Phosphodiesterase type-5 (PDE-5) inhibitors are novel and important options for the treatment of pulmonary arterial hypertension (PAH). Therefore, we aimed to examine effects of vardenafil, a PDE-5 inhibitor, on the pulmonary arteries isolated from rats with monocrotaline- (MCT-) induced pulmonary hypertension. MCT (60 mg/kg) or its vehicle was administered by a single intraperitoneal injection to 6-week-old male Sprague Dawley rats. Rats were sacrificed 21 days after MCT injection, and the main pulmonary arteries were isolated and then mounted in 20 mL organ baths. Concentration-response curves for vardenafil (10−10–10−5 M) were constructed in phenylephrine- (Phe-) precontracted rings. PAH caused marked rightward shift in the curves to vardenafil whereas maximal responses were not affected. Inhibition of NO synthase (L-NAME, 10−4 M) or guanylyl cyclase (ODQ, 10−5 M) caused similar attenuation in responses evoked by vardenafil. Moreover, contraction responses induced by CaCl2 (3×10−5–3×10−2 M) were significantly reduced in concentration-dependent manner by vardenafil. In conclusion, vardenafil induced pulmonary vasodilatation via inhibition of extracellular calcium entry in addition to NO-cGMP pathway activation. These results provide evidence that impaired arterial relaxation in PAH can be prevented by vardenafil. Thus, vardenafil represents a valuable therapeutic approach in PAH besides other PDE-5 inhibitors.
Pulmonary arterial hypertension (PAH) is a severe disease with a poor prognosis. In untreated patients, there is a progressive increase in pulmonary vascular resistance that leads to intractable right ventricular failure and premature death [
The pathogenesis of PAH is multifactorial [
To the best of our knowledge, studies investigating the efficacy of vardenafil to treat PAH are still limited to few case reports and clinic studies [
This study was approved by the Animal Ethics Committee of Akdeniz University Medical Faculty.
Vardenafil was provided by Bayer Health Care AG, Leverkusen, Germany. Acetylcholine (Ach), ethylene glycol tetraacetic acid (a chelator agent for calcium ion, EGTA),
Monocrotaline is a member of the alkaloid family of plant toxins that induces a delayed, yet progressive vascular injury resulting in pulmonary hypertension in rats, dogs, and monkeys [
MCT was dissolved in 1.8 mL 1 M HCl, and 3-4 mL of distilled water was added, as described previously [
After 21 days, each animal was anesthetized with pentobarbital (45 mg/kg). The heart and lungs were removed by en bloc resection. Right and left main pulmonary arteries were dissected and harvested for vascular reactivity. After removal of the arteries, right ventricle (RV) and left ventricle with septum (LV + S) were separated and weighed. Also in some animals both from control (
Left and right lungs of rats were removed from the thoracic cavity, and histological specimens were fixed overnight by instillation of 10% buffered formalin into the airways and vascular structures. And then, representative cross-sections of the lungs which include the peripheral and the central pulmonary arteries were sampled and embedded in paraffin blocks. Serial sections (5-
Experiments were conducted on isolated main left and right pulmonary arteries. Main pulmonary artery branches were rapidly removed, gently cleaned of fat and connective tissues taking care not to damage the endothelium and cut into rings of about 3 mm then carefully suspended by two stainless-steel clips passed through the vessel lumen in a 20 mL organ bath containing Krebs solution (in mM: NaCl 136.9, KCl 5.4, CaCl2 1.5, MgCl2 1.0 and glucose 5.5.) at 37°C, and was continuously aerated with 95% O2 and 5% CO2 to obtain a pH of 7.4. Isometric tension was continuously measured with an isometric force transducer (FDT-05 Force Displacement Transducer, BioPac), connected to a computer-based data acquisition system (MP30 Transducer Data Acquisition System, BioPac). The tissue was equilibrated for 60 min under a resting tension of 1 g. During this time, Krebs solution was replaced every 15 minutes with fresh solution. After the 1 hour equilibration period, pulmonary rings were challenged with 80 mM KCl (the same composition as Krebs’ solution with NaCl replaced by equimolar KCl) to check tissue viability. Next, the endothelial integrity of the preparations was determined by verifying the responsiveness to ACh (10−6 M) in vessels precontracted with Phe (10−6 M). Rings were then washed several times to restore tension to the baseline level.
In the first set of experiments, concentration-response curves (10−10–10−5 M) for vardenafil were constructed in precontracted (Phe, 10−6 M) control or pulmonary hypertensive rat pulmonary artery rings in the absence or in the presence of L-NAME (
Experimental values of relaxation or contraction were calculated relative to the maximal changes from the contraction produced by Phe and KCl, respectively, taken as 100% in each tissue. In order to evaluate the effects of vardenafil; maximum response (
As shown in Table
Effects of monocrotaline treatment on rat body weight and cardiac weight. Results are expressed as mean ± s.e.mean. *Significantly different from control rat at
Control ( | MCT ( | |
---|---|---|
BW (0 day, gr) | 195 ± 18 | 170 ± 3 |
BW (21.day, gr) | 317± 15 | 225 ± 3* |
RV/BW (gr/kg) | 0.57 ± 0.02 | 1.15 ± 0.03* |
RV/LV + S | 0.23 ± 0.01 | 0.59 ± 0.01* |
Monocrotaline-induced morphometric changes in pulmonary arteries. Results are expressed as mean ± s.e. mean. *Significantly different from control rat at
Control ( | MCT ( | |
---|---|---|
WT ( | 9.7 ± 0.5 | 19.2 ± 1.2* |
ED ( | 84 ± 6 | 95 ± 5 |
% WT | 21.1 ± 1.2 | 43.1 ± 1.1* |
Pulmonary arteries were demonstrated in lungs from control group (a) and from the rat with monocrotaline-induced pulmonary hypertension (b).
Vardenafil (10−10–10−5 M) induced concentration-dependent relaxation response (pD2
Concentration- response curves (10−10–10−5 M;
Effects of L-NAME (
The second set of experiment was performed in Ca+2-free Krebs solution containing EGTA. Vardenafil (10−7–10−6 M) concentration dependently inhibited contractions evoked by CaCl2 (3 × 10−5–3 × 10−2 M) with reducing the maximum response to CaCl2 (Figure
Concentration-response curves to CaCl2 in the absence or presence of vardenafil (10−7–10−6 M;
This is the first analysis of the pharmacological profiles of specific PDE-5 inhibitor vardenafil, in the rat MCT-induced pulmonary artery hypertension model. In our study, by examining the vasorelaxant properties of vardenafil, we demonstrated that vardenafil potently caused pulmonary artery relaxation in a dose-dependent fashion through mechanisms involving both NO/cGMP-dependent and -independent pathways.
Pulmonary arterial hypertension is characterized by increased pulmonary vascular resistance, narrowing of pulmonary vascular lumen due to thickening of the vessel media, changes in functional parameters of the lung vasculature, and right ventricular hypertrophy [
Although the underlying mechanisms of PAH have not been fully elucidated, it has been shown that vasodilator therapies including PDE-5 inhibition, can reduce the increase in pulmonary vascular resistance and cause vasodilatation. [
Our findings confirm that vardenafil is an effective pulmonary vasodilator and the incubation with NO-cGMP pathway inhibitors (L-NAME or ODQ) attenuates the vasodilator action of vardenafil. This finding is in agreement with previous studies in that PDE-5 is implicated in the process via inactivating cGMP [
Calcium (Ca+2) influx through channels represents one of the major pathways to control the vascular tone [
In conclusion, the data reported herein show that vardenafil potently relaxes pulmonary artery rings through NO-cGMP-dependent and -independent mechanisms. In addition, vardenafil-induced relaxations seem to involve blockade of Ca+2 entries. The results in this suggest that vardenafil may be more effective than sildenafil and tadalafil for the treatment of pulmonary hypertension. Also, the clinical implications of this research require further randomized, placebo-controlled studies to precisely define the long-term efficacy, side effects, and drug interactions of vardenafil in order to emphasize its place in the management of PAH.
This study was supported by “The Scientific Research Projects Coordination Unit of Akdeniz University” (2006.04.0103.006).