Protection of Endothelial Cells, Inhibition of Neointimal ... - Springer Link

2 downloads 0 Views 449KB Size Report
May 24, 2011 - Protection of Endothelial Cells, Inhibition of Neointimal. Hyperplasia by β-elemene in an Injured Artery. Lingyan Wu & Guixue Wang & Shutang ...
Cardiovasc Drugs Ther (2011) 25:233–242 DOI 10.1007/s10557-011-6305-9

Protection of Endothelial Cells, Inhibition of Neointimal Hyperplasia by β-elemene in an Injured Artery Lingyan Wu & Guixue Wang & Shutang Tang & Guang Long & Tieying Yin

Published online: 24 May 2011 # Springer Science+Business Media, LLC 2011

Abstract Aims It is generally accepted that the oxidative stress and the proliferative activity of vascular smooth muscle cells (VSMCs) contribute to the pathogenesis of neointimal hyperplasia after vascular injury. Although β-elemene (β1-methyl-1-vinyl-2, 4-diisopropenyl-cyclohexane) has been used as an antitumour drug, its therapeutic effect on vascular diseases has not yet been determined. In this study, we investigated whether β-elemene could inhibit oxidative damage of vascular endothelial cells, suppress VSMCs growth and prevent neointimal hyperplasia. Methods and results β-elemene can increase the survival rate of human umbilical vein endothelial cells in vitro. By measuring the malondialdehyde content, total antioxidant capacity, superoxide dismutase activity, catalase activity, glutathione peroxidase activity and nitric oxide levels, we assessed the protective effect of β-elemene in the vascular endothelium model against oxidant-induced injury. Μoreover, β-elemene inhibited cell proliferation and induced apoptosis in cultured VSMCs. In a flow culture system, β-elemene reduced the migration distance of VSMCs. By transwell migration assay, β-elemene was found to reduce the migration cell number of VSMCs, but not affect the HUVECs migration. In a rat carotid artery balloon injury model, administration of β-elemene significantly reduced the ratio of intimal area to medial area and neointima formation. Conclusions Our results indicate that β-elemene is effective in protecting the endothelial cells from injury induced by hydrogen peroxide in vitro, inhibiting smooth muscle L. Wu : G. Wang (*) : S. Tang : G. Long : T. Yin Key Laboratory of Biorheological Science and Technology (Chongqing University), Ministry of Education, Chongqing Engineering Lab. in Vascular Implants, Bioengineering College of Chongqing University, Chongqing 400044, China e-mail: [email protected]

cell proliferation/migration and inhibiting neointima formation in vivo after vascular injury. Key words β-elemene . Vascular endothelial cell . Vascular smooth muscle cell . Rat . Neointimal hyperplasia

Introduction The malformation and malfunction of vascular endothelial cells (VECs) is one of the major pathophysiological mechanisms contributing to the cardiovascular diseases [1, 2]. The endothelium is located within the blood vessel wall and acts as a barrier between the blood and the vascular smooth muscle cells (VSMCs). Therefore, the functional integrity of the endothelium monolayer is essential to prevent vascular leakage and formation of neointimal hyperplasia [3]. Many researchers consider that the injury and the malfunction of VECs are closely related to atherosclerosis and neointimal hyperplasia. Prevention of endothelial cells from injury may improve their function [4]. Oxidative damage to VECs is the most important cause of the genesis of cardiovascular diseases. Various kinds of regulation factors, which are produced and expressed, play a major role in regulating the function of blood vessels [5]. There is evidence to suggest that free radicals, active oxygen and hydrogen peroxide (H2O2) are able to evoke the injury of VECs [6]. Oxidative injury can induce endothelial cell apoptosis, which is partially prevented by antioxidants and free radical scavengers [7–10]. Therefore, antioxidants are believed to be important agents against endothelial cell apoptosis and might be used to develop novel therapeutic strategies to protect endothelial cells and to induce reendothelialisation, key mechanisms for the prevention of cardiovascular diseases.

234

The pathogenesis of neointimal hyperplasia is associated with not only a cascade of activated cellular phenotypes triggered by endothelial cell damage [11] but also the accumulation and proliferation of VSMCs [12, 13], which migrate from the medial layer and result in lesion progression and encroachment in the coronary vascular lumen [14]. Numerous factors associated with neointimal hyperplasia and restenosis are known to trigger the activation, proliferation and migration of VSMCs [15–17]. β-elemene (β-1-methyl-1-vinyl-2,4-diisopropenyl-cyclohexane), isolated from the Chinese medicinal herb Curcuma wenyujin [18], was shown to act against cell apoptosis and inhibit human and murine tumour cell growth in vitro and in vivo [19–26]. Efficacy of β-elemene injection, a broadspectrum antitumour drug, was studied in China in clinical trials in cancer patients, and β-elemene is being applied in clinical studies in the United States. However, its protective effect against oxidative stress and its ability to inhibit the proliferation and migration of VSMCs have not been reported. Therefore, it prompted us to test whether β-elemene has a protective effect on H2O2-induced injury in human umbilical vein endothelial cells (HUVECs). Moreover, we investigated whether β-elemene could suppress the growth and migration of VSMCs in vitro and reduce neointimal hyperplasia in a rat carotid artery balloon injury model. In this study, we confirmed that the β-elemene has a protective effect on H2O2-induced injury in HUVECs, and that the possible mechanism was associated with the enzyme activities, which also suggests a role of the drug in preventing and curing neointimal hyperplasia and cardiovascular diseases.

Cardiovasc Drugs Ther (2011) 25:233–242

mycin (100 μg/ml) in a 96-well plate and were incubated at 37°C. VSMCs (A7r5) (ATCC, USA) were cultured at a density of 4×104 to 5×104/ml in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% FBS, penicillin (100 U/ml) and streptomycin (100 μg/ml) in a 96-well plate and were incubated at 37°C. Drug treatments were usually done 24 h after seeding the cells. The absorbance values were measured using an ELISA assay reader. H2O2 injury and viability analysis of HUVECs To induce oxidative stress, HUVECs were cultured at a density of 5×104/ml for 24 h. Cells were incubated with various concentrations of H2O2 (0.125 mM, 0.25 mM, 0.5 mM, 1 mM and 2 mM) for different time intervals (1 h, 2 h and 4 h). The viability of HUVECs was measured by MTT assay. The cell viability was expressed in percentage of survival relative to the control cell samples. To study the protective effect of β-elemene against H2O2 toxicity, HUVECs were seeded (2×104/well) in a 96-well plate. After 24 h, the cells were treated with various concentrations of β-elemene (5 μg/ml, 10 μg/ml, 20 μg/ml and 40 μg/ml). After 24 h, culture medium was discarded and cells were incubated with 1 mM H2O2. After 1 h, the viability of HUVECs was measured by MTT assay. To study the therapeutic effects of β-elemene against H2O2 toxicity, HUVECs were seeded (2×104/well) in a 96-well plate. After 24 h, HUVECs were incubated with 1 mM H2O2. After 1 h, the cells were treated with various concentrations of β-elemene (5 μg/ml, 10 μg/ml, 20 μg/ml and 40 μg/ml). After 24 h, the viability of HUVECs was measured by MTT assay.

Materials and methods Assay of enzymatic activities Animals and reagents All scientific protocols involving the use of animals were approved by the Institutional Animal Care and Use Committee of China, and institutional guidelines for animal welfare and experimental conduct were followed. Animals were housed in a secured animal facility at Nanhua University School of Medicine. β-elemene emulsion was purchased from Dalian Jingang Pharmaceutical Co. (5 mg/ml, Dalian, China). MTT [3-(4,5-dimethyl tiazol-2-yl)-2,5 diphenyltetrazolium bromide] was purchased from Sigma (Shanghai, China). H2O2 was purchased from Chongqing Chemical Agent Co. (Chongqing, China).

HUVECs, exposed to various concentrations of β-elemene (5 μg/ml, 10 μg/ml, 20 μg/ml and 40 μg/ml) for 24 h, were treated with 1 mM H2O2 for additional 1 h. Cells were collected and homogenised by ultrasonic, and supernatant was collected to assay some oxidative biomarkers. The total antioxidant capacity (T-AOC) was measured using 2, 2′azino-bis (3-ethylbenzthiazoline-6-sulfonic acid) method. Malondialdehyde (MDA) content, NO content and the enzyme activities of superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GSH-Px) were detected using the kits according to the manufacturer’s instructions (Nanking Jiancheng Biological Research Institute, Nanking, China).

Cell culture and experimental treatment Proliferation assay HUVECs (ATCC, USA) were cultured at a density of 4×104 to 5×104/ml in RPMI-1640 medium supplemented with 10% fetal bovine serum (FBS), penicillin (100 U/ml) and strepto-

VSMCs, cultured for 24 h, were exposed to various concentrations of β-elemene (5 μg/ml, 10 μg/ml, 20 μg/ml, 40 μg/ml

Cardiovasc Drugs Ther (2011) 25:233–242

and 100 μg/ml) for different time intervals (24 h, 48 h and 72 h). The viability of cells was measured by MTT assay. Twenty microlitres of MTT (5 mg/mL) was added to each well and incubated for 4 h at 37°C. Then, the supernatant was discarded and 150 μl of dimethyl sulfoxide was added to each well. The absorbance was measured using an ELISA assay reader. Real-time PCR Total RNAs were extracted from VSMCs, which exposed to 20 μg/ml β-elemene for 24 h. For semiquantitative RTPCR assay, 1 μg of total RNAs was used to detect mRNA expression by Super-Script One-Step RT-PCR kit (Invitrogen, Carlsbad, CA). The reverse transcription reaction was performed at 25°C for 10 min, 48°C for 30 min, and 95°C for 5 min. PCR cycles (28 cycles) include 94°C for 30 s, 55°C for 30 s, and 72°C for 30 s. Primers for amplification of caspase3, caspase7, caspase9 and GAPDH are listed as follows. The PCR product were subjected to 1% agarose gel electrophoresis and stained with ethidium bromide. The relative levels were quantified by Matrox Inspector V.4.1 software. Casp3 Upstream primer: 5′-TGC TTA CTC TAC AGC ACC TGG TTA CT-3′ Downstream primer: 5′-TGA ACC ACG ACC CGT CCT T-3′ Casp7 Upstream primer: 5′-CCG TCC ACA ATG ACT GCT CTT-3′ Downstream primer: 5′-GGT CCT CCT CAG AGG CTT TTC-3′ Casp9 Upstream primer: 5′-AAC GAC CTG ACT GCC AAG AAA-3′ Downstream primer: 5′-GGT TCC GGT GTG CCA TCT C-3′ GAPDH: Upstream primer: 5′-GGCCATCAAGCCAGAGCTT-3′, Downstream primer: 5′-CCAAACCATCACTGA CACTCAGA-3′. Migration distance of VSMCs in flow culture system The flow culture system is composed of a parallel-plate flow chamber, a perfusion-assisted system, a mixed gas supply part and a biochemical thermostat incubator (Fig. 4a). Parallel-plate flow chamber is composed of a

235

polycarbonate plate attached to a rectangular silicone gasket of 0.2-mm thickness (specifications: 25.0 mm×15.0 mm× 0.2 mm), and perfusion support system is composed of upper and lower glass reservoir bottles, silicone tube and constant-current pump. Mixture contains 95% air and 5% CO2. Flow shear stress to which VSMCs are exposed was calculated using the formula: τ=6ηQ/wh2 (η, the perfusion fluid viscosity; Q, flow rate; h, flow chamber height). Flow chamber, filling pipes and storage tank were sterilised and were assembled in an incubator at 37°C. In this study, η is 1.2 mPa·s and shear stress is 24 dynes/cm2. The VSMCs were seeded on the cover slip coated with 2 μg/ml polylysine and 2 μg/ml collagen. After 24 h of culture, the cells were treated with different concentrations of β-elemene for 24 h. Cells were scraped along the edge of the cover slip, and then the excessive cells were washed away in the culture medium. This cover slip was set in flow chamber and subjected to flow shear stress. The DMEM medium was the circulating fluid. The cell migration pictures were collected at 0 h and 6 h. Cell migration distance was measured by image processing software (IMAGE-PRO PLUS a computer image analysis system). The cover slip edge was set as the base line and used to normalise the cell migration distance (Fig. 4b). T0 is the distance of the original cells to the cover slip edge, and T1 is the distance of the migrated cells to the cover slip after β-elemene treatment. Cell migration distance is calculated as (T0−T1). Transwell migration assay of VSMCs and HUVECs VSMCs (5×104 cells/chamber) were seeded into the upper chamber (Boyden). Each well was separated into two chambers by an insert membrane with 8 μm pores. Chemotaxis was achieved in the presence of 100% FBS in the bottom chamber. For HUVECs, as described previously [27], the assay was modified to use a 3 μm pore size and a chemoattractant stimulus of EGM-2MV medium with 5% FBS and 50 ng/ml vascular endothelial growth factor (BD bioscience, San Jose, CA). After 8 h, the transwell chambers were rinsed in PBS, and the cells at the bottom of the transwell membrane were fixed with 4% paraformaldehyde at 37°C for 15 min, and then rinsed with PBS. To quantify the number of migrating cells at the bottom of the transwell, the cells were labelled with 4′, 6diamidino-2-phenylindole, and then the cells that had migrated were counted manually using a fluorescent microscope. The migration was quantified by the analysis of at least 10 random fields per filter. Carotid artery injury model and drug treatment Male Sprague–Dawley rats (300±20 g) were anaesthetised, and a neck incision was made. A 20-mm section of the right

236

Histology and morphometry The arteries from the treated rats were fixed and embedded in paraffin. Then, the paraffin-embedded arteries were cut into ten sections for analysis. Morphometric analysis was carried out on HE-stained arteries. For quantitative characterisation, the images (20×) were transferred to a computer using a video camera. For rat balloon-injury artery, 10 sections were randomly selected for analysis. The relative parameters were determined using the Imaging software by selecting appropriate functions. The areas encompassed by the lumen surface (lumen area), internal elastic lamina– lumen surface (intima area) and External elastic lamina (EEL) perimeter were measured. For the evaluation of the degree of intimal hyperplasia, intima and media wall thickness and the ratio of intimal area to medial area (I/M ratio) were calculated and compared using the digital imaging software (Image-pro Plus). Statistics Data are expressed as means ± SEM. Statistical analyses were performed by one-way ANOVA, followed by Student’s ttest for multiple comparisons. Statistical significance was set P