Contributed equally
Pulmonary arterial hypertension (PAH) is a serious pulmonary vascular disease. The changes in the structure, function and metabolism of endothelial cells are some of the important features of PAH. Previous studies have demonstrated that the mevalonate pathway is important in cardiovascular remodeling. However, whether the mevalonate pathway is involved in the development of PAH remains to be elucidated. The present study aimed to investigate the expression pattern of mevalonate pathway-related enzymes in monocrotaline (MCT)-induced PAH. F344 rats were randomly divided into two groups (n=6/group): Control group rats were injected with a single dose of saline, and MCT group rats were injected with a single dose of MCT (60 mg/kg). After 4 weeks, the right ventricular systolic pressure (RVSP) was measured, and lung and pulmonary artery tissue samples were collected. It was demonstrated that the RVSP increased and pulmonary vascular remodeling was detected in the PAH group. The expression levels of the enzymes farnesyldiphosphate synthase farnesyltransferase α and geranylgeranyltransferase type I increased in the PAH group, which suggested that the mevalonate pathway may be involved in the pathological development of PAH.
Pulmonary arterial hypertension (PAH) is a serious pulmonary vascular disease, which is defined by a mean pulmonary arterial pressure ≥25 mmHg (
A typical feature of endothelial injury is an imbalance of nitric oxide (NO)-reactive oxygen species (ROS) levels, which is caused by reductions in endothelium-derived NO synthesis, release and activity, and is increased by ROS generation and release (
Previous studies have demonstrated that the mevalonate pathway is involved in small G-protein activation (
At present, the effects of key enzymes, including farnesyldiphosphate synthase (FDPS), farnesyltransferase α (FNTA), farnesyltransferase β (FNTB) and geranylgeranyltransferase type I (GGTase-I) on endothelial dysfunction have not been reported, with the exception of the initial enzyme, HMGR. The pathway downstream of FPP produced in the mevalonate pathway has three branches: Asterol branch, which primarily contributes to cholesterol synthesis, and two non-sterol branches, which regulate signal transduction proteins, including those of the Ras and Rho families (
Based on previous studies, the present study aimed to determine whether the expression of key enzymes, including HMGR, FDPS, FNTA, FNTB and GGTase-I, in the mevalonate pathway are altered in MCT-induced PAH rats, which may potentially serve as novel therapeutic targets for PAH.
The present study was performed in adherence with the National Institutes of Health Guidelines for the Care and Use of Laboratory Animals andapproved by the ethics committee of Zhejiang University (approval no. ZJU20170874; Hangzhou, China). A total of 100 F344 male rats (6-weeks old; weighing 200±10 g) were obtained from Shanghai Laboratory Animal Center (Chinese Academy of Sciences, Shanghai, China). A total of 5 rats/cage, temperature of 22°C, light and dark cycle 12-h, free access to drinking water and normal feed. The rat PAH model was induced by injecting a single dose of MCT (60 mg/kg, dissolved in 1N HCl, neutralized with 1N NaOH, diluted with saline), purchased from Sigma-Aldrich; Merck Millipore (Darmstadt, Germany) and fed for 4 weeks.
The rats were randomly divided into two groups (n=6); in the control group, each rat was injected with a single dose of saline; in the PAH group, each rat was injected with a single dose of MCT (60 mg/kg). On day 28, all rats were sacrificed.
The rats were injected with 8% chloral hydrate following weighing (1 ml/200 g), and fixed on an autopsy table. Right ventricular systolic pressure (RVSP) was measured by insertion of a PE50 pipe through the jugular vein to the right ventricle; the RVSP was transferred into an electric signal and recorded using MedLab software (version 6.3; Nanjing Medease Science and Technology, Nanjing, China).
All rats were sacrificed following measurement of pulmonary arterial pressure, and the hearts, lungs and pulmonary arteries were harvested. The blood was removed in cold PBS. The right ventricle (RV) was isolated from the left ventricle (LV) and septum (S), and these two components were weighted separately. RVH was determined as the ratio of RV weight to (LV+S) weight.
A sample of lung tissue was fixed in prepared 4% paraformaldehyde for 24 h, made into paraffinized sections (4-µM thick), and then stained with hematoxylin and eosin. A fluorescence microscope (Nikon Eclipse 80i; Nikon, Tokyo, Japan) was used to observe the pulmonary arteries in the stained sections.
The pulmonary arteries were cleared in cold PBS and frozen in liquid nitrogen prior to being stored at −80°C. The pulmonary artery tissues were homogenized in lysis buffer (radioimmunoprecipitation assay buffer, PMSF; 100:1) and then centrifuged at 13,800 × g for 15 min at 4°C. The protein concentrations were determined using a bicinchoninic protein assay kit, and 30 µg were separated on a 10% SDS-PAGE gel, followed transferal onto a polyvinylidene difluoride membrane. The membrane was blocked in 5% skim milk (5 g skim milk dissolved in 100 ml Tris-buffered saline Tween solution) at room temperature for 1 h. The membrane was incubated with the following antibodies: HMGR (cat. no. ab174830, 1:2,000), FDPS (cat. no. ab189874, 1:1,000), FNTA (cat. no. ab109738, 1:1,000), and FNTB (cat. no. ab109748, 1:1,000) (all from Abcam, Cambridge, UK), GGT-I (cat. no. sc18996, 1:200; Santa Cruz Biotechnology Co., Ltd., Dallas, TX, USA), phosphorylated (p)-eNOS (cat. no. 95719, 1:1,000), eNOS (cat. no. 9586, 1:1,000), and RhoA (cat. no. ARH04, 1:1,000) (all from CST Biological Reagents Company Limited, Shanghai, China), Rac1 (cat. no. ARC03, 1:1,000; BD Biosciences, Franklin Lakes, NJ, USA) at 4°C for 16 h. The membranes were then incubated with the appropriate secondary antibody: Goat-anti-rabbit immunoglobulin G (IgG) (cat. no. 1268, 1:2,500), goat-anti-mouse IgG (cat. no. 1265, 1:2,500), and rabbit-anti-goat IgG (cat. no. M1102, 1:2,500) (all from Biovision, Inc., Milpitas, CA, USA) for 2 h at room temperature. The target protein bands were visualized using chemiluminescence and quantified using ImageJ software (version 1.47; National Institutes of Health, Bethesda, MD, USA). GAPDH was used as an endogenous control (cat. no. 377R, 1:5,000; Biovison, Inc.). All antibodies were diluted in 5% BSA (HuaBio, China).
The activation of NADPH oxidase was detected using a Tissue NADPH Oxidase Activation Assay kit (Genmed Scientifics, Inc., Arlington, MA, USA). The pulmonary arteries were homogenized in lysis buffer and protein concentrations were determined using a BCA protein assay kit. According to the manufacturer's instructions, this was finally detected at 550 nm using a microplate reader (Thermo Fisher Scientific, Inc., Waltham, MA, USA.).
A Tissue ROS Kinase Activation Assay kit (Genmed Scientifics, Inc.) was used to measure the level of ROS in the pulmonary artery. The pulmonary arteries were homogenized in lysis buffer and protein concentrations were determined using a BCA protein assay kit. The results were detected at 340 nm using a microplate reader (Thermo Fisher Scientific, Inc.).
The blood samples were collected and centrifuged at 13,8000 × g, 4°C for 15 min. The serum was then removed into a new EP tube, and serum NO levels were determined using a Nitric Oxide Fluorometric Assay kit (Nanjing Jiancheng Bioengineering Institute, Nanjing, China) according to the manufacturer's instructions. The results were final detected using a microplate reader (Thermo Fisher Scientific, Inc.).
All values are expressed as the mean ± standard error of the mean. Statistical significance was measured using one-way analysis of variance. Software used for analysis was GraphPad Prism (version 6.0c; GraphPad Software, Inc., La Jolla, CA, USA.). P<0.05 was considered to indicate a statistically significant difference.
The PAH model was induced by injection of a single dose of MCT (60 mg/kg). After 4 weeks, RVSP was measured by insertion of a PE50 pipe through the jugular vein to the right ventricle. The pressures were transferred into electric signals and collected using MedLab software. The RVSP of the PAH group was significantly increased, compared with that in the control (36.9±1.1 vs. 20.9±1.1; P<0.01) as exhibited in
The present study aimed to determine whether the expression levels of key enzymes in the mevalonate pathway are altered in PAH. This involved comparing the expression levels of key enzymes in the pulmonary artery between the PAH and control groups.
HMGR is an initial enzyme in the mevalonate pathway, and the present study found no significant difference in its expression between the PAH and control group (
Small G-proteins are important in regular specific cell function and gene expression (
NADPH oxidase is the downstream effector of small G-protein, and its activation depends on the Rac protein (
NADPH oxidase is a resource for generating ROS (
The eNOS enzyme catalyzes the biosynthesis of NO, and NO in its molecular form has been reported to be important for the development of PAH (
NO is an important molecule for cardiovascular health. It is released as an endothelium-derived relaxing factor (
PAH is a serious pulmonary vascular disease, which can lead to right heart failure following qualitative changes in the artery and lumen, blood flow and pressure, and cardiac muscle (
In the present study, it was demonstrated that the expression of key enzymes in the mevalonate pathway, including FDPS, FNTA and GGTase-I, were significantly increased in the pulmonary artery of MCT-induced PAH rats. The expression of small G-protein Rac1 and RhoA were also increased, which was consistent with the results of GGTase-I and FTase. Small G-proteins downstream effectors, including NADPH and ROS, were also examined; significant increases in ROS and NADPH oxidase activity were demonstrated, whereas the protein expression of eNOS and release of serum NO decreased.
HMGR is the initial enzyme in cholesterol synthesis. The therapeutic effect of statins on pulmonary hypertension is controversial in clinical trails. In the present study, no significant change was demonstrated in HMGR in the PAH rats, consistent with a previous meta-analysis, which reported that HMGR inhibitor statins had no benefit in patients with PAH (
The higher level of FDPS induced the accumulation of downstream products, including GPP and FPP. As an intermediate product, FPP is an important precursor in the synthesis of sterols, dolichols and ubiquinones (
The Rho family, including Rac and Rho, can regulate the function of the cytoskeleton (
NADPH oxidase is a membrane-bound enzyme complex. Several studies have shown that the activation of NADPH oxidase depends on the Rac protein and two other cytosolic proteins, p47phox and p67phox (
NADPH oxidase is a source of superoxides, and superoxides undergo further reactions to generate ROS (
In several heart diseases, NO is an important molecule in vasodilation. The present study found decreased activity of eNOS and serum levels of NO in the PAH group. Although the bio-activity of eNOS was weaker in PAH, no differences in the expression of eNOS were demonstrated between PAH and the control group. This contradicted the results of a previous study, which reported that the expression level of eNOS decreased in the PAH model (
The present study detected changes in the expression of key enzymes of the mevalonate pathway in an MCT-induced PAH model, which may serve as drug targets for PAH treatment. The inhibitors of certain enzymes, including FDPS and GGTase-I, have been used in the treatment of certain human diseases. However, whether treatment with the inhibitors of altered enzymes in PAH can attenuate persistent pulmonary artery remodeling and high pressure remains to be elucidated, for which further investigations are required.
This study was financially supported by the National Natural Sciences Foundation of China (grant no. 81400277) and the Natural Sciences Foundation of Zhejiang Province (grant no. LY17H020006). The abstract was presented at the 15th meeting of China Interventional Therapeutics March 30 - April 2, 2017 in Beijing, China (abstract no. AS-0341).
Development of PAH 4 weeks following a single injection of monocrotaline (60 mg/kg). (A) RVSP in control and PAH groups. Data are expressed as the mean ± standard deviation. **P<0.01 compared with the control group. (B) RV/(LV+S) in each group. (C) Hematoxylin and eosin staining of lung tissues. The arrows indicate pulmonary micro-arteries (original magnification, ×200). RVSP, right ventricular systolic pressure; PAH, pulmonary arterial hypertension; RV, right ventricle; LV, left ventricle; S, septum.
Expression levels of HMGR, FDPS, FNTA, FNTB and GGTase-I in the MCT-induced PAH rat pulmonary artery. The proteins were extracted from pulmonary arteries of each group. (A) Western blot analyses demonstrate the expression of HMGR, FDPS, FNTA, FNTB and GGTase-I in the control and MCT-induced PAH rats. GAPDH was used as the endogenous loading control. Graphs demonstrate the relative changes in (B) HMGR, (C) FDPS, (D) FNTA, (E) FNTB and (F) GGTase-I in the control and MCT groups. Data are expressed as the mean ± standard deviation. *P<0.05 and **P<0.01 compared with the control group. PAH, pulmonary arterial hypertension; MCT, monocrotaline; HMGR, 3-hydroxy-3-methylglutaryl-coenzyme A; FDPS, farnesyldiphosphate synthase; FNTA, farnesyltransferase α; FNTB, farnesyltransferase β; GGTase-I, geranylgeranyltransferase type I.
Oxidative stress and small-G protein expression in control and PAH rats. Proteins were extracted from the pulmonary artery of control and PAH rats. (A) Western blot analyses demonstrate the expression of RhoA and Rac1 in the control and PAH rats. GAPDH was used as an endogenous loading control. Graphs demonstrate the relative changes of (B) RhoA and (C) Rac1 in each group. (D) Changes in NADPH oxidase activity in each group were detected using a tissue NADPH oxidase activation assay kit. (E) ROS changes in each group were detected using a tissue ROS kinase activation assay kit. Data are expressed as the mean ± standard deviation. *P<0.05 and **P<0.01 compared with the control group. PAH, pulmonary arterial hypertension; ROS, reactive oxygen species; RhoA, Ras homolog family, member A; Rac1, Ras-related C3 botulinum toxin substrate 1; C, control.
Alterations in eNOS activity and serum NO release in control and PAH rats. Proteins were extracted from the pulmonary artery of animals each group. (A) Western blot analyses demonstrate the expression of P-eNOS and T-eNOS in the control and PAH groups. GAPDH was used as an endogenous loading control. Graphs demonstrate the relative changes of (B) P-eNOS and (C) T-eNOS in each group. (D) Graph demonstrating the ratio of P-eNOS to T-eNOS. (E) Graph demonstrating the changes in serum NO release in the control and PAH groups. Data are expressed as the mean ± standard deviation. *P<0.05 and **P<0.01 compared with the control group. PAH, pulmonary arterial hypertension; NO, nitric oxide; eNOS, endothelial NO synthase; P, phosphorylated; T, total.