Therapeutic efficacy of valproic acid in a combined monocrotaline and chronic hypoxia rat model of severe pulmonary hypertension

Abstract

Background: Pulmonary hypertension (PH) is a serious disease with poor prognosis. Reports show that cells in remodeled pulmonary arteries of PH patients have similar characteristics to cancer cells, such as exuberant inflammation, increased proliferation, and decreased apoptosis. An ideal strategy for developing PH therapies is to directly target pulmonary vascular remodeling. High levels of histone deacetylase (HDAC) expression and activity are found in certain cancers, and research has shown the potential of HDAC inhibitors in repressing tumor growth via anti-inflammatory and anti-proliferative effects. To date, little is known about the effectiveness of HDAC inhibitors against pulmonary vascular remodeling in severe PH.

Objective: To investigate whether class I HDAC inhibitors suppress or reverse the development of severe PH in rats.

Methods: Male Sprague-Dawley rats were injected with a single, subcutaneous dose of monocrotaline (60 mg/kg), and were exposed to chronic hypoxia to induce severe PH. Valproic acid, a class I HDAC inhibitor, was administered to rats daily via gastric gavage (300 mg/kg) in a PH prevention study (during the first 3 weeks) or a PH reversal study (from 3 to 5 weeks). At the end of experiment, hemodynamic indices were measured, ventricular hypertrophy indices were calculated and vascular remodeling phenotypes were analyzed.

Results: After 3 weeks exposure to a combined stimulation of monocrotaline and chronic hypoxia, rats exhibited a reduced body weight, elevated right ventricular systolic pressure, an increased Fulton index, right ventricle weight ratio, medial wall thickness and muscularized peripheral pulmonary arteries. These parameters for PH evaluation were exacerbated from 3 to 5 weeks. Daily administration of valproic acid therapy prevented and partially reversed the development of severe PH in rats, and decreased inflammation and proliferation in remodeled pulmonary arteries.

Conclusion: These data show that class I HDAC inhibitors may be effective for treating severe PH.

Fig 1. A combination method increased severity of pulmonary hypertension (PH) in rats.

(A) Schematic PH model protocols for control, chronic hypoxia (CH), monocrotaline (MCT), and combination (MCT/CH) groups. Comparison of (B) body weight change ratio, (C) systemic blood pressure (SBP), (D) right ventricular systolic pressure (RVSP), (E) Fulton index, (F) the ratio of right ventricle weight to body weight (RV/BW), (G) medial wall thickness and (H) muscularization ratio of small pulmonary arteries among different treatment groups (n = 6 per group). + p < 0.05 vs. control group; # p < 0.05 vs. MCT/CH group.

Fig 2. Morphological and immunohistochemical analysis of pulmonary arteries (PAs) in severe PH rats.

MCT/CH resulted in vascular occlusive neointimal lesions. (A) Victoria blue staining and α-smooth muscle actin (SMA) immunohistochemical staining were used to delineate the elastic membrane and media of PAs in control and MCT/CH rats at 3 weeks (MCT/CH 3w), 4 weeks (MCT/CH 4w), and 5 weeks (MCT/CH 5w). (B) Immunohistochemical staining for (a) fetal liver kinase 1 (FLK1), (b) ED1, (c) proliferating cell nuclear antigen (PCNA) and (d) cleaved caspase-3 in lung tissue sections (arrows) from rats with severe PH (sections from control rats are not shown). Scale bar, 50 μm. (C) Occlusive neointimal lesions occurred distal to the branch points of small muscularized PAs, and showed positive ED1 and PCNA staining. Scale bar, 50 μm.

Fig 3. Therapeutic effects of valproic acid (VPA) on severe PH.

(A) Schematic of therapeutic protocols used in the control, prevention, and reversal studies. The comparison of (B) body weight change ratio, (C) systemic blood pressure (SBP), (D) right ventricular systolic pressure (RVSP), (E) Fulton index, (F) the ratio of right ventricular weight to body weight (RV/BW), (G) medial wall thickness, (H) muscularization ratio and (I) the vascular occlusion score (VOS) of small PAs between the Vehicle-treated group and the VPA-treated group (n = 6 per group). * p < 0.05; ** p < 0.01; *** p < 0.001.

Fig 4. Histone deacetylase (HDAC) activity inhibition by VPA.

(A) HDAC1, HDAC2, and HDAC3 expression levels in PH models were determined by western blot analysis. (B) Representative immunohistochemical staining of HDAC1 in MCT/CH rats. (C) Quantification of HDAC1, HDAC2 and HDAC3 expression in different PH model groups. Comparisons of HDAC1, HDAC2 and HDAC3 expression (D) in the prevention study and (E) the reversal study. (F) Acetylated-histone 3 expression in nuclear protein extracts. All western blots were quantified with a lumino-analyzer, and expression is shown as fold increases normalized to the expression of β-actin or lamin A/C. + p < 0.05 vs. control; # p < 0.05 vs. MCT/CH.

Fig 5. Effects of VPA on proliferation and inflammation.

(A) PCNA, (B) SMA, and (C) ED1 immunohistochemical staining quantitative analysis of (D) the index of proliferation (the number of PCNA-positive cells per pulmonary vessel) and (E) the index of inflammation (the number of ED1-positive cells per pulmonary vessel) were used to show the regulation of proliferation and inflammation by VPA in the peripheral pulmonary vessels. Scale bar, 50 μm. * p < 0.05; *** p < 0.001.

Fig 6. Effects of VPA on gene transcription.

(A) HIF1a, (B) P21, (C) MCP1, (D) Casp3, (E) Bcl2, (F) Bcl-xl mRNA levels were assessed using real-time reverse transcription-polymerase chain reaction (RT-PCR) and are shown as fold change relative to the expression level of the control group. * p < 0.05; ** p < 0.01; *** p < 0.001. Bcl2, B-cell lymphoma 2; Bcl-xl, B-cell lymphoma-extra large; MCP1, monocyte chemoattractant protein 1; HIF1a, hypoxia induced factor 1a.