Chronic hypertension induces vascular and cardiac remodeling. OW1 is a novel imperatorin derivative that was previously reported to inhibit vascular remodeling and improve kidney function affected by hypertension. In the present study, the effect of OW1 on the cardiac remodeling induced by hypertension was investigated. OW1 inhibited vascular smooth muscle cell (VSMC) proliferation and the phenotypic modulation of VSMCs induced by angiotensin II (Ang II). The OW1-induced vasodilatation of rat cardiac arteries was evaluated in vitro. Renovascular hypertensive rats were developed using the two-kidney one-clip method and treated with OW1 (40 or 80 mg/kg/day) or nifedipine (30 mg/kg per day) for 5 weeks. OW1 markedly reduced the systolic and diastolic blood pressure compared with that in the hypertension group or the respective baseline value during the first week. OW1 also reduced cardiac weight, and the concentrations of Ang II, aldosterone and transforming growth factor-β1 (TGF-β1). Histological examination demonstrated that OW1 exerted an inhibitory effect on vascular and cardiac remodeling. These inhibitory effects were associated with decreased cardiac levels of Ang II, matrix metalloproteinase-2 and TGF-β1 in the hypertensive rats. In summary, OW1 exhibited a clear antihypertensive effect. More importantly, it inhibited vascular and cardiovascular remodeling, which may reduce the risk of hypertension-induced cardiovascular diseases. These results have potential implications in the development of new antihypertensive drugs.
Chronic hypertension induces vascular and cardiac remodeling (
Numerous treatments for hypertension have been investigated, with a focus on reducing the risk of hypertension-induced cardiovascular diseases. In the present study, a novel imperatorin derivative, 2-(3,4-dimethoxyphenyl)-2-isopropyl-5-[(2-[(7-oxo-7H-furo[3,2-g] chromen-4-yl)-oxy]ethyl)amino]pentanenitrile (OW1;
A total of 55 male Sprague-Dawley (SD) rats (age, 6–8 weeks; body weight, 200–230 g) and two male SD rats (age, 3–4 weeks) were obtained from the Animal Center of Xi'an Jiaotong University (Xi'an, China). The rats were maintained at room temperature under a 12-h light/dark cycle with
OW1 was prepared in the Natural Drug Research and Engineering Center of Xi'an Jiaotong University using xanthotoxin (Meryer Chemical Technology Co., Ltd., Shanghai, China) as the starting material. OW1 was synthesized via a previously reported method comprising six reaction steps, with characterization of the chemical structures (
A total of 10 male SD rats aged 6–8 weeks were anesthetized by intraperitoneal injection with 10% chloral hydrate (350 mg/kg body weight; Shanghai Jinjinle Industry Co., Ltd., Shanghai, China) and sacrificed by decapitation. The superior CAs (2 mm) were dissected free of fat and connective tissue and mounted in a Multi Wire Myograph System (Danish Myo Technology A/S, Inc., Skejbyparken, Denmark). The vessels were maintained at 37°C in physiological Krebs-Henseleit solution (pH 7.4) through which a gaseous mixture of 95% O2 and 5% CO2 was bubbled. Following a 30 min equilibration period, the CA segments were equilibrated for 1.5 h with a resting tension of 3 mN prior to the initiation of testing. The contractile capacity of each CA segment was tested by exposure to a K+-rich Krebs-Henseleit solution (containing 4.45 g/l KCl), in which NaCl was exchanged for an equimolar concentration of KCl. When two reproducible contractions were achieved the CA segments were further evaluated. Following equilibration, the segments were pre-contracted with KCl (60 mM). Once the sustained tension was obtained, OW1 (0.1 µM-0.1 mM) was added at increasing concentrations to the baths, and concentration-response curves were constructed.
The SD rats aged 3–4 weeks were sacrificed by decapitation and the thoracic aortas were removed. Vascular smooth muscle cells (VSMCs) were isolated from the thoracic aorta as previously described (
A total of 45 male SD rats aged 6–8 weeks were used to establish the 2K1C rat model of hypertension. Surgical procedures were performed under 10% chloral hydrate anesthesia (350 mg/kg body weight, intraperitoneally). 2K1C modeling was conducted by separating the left renal artery through an abdominal approach and placing it in a 0.3 mm silver clip. Rats in the sham-operated group underwent the same procedure, with the exception that the renal artery was not clipped. The rats were placed into single cages and their airways were kept open until they regained consciousness (
On week 6 after the surgery, rats with a systolic blood pressure (SBP) ≥140 mmHg were considered to be hypertensive. At the end of week 6, the SBP was very stable and rats with a SBP >160 mmHg were randomly divided into four groups: i) Hypertension group, comprising rats intragastrically treated with vehicle (ultrapure water); ii) nifedipine group (30 mg/kg/day); iii) OW1 high dose group (OW1-H; 80 mg/kg/day); iv) OW1 low dose group (OW1-L; 40 mg/kg/day). A sham-operated group was also established. Drugs or vehicle were administered by gavage once a day for 5 weeks. The blood pressure of the rats in each group was measured by the tail-cuff method.
When the treatment period was complete, the rats were sacrificed with 20% urethane (1 g/kg, intraperitoneally; Yangzhou Xinhua Chemical Industry Co., Ltd., Yangzhou, China) in the morning after overnight fasting, and blood samples were then collected in vacuum tubes with ethylenediamine tetraacetic acid as an anticoagulant. The blood samples were centrifuged at 1,000 × g for 15 min at 4°C to separate the plasma, which was then frozen at −80°C until required for testing. Levels of aldosterone (ALD) in plasma were detected using a radioimmunoassay kit (Beijing Sino-UK Institute of Biological Technology, Beijing, China). Levels of TGF-β1 in the plasma were quantified using an enzyme-linked immunosorbent assay kit (ML-Elisa-0014; Shanghai Fuzhong Bio Tech Co., Ltd., Shanghai, China).
When the treatment period was complete, the rats were anesthetized with urethane as described above. The thoracic cavity was opened to expose the heart, which continued to beat. The heart was rapidly removed, rinsed in ice-cold 0.9% saline solution, blotted and weighed. The two ventricles from the heart were isolated, cut into two fragments by a mid-ventricular coronal section and stored in phosphate-buffered 10% formalin (pH 7.3) for histological examination. The remainder of the heart was frozen and stored at −80°C until required for biochemical examination. The levels of Ang II in the heart were detected using a radioimmunoassay kit (Beijing Sino-UK Institute of Biological Technology).
The thoracic aorta and heart were stained with hematoxylin and eosin (H&E) and Masson's trichrome respectively. Images were viewed and captured using the Leica Q550CW Image Analysis System (Leica Microsystems GmbH, Wetzlar, Germany) and analyzed using ImagePro Plus version 3.0 (Media Cybernetics,. Rockville, MD, USA) to determine the aortic cross-sectional area (CSA) and media to lumen (M/L) ratio.
VSMCs exposed to Ang II in the presence or absence of OW1 (5, 10 or 20 µM) as in the proliferation assay, or thoracic aortas from individual rats in each group were lysed in radioimmunoprecipitation assay lysis buffer containing 0.1 M phenylmethylsulfonyl fluoride and centrifuged at 13,400 × g for 20 min. The protein concentrations of the supernatants were determined by bicinchoninic acid (BCA) Protein Assay (Applygen Technologies, Inc., Beijing, China). Extracts were boiled in a 1:1 ratio with loading buffer containing Tris (125 mmol/l, pH 6.8), 4% w/v sodium dodecylsulfate, 10% v/v glycerol, 4% v/v 2-mercaptoethanol, and 2 mg/ml bromophenol blue. Equal amounts of protein (30–60 µg) were resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE; 10%) and transferred to nitrocellulose membranes (GE Healthcare Life Sciences, Logan, UT, USA). The membranes were incubated overnight at 4°C with mouse anti-α-smooth muscle actin polyclonal antibody (α-SMA; 1:200; BM0002; Wuhan Boster Biological Technology, Ltd., Wuhan, China), rabbit anti-MMP-2 polyclonal antibody (1:500; ab37150; Abcam, Hangzhou, China), rabbit anti-collagen I polyclonal antibody (1:100; bs-10423R; Beijing Biosynthesis Biotechnology Co., Ltd., Beijing, China), and rabbit anti-glyceraldehyde-3-phosphate dehydrogenase polyclonal antibody (GAPDH; 1:500; AB-P-R 00; Hangzhou Xianzhi Bio Tech Co., Ltd., Hangzhou, China) as an internal control. After wshing the membranes three times with phosphate-buffered saline containing Tween-20, the membranes were incubated with horseradish peroxidase-conjugated secondary antibodies (1:2,000; 31460; Shaanxi Xianfeng Biotechnology Co., Ltd., Shaanxi, China) for 45 min at 37°C, followed by incubation with Western Blotting Luminol Reagent (Shanghai Tuoran Technology Co., Ltd., Shanghai, China). Images of the membranes were captured using an image acquisition and analysis system (ChampGel™ 6000; Beijing Sage Creation Science Co., Ltd., Beijing, China), and the relative expression levels were determined using ImageJ software, version 1.46r (National Institutes of Health, Bethesda, MA, USA).
Data are expressed as the mean ± standard error of the mean. One-way analysis of variance followed by a Tukey's multiple comparison test was used to test the significance between three or more groups. P<0.05 was considered to indicate a statistically significant difference.
OW1 (0.1 µM-0.1 mM) relaxed the CA segments that were pre-contracted with KCl in a concentration-dependent manner. The maximum relaxation effect (Emax) of OW1 on the CA segments was 114.1±2.3% and the median effective concentration (EC50) was 1.00±0.05 µM (
The MTT assay showed that OW1 (5 µM) inhibited Ang II-induced VSMC proliferation (
The baseline blood pressure values among the groups were not observed to differ significantly, with the exception that the sham-operated group had a lower SBP and diastolic blood pressure (DBP) compared with the groups subjected to the 2K1C modeling procedure (P<0.01;
The mean heart weight was higher in the hypertension group compared with the sham group (P<0.05). Compared with the hypertension group, the heart weight of the OW1-H group was significantly decreased (P<0.05;
Cardiac remodeling was observed in the hypertension groups, with significant myocardial fibrosis (
Serious arterial wall hypertrophy was observed in the hypertension group (
The 2K1C renovascular hypertensive rat is a widely used model of chronic hypertension; it resembles human renovascular hypertension, with activation of the RAAS and increased Ang II levels, which promotes vascular hypertrophy and cardiac remodeling (
In the present study, the blood pressure in the hypertensive rats was markedly reduced by OW1, and the antihypertensive effects of high-dose OW1 were comparable with those of nifedipine. The effect of OW1 on VSMC proliferation was investigated
The results of the present study suggested that OW1 had antihypertensive and inhibitory effects on vascular and cardiovascular remodeling. Therefore, OW1 may reduce the risk of hypertension-induced cardiovascular diseases, which has potential clinical implications.
The present study was supported by the National Natural Science Foundation of China (grant nos. 81230079, 81227802 and 81202494).
Chemical structure of OW1.
OW1 induced vasodilatation in rat cardiac arteries and had an inhibitory effect on Ang II-induced VSMC proliferation
Effect of OW1 on blood pressure in 2K1C hypertensive rats. Effect of OW1-H (80 mg/kg/day) and OW1-L (40 mg/kg/day) on (A) systolic blood pressure (SBP) and (B) diastolic blood pressure (DBP). All values are means ± standard error of the mean (n=6). *P<0.05, **P<0.01 vs. the hypertension group; ##P<0.01 vs. the Nifedipine group; $$P<0.01 vs. the OW1-H group; ΔP<0.05 vs. the OW1-L group and ΔΔP<0.01 vs. the OW1-L group. 2K1C, two-kidney one-clip; OW1-H, OW1 high dose group; OW1-L, OW1 low dose group.
OW1 reduces cardiac weight, and Ang II, ALD and TGF-β1 concentrations. Effect of OW1-H (80 mg/kg/day) and OW1-L (40 mg/kg/day) on (A) cardiac weights, (B) concentration of Ang II in cardiac tissue, and plasma levels of (C) TGF-β1 and (D) ALD. All values are means ± standard error of the mean (n=8). **P<0.01, *P<0.05 vs. the hypertension group. OW1, an imperatorin derivative; Ang II, angiotensin II; ALD, aldosterone; TGF, transforming growth factor.
OW1 inhibits cardiac remodeling in 2K1C hypertensive rats
OW1 inhibits vascular remodeling in 2K1C hypertensive rats