Background
Pulmonary arterial hypertension (PAH) is associated with dramatic structural remodeling of small pulmonary arteries (PAs) [
1]. The remodeling process is due to excessive proliferation of fibroblasts, endothelial cells and smooth muscle cells, and correlates with vascular inflammation and adventitial fibrosis. Occlusion of PAs, coupled with aberrant vasoconstriction, causes severe increases in pulmonary vascular resistance (PVR) and often culminates in right-sided heart failure. As such, it has been proposed that anti-proliferative agents should be used in combination with vasodilators for the treatment of PAH. To address this hypothesis, a Phase III clinical trial was performed with the anti-cancer agent imatinib in patients with PAH and receiving background standard-of-care (SOC) therapy [
2]; current SOC for PAH typically involves the use of vasodilators, including endothelin receptor antagonists (ERAs), phosphodiesterase-5 (PDE-5) inhibitors, and prostacyclins [
3]. Imatinib treatment improved exercise tolerance and pulmonary hemodynamics in PAH patients. However, despite functional improvements, imatinib caused serious adverse side effects, and thus will not be developed further for the PAH indication [
2]. As such, there remains a significant unmet medical need with regard to treatment of PAH in humans.
To model PAH pre-clinically, rodents are often exposed to chronic hypoxia, which causes pulmonary vasoconstriction and right ventricular (RV) hypertrophy [
4],[
5]. An alternative rodent model is based on administration of the plant alkyloid, monocrotaline, which is thought to trigger PAH by altering pulmonary artery endothelial cell function [
6]. Despite their widespread use and utility, neither model exhibits the obliterative vascular lesions found in human PAH. A breakthrough in PAH research was provided by the discovery that combining hypoxia with the VEGF receptor inhibitor, SU5416, in rats results in progressive and severe PAH characterized by occlusive neointima and complex plexiform lesions reminiscent of those found in lungs of patients with PAH [
7],[
8]. In this model, which will hereafter be referred to as SU-Hx, rats develop RV failure and PAH that is directly correlated with the degree of occluded vessels [
9].
SOC therapies for PAH have exhibited only minimal efficacy in the SU-Hx model, which is consistent with the palliative actions of approved PAH drugs, and the inability of these drugs to significantly prolong lifespan in PAH patients. For example, the soluble guanylate cyclase (sGC) agonist riociguat lowered PA pressure (PAP) by approximately 15% in SU-Hx rats, and the PDE-5 inhibitor, sildenafil, was even less effective in the model [
10]. The ERA bosentan modestly reduced RV hypertrophy in SU-Hx rats, and prevented further increases in RV systolic pressure (RVSP) when delivered starting on day 10 of a 21-day study [
11].
Ex vivo, BQ123, a peptide ERA, was able to partially block spontaneous vasoconstriction in blood-perfused lungs excised from SU-Hx rats [
9].
We hypothesized that, relative to monotherapy, simultaneous use of two FDA-approved PAH drugs that target distinct but redundant biochemical pathways will provide superior efficacy in the SU-Hx model. To test this hypothesis, the PDE-5 inhibitor, tadalafil (TAD) [
12],[
13], was tested in combination with the type A endothelin receptor (ET
A) antagonist, ambrisentan (AMB) [
14],[
15], for ability to reverse pre-existing PAH in SU-Hx rats. The data presented here reveal profound and unparalleled efficacy of combined TAD/AMB in the rat SU-Hx model.
Methods
Experimental animals
Animal experiments were approved by the Institutional Animal Care and Use Committee at the University of Colorado Denver. Ten week-old male Sprague Dawley rats (Charles River Laboratories) were used for all studies. A single dose of SU5416 (Tocris Bioscience; 20 mg/kg) or vehicle control (50% DMSO and 50% of a solution containing 0.5% carboxymethylcellulose sodium, 0.9% sodium chloride, 0.4% Tween-80, 0.9% benzyl alcohol in deionized water) was administered at day zero. Rats receiving SU5416 and were housed in a hypobaric chamber to simulate an altitude of 18,000 feet above sea level and create a hypoxic environment (10% 02). Normoxic control rats were maintained in chambers simulating sea level (21% 02). After three weeks, all animals were transferred to Denver altitude and treated daily for four weeks by oral gavage with vehicle (0.5% hydroxypropyl-methyl cellulose), tadalafil (10 mg/kg), ambrisentan (10 mg/kg), or a combination of tadalafil and ambrisentan (10 mg/kg of each compound). Tadalafil and ambrisentan were obtained from Sequoia Research Products.
Hemodynamic analysis
Echocardiographic analyses were performed using a Vevo770 (VisualSonics). Animals were anesthetized using 2% isoflurane and their body temperature was maintained at 37°C. Pulse-wave Doppler of pulmonary outflow was recorded in the parasternal short-axis view at the level of the aortic valve. Baseline measurements were obtained one day prior to placing animals in chambers and serially thereafter. RV hemodynamics were assessed at end-point and after the last ultrasound analysis using a pressure-volume system (Scisense); rats were ventilated with 100% oxygen and 2% isoflurane (Hallowell). Systemic blood pressure was monitored with another pressure catheter inserted in the femoral artery and steady-state hemodynamics were recorded. PAP was measured with the same catheter advanced to the main pulmonary artery; correct placement of the catheter was confirmed by observing a significant rise in diastolic pressure as the catheter moved out of the ventricle. For data from all in vivo studies, GraphPad Prism software was used to generate graphs and analyze data. ANOVA with Bonferroni's post-test (P < 0.05) was used to determine statistical differences between groups.
Tissue procurement and analysis
After end-point hemodynamic measurements were obtained, rats were sacrificed by exsanguination. Heparinazed blood was used to measure blood gases (ABL825 Flex, Radiometer). Lungs were flushed with cold saline and the left lobe was inflated with a mixture of 50:50 cryoprotective embedding medium (OCT) and 30% sucrose, then cut longitudinally and snap-frozen in a block containing OCT. For assessment of pulmonary vascular remodeling, lung sections were stained with hematoxylin and eosin. Stereological assessment of intima (i.e., obliterative) and media (i.e., thickening) was performed using a sampling strategy outlined in Stacher et al. [
16], which determined that ~40 histological fields provide ~100 hitting points in intima using a 512 point grid. The structure-specific hitting points were normalized by grid points hitting alveolar septa, which should not change in untreated vs. treated SU-Hx rats, therefore providing a key measure of the reference tissue. This provides the volume density of intima or media in relation to alveolar septa, which is dimensionless. Stereological assessment was performed with coded slides, which were assigned using a random number generator.
Frozen lung sections were fixed with 4% paraformaldehyde for 15 minutes prior to staining for von Willebrand factor (vWF; endothelial cells) and α-smooth muscle actin (αSMA; smooth muscle cells). Antibodies: anti-vWF (Abcam, ab6994), Cy3-anti-αSMA (Sigma, C6198), and FITC-anti-rabbit IgG (Invitrogen, A11034). Vectashield mounting medium with DAPI (Vector, H-1500) was used to mount coverslip to slides.
RV was dissected from LV by cutting along the septum and the outer wall of the LV and weighed. Fifty milligram biopsies of RV free wall were flash-frozen in liquid nitrogen for biochemical analysis. Protein lysates were prepared in PBS (pH 7.4) containing 0.5% Triton X-100, 300 mM NaCl and protease/phosphatase inhibitor cocktail (Thermo Fisher) using a Bullet Blender homogenizer (Next Advance). Proteins were resolved by SDS-PAGE, transferred to nitrocellulose membranes (BioRad) and probed with antibodies for RCAN-1 [
17] or calnexin (Santa Cruz Biotechnology, sc-11397). Proteins were detected using a SuperSignal West Pico chemiluminescence system (Thermo Scientific) and a FluorChem HD2 imager (Alpha Innotech).
Conclusions
Morbidity and mortality rates for individuals with PAH remain unacceptably high, underscoring the need for additional therapeutic options [
19]. In the current study, monotherapy with 10 mg/kg tadalafil failed to reduce PAP or RV hypertrophy in the rat SU5416 plus hypoxia model of PH, while the same dose of ambrisentan was mildly efficacious. In sharp contrast, combined treatment with tadalafil and ambrisentan (10 mg/kg each), starting when SU-Hx rats had pre-existing PH and RV hypertrophy, dramatically reversed multiple disease endpoints in the lung and right side of the heart. The findings underscore the promise of combinatorial therapy for PAH based on simultaneous targeting of redundant signaling effectors, PDE-5 and ET
A, which serve crucial roles in the pathogenesis of PAH.
AMBITION is a Phase III clinical trial designed to assess whether treatment with a combination of TAD and AMB provides superior efficacy over monotherapy in newly diagnosed patients with PAH [
20]; preliminary findings from the study were recently announced at the European Respiratory Society meeting in Munich, Germany. Participants in the trial include patients with idiopathic pulmonary arterial hypertension (i.e., PAH or World Health Organization classified Group I PH), as well as individuals with PH due to insults such as structural lung disease, toxin exposure and HIV infection. Based on data presented here, it will be particularly enlightening to determine whether TAD/AMB is efficacious in severe, angioobliterative disease. Additionally, a comparison of AMBITION data with our results will provide an unprecedented opportunity to address the predictive value of the SU-Hx rat model with regard to translating results to the clinical setting. Such comparisons could have a profound impact on the success rate of future drug discovery efforts with experimental compounds for PAH, such as inhibitors of leukotriene B4 biosynthesis [
21].
Authors' contributions
MAC, KMD-D, and KBS conducted all in vivo studies. WWB and RMT performed histological analysis, and MSS conducted biochemical studies. MAC and TAM wrote the manuscript, with critical input from all authors. All authors read and approved the final manuscript.
Acknowledgements
We thank D. Irwin for assistance with blood gas measurements. KBS and MSS were supported by a T32 training grant from the NIH (5T32HL007822-12), and WWB was funded by the University of Colorado Denver Pharmacology Program NIH T32 Training Grant (GM007635). TAM was supported by NIH (HL116848 and AG043822) and the American Heart Association (Grant-in-Aid, 14510001).
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Competing interest
The authors declare that they have no competing interest.