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        Molecular mechanism of green microalgae, Dunaliella salina, involved in attenuating balloon injury-induced neointimal formation
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        Molecular mechanism of green microalgae, Dunaliella salina, involved in attenuating balloon injury-induced neointimal formation
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Abstract

The pathological mechanism of restenosis is primarily attributed to excessive proliferation of vascular smooth muscle cells (VSMC). The preventive effects of ethanol extract of Dunaliella salina (EDS) on balloon injury-induced neointimal formation were investigated. To explore its molecular mechanism in regulating cell proliferation, we first showed that EDS markedly reduced the human aortic smooth muscle cell proliferation via the inhibition of 5′-bromo-2′-deoxyuridine (BrdU) incorporation at 40 and 80 μg/ml. This was further supported by the G0/G1-phase arrest using a flow cytometric analysis. In an in vivo study, EDS at 40 and 80 μg/ml was previously administered to the Sprague–Dawley rats and found that the thickness of neointima, and the ratio of neointima:media were also reduced. EDS inhibited VSMC proliferation in a dose-dependent manner following stimulation of VSMC cultures with 15 % fetal bovine serum (FBS). Suppressed by EDS were 15 % FBS-stimulated intracellular Raf, phosphorylated extracellular signal-regulated kinases (p-Erk) involved in cell-cycle arrest and proliferating cell nuclear antigen. Phosphorylated focal adhesion kinase (p-FAK) was also suppressed by EDS. Also active caspase-9, caspase-3 and cleaved poly(ADP-ribose) polymerase (PARP) protein expression levels were increased by administration with EDS; the apoptotic pathway may play an important role in the regulatory effects of EDS on cell growth. These observations provide a mechanism of EDS in attenuating cell proliferation, thus as a potential intervention for restenosis.

Dunaliella salina, Teod. (Chlorophyceae), the unicellular halophilic green microalga, is known as a major source of β-carotene. Administration of D. salina decreased the levels of cholesterol and lactate dehydrogenase as well as increasing the activities of catalase, superoxide dismutase, serum aspartate aminotransaminase and serum alanine aminotransferase( 1 ). Aside from being a precursor for vitamin A, D. salina has also been known to possess a potent antioxidant activity, as shown in an in vivo study( 2 ). Analysing the constituents of an ethanol extract of D. salina (EDS) in our previous study demonstrated 6 % of β-carotene, 0·12 % of α-carotene, 0·2 % of xanthophyll, 0·3 % of zeaxanthin, and scarse amounts of lycopene and chlorophyll( 3 ). It has been shown that 9-cis β-carotene-rich powder of the alga D. bardawil increases plasma HDL-cholesterol in fibrate-treated patients( 4 ). Levy et al. found a significant increase in the lag time of oxidising LDL-cholesterol following a 3-week β-carotene supplementation (60 mg/d), suggesting the antioxidant effects of β-carotene( 5 ).

Percutaneous transluminal coronary angioplasty (PTCA) has been used in patients with angina and acute myocardial infarction( 6 ). However, restenosis in about 30 % of patients within 6 months following the angioplasty procedure has been a major disadvantage of this therapy( 7 ). Stents were then developed to decrease restenosis rate; however, 20 to 30 % of the patients are still affected by restenosis after coronary stenting( 8 ). The regulation of this pathological process remains elusive. One of the major causes leading to arterial reocclusion after PTCA has been linked to the outgrowth of vascular smooth muscle cells (VSMC)( 9 , 10 ). During this time, growth and prothrombotic factors released from platelets and leucocytes trigger the VSMC cell cycle from the G1 to S phase( 11 ). Preventing the cell cycle of VSMC from the G1 to S phase may be beneficial in reducing cell proliferation or migration( 12 ). For this reason, drugs associated with cell-cycle blocking are considered as potential candidates to reduce the incidence of restenosis( 13 ). Restenosis emerges from the proliferation and migration of smooth muscle cells from the arterial media to the intima in conjunction with the formation of extracellular matrix, thereby resulting in a reduced diameter of the vessel lumen( 14 , 15 ).

Since earlier studies have shown that pretreatment with antioxidants can significantly reduce balloon injury-induced neointima formation( 16 ), EDS containing many antioxidants including β-carotene and lycopene may be developed as another potential candidate to prevent restenosis. To test whether EDS can be an effective therapeutic intervention for balloon injury, the molecular and cellular mechanisms of EDS in preventing abnormal cell proliferation were evaluated at various concentrations in both in vitro and in vivo studies. The protein levels of proliferating cell nuclear antigen (PCNA), Raf, focal adhesion kinase (FAK), extracellular signal-regulated kinase (Erk), caspase-9, caspase-3 and poly (ADP-ribose) polymerase (PARP) were evaluated to explore its inhibitory mechanism on neointimal formation. The present study provides a general insight into the pharmacological mechanism of EDS in preventing the outgrowth of smooth muscle cells, which is a potential intervention for balloon injury-induced neointimal formation.

Materials and methods

Cell culture

Human aortic smooth muscle cells (HASMC) were purchased from the Food Industry Research and Development Institute, Hsinchu, Taiwan (CCRC 60 293). They were maintained in Ham's F12K medium containing 10 % fetal bovine serum (FBS), 2 mm-l-glutamine, sodium bicarbonate (1·5 g/l), 10 mm-HEPES, 10 mm-(N-tris)hydroxymethyl-2-aminoethanesulfonic acid, ascorbic acid (0·05 mg/ml), transferrin (0·01 mg/ml), insulin (0·01 mg/ml), sodium selenite (10 ng/ml) and epidermal growth factor (0·03 mg/ml). All experiments were performed with HASMC in passages 21–31, which had been grown to 80–90 % confluence and made quiescent by serum starvation (0·1 % FBS) for at least 24 h. Treatment was with 5, 10, 20, 40 and 80 μg/ml of EDS in F12K containing 15 % FBS for 24 h. The control of the experiments was 15 % FBS.

Cytotoxicity assay

The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay was performed to measure the cytotoxicity of EDS on HASMC. Cells were seeded in ninety-six-well plates with 1 × 104 cells/well in F12K supplemented with 15 % FBS. After 24 h, cells were washed with PBS and then exposed to either 15 % FBS alone or serial dilutions (5, 10, 20, 40 and 80 μg/ml) of EDS. After 12, 24, 48 and 72 h, the number of viable cells was determined2. Briefly, MTT (3 mg/ml in PBS) was added to each well (25 μl per 200 ml medium), and the plate was incubated at 37°C for 2 h. Cells were then spun at 300  g for 5 min, and the medium was carefully aspirated. A 50 μl sample of dimethylsulfoxide was added, and the absorbance at 570 nm was measured for each well on an ELISA reader (Anthos 2001; Anthos Labtec, Salzburg, Austria).

5′-Bromo-2′-deoxyuridine incorporation for DNA synthesis

HASMC cultured in six-well plates were incubated with 5, 10, 20, 40 and 80 μg/ml of EDS in F12K containing 15 % FBS for 48 h. The cells were then subjected to 10 mm-5′-bromo-2′-deoxyuridine (BrdU) incubation for 3 h. BrdU incorporation into DNA was measured by utilising a colorimetric reaction with peroxidase-linked anti-BrdU antibody using a cell proliferation ELISA kit according to the manufacturer's instructions (Boehringer Mannheim, Ingelsheim, Germany).

Flow cytometric analysis

Cellular total DNA contents of the treated cells were assessed using flow cytometry following propidium iodide (PI) staining. Cells were harvested with trypsin–EDTA, washed twice with 10 ml ice-cold PBS, fixed in 70 % ethanol, and kept at 4°C before fluorescence activated cell sorting (FACS) analysis. For DNA content analysis, cells were centrifuged and re-suspended in 0·3 ml of DNA staining solution (100 Ag/ml PI, 0·2 % Nonidet P-40, and 1 mg/ml RNase A (DNase-free) in PBS lacking Ca2+ and Mg2+; at a 1:1:1 ratio by vol.). The cell suspension was stored on ice in a dark room for a minimum of 30 min and analysed within 2 h. Cells were analysed using a FACScan flow cytometer (Becton Dickinson, San Jose, CA, USA). PI fluorescence was linearly amplified and both the area and width of the fluorescence pulse were measured. Ten thousand events were acquired, and the percentages of hypodiploid (apoptotic, sub-G1) events and percentages of cells in the G0/G1, S and G2-M phases were determined using the DNA analysis software ModFitLT, version 2.0 (Verity Software, Topsham, ME, USA).

DNA gel electrophoresis assay

The genomic DNA extracted from smooth muscle cells was prepared according to the protocol provided by the Genomic DNA Isolation kit (BioVision Inc., Mountain View, CA, USA). After the cells had been cultured with various concentrations (20, 40 and 80 μg/ml) of EDS for 48 h, about 1·85 ml of cell suspension solution was added with 50 μl RNase mix and 100 μl cell lysis/denaturing solution in a 55°C water-bath for 30 min, followed by addition of 25 μl protease mix in the 55°C water-bath for 60 min. The mixture was then gently mixed and centrifuged at 10 000  g for 10 min. The supernatant fraction was added with 2 ml 2-amino-2-hydroxymethyl-propane-1,3-diol–EDTA (Tris–EDTA; TE) buffer and 8 ml absolute ethanol at 1000  g for 10 min. After pouring out the supernatant fraction, the pellet was then air dried by re-suspension in 40–90 μl TE buffer. The DNA-containing solution was then subjected to electrophoresis. This experiment was repeated three times. Approximately 20 μg genomic DNA was loaded in each well, visualised under UV light and photographed.

Annexin-V–propidium iodide double staining

For annexin-V (BioSource, Camarillo, CA, USA) staining, a commercially available kit by the FACS Calibur™ system was used. In brief, the cells were washed twice in PBS buffer and re-suspended in 400 μl 1 × annexin-V binding buffer per tube. The cells were then stained with 5 μl annexin-V fluorescein isothiocyanate and 10 μl of PI buffer to each tube. After incubation for 15 min in the dark at room temperature, cells were diluted with 400 μl 1 × annexin-V binding buffer and then measured without gating within 1 h with the FACS Calibur™ system (Becton Dickinson, San Jose, CA, USA).

Western blotting analysis

HASMC cultured in six-well plates were incubated with EDS at 5, 10, 20, 40 and 80 μg/ml in F12K containing 15 % FBS for 24 h. The cells were then lysed in a buffer containing 2 % SDS, 50 mm-dithiothreitol, 62·5 mm-Tris-HCl, pH 6·8, followed by incubation at 95°C for 5 min. Samples were separated using SDS-PAGE, transferred to polyvinylidene fluoride (PVDF) membranes, blocked with 5 % non-fat dry milk in PBS–Tween for 1 h, and then probed with the desired antibodies (anti-PCNA, anti-Raf, anti-phosphorylated Erk, anti-phosphorylated focal adhesion kinase (p-FAK), anti-capase-9 and anti-caspase-3; Novus Biologicals, Littleton, CO, USA) overnight at 4°C. The blots were then incubated with horseradish peroxidase-linked secondary antibody for 1 h followed by development with the electrochemical luminescence (ECL) reagent and exposure to Hyperfilm (Amersham, Arlington Heights, IL, USA).

Balloon angioplasty

Fourty male Sprague–Dawley rats weighing 350–400 g were purchased from National Science Council (Taipei, Taiwan). Forty-eight rats were divided into five groups including total injury control without EDS (n 8), and 10 mg/kg, 20 mg/kg, 40 mg/kg (n 8) and 80 mg/kg (n 8) of EDS-treated groups. Animals were housed in a 12 h light–dark cycle with free access to food and water. All experimental procedures involving animals were approved by the ethics committee of the Institutional Animal Care and Use Committee of China Medical University. The rats were anaesthetised with 3·6 % (w/v) chlorohydrate (1 ml/100 g, intraperitoneally). Angioplasty of the carotid artery was performed using a balloon embolectomy catheter as described previously( 18 ). In brief, the balloon catheter (2F Fogarty; Becton-Dickinson, Franklin Lakes, NJ, USA) was introduced through the right external carotid artery into the aorta, and the balloon was inflated at 1·3 kg/cm2 using an inflation device. An inflated balloon was pushed and pulled through the lumen three times to damage the vessel. The six groups of the animals include sham (no angioplasty), balloon-injured alone, and four doses of EDS (10, 20, 40 and 80 mg/kg) given to rats daily for 2 weeks before and after balloon injury via gastric intubation( 7 , 19 ). At 2 weeks after balloon injury, rats were killed with an over dose of pentobarbital by injection. Tissue sectioning was performed at a desktop microtone with 7 μm thickness. Ten sections from each group were averaged to evaluate the area ratio of neointima:media layers. After staining with Weigert's method using Weigert's Iron Hematoxylin solution, Resorcin–Fuchsin solution and Van Gieson's solution were used to delineate the elastic fibres over which are the neointima layers. After staining, the pictures of the sections were captured for image analysis via the digital program Matrox Inspector (Matrox Electronic Systems Ltd, Montreal, Quebec, Canada).

Immunohistochemistry demonstration of proliferating cell nuclear antigen

Each tissue sample of the rat artery was cut into 7 μm thick sections and mounted on glass slides for immunohistochemistry. The antibodies were monoclonal mouse antibody PCNA (1:2000 dilution; Novus Biologicals, Littleton, CO, USA).

Statistical analysis

Results are shown as mean values with their standard errors. Statistical analyses of MTT were performed using one-way ANOVA performed for statistical analysis of continuous variables followed by the Newman–Keuls test. P < 0·05 was considered statistically significant.

Results

Effects of extract of Dunaliella salina on human aortic smooth muscle cell viability

Since outgrowth of VSMC has been regarded as the major factor leading to restenosis, we performed the MTT assay to determine the inhibitory effects of EDS on cell viability of HASMC VSMC. As shown in Fig. 1, EDS inhibited HASMC viability in a dose- and time-dependent manner. The inhibitory effect of EDS on cell viability became significant at 40 μg/ml (42·1 %; P < 0·01) and 80 μg/ml (50·8 %; P < 0·01) after 48 h incubation.

Fig. 1 Effects of extract of Dunaliella salina (EDS) on cell growth of human aortic smooth muscle cells by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. The cells were incubated for 12, 24, 48 and 72 h with 15 % fetal bovine serum alone (–●–; control) or with different concentrations of EDS: 5 μg/ml (–○–), 10 μg/ml (–▾–), 20 μg/ml (–△–), 40 μg/ml (–■–) and 80 μg/ml (–□–). Values are means of three separate experiments, with standard errors represented by vertical bars. Mean value was significantly different from that of the control group at 0 h: ** P < 0·01, *** P < 0·001.

Effects of extract of Dunaliella salina on DNA synthesis

To further elucidate the inhibitory effects of EDS on HASMC viability, we determined DNA synthesis in HASMC treated with EDS. Serum-stimulated HASMC cells were treated with EDS at 5, 10, 20, 40 and 80 μg/ml for 48 h to evaluate the effects on DNA synthesis by the BrdU incorporation assay. Fig. 2 shows that treatment with EDS at 40 and 80 μg/ml significantly decreased serum-induced BrdU incorporation by 35·6 % (P < 0·01) and 55·6 % (P < 0·001), respectively, as compared with the serum control.

Fig. 2 Effects of extract of Dunaliella salina (EDS) on 5′-bromo-2′-deoxyuridine (BrdU) incorporation of human aortic smooth muscle cells. Control (15 % fetal bovine serum) and various concentrations of EDS (5, 10, 20, 40 and 80 μg/ml) were applied to A10 cells to determine its effects on DNA synthesis for 48 h. Each individual experiment included three experiments of the duplicated test. Values are means, with standard errors represented by vertical bars. Mean value was significantly different from that of the control group at 0 h: ** P < 0·01, *** P < 0·001.

Effects of extract of Dunaliella salina on cell cycle

Since the MTT and DNA synthesis assays showed that EDS at both 40 and 80 μg/ml significantly suppressed cell viability as well as cell proliferation, we postulated that the inhibitory effects of EDS on cell proliferation might be mediated by apoptosis. We chose EDS at 80 μg/ml to determine its effects on cell cycle arrest and apoptosis. The results demonstrate that EDS could arrest the cell cycle at the G0/G1 phase (Figs. 3 and 4). Treatment for 24 and 48 h with EDS at 80 μg/ml significantly increased the cells apoptotic in the sub-G1 phase (Figs. 3 and 4).

Fig. 3 Flow cytometric analysis of the extract of Dunaliella salina (EDS) on the cell cycle of human aortic smooth muscle cells. All the cells were treated with 15 % fetal bovine serum with the addition of EDS at 80 μg/ml for 0 h (a), 6 h (b), 12 h (c), 24 h (d) and 48 h (e). Each phase (G0/G1, S and G2/M) of the cell cycle was determined. The value on the x axis represents the DNA content, while the shaded area ( ) indicates the percentage of cells at the S phase. PE-A, phycoerythrin-A.

Fig. 4 Human aortic smooth muscle cells were treated with extract of Dunaliella salina (EDS) at 80 μg/ml for 0, 6, 12, 24 and 48 h. The y axis represents the percentage of cells at the sub-G1 ( ), G0/G1 ( ), S ( ) and G2/M ( ) phases. Values are means, with standard errors represented by vertical bars. Mean value was significantly different from that of the control group at 0 h: * P < 0·05, *** P < 0·001.

DNA fragmentation induced by extract of Dunaliella salina in a higher dose

We postulated that the inhibitory effects of EDS on cell proliferation might be mediated by apoptosis. By performing DNA laddering assays, we found that EDS at 80 μg/ml induced DNA laddering in HASMC. This finding suggests that EDS-induced apoptosis in HASMC is only at the highest concentration (80 μg/ml), while lower concentrations of EDS could not induce apoptosis of HASMC (Fig. 5).

Fig. 5 The DNA laddering demonstrates the apoptotic effects of extract of Dunaliella salina (EDS) on human aortic smooth muscle cells. All the cells were cultured in 15 % fetal bovine serum (FBS) with the addition of EDS at 20, 40 and 80 μg/ml for 48 h. The DNA laddering was only observed at the highest concentration of EDS at 80 μg/ml but not at 20 and 40 μg/ml. n 3. M, DNA 100 bp ladder; C, control A10 cells, 15 % FBS; D, A10 cells treated with dimethylsulfoxide as vehicle control.

Effects of extract of Dunaliella salina on the annexin V–propidium iodide double staining of human aortic smooth muscle cells

We further applied the annexin V–PI double staining method to verify that EDS induced apoptosis. The results showed that the proportion of early apoptotic cells (lower right) increased from 0·2 % at 0 h to 3·9 % at 24 h and in EDS (80 μg/ml)-treated HASMC. The results also showed that the proportion of late apoptotic cells (upper right) increased from 1·8 % at 0 h to 8·2 % at 24 h and in EDS (80 μg/ml)-treated HASMC. These results suggest that apoptosis might contribute to the EDS-induced death of HASMC (Fig. 6). After 48 h treatment with pipoxolan, the proportions of early and late (lower right) apoptotic cells (upper right) demonstrate 5·5 and 10·9 %, respectively. After 48 h treatment with EDS, more necrosis of the HASMC was found (Fig. 6). However, few cells undergo apoptosis, which is evident from Fig. 5.

Fig. 6 Annexin V–propidium iodide (PI) double staining demonstrates the apoptotic effects of extract of Dunaliella salina (EDS) on human aortic smooth muscle cells. All the cells were cultured in 15 % fetal bovine serum with the addition of EDS at 80 μg/ml for 6, 12, 24 and 48 h. PE-A, phycoerythrin-A; FITI,; FITC-A, fluorescein isothiocyanate-A.

Effects of extract of Dunaliella salina on protein expression levels of proliferating cell nuclear antigen, Raf, phosphorylated focal adhesion kinase and phosphorylated extracellular signal-regulated kinase

Serum-stimulated HASMC were treated with EDS at 5, 10, 20, 40 and 80 μg/ml for 24 h. Total proteins were extracted from cells and subjected to Western blotting analysis with antibodies against PCNA, Raf, phosphorylated Erk and p-FAK. The present results demonstrated that EDS at 80 μg/ml significantly reduced the protein expression levels of PCNA, Raf, phosphorylated Erk and p-FAK by approximately 17, 25, 44 and 26 %, respectively. The data shown here represent the ratio of each protein expression level normalised by β-actin (Fig. 7).

Fig. 7 Effects of extract of Dunaliella salina (EDS) on protein levels of proliferating cell nuclear antigen (PCNA), Raf, phosphorylated extracellular signal-regulated kinase (p-Erk) and phosphorylated focal adhesion kinase (p-FAK) in human aortic smooth muscle cells (HASMC). HASMC were treated with several different concentrations of EDS (5, 10, 20, 40 and 80 μg/ml) for 24 h. Cells receiving 15 % fetal bovine serum served as positive controls (C). The values indicate the density proportion of each protein compared with control. A typical immunoblot from three independent experiments with similar results is shown.

Effects of extract of Dunaliella salina on protein expression levels of caspase-9, caspase-3 and poly(ADP-ribose) polymerase

HASMC stimulated by 15 % FBS were treated with EDS at 80 μg/ml for 12, 24, 48 and 72 h. Total proteins were extracted from cells and subjected to Western blotting analysis with antibodies against caspase-9, caspase-3 and PARP. The present results demonstrated that EDS at 80 μg/ml significantly increased the protein expression levels of active caspase-9, active caspase-3 and cleaved PARP (Fig. 8).

Fig. 8 Extract of Dunaliella salina (EDS) induced the expression of the cleavage of caspase-9, caspase-3 and poly(ADP-ribose) polymerase (PARP) in human aortic smooth muscle cells (HASMC). HASMC were treated with several different concentrations of EDS (5, 10, 20, 40 and 80 μg/ml) for 24 h. Cells receiving 15 % fetal bovine serum served as positive controls (C). The numbers indicate the density proportion of each protein compared with control. A typical immunoblot from three independent experiments with similar results is shown.

Effect of extract of Dunaliella salina on balloon injury-induced neointimal formation on the carotid artery

To test the efficacy of EDS in inhibiting neointimal formation, Sprague–Dawley rats were fed with different concentrations of EDS (40 and 80 mg/kg) for 14 d following balloon injury. After 2 weeks of balloon injury, the injured arteries were harvested and subjected to histological analysis for neointimal formation assay. Intimal hyperplasia induced by balloon injury was evident as compared with the normal control (Fig. 9). The present results showed that both the doses of EDS (40 and 80 mg/kg) were effective in preventing neointimal formation (Fig. 9). However, EDS at 10 and 20 mg/kg did not show any influence on balloon injury-induced neointimal formation (data not shown). Using computerised image analysis, we calculated the area ratio of intimal and media layers; we found a reduction of 45·58 and 70·98 % in the area ratio of EDS-treated groups as compared with the balloon-injured control group by EDS at 40 and 80 mg/kg of EDS, respectively (Fig. 10).

Fig. 9 Responses of rat carotid arteries to balloon injury, and the effects of extract of Dunaliella salina (EDS) on balloon injury. The left panel represents the low-power (100 × ) observations from a balloon-injured vessel (a), a balloon-injured vessel treated with EDS at 40 μg/ml (b) and a balloon-injured vessel treated with EDS at 80 μg/ml (c). The right panel represents the high-power (400 × ) observations from a balloon-injured vessel (d), a balloon-injured vessel treated with EDS at 40 μg/ml (e) and a balloon-injured vessel treated with EDS at 80 μg/ml (f). L, lumen; N, neointima; M, media.

Fig. 10 The neointima:media area ratio in balloon injured rat carotid arteries (400 × ). The control group (CONT) shows a significantly higher area ratio as compared with the groups treated with extract of Dunaliella salina (EDS) at a lower concentration (40 mg/kg) or a higher concentration (80 mg/kg). Values are means of three separate experiments, with standard errors represented by vertical bars. Mean value was significantly different from that of the control group: ** P < 0·01, *** P < 0·001.

Effects of extract of Dunaliella salina on proliferating cell nuclear antigen immunostaining

Fig. 11 shows the effect of two different doses of EDS (40 and 80 mg/kg) on PCNA immunostaining after balloon injury. PCNA-positive cells were abundant in the balloon injury group (Fig. 11(a)). However, the PCNA immunostaining of cells treated with EDS at 40 mg/kg (Fig. 11(b)) and 80 mg/kg (Fig. 11(c)) was less observed.

Fig. 11 Cross-sections from Sprague–Dawley rat coronary arteries after balloon injury and stenting. Immunostaining with proliferating cell nuclear antigen (PCNA): (a) a balloon-injured vessel (b); a balloon-injured vessel treated with extract of Dunaliella salina (EDS) at 40 μg/ml; (c) a balloon-injured vessel treated with EDS at 80 μg/ml. Each tissue sample of the rat artery was cut into 7 μm thick sections and mounted on glass slides for immunohistochemistry. The antibodies were monoclonal mouse antibody PCNA (1:2000 dilution). → , Positive cells.

Discussion

According to the previous study( 20 ), EDS have been prepared at two different temperatures (40 and 100°C) and various times (5, 17·5 and 30 min). These results showed that EDS treatment at 100°C for 30 min was the most optimal condition to demonstrate its antioxidant activity. This was consistent with our previous studies, as EDS and the standard control (all trans-β-carotene) showed a similar peak at the retention time at about 24 min by HPLC analysis. The chromatogram indicated that all-trans β-carotene can be the active ingredient of EDS. We also found that 9-cis-β-carotene was evidently shown in the fingerprint. In addition to this approximate 6 % of β-carotene in EDS, there are still 0·12 % of α-carotene, 0·2 % of xanthophyll, 0·3 % of zeaxanthin, and scarse amounts of lycopene and chlorophyll found in EDS( 13 ). The correlation of certain diets with CVD has been reported from several epidemiological and clinical studies( 18 , 21 ). The inhibitory mechanism of EDS on serum-induced VSMC behaviour remains poorly understood. In the present study, for the first time we show that EDS attenuates neointima hyperplasia after angioplasty and inhibits proliferation and migration of VSMC by interfering with Raf and Erk.

Restenosis of the artery shortly following PTCA is a major limitation to the success of the procedure and is primarily due to smooth muscle cell accumulation in the artery wall at the site of balloon injury. Therefore, modulation of VSMC growth has critical therapeutic implications( 22 ). In the present study, we demonstrated that oral administration of EDS led to a significant reduction of neointimal growth 14 d following arterial injury. The intima:media (I:M) ratios of arterial samples from animals treated with EDS were significantly lower than those of the control tissues (Fig. 10). VSMC proliferation and migration are important contributors to neointima formation after balloon injury. We first demonstrated that EDS exerted potent inhibitory effects on the growth of HASMC (Fig. 1). The antiproliferative effects of EDS were demonstrated by the inhibition of BrdU incorporation (Fig. 2). In view of our previous reports showing that the ras gene was involved in the underlying mechanisms for neointimal formation by balloon injury( 7 ), several proteins involved in the Ras pathway if affected by EDS were therefore investigated in the present study. There has been a consensus that inhibition of smooth muscle proliferation can reduce intimal hyperplasia after angioplasty( 18 , 23 ). PCNA, a cofactor for DNA polymerase δ( 24 , 25 ), is required for DNA synthesis and, therefore, cell proliferation( 25 ). This protein combines with other key cell-cycle control proteins, such as the cyclins and cyclin-dependent kinase( 26 ). Wei et al. showed that PCNA was markedly induced after balloon injury using a rat carotid-injury model( 27 ). FAK is a protein involved in transducing extracellular growth signal from matrix via integrin interaction. Down-regulation of FAK may result in cell-cycle arrest( 28 ). Raf, an important protein in the mitogen-activating protein kinase (MAPK) pathway, is responsible for signal transduction from Ras to Erk. Along the pathway, signalling of phosphorylated-Erk 1/2 is also an essential element for cell proliferation. Therefore, the protein expression levels of PCNA, Raf, FAK and Erk were all evaluated in the present study to explore the mode of preventive action of EDS against neointimal formation by balloon injury. In the present study, down-regulation of PCNA translational levels by EDS suggested its role in arresting cells at the G0/G1 phase (Figs. 3 and 4). Suppressing Raf expression levels suggested an inhibitory effect of EDS on MAPK-mediated signalling, known as a key pathway leading to cell proliferation (Fig. 7). We also demonstrated in the present study that the level of phosphorylated-Erk was down-regulated by EDS and was in accordance with the well-known function of Erk as a critical signalling molecule leading to cell proliferation and survival( 28 30 ).

Apoptosis is another mechanism that prevents cells from abnormal outgrowth. RIP is a death domain-associated protein possessing serine/threonine kinase activity. It has been reported that RIP complexes with a death receptor, CD95 (Fas), with subsequent activation of the proenzymic caspase-2 leading to a caspase cascade( 31 ). As a member of the cysteine protease family, caspase-3 is one of the most important components in this cascade. Upon activation, two caspase-3 molecules dimerise to induce an irreversible apoptotic process and cause cell death( 32 ). In the present study, Western blotting studies suggest that apoptosis induction occurs as the cleavage of inactive pro-caspase-9, resulting in 35–37 kDa active fragments (Fig. 8). We also analysed the effect of EDS on hydrolysis of the zymogen by the Western blotting assay. Cleavage of pro-caspase-3 into 17–19 kDa fragments significantly increased in EDS-treated cells (Fig. 8) and PARP, a known substitute for caspase-3, was effectively hydrolysed to the 85 kDa fragment.

To further evaluate if EDS was effective in suppressing neointimal formation following balloon angioplasty, an in vivo study using rat carotid artery as a model was conducted in the present study. A balloon catheter was first surgically inserted into rat carotid arteries to induce injury. At 2 weeks after balloon injury, the arteries were subjected to histological analysis and EDS was found to significantly reduce neointimal formation (Fig. 10). To further evaluate the effects of EDS on the regulation of the neointimal formation at the nuclear level, we examined immunohistochemical PCNA. PCNA is synthesised in the early G1- and S-phases of the cell cycle and is required for cells to progress from the G1-phase to the S-phase. PCNA can therefore be employed as a marker for proliferating cells in both normal and disease states( 33 ). Here, we showed that EDS suppressed PCNA expression, suggesting that EDSmay affect neointimal formation (Fig. 11).

Taking together the above reported results, the present study demonstrated that balloon injury-induced neointimal formation could be markedly reduced by EDS. Its pharmacological mechanism may be associated with the down-regulation of PCNA, FAK, ERK phosphorylation and Raf protein levels. Also, caspase-9, caspase-3 and PARP could be involved in the progress of apoptosis. The present results detailed the molecular mechanisms of EDS in preventing the smooth muscle cell proliferation either in vitro or in vivo. In the present study, we found that treatments with EDS at 40 and 80 mg/kg both significantly reduced the neointimal formation in rat carotid arteries after balloon injury. Our findings regarding the inhibitory effects of EDS on smooth muscle cells may shed light onto the conjunctive roles of EDS with some other pharmacological agents in preventing restenosis. This was evident by cell-cycle arrest as well as down-regulation of PCNA protein level by EDS. Apoptotic activation was also another mechanism of EDS to suppress outgrowth of smooth muscle cells. DNA laddering induced by EDS may provide this evidence of programmed cell death. A further study on larger animal models or even a clinical evaluation needs to be conducted to confirm the proposed approach in this aspect.

Acknowledgements

We are grateful to Gong Bih Enterprise Co., Ltd for providing dry D. salina powder. We also appreciate the technical support from Ms Shih Chen-I. We are also grateful to Jeffery Conrad for English writing assistance. The present study was partially supported by CMU95-247, CMU97-141 and CMU98-S-08 from China Medical University, Taichung, Taiwan.

M.-J. S. participated in the design of the study, carried out the MTT assay, BrdU assay, animal study and immunohistochemistry of PCNA. H.-C. C. carried out the animal study and flow cytometry analysis. Y.-C. C. carried out the animal study and flow cytometry analysis. P.-Y. C. and G.-J. H. participated in the DNA gel electrophoresis study. J.-S. C. and S.-Y. L. carried out the annexin V–PI double staining assay. C.-H. W. was responsible for the Western blotting assay.

M.-J. S. and H.-C. C. contributed equally to the present study.

There are no conflicts of interest.

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