherlands).
RNA Extraction and Real-Time qPCR analysis
The total RNA was extracted from cryopreserved tissue samples, namely the pulmonary artery, using the miReasy Micro Kit (QIAGEN). For one sample, approximately 10 mg of tissue were minced very well with fine scissors on a metal support held in liquid nitrogen, 700µl lysis buffer (QIAzol Lysis Reagent) were added, kept in the freezer for 10 minutes and then 1.4mm zirconium oxide beads (Precellys) were added. The sample was subjected to grinding cycles at 1800rpm, 6 times for 1 min on ice. Centrifugation was performed at 10,000 x g for 5 min at 4 ºC, and the extract was transferred to a new tube where the total RNA was extracted with chloroform and precipitated with isopropanol and processed according to the manufacturer's recommendations. The elution of the RNA was done in a final volume of 16µl of RNase-free water, and the RNA thus obtained was kept at − 80°C until examinations. The purity and concentration of the RNA was read by spectrophotometry using NanoDrop 2000c (ThermoFisher). TaqMan MicroRNA Reverse Transcription Kit (Applied Biosystems) was used for reverse transcription of RNA into cDNA synthesis in combination with TaqMan-Gene Expression Master Mix according to the instructions of the manufacturer on a Veriti real-time PCR system (Applied Biosystems). A total of six miRNAs were analysed, each reaction per sample done in triplicate: hsa-miR-21 (ID:0000397), hsa-miR-124 (ID:000446), hsa-miR-204 (ID:000508), hsa-miR-214 (ID:002306), hsa-miR-210 (ID:000512), hsa-miR-145 (ID:002278). U6 small nucleolar RNA snRU6 (ID:00197) was used to normalize the expression level of miRNA and quantified using the 2–∆∆Ct calculation method. The VIIA7 Software v1.2 (Applied Biosystems) with the automatic quantification cycle (Cq) setting was used to analyse the data.
Tissue processing and histological examination
After sampling the organs of interest (pulmonary artery, lung, right ventricle and liver), previously washed well with PBS with Ca2+, the tissues were prepared for histopathological examination. For the paraffin sections, the samples were fixed in 4% paraformaldehyde for 24 hours at RT, dehydrated in successive ethanol baths, embedded in paraffin, and subsequently they were cut with a microtome at a thickness of 5 µm. Before staining, the sections were kept in oven at 60ºC, deparaffinized and rehydrated in decreasing graded xylene and ethanol baths (from 100–70%), washed with distilled water and then mounted on SUPERFROST PLUS glass slides (ThermoScientific) treated with Poly-L-Lysine. The paraffin sections were then subjected to Hematoxylin&Eosin (H&E) staining to observe the presence of collagen, the main indicator of fibrosis, but also to observe vascular remodelling. Hematoxylin is a basic dye with affinity for acidic components of the cell and Eosin is an acidic dye with affinity for basic cellular components. All slides were observed and imaged using bright-field microscope (Leica DMi8, software LAS X) at 20X magnification.
Immunofluorescence analysis
Immunofluorescent staining was performed on tissue samples (pulmonary artery, lung, right ventricle and liver) in order to monitor inflammatory and fibrotic markers. Immediately after harvesting, they were fixed in cryoprotection solution of 2% paraformaldehyde in 0.1M phosphate buffer and left at 4ºC overnight. To prepare the histological sections, the tissue was passed through consecutive baths of glycerol of different concentrations (5% for 15 min at RT, 10% for 1h at 4ºC, 20% overnight at 4ºC and 50% for 1h at 4ºC), then washed with a solution of 3% sucrose, 6 times for 15 min, and immersed in OCT (Tissue-Tek, Sakura) for 30 min. To be mounted on the cutting support, the samples were quickly frozen in liquid nitrogen and cut with a cryotome (Leica CM1850) using special blades (MX35 Ultra, ThermoScientific), into 5µm thick sections and attached to special SUPERFROST PLUS glass slides (ThermoScientific) treated with Poly-L-Lysine. The immunostaining process consisted of acclimatization of the sections at RT, fixation in methanol (-20ºC), quenching of autofluorescence with sodium borohydride for 1h at 4 ºC, permeabilization with 0.2% Triton X-100 (ROTH) in PBS with 0.05% Tween 20 (AppliChem) for 30 min at RT and blocking non-specific sites with 10% goat serum (Invitrogen). The sections were separated on the slide using a lipid pen (Invitrogen) and labeled overnight at 4ºC with the following primary antibodies (diluted in PBS with 1% BSA) against: COL1A (1:250, Santa Cruz Biotechnology), α-SMA (1:200, Cell Signaling Technology), Cx43 (1:200, Thermo Fisher Scientific), MMP-9 (1:200, Santa Cruz Biotechnology), Phalloidin-FITC (5µg/ml, Sigma-Aldrich), PECAM-1 (CD31) (1:200, Santa Cruz Biotechnology), VE-cadherin (1:200, Santa Cruz Biotechnology), OB-cadherin (1:200, Santa Cruz Biotechnology), CTGF (1:200, Santa Cruz Biotechnology), Fibronectin (1:200, Invitrogen), Vimentin (1:200, Santa Cruz Biotechnology), washed 3 times with PBS, incubated with secondary antibodies, Alexa Fluor 647 donkey anti-mouse IgG (H + L) and Alexa Fluor 488 goat anti-rabbit IgG (H + L) (1:500, Invitrogen) for 1h, and then washed 3 times with PBS. Nuclei were stained with 4’,6-Diamino-2-phenylindole (DAPI) solution (5mg/ml in PBS − 10mM) for 5 min, washed 3 times with PBS and mounted with ProLong solution (Invitrogen) and allowed 24 hours to polymerize at RT. To assess ROS (reactive oxygen species) levels, 6µM of fluorochrome dihydroethidium (DHE) (Sigma-Aldrich) were added on tissue sections for 30 min at RT.
All images were captured and analyzed under a fluorescence microscope (Axio Vert.A1 Fl, Carl Zeiss, software Axio Vision Rel 483SE64-SP1) at 20X magnification. Image analysis was performed using ImageJ program.