Inhaled tolafentrine reverses pulmonary vascular remodeling via inhibition of smooth muscle cell migration

Experiments were performed on male CD rats 300 – 350 g body weight, Charles River, Sulzfeld, Germany). Pulmonary hypertension was induced by a single subcutaneous injection of monocrotaline (MCT, 60 mg/kg, Sigma, Deishofen, Germany), dissolved in 0.1 M NaOH, adjusted to pH 7.4 with 0.1 M HCl, according to the previous reports [7, 10, 21, 22].

The experiments were performed in accordance with the National Institutes of Health Guidelines on the Use of Laboratory Animals. Both the University Animal Care Committee and the Federal Authorities for Animal Research of the Regierungspräsidium Giessen (Hessen, Germany) approved the study protocol.

The animals were classified into the following four groups: 1) rats injected with saline sacrificed after 28 days (Control, n = 8); 2) MCT-injected rats sacrificed after 28 days (MCT_[28d], n = 12); 3) MCT-injected sacrificed after 42 days, with nebulized vehicle being administered from day 28 to day 42 (MCT_[42d], n = 14); 4) MCT-injected rats sacrificed after 42 days, with nebulized tolafentrine being administered from day 28 to day 42 (MCT_[42d]/Tola, n = 10). For acute hemodynamic studies 12 MCT_[28d] rats (MCT injected for 28 days) were used (inhalation of saline, n = 6; and inhalation of tolafentrine in two different doses, n = 6 each).

Four weeks after a single MCT injection, rats were subjected to inhalation of tolafentrine or sham nebulization in an unrestrained, whole body aerosol exposure system as described [23]. For assessment of chronic effects of inhaled saline or tolafentrine (dose deposited in the lungs ~ 120 μg/kg day), 15 min nebulization maneuvers using a jet nebulizer with a constant flow rate of 6 l/min (Pari LC Star, Pari, Starnberg, Germany) were repeated twelve times per day for 2 weeks (day 28 – 42). Particles generated by the jet nebulizer were characterized by a mass median aerodynamic diameter (MMAD) of 2.8 μm and a geometric standard deviation (GSD) of 2.5 (determined by laser difractometric measurements, as described [24]. Preliminary experiments with nebulization of ⁹⁹Tc-DTPA determined the total lung deposition of nebulized material to range at 0.5 %, in accordance with previous studies in this model [22].

To assess the acute effects of inhaled tolafentrine, hemodynamic testing in response to the drug was performed in animals that had previously received a MCT injection 4 weeks prior to study. Hemodynamics were measured before and after a single inhalation of the tolafentrine (130 and 650 μg/kg min for 10 min) by an ultrasonic nebulizer with MMAD of 4.0 μm and GSD of 2.1 as described previously [22, 23, 25].

For measurement of hemodynamic parameters, rats were anaesthetized with an i.p. injection of ketamine (9 mg/kg body mass) and medetomidine (100 μg/kg body mass), followed by an i.m. injection of atropine (250 μg/kg body mass). The rats were tracheotomized and ventilated with a frequency of 60 breaths/min. Positive end expiratory pressure was set at 1 cm H₂O. A polyethylene catheter was inserted into the left carotid artery to measure arterial pressure. A right heart catheter (PE 50 tubing) was inserted into the right ventricle through the right jugular vein for measurement of right ventricular systolic pressure with fluid-filled force transducers [10, 11]. Cardiac output (CO) was measured by thermodilution technique as described [10, 11]. Briefly, a thermistor (1.5 F) was placed into the ascending thoracic aorta via the right carotid artery for the measurement of transpulmonary thermodilution cardiac output (Cardiotherm 500-X, Hugo-Sachs Electronic – Harvard Apparatus GmbH, March-Hugstetten, Germany). The CO was averaged from three consecutive determinations and indexed to the weight of the animal to obtain cardiac index (CI). After exsanguination, the left lung was fixed for histology in 10% neutral buffered formalin and the right lung was snap frozen in liquid nitrogen.

The RV was dissected from the left ventricle (LV) and the septum (S) and weighed to determine the extent of RV hypertrophy as follows: RV/ (LV+S).

Paraffin lung sections (3 μm) were double-stained with anti-α-smooth muscle actin antibody (dilution 1:900, clone 1A4, Sigma, Saint Louis, Missouri) and anti-human von Willebrand factor antibody (vWF, dilution 1:900, Dako, Hamburg, Germany). Sections were counterstained with hematoxylin and examined by light microscopy using a computerized morphometric system (Qwin, Leica, and Wetzlar, Germany) for assessing the degree of muscularization of small peripheral pulmonary arteries [10, 11, 23]. In addition, lung sections were stained for Elastin-Nuclear Fast Red to assess the medial wall thickness.

Categorization of pulmonary arteries based on the degree of muscularization and on the external diameter and the percentage of medial wall thickness were performed as previously described [10, 11, 23].

Total RNA extraction was performed using the Trizol reagent (Burlington, Ontario, Canada) according to the instructions given by the manufacturer from control, monocrotaline (MCT_[42d]) and monocrotaline plus tolafentrine (MCT_[42d]/Tola) treated lungs. Samples were treated with DNase I according to the RNase-free DNase set (QIAGEN, Hilden, Germany). Total RNA (5 μg) from each sample was used to generate Biotin-16-dUTP labeled cDNA and hybridized to the extracellular matrix and adhesion molecules gene array (GEArray Q series, Superarray, MD, USA). After hybridization, the membranes were developed according to the manufacturer's recommendations to yield luminescent signals, which were then captured on X-ray film.

The resulting image data were analyzed for differential gene expression patterns using GE Array Analyzer 1.2 (Super Array Bioscience Corp., Frederick, MD) software. Loading was adjusted on the basis of the intensity of hybridization signals relative to the housekeeping gene GAPDH.

Quantitative real-time PCR was performed as described [26]. Briefly, total RNA was isolated from frozen lungs using Trizol Reagent according to the manufacturer's instructions. For the generation of cDNA, equal amounts of RNA from each sample were used as templates for reverse transcription of first-strand cDNA using the qPCR™ Mastermix (Euro-genetec, Seraing, Belgium) according to the manufacturer's protocol. For quantitative real-time RT-PCR analysis, 2 μl cDNA was placed into 50 μl reaction volume containing SYBR Green PCR mix and sequence-specific oligonucleotide primers. The thermal cycle conditions used for all reactions were as follows: activation, 50°C for 2 min; denaturation, 95°C for 10 min; and cycle, 95°C for 10 s and 60°C for 5 s (40 times). Specific primers used for sequence detection were: rGAPDH,5'-GTG ATG GGT GTG AAC CAC GAG-3' (forward) and5'-CCA CGA TGC CAA AGT TGT CA-3'(reverse); forMMP2,5'-ATC TGC AAG CAA GAC ATT GTC TT-3'(forward)and 5'-GCC AAA TAA ACC GAT CCT TGA A-3' (reverse);for MMP8, 5'-CGG GAA GAC ATA CTC TTC GAA-3'(forward)and 5'-CAT GGA TCT TCT TTG ATT GTC G-3' (reverse);for MMP9, 5'-GTA ACC CTG GTC ACC GGA CTT -3'(forward)and 5'-ATA CGT TCC CGG CTG ATC AG-3' (reverse); for MMP10, 5'- GGA GAT GCT CAC TTC GAT GAT-3' (forward)and 5'-CAG CAA CCA GGA ATA AAT TGG-3' (reverse); for MMP11, 5'-TTC TGA GAT TGA TGC TGC TTT C-3'(forward)and 5'-TGT CCA CGA AGG AAG TAG GC-3' (reverse); for MMP12, 5'-GCT GTC ACA ACA GTG GGA GA-3' (forward)and 5'-GTA ATG TTG GTG GCT GGA CTC-3' (reverse); for MMP20, 5'-GAA TAA ACT CTG GGG AAG CAG A-3'(forward)and 5'-TGA TTG GAT TAA GGC CTC GT-3' (reverse); for Icam, 5'-CAG CTG CGC TGT GTT TTG-3'(forward)and 5'-GGA TGG GAG CTG AAA AGT TG-3' (reverse); For Itgax, 5'-GCC TCG AGA CTG GAG ATC AT-3'(forward)and 5'-GGA GAG CTG GGA GCC AGT-3' (reverse);for Ncam1, 5'-ACC ATG AGC TGG ACA AAG GA-3'(forward)and 5'-CAC TGA AGA TGT GCT TCT CGT C-3' (reverse); for Plat, 5'-AAT GAA GGG AGA GCT GTT GTG-3'(forward) and 5'-TCC TCT TCT GAA CCT CCT GTG-3' (reverse); for Serpinb2, 5'-CGA AAG GGA TTT TGT GAT GTC-3' (forward)and 5'-GTG GGA AAT GGG AAT TCG T-3' (reverse). All real-time reactions were carried on an ABI 7700 sequence Detection System (Applied Biosystems, Foster City, CA, USA), and analysis was performed with the accompanying software. At the end of the PCR cycle, a dissociation curve was generated to ensure the amplification of a single product and the threshold cycle time (Ct values) for each gene was determined. Relative mRNA levels were calculated based on the Ct values and normalized to house keeping gene GAPDH.

Pulmonary artery smooth muscle cell (PASMC) migration was examined in Transwell cell culture chambers with gelatin-coated polycarbonate membranes as described previously [27]. For this assay, control and MCT-treated rat PASMCs were isolated and added to the upper well of a Transwell (Corning Costar, Cambridge, MA) at 2 × 10⁵ cells/well. The platelet-derived growth factor – BB (PDGF-BB) was added to the lower chamber at a concentration of 25 ng/ml and cells were allowed to migrate for 8 and 24 hours. In experiments with the combined selective PDE3/4 inhibitor tolafentrine (0.05, 0.1 and 0.3 μM), MCT-treated rat PASMCs were pre-incubated with tolafentrine in the upper well of a Transwell for 30 min before the addition of PDGF-BB into the lower chamber. Migration was quantified by staining the cells with crystal violet (Sigma, Deishofen, Germany) following microscopic cell counts on five random fields in each well. Experiments were performed in triplicate and were repeated at least three times.

All data are given as mean ± SEM. Differences between the groups were assessed by analysis of variance (one-way ANOVA) and Student-Newman-Keuls test for multiple comparisons with a p value < 0.05 regarded to be significant.

Aerosolized tolafentrine reduced right ventricular systolic pressure [RVSP] in MCT_[28d] rats in a dose-dependent manner (Figure 1). As depicted, this pulmonary vasodilatation was accompanied by less pronounced decrease in systemic arterial pressure.

After injection of monocrotaline, pulmonary hypertension developed (right ventricular systolic pressure on day 28 = 66.5 ± 3.2 mm Hg (n = 11) and on day 42 = 74.9 ± 5.1 mm Hg (n = 9), as compared to 25.9 ± 4.0 mm Hg in the control animals (n = 10)) (Figure 2). No significant changes in systemic arterial pressure occurred. As compared to control animals (36.5 ± 3.5 ml/min 100 g body weight), cardiac index was slightly decreased on day 28 (31.8 ± 1.3 ml/min/100 g body weight) and significantly decreased on day 42 (28.1 ± 2.5 ml/min/100 g body weight). Aerosolized tolafentrine treatment significantly lowered right ventricular pressure to 48.4 ± 2.1 mmHg (p < 0.05 versus MCT_[42d] and MCT_[28d]). No significant changes in systemic arterial pressure occurred in animals undergoing long-term tolafentrine inhalation (n = 8), while cardiac output was significantly increased in the MCT_[42d]/Tola group.(42.1 ± 5.1 ml/min/100 g body weight; p < 0.05 versus MCT_[42d]). The total pulmonary resistance index, calculated from the RVSP and cardiac output values, was even fully normalized in the MCT_[42d]/Tola animals.

Four weeks after injection of MCT, animals demonstrated significant right heart hypertrophy, as indicated by an increase in the right ventricular to left ventricular plus septum weight ratio (RV/LV+S) from 0.29 ± 0.02 (control animals) to 0.60 ± 0.02 (Figure 3). Rats that received inhaled vehicle for 2 weeks demonstrated further progression of right ventricular hypertrophy (RV/LV+S = 0.71 ± 0.05). Inhaled tolafentrine reversed established right ventricular hypertrophy (MCT_[42d]/Tola = 0.47 ± 0.03; p < 0.05 versus MCT_[28d] and MCT_[42d]) (Figure 3).

In this study, rats were randomized to receive vehicle or inhaled tolafentrine beginning four weeks after MCT treatment. In the MCT_[28d] group a survival of 78% was noted, while survival decreased to 60% in the MCT_[42d] group. In the MCT_[42d]/Tola 80 % survived the treatment protocol. However, due to insufficient number of experiments the significant effects on this parameter were not shown in detail.

Elastin staining and subsequent morphometric analysis of pulmonary arteries demonstrated a markedly increased medial wall thickness in both the MCT_[28d] and the MCT_[42d] groups, when compared with the saline-treated group (Figure 4). In comparison to control animals, the percentage of medial wall thickness of arteries sized between external diameters of 25 to 50 μm was increased significantly from 18.3 ± 0.42 to 28.5 ± 0.33 (MCT_[28d]) and 29.1 ± 0.48 (MCT_[42d]) (Figure 5A). To a similar extent, the percentage of medial wall thickness of arteries with external diameters of 51–100 μm increased from 17 ± 0.49 (control) to 22.2 ± 0.31 (MCT_[28d]) and 25.1 ± 0.77 (MCT_[42d]) (Figure 5B). In arteries larger than 100 μm, the corresponding data were 14.8 ± 0.95 (control), 18.1 ± 0.57 (MCT_[28d]) and 20.6 ± 1.44 (MCT_[42d]) (Figure 5C), respectively. The evaluation of the extent of muscularization of pulmonary arteries with external diameters of 15 to 50 μm demonstrated a significant reduction in non-muscularized pulmonary arteries (62.6 ± 5.5% in controls, 3.7 ± 2.1 % in MCT_[28d], 1.6 ± 0.7 % in MCT_[42d]). Concomitantly, a significant increase in fully muscularized vessels from 2.4 ± 0.62 (control) to 51.1 ± 6.85 (MCT_[28d]) and 71.6 ± 2.63 (MCT_[42d]) was noted (Figure 6).

Most impressively, both, medial wall thickness (22.5 ± 0.65%) and percentage of fully muscularized pulmonary arteries (32.5 ± 5) were significantly reduced by long term treatment of aerosolized tolafentrine, while the percentage of non-muscularized pulmonary arteries significantly increased.

Migration of control and MCT-treated rat PASMCs was examined at 8 and 24 h in the presence of the chemo-attractant PDGF-BB (Figure 7). In the presence of PDGF-BB, the migration rate of PASMCs derived from MCT rats ranged at 207% and 155% of that of the PASMCs derived from control rats (8 hr and 24 hr data, respectively).

Inhibitory effects of tolafentrine on MCT- treated rat PASMCs migration were examined using a migration assay with modified Boyden chamber. The PDGF caused strong enhancement of PASMC migration. Treatment with tolafentrine inhibited PDGF induced PASMC migration in a dose-dependent fashion: at 0.05, 0.1, and 0.3 μM tolafentrine, PASMC migration was reduced by 34 %, 72 %, and 92 % (Figure 8).

To study the mechanisms underlying tolafentrine-inhibited PASMC migration, lung samples from control, MCT_[42d] and MCT_[42d]/Tola rats were analyzed on 96 gene arrays encoding key extracellular matrix and adhesion molecules (representative arrays given in Figure 9). The analysis shows expression of 12 of the 96 extracellular matrix and adhesion related genes in the control lungs. Further, a total of 12 genes were differentially expressed and modulated in the MCT-treated lungs. The up-regulated genes include 7 MMPs (MMP 2, MMP 8, MMP 9, MMP 10, MMP 11, MMP 12, MMP 20), 1 serine protease (Plat), 3 cell adhesion molecules (ICAM-1, NCAM-1, Itgax), and 1 protease inhibitor (Serpinb2).

To validate these findings, the expression of 12 genes was investigated by quantitative real-time PCR. These results confirmed the array data for most of the proteases investigated (Figure 10). Monocrotaline treatment for four weeks caused an upregulation of the gelatinases MMP 2 and MMP 9 by a factor of 3.39 ± 0.65 and 3.92 ± 0.77, respectively. MMP 8, the interstitial collagenase, was elevated 4.77 ± 1.44 fold with MCT treatment. In addition, the stromelysins MMP 10, MMP 11, MMP 12 and MMP 20 were significantly upregulated by factors of 4.65 ± 1.13, 5.27 ± 1.03, 2.27 ± 0.63 and 6.67 ± 0.75, respectively.

Several adhesion molecules were also differentially expressed at the mRNA level. We observed a significantly increased expression of Icam and Itgax (4.34 ± 1.22 and 3.44 ± 0.33 fold). In contrast, Ncam was not changed upon MCT treatment. An upregulation of tissue plasminogen activator (plat) by 3.74 ± 0.83 and plasminogen activator inhibitor-2 (Serpinb2) by 2.68 ± 0.83 foldwith MCT treatment was also observed.

Most impressively, virtually all alterations in extracellular matrix and adhesion molecule gene expression induced by monocrotaline were fully or partially abrogated by tolafentrine (12 genes given in Figure 10).