J Huazhong Univ Sci Technol［Med Sci］ 34(6):791-795,2014 DOI 10.1007/s11596-014-1354-5 J Huazhong Univ Sci Technol［Med Sci］ 34(6):2014
791
Inhibitory Effects of Roscovitine on Proliferation and Migration of Vascular Smooth Muscle Cells In Vitro* Shuang-shuang ZHANG (张双双)1, 2, Wei WANG (王 伟)3, Chong-qiang ZHAO (赵崇强)1, Min-jie XIE (谢敏杰)3, Wen-yu LI (李闻宇)1, Xiang-li YANG (杨向俐)1，Jia-gao LV (吕家高)1# 1 Department of Cardiovascular Medicine, 3Department of Neurology, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430030, China 2 Department of Cardiovascular Medicine, The Central Hospital of Wuhan, Wuhan 430014, China © Huazhong University of Science and Technology and Springer-Verlag Berlin Heidelberg 2014
Summary: Abnormal proliferation and migration of vascular smooth muscle cells (VSMCs) are the major cause of in-stent restenosis (ISR). Intervention proliferation and migration of VSMCs is an important strategy for antirestenotic therapy. Roscovitine, a second-generation cyclin-dependent kinase inhibitor, can inhibit cell cycle of multiple cell types. We studied the effects of roscovitine on cell cycle distribution, proliferation and migration of VSMCs in vitro by flow cytometry, BrdU incorporation and wound healing assay, respectively. Our results showed that roscovitine increased the proportion of G0/G1 phase cells after 12 h (69.57±3.65 vs. 92.50±1.68, P=0.000), 24 h (80.87±2.24 vs. 90.25±0.79, P=0.000) and 48 h (88.08±3.86 vs. 88.87±2.43, P=0.427) as compared with control group. Roscovitine inhibited proliferation and migration of VSMCs in a concentration-dependent way. With the increase of concentration, roscovitine showed increased capacity for growth and migration inhibition. Roscovitine (30 μmol/L) led to an almost complete VSMCs growth and migration arrest. Combined with its low toxicity and selective inhibition to ISR-VSMCs, roscovitine may be a potential drug in the treatment of vascular stenosis diseases and particularly useful in the prevention and treatment of ISR. Key words: roscovitine; vascular smooth muscle cells; cell cycle; cell proliferation; cell migration; restenosis
Since the first percutaneous transluminal coronary angioplasty (PTCA) was performed in 1977, there is a great progress in coronary interventional therapy[1, 2]. Implantation of intracoronary stents reduces the incidence of restenosis following percutaneous coronary intervention (PCI) compared to conventional balloon angioplasty, but in-stent restenosis (ISR) remains an important problem[3, 4]. The main contributor of ISR is neointima formation, resulting from migration from the vessel media to the intima and proliferation of vascular smooth muscle cells (VSMCs) followed by deposition of extracellular matrix[5, 6]. Therefore, intervention in VSMCs proliferation and migration is an important strategy to reduce ISR. Among efforts to reduce ISR, drug eluting stent (DES) is the most successful. The first generation DES releasing antiproliferative drug rapamycin or paclitaxel respectively via a nonbiodegradable polymer made the restenosis rate fall to about 5%[7–18]. DES significantly improves the prognosis of interventional treatment, but it also brings many new problems. The synthetic non-biodegradable polymer coated on the surface of the stent induces inflammation and delays wound healing. Coating drugs inhibit regeneration of endothelial cells at the same time of suppression of VSMCs, which
results in delayed reendothelialization of stent and vessel wall. These responses eventually increase the late stent thrombosis (LST) especially the very late stent thrombosis. LST occurs with an incidence of at least 0.35% in patients treated with DES. Although rare, these events generate high case-fatality rate[19–22]. In addition to improving stent and coating materials, it is necessary to seek low-toxic drug candidates interfering in VSMCs proliferation and migration. R-roscovitine (also named CYC202, seliciclib), a purine analogue, is a potent second-generation cyclindependent kinase inhibitor. It inhibits the cyclin-dependent kinases (CDKs) cdc2, cdk2 and cdk5. It has anti-tumour activity against a broad range of cancer cell lines[23–27]. Selective inhibition of cell cycle progression and low toxicity make it a potent anti-cancer compound that is presently in phase Ⅱ studies in patients with nasopharyngeal carcinoma and non-small-cell lung cancer[28, 29]. Previous study has shown that roscovitine inhibited ISR-VSMCs proliferation more potently than medial VSMCs[30]. We intend to observe the effect of roscovitine on cell cycle progression, proliferation and migration of VSMCs in vitro and explore the possibility of roscovitine preventing and treating ISR.
Shuang-shuang ZHANG, E-mail: [email protected] # Corresponding author, E-mail: [email protected] * This project was supported by grants from the National Natural Science Foundation of China (Nos. 30870641 and 81030021), and the National Basic Research of China “973” Program (No. 2011CB504403).
1 MATERIALS AND METHODS 1.1 Cell Culture The rat VSMC line A10 was purchased from American Tissue Type Collection (ATCC, CRL-1476, USA). Cells were grown in complete medium of Dul-
792 becco’s modified eagle medium (DMEM) supplemented with 10% fetal bovine serum (FBS), 100 U/mL penicillin, and 100 μg/mL streptomycin. Cultures were maintained at 37ºC in a humidified 95% air and 5% CO2 atmosphere. Cells were subcultured with 0.25% trypsin every 3–4 days. Passages 5–10 were used. 1.2 Cell Cycle Analysis Cell cycle distribution of VSMCs was determined by flow cytometry according to the method reported previously[31]. In brief, VSMCs were seeded into 6-cm dishes. Growth was arrested at about 70% confluence for 24 h by serum deprivation. Cells were treated with complete medium in the presence of roscovitine (LC Laboratories, USA) (0 or 20 μmol/L) for indicated time. Cells were trypsinized, washed with 0.01 mol/L phosphate-buffered saline (PBS), and then fixed in cold 80% ethanol at –20ºC overnight. One h before analysis, ethanol was removed by washing with PBS and centrifuged. The cells were then re-suspended in 200 μL PBS containing 50 μg/mL RnaseA (Sigma-Aldrich, USA) and incubated for 30 min at 37°C. PI (50 μg/mL) was added and the cells were incubated for 30 min at 4ºC before FACS analysis on the FACSort (BD Biosciences, USA). 1.3 BrdU Incorporation Assay As a measurement of DNA synthesis and marker of cell proliferation, 5-bromo-2’-deoxyuridine (BrdU) incorporation was determined. VSMCs were seeded into 12-well plates (2×104/mL, containing coverslips coated with polylysine), then growth was arrested at 70% confluence for 24 h with DMEM without FBS. After that, VSMCs were treated with 0, 10, 20 or 30 μmol/L roscovitine for 9, 21, and 45 h respectively. BrdU (1 μmol/L) was added, and cells were further cultured for 3 h. Cells were fixed with cold 100% methyl alcohol and stained with BrdU antibody (1:200, Santa Cruz, USA) and 4’, 6-diamidino-2-phenylindole (DAPI) (Roche, USA) successively. Images were captured using fluorescence microscope (Olympus BX51) and analyzed using IPP 6.0 software. 1.4 Wound Healing Assay The effect of roscovitine on VSMCs migration was measured using wound healing assay[32]. An injury line was made using a pipette tip on a confluent monolayer of cells in 6-well plate. After rinsing with PBS, cells were subsequently incubated with DMEM in the absence (control) or presence of roscovitine at different concentrations. Photographs were taken (×10) immediately (0), 6 and 12 h after wounding in the same scratch region and analyzed using IPP 6.0 software. 1.5 Statistical Analysis The data were expressed as ±s and compared by Dunnett-t test using SPSS 16.0 software. Statistical significance was accepted when P
791
Inhibitory Effects of Roscovitine on Proliferation and Migration of Vascular Smooth Muscle Cells In Vitro* Shuang-shuang ZHANG (张双双)1, 2, Wei WANG (王 伟)3, Chong-qiang ZHAO (赵崇强)1, Min-jie XIE (谢敏杰)3, Wen-yu LI (李闻宇)1, Xiang-li YANG (杨向俐)1，Jia-gao LV (吕家高)1# 1 Department of Cardiovascular Medicine, 3Department of Neurology, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430030, China 2 Department of Cardiovascular Medicine, The Central Hospital of Wuhan, Wuhan 430014, China © Huazhong University of Science and Technology and Springer-Verlag Berlin Heidelberg 2014
Summary: Abnormal proliferation and migration of vascular smooth muscle cells (VSMCs) are the major cause of in-stent restenosis (ISR). Intervention proliferation and migration of VSMCs is an important strategy for antirestenotic therapy. Roscovitine, a second-generation cyclin-dependent kinase inhibitor, can inhibit cell cycle of multiple cell types. We studied the effects of roscovitine on cell cycle distribution, proliferation and migration of VSMCs in vitro by flow cytometry, BrdU incorporation and wound healing assay, respectively. Our results showed that roscovitine increased the proportion of G0/G1 phase cells after 12 h (69.57±3.65 vs. 92.50±1.68, P=0.000), 24 h (80.87±2.24 vs. 90.25±0.79, P=0.000) and 48 h (88.08±3.86 vs. 88.87±2.43, P=0.427) as compared with control group. Roscovitine inhibited proliferation and migration of VSMCs in a concentration-dependent way. With the increase of concentration, roscovitine showed increased capacity for growth and migration inhibition. Roscovitine (30 μmol/L) led to an almost complete VSMCs growth and migration arrest. Combined with its low toxicity and selective inhibition to ISR-VSMCs, roscovitine may be a potential drug in the treatment of vascular stenosis diseases and particularly useful in the prevention and treatment of ISR. Key words: roscovitine; vascular smooth muscle cells; cell cycle; cell proliferation; cell migration; restenosis
Since the first percutaneous transluminal coronary angioplasty (PTCA) was performed in 1977, there is a great progress in coronary interventional therapy[1, 2]. Implantation of intracoronary stents reduces the incidence of restenosis following percutaneous coronary intervention (PCI) compared to conventional balloon angioplasty, but in-stent restenosis (ISR) remains an important problem[3, 4]. The main contributor of ISR is neointima formation, resulting from migration from the vessel media to the intima and proliferation of vascular smooth muscle cells (VSMCs) followed by deposition of extracellular matrix[5, 6]. Therefore, intervention in VSMCs proliferation and migration is an important strategy to reduce ISR. Among efforts to reduce ISR, drug eluting stent (DES) is the most successful. The first generation DES releasing antiproliferative drug rapamycin or paclitaxel respectively via a nonbiodegradable polymer made the restenosis rate fall to about 5%[7–18]. DES significantly improves the prognosis of interventional treatment, but it also brings many new problems. The synthetic non-biodegradable polymer coated on the surface of the stent induces inflammation and delays wound healing. Coating drugs inhibit regeneration of endothelial cells at the same time of suppression of VSMCs, which
results in delayed reendothelialization of stent and vessel wall. These responses eventually increase the late stent thrombosis (LST) especially the very late stent thrombosis. LST occurs with an incidence of at least 0.35% in patients treated with DES. Although rare, these events generate high case-fatality rate[19–22]. In addition to improving stent and coating materials, it is necessary to seek low-toxic drug candidates interfering in VSMCs proliferation and migration. R-roscovitine (also named CYC202, seliciclib), a purine analogue, is a potent second-generation cyclindependent kinase inhibitor. It inhibits the cyclin-dependent kinases (CDKs) cdc2, cdk2 and cdk5. It has anti-tumour activity against a broad range of cancer cell lines[23–27]. Selective inhibition of cell cycle progression and low toxicity make it a potent anti-cancer compound that is presently in phase Ⅱ studies in patients with nasopharyngeal carcinoma and non-small-cell lung cancer[28, 29]. Previous study has shown that roscovitine inhibited ISR-VSMCs proliferation more potently than medial VSMCs[30]. We intend to observe the effect of roscovitine on cell cycle progression, proliferation and migration of VSMCs in vitro and explore the possibility of roscovitine preventing and treating ISR.
Shuang-shuang ZHANG, E-mail: [email protected] # Corresponding author, E-mail: [email protected] * This project was supported by grants from the National Natural Science Foundation of China (Nos. 30870641 and 81030021), and the National Basic Research of China “973” Program (No. 2011CB504403).
1 MATERIALS AND METHODS 1.1 Cell Culture The rat VSMC line A10 was purchased from American Tissue Type Collection (ATCC, CRL-1476, USA). Cells were grown in complete medium of Dul-
792 becco’s modified eagle medium (DMEM) supplemented with 10% fetal bovine serum (FBS), 100 U/mL penicillin, and 100 μg/mL streptomycin. Cultures were maintained at 37ºC in a humidified 95% air and 5% CO2 atmosphere. Cells were subcultured with 0.25% trypsin every 3–4 days. Passages 5–10 were used. 1.2 Cell Cycle Analysis Cell cycle distribution of VSMCs was determined by flow cytometry according to the method reported previously[31]. In brief, VSMCs were seeded into 6-cm dishes. Growth was arrested at about 70% confluence for 24 h by serum deprivation. Cells were treated with complete medium in the presence of roscovitine (LC Laboratories, USA) (0 or 20 μmol/L) for indicated time. Cells were trypsinized, washed with 0.01 mol/L phosphate-buffered saline (PBS), and then fixed in cold 80% ethanol at –20ºC overnight. One h before analysis, ethanol was removed by washing with PBS and centrifuged. The cells were then re-suspended in 200 μL PBS containing 50 μg/mL RnaseA (Sigma-Aldrich, USA) and incubated for 30 min at 37°C. PI (50 μg/mL) was added and the cells were incubated for 30 min at 4ºC before FACS analysis on the FACSort (BD Biosciences, USA). 1.3 BrdU Incorporation Assay As a measurement of DNA synthesis and marker of cell proliferation, 5-bromo-2’-deoxyuridine (BrdU) incorporation was determined. VSMCs were seeded into 12-well plates (2×104/mL, containing coverslips coated with polylysine), then growth was arrested at 70% confluence for 24 h with DMEM without FBS. After that, VSMCs were treated with 0, 10, 20 or 30 μmol/L roscovitine for 9, 21, and 45 h respectively. BrdU (1 μmol/L) was added, and cells were further cultured for 3 h. Cells were fixed with cold 100% methyl alcohol and stained with BrdU antibody (1:200, Santa Cruz, USA) and 4’, 6-diamidino-2-phenylindole (DAPI) (Roche, USA) successively. Images were captured using fluorescence microscope (Olympus BX51) and analyzed using IPP 6.0 software. 1.4 Wound Healing Assay The effect of roscovitine on VSMCs migration was measured using wound healing assay[32]. An injury line was made using a pipette tip on a confluent monolayer of cells in 6-well plate. After rinsing with PBS, cells were subsequently incubated with DMEM in the absence (control) or presence of roscovitine at different concentrations. Photographs were taken (×10) immediately (0), 6 and 12 h after wounding in the same scratch region and analyzed using IPP 6.0 software. 1.5 Statistical Analysis The data were expressed as ±s and compared by Dunnett-t test using SPSS 16.0 software. Statistical significance was accepted when P
