Tetrahydrobiopterin improves pulmonary hypertension associated to pulmonary fibrosis in wistar rats.

P Almudéver1, J Milara3, A Serrano1, T Peiró2, G Hernández2, J Cortijo1,2. 1University of Valencia, Faculty of Medicine and Odontology. Pharmacology Department. 46010, Spain, 2Research Fundation of Valencia General Hospital, Respiratory Pharmacology.46014, Spain, 3Carlos III Health Institute, Research Area. 28029, Spain

Background/Objective : Pulmonary hypertension in pulmonary fibrosis portends a poor prognosis. Recent evidence suggests that tetrahydrobiopterin (BH4), the cofactor of nitric oxide synthase, is involved in pulmonary hypertension. However the role of BH4 in pulmonary hypertension secondary to pulmonary fibrosis is unknown. The current study investigated the role of BH4 on vascular remodelling and pulmonary hypertension in an animal model of bleomycin-induced lung fibrosis.

Methods : Wistar rats were instilled intratracheally with a single dose of bleomycin (3.75 U/kg; n=10) to induce lung injury or with bleomycin vehicle as a control group (n=10). BH4 (20mg/kg/day; n=10) or BH4 vehicle (control group) was administered orally once a day, during 21 days. 24h after the last dosage, jugular vein was cannulated to reach the pulmonary artery through the right ventricule. Rats were sacrificed and plasma, lungs and heart were removed. Plasmatic BH4 concentration was measured by fluorescence-high performance liquid chromatography. The right ventricular (RV) wall of the heart was dissected free and weighed along with the left ventricle wall plus septum (LV + S), and the resulting weights were reported as RV/LV + S ratio to provide the right ventricular hypertrophy index. The wall thickness was calculated by dividing the arterial wall area by the external perimeter (n=30, p=3). Tgf-β1 and Et-1 gene and protein expression were both measured by real time PCR and WB in lung homogenates as pulmonary vascular remodeling markers. Relative quantification of Tgf-β1 and Et-1 was determined with the 2- ΔΔ Ct method using GAPDH as endogenous control and normalized to control group. Data analysis was performed by one-way ANOVA with Bonferroni test and described as mean ± SEM using GraphPad Software Inc (San Diego, CA, U.S.A.). Significance was accepted when p<0.05.

Results: Bleomycin lung injury reduced the BH4 endogenous plasmatic levels (27,6±4,6 ng/mL) related to control group (44,8±2,6 ng/mL). Pulmonary hypertension was increased in bleomycin (27,1±0,4 mmHg) compared to control (16,2±2,6 mmHg) and reduced by daily oral BH4 administration (19,7±1,8 mmHg). Right ventricular hypertrophy index was increased in bleomycin (0,39±0,03) compared to control (0,23±0,02) and improved in bleomycin rats that were supplemented with BH4 20 mg/Kg/day (0,29 ± 0,03). In genetic expression, normalizing control group to the value one, we observed overexpression of ET-1 (2,53±0,4) and TGF-β1 (3,5±0,5) in bleomycin rats, that was counteracted with BH4 treatment (1,2±0,2 and 1,6±0,3 respectively). Arteries wall thickness increased in bleomycin group (23,6±1,0) compared to control group (16,7±0,7) and was normalized in BH4 treated rats.

Conclusions: BH4 inhibits bleomycin-induced pulmonary hypertension and vascular remodelling in wistar rats.