Critical Role of Mst1 in Vascular Remodeling After Injury
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动脉硬化血栓血管生物学 2005年第9期
From the Department of Cardiovascular Medicine, Kyushu University Graduate School of Medical Sciences, Fukuoka, Japan; and the Cardiovascular Research Institute, University of Medicine and Dentistry of New Jersey, Newark.
Correspondence to Toshihiro Ichiki, MD, Department of Cardiovascular Medicine, Kyushu University Graduate School of Medical Sciences, 3-1-1 Maidashi, Higashi-ku, 812-8582 Fukuoka, Japan. E-mail ichiki@cardiol.med.kyushu-u.ac.jp
Abstract
Objective— Apoptosis of vascular smooth muscle cells (VSMCs) is observed in chronic vascular lesions such as atherosclerotic plaques and is believed to contribute to the vascular remodeling process. Mst1 is a ubiquitously expressed serine/threonine kinase known to be activated in response to a wide variety of nonphysiological apoptotic stimuli. However, little is known of the physiological function of Mst1, and its role in VSMCs has never been examined.
Methods and Results— Treatment of VSMCs with staurosporine induced apoptosis and cleavage of Mst1, which is a marker of its activation, as well as activation of caspase 3. Adenovirus-mediated overexpression of wild-type Mst1 (AdMst1) in VSMCs increased apoptotic cells with activation of caspase 3. Mst1 was induced and activated in the balloon-injured rat carotid artery. Infection with AdMst1 in balloon-injured rat carotid artery suppressed neointimal formation compared with infection with AdLacZ. Infection with AdMst1 significantly increased the apoptotic cell number in the neointima compared with infection with AdLacZ without affecting BrdU incorporation.
Conclusion— Our results suggest that Mst1 plays an important role in the induction of apoptosis of VSMCs, mediating the vascular remodeling process, and may be a potential therapeutic target for vascular proliferative diseases.
Apoptosis of vascular smooth muscle cells (VSMCs) is believed to contribute to the vascular remodeling process. Mst1 mediated VSMC apoptosis in vitro and vivo and suppressed neointimal formation in balloon-injured artery, suggesting that Mst1 mediates the vascular remodeling process and may be a potential therapeutic target for vascular proliferative diseases.
Key Words: Mst1 ? caspase 3 ? apoptosis ? vascular smooth muscle cell ? balloon injury
Introduction
Apoptosis, or programmed cell death, is an active process fundamental to the development and homeostasis of multicellular organisms. Many studies have documented apoptosis of vascular smooth muscle cells (VSMCs) in human advanced atherosclerotic plaques.1–3 Apoptosis of VSMCs has also been documented in various animal models of acute vascular injury.4–6 These studies suggest that the balance between proliferation and apoptosis of VSMCs is one of the critical determinants of atherosclerotic lesion formation and the vascular remodeling process.7–9 Therefore, knowledge of the key regulators of apoptosis may offer novel therapeutic targets in both the prevention and treatment of atherosclerosis.
Mammalian sterile 20-like kinase 1 (Mst1) is a ubiquitously expressed serine/threonine kinase10 which belongs to a mammalian sterile 20-like kinase (Ste20) family.11 Several Ste20 group kinases, including Mst1, have been reported to be involved in apoptotic response and cytoskeletal regulation.11 Mst1 is cleaved by caspase 3 and this cleavage increases kinase activities of Mst1 by removal of the regulatory C-terminal region, which in turn activates caspase 3.12,13 Thus, Mst1 contributes to a positive feedback loop that amplifies apoptotic responses. Most Ste20 group kinases activate the mitogen-activated protein kinase (MAPK) cascades and act as MAP kinase kinase kinase kinase (MAP4K).11 It has been reported that Mst1 activates the p38-MAPK and the c-jun N-terminal kinase (JNK) pathways.12 Mst1 is known to be stimulated in response to a wide variety of nonphysiological apoptotic stimuli such as staurosporine, UV, etoposide, and serum starvation;12–16 however, few physiological activators of Mst1 are known other than engagement of Fas ligand. Recently it was reported that overexpression of Mst1 in cardiac myocytes caused dilated cardiomyopathy by stimulating apoptosis.17 However the role of Mst1 in VSMCs or blood vessel has never been examined.
We report in the present study that overexpression of Mst1 induces VSMC apoptosis both in vitro and in vivo and suppressed neointimal formation in balloon-injured artery.
Materials and Methods
Materials
Dulbecco’s modified Eagle’s medium (DMEM) and fetal bovine serum (FBS) were purchased from GIBCO BRL. Bovine serum albumin (BSA), staurosporine, and SP600125 were purchased from Sigma Chemical Co. PD98059 was purchased from BIOMOL Research Laboratories Inc. SB203580 was a generous gift from GlaxoSmithKline (Middlesex, UK). Horseradish peroxidase conjugated secondary antibodies (anti-rabbit or anti-mouse IgG) were purchased from VECTOR Laboratories Inc. Other antibodies used in the experiments were obtained from Cell Signaling Technology. Ac-DEVD-CHO was purchased from BD Biosciences. Other chemical reagents were purchased from Wako Pure chemicals unless specifically mentioned.
Cell Culture
VSMCs were isolated from the thoracic aorta of Sprague–Dawley rats and grown in a humidified atmosphere of 95% air/5% CO2 at 37°C in DMEM. Cells were grown to confluence and growth-arrested in DMEM with 0.1% BSA for 2 days before use. Passages between 5 and 14 were used for the experiments.
Adenovirus Vector Expressing Wild-Type Mst1 and LacZ
A recombinant adenovirus vector expressing wild-type Mst1 (AdMst1) was reported previously.17 Confluent VSMCs were washed 2 times with PBS and incubated with AdMst1 or adenovirus vector expressing LacZ (AdLacZ) under gentle agitation for 2 hours at room temperature. Then the cells were washed 3 times, cultured in DMEM with 0.1% BSA for 2 days, and used for the experiments. Multiplicity of infection (moi) indicates the number of virus per cell added to a culture dish.
Detection of Apoptosis
After floating cells in the supernatant were collected, attached cells were harvested through trypsinization. Collected cells were stained with Hoechst 33258. The number of apoptotic cells (cell shrinkage, chromatin condensation, and nuclear fragmentation) was counted from 500 cells under fluorescence microscopy. Cells were incubated with cold 70% ethanol and stained with propidium iodine (PI) in the presence of RNase. The fluorescence of PI from 10 000 cells was measured by flow cytometry (EPICS ALTRA MultiCOMP, Beckman Coulter) as described previously.18
Western Blot Analysis
VSMCs were lysed in a sample buffer (5 mmol/L EDTA, 10 mmol/L Tris-HCl, pH 7.6, 1% Triton X-100, 50 mmol/L NaCl, 30 mmol/L sodium phosphate, 50 mmol/L NaF, 1% aprotinin, 0.5% pepstatin A, 2 mmol/L phenylmethylsulfonyl fluoride and 5 mmol/L leupeptin). Western blot analyses of Mst1, cleaved caspase 3, extracellular signal-regulated protein kinase (ERK), p38-MAPK, or JNK were performed as described previously.19
Balloon Injury Model and Infection With Adenovirus
All procedures were approved by the institutional animal use and care committee and were conducted in conformity with institutional guideline. Balloon injury and infection with adenovirus were performed as described previously.18 A male Wistar rat (300 to 350 g) was anesthetized by intraperitoneal administration of pentobarbital sodium. The left common carotid artery was denuded of the endothelium with a 2F Fogarty balloon catheter (Baxter) that was introduced through the external carotid artery. Inflation and retraction of the balloon catheter were repeated 5 times. AdMst1 or AdLacZ was introduced into the lumen, and the carotid artery was incubated for 20 minutes without blood flow. Then viral solution was removed, and blood flow was restored. ?-galactosidase activity was observed in both the neointima and media in the AdLacZ-infected artery (data not shown).
Morphometry and Immunohistochemistry
Morphometry was performed as described previously.18 Immunohistochemistry for Mst1 was performed in frozen cross sections. In brief, after the rats were killed, the carotid artery was embedded in Optimal Cutting Temperature compound and quick-frozen in liquid nitrogen. The samples were sectioned serially at 4 μm thickness, fixed in acetone, and stained immunohistochemically with Mst1 antibody.
Detection of Apoptosis and DNA Synthesis In Vivo
Apoptotic cells were detected by the terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick end-labeling (TUNEL) method with an apoptosis in situ detection kit (Wako Pure Chemicals) as described previously.18 The counterstain was hematoxylin. In vivo labeling with BrdU (0.5 mg/kg), a thymidine analogue that was injected intraperitoneally 3 hours before preparation of the artery, was performed to identify replicating cells by detection of DNA synthesis. Incorporated BrdU was detected immunohistochemically with an anti-BrdU antibody (cell proliferation kit, Amersham Pharmacia Biotech). Quantitative analysis was performed from 500 cells in independent sections from each rat (n=5). The ratio of TUNEL- or BrdU-positive cells to total nucleated cells was expressed as the TUNEL index or BrdU labeling index, respectively.
Statistical Analysis
Statistical analysis was performed with 1-way ANOVA and Fisher test if appropriate. P<0.05 was considered to be statistically significant. Data are shown as mean±SEM.
Results
Mst1 Was Activated by Apoptotic Stimuli in VSMCs
Staurosporine is a potent inducer of apoptosis in many cells. We examined whether staurosporine induced apoptosis in VSMCs. Staining with Hoechst 33258 showed that staurosporine increased the number of apoptotic cells, characterized by chromatin condensation and nuclear fragmentation (Figure 1a). Flow cytometric analysis of DNA content by PI staining showed that staurosporine increased hypodiploid cells, indicating an increase in DNA fragmentation (Figure I, available online at http://atvb.ahajournals.org). Increases in the number of hypodiploid cells by staurosporine were time- and dose-dependent (Figure I). Staurosporine induced cleavage of caspase 3 with a peak at 12 hours (Figure 1b), which indicates activation of caspase 3. These results suggest that staurosporine induced VSMC apoptosis. Mst1 cleavage was induced by staurosporine with a peak at 12 hours, which is the same peak as cleavage of caspase 3 (Figure 1b; Figure II, available online at http://atvb.ahajournals.org). Pretreatment with Ac-DEVD-CHO, a specific inhibitor of caspase 3,20,21 inhibited Mst1 cleavage (Figure 1c). Although Ac-DEVD-CHO almost completely inhibited Mst1 activation, it partially suppressed staurosporine-induced apoptosis (Figure 1c). These data suggest that staurosporine-induced apoptosis is partially dependent on Mst1 pathway. Previous report demonstrated that H2O2 causes VSMC apoptosis22. H2O2 induced Mst1 cleavage with the same peak as cleavage of caspase 3 (Figure 1d). Mst1 cleavage induced by H2O2 was also inhibited by Ac-DEVD-CHO (data not shown). These data suggest that Mst1 was activated by apoptotic stimuli downstream from caspase 3 in VSMCs.
Figure 1. Mst1 was activated by apoptotic stimuli. a, VSMCs were stimulated with staurosporine (1 μmol/L) for 24 hours and stained with Hoechst 33258, which stains nuclei. Proportion of apoptotic cells indicated by nuclear fragmentation (indicated by arrows) is shown as bar graph (n=4). **P<0.01 vs control. b, VSMCs were stimulated with staurosporine (1 μmol/L) for varying periods as indicated in the figure, and expression of cleaved and uncleaved Mst1 and cleaved caspase 3 was detected by Western blot analysis. Density of the specific band was scanned and quantified with an imaging analyzer. The statistical analysis of densitometric measurements of uncleaved and cleaved Mst1 expression is shown in Figure II (n=5). c, VSMCs were preincubated with Ac-DEVD-CHO (100 μmol/L) for 6 hours then stimulated with staurosporine (1 μmol/L) for 12 hours. Expression of Mst1 and cleaved caspase 3 were detected by Western blot analysis. The same results were obtained in other independent experiments and a representative autoradiogram is shown (n=4). Proportion of apoptotic cells is shown in the right panel (n=4). **P<0.01 vs control. d, VSMCs were simulated with H2O2 (1 mmol/L) for varying periods as indicated in the figure, and expression of Mst1 and cleaved caspase 3 was detected by Western blot analysis. The same results were obtained in other independent experiments, and a representative autoradiogram is shown (n=4).
Mst1 Induced VSMC Apoptosis
To clarify the role of Mst1 in VSMCs, we overexpressed Mst1, which induces activation of Mst1 by unknown mechanism.12,23 Infection with AdMst1 dose-dependently increased expression of both uncleaved and cleaved Mst1 (Figure 2a). AdMst1 dose-dependently induced cleavage of caspase 3 (Figure 2a), suggesting that Mst1 activated the caspase pathway. Staining with Hoechst 33258 and PI showed that AdMst1 dose-dependently increased apoptotic cells (Figure 2b). The magnitude of the apoptotic effect of Mst1 overexpression is 29% to 44% of the apoptosis induced by staurosporine. Although infection of AdLacZ slightly increased hypodiploid cells (Figure 2b), it did not activate Mst1 or caspase 3 (Figure 2a) suggesting that the effect of AdLacZ may be nonspecific.
Figure 2. Overexpression of Mst1 induced VSMC apoptosis. a, VSMCs were infected with AdMst1 (1 to 10 moi) or AdLacZ (10 moi) for 48 hours, and expression of Mst1 and cleaved caspase 3 was detected by Western blot analysis. The same results were obtained in other independent experiments, and a representative autoradiogram is shown (n=4). b, VSMCs were infected with AdMst1 (1 to 10 moi) or AdLacZ (10 moi) for 96 hours, and apoptotic cells were detected with Hoechst 33258 and PI staining as described in the legend to Figure 1. Proportion of apoptotic cells is shown as a bar graph (n=4). **P<0.01 vs control and AdLacZ. *P<0.01 vs control
Role of MAPKs
Many mammalian Ste20 homologs have been shown to function as MAP4K.11 Overexpression of Mst1 induced phosphorylation of p38-MAPK and JNK; however, it did not affect phosphorylation of ERK in VSMCs (Figure IIIa, available online at http://atvb.ahajournals.org). This suggests that p38-MAPK and JNK were activated downstream from Mst1. Staurosporine induced phosphorylation of ERK, p38-MAPK, and JNK (Figure IIIb). We therefore examined which pathway was responsible for Mst1 and caspase 3 activation by staurosporine. SB203580, a p38-MAPK inhibitor, partially decreased staurosporine-induced Mst1 and caspase 3 cleavage. However, PD98059, an ERK kinase inhibitor, and SP600125, a JNK inhibitor, did not affect them (Figure IIIc). These data suggest that p38-MAPK is, at least in part, involved in staurosporine-induced Mst1 activation and apoptosis.
Activation of Mst1 in Balloon-Injured Artery
It was reported that both proliferation and apoptosis were increased in the neointima of the balloon-injured rat carotid artery, and that the number of apoptotic cells in neointima peaked after 7 to 14 days.24 We therefore examined whether Mst1 was activated in balloon-injured rat carotid artery. Immunohistochemistry revealed that immunoreactive Mst1 was hardly detectable in intact artery. At 14 days after balloon injury, expression of Mst1 was observed in the nuclei and, to a lesser extent, in the cytoplasm of the neointimal cells (Figure 3a), most of which are positive for -SM actin.18 Western blot analysis showed that expression of both uncleaved and cleaved Mst1 was significantly increased at 14 days after balloon injury, along with cleavage of caspase 3 (Figure 3b). This suggests that Mst1 was increased and activated in the process of vascular remodeling after balloon injury. We overexpressed Mst1 in balloon-injured artery by infection with AdMst1. Infection with AdMst1 suppressed neointimal formation (I/M ratio) compared with infection with AdLacZ 14 days after balloon injury (Figure 4a and 4b). The intimal area was suppressed in AdMst1-infected artery compared with AdLacZ-infected one without affecting the medial area (Figure 4b). It was previously reported that the TUNEL index peaked after 7 to 14 days of vascular injury, and the BrdU labeling index peaked after 7 days.24 TUNEL index in the neointima of AdMst1-infected arteries was significantly increased compared with that of AdLacZ-infected arteries, whereas AdMst1 did not affect the TUNEL index in medial cells at 14 days after balloon injury (Figure 4c). Infection with AdMst1 did not affect the BrdU labeling index in either the neointima or media at 7 days after injury (Figure 4c).
Figure 3. Expression of Mst1 in balloon-injured artery. a, Representative microphotographs of immunohistochemistry for Mst1 in intact and injured carotid artery at 14 days after injury (n=5). b, Western blot analysis of Mst1 and cleaved caspase 3 in balloon-injured rat carotid artery at 14 days after injury is shown. The ratio of uncleaved Mst1 (white bars) or cleaved Mst1 (black bars) to -tubulin is shown in the right panel (n=6). The values are expressed as mean±SEM, **P<0.01 vs control, *P<0.05 vs control.
Figure 4. Overexpression of Mst1 in balloon-injured artery. a, Representative microphotographs of hematoxylin-eosin staining of cross sections of carotid arteries 14 days after balloon injury. The same results were obtained in other independent experiments (n=5). b, The intima/media area ratio (I/M ratio) and cross-section area (CSA) of intima, media, lumen, and total artery in injured artery are shown as bar graphs (n=5). The values are expressed as mean±SEM, *P<0.05 vs control or AdLacZ. c, TUNEL or BrdU labeling were performed in cross sections of carotid arteries 14 or 7 days after balloon injury. TUNEL index of intima (black bars) or media (white bars) is indicated in a left bar graph (n=5). BrdU labeling index of intima (white bars) or media (black bars) is indicated in a right bar graph (n=5). The values are expressed as mean±SEM, **P<0.01 vs control or AdLacZ; N.S.=not significant.
Discussion
In the present study, we showed that overexpression of Mst1 induced VSMC apoptosis in vitro and in vivo and suppressed neointimal formation in balloon-injured artery. This is the first report examining the function of Mst1 in VSMCs and in balloon-injured artery. A recent study reported that cardiac-specific overexpression of Mst1 causes dilated cardiomyopathy,17 suggesting that apoptosis of cardiac myocytes impairs cardiac function. However, overexpression of Mst1 in injured artery has anti-proliferative effect by stimulating apoptosis, which may be used as a therapeutic tool for vascular proliferative lesion such as in-stent restenosis.
Numerous animal models of acute balloon injury of artery have documented apoptotic VSMC death. Several studies demonstrated that balloon injury of vessels induces two waves of VSMC apoptosis. The first wave of apoptosis occurred in the media within hours of the injury.5 The second wave occurs at much later times after injury (days to weeks). VSMC accumulation in the neointima of injured rat carotid arteries reached a maximal level at 2 weeks after injury; however, cellular proliferation continues for up to 12 weeks.25 Therefore, it is thought that this second wave of apoptosis limits lesion growth. The rates of neointimal VSMC death and proliferation are in equilibrium at 2 weeks, thereby preventing further increase in lesion size. Expression of both the uncleaved and cleaved forms of Mst1 was increased after balloon injury. Upregulation of uncleaved and cleaved Mst1 indicated that Mst1 kinase activity was increased and may contribute to the delayed enhancement of apoptosis in the neointimal formation. Although genotoxic agents are known to activate Mst1, this is the first report showing that clinically relevant stimuli such as balloon injury activate Mst1 in blood vessel. An in vitro study suggested that reactive oxygen species may be involved in the activation of Mst1 in injured artery. Regulation of Mst1 expression has not been examined in detail, and the mechanism of Mst1 upregulation and activation in injured rat carotid artery is not clear at this point. Further studies are necessary to determine the mechanism of Mst1 upregulation and activation in injured blood vessel.
We showed in the present study that Mst1 was activated in apoptotic response by staurosporine and that overexpression of Mst1 induced apoptosis in VSMCs. Several studies have demonstrated that Mst1 is necessary for the induction of apoptosis. A kinase-negative mutant of Mst1 in which Lys-59 is changed to Arg inhibited staurosporine-induced chromatin condensation26 and chelerythrine-induced DNA fragmentation.17 Suppression of endogenous Mst1 by cardiac-specific overexpression of the kinase-negative mutant of Mst1 in transgenic mice prevented myocyte death induced by ischemia/reperfusion.17 These data suggest that Mst1 plays an essential role in mediating apoptotic response, which may be true in blood vessel as well.
Most Ste20 group kinases act as MAP4K, and it has been reported that Mst1 activates the p38-MAPK and JNK pathways.12 Mst1 has been shown to function upstream from MEKK1 and MKK7 in the JNK pathway12,27 and upstream from MKK6 in the p38-MAPK pathway.12 These results are consistent with Mst1 function as a MAP4K. However, there are conflicting evidences as to which pathway mediates apoptosis downstream of Mst1. Graves et al reported that the p38-MAPK pathway may mediate Mst1-induced apoptosis because SB203580 inhibited Mst1-induced morphological changes in 293T cells.12 Another group reported that a dominant-negative mutant of JNK inhibited Mst1-induced morphological changes as well as caspase activation, while a dominant-negative p38-MAPK did not.23 In VSMCs, p38-MAPK and JNK were both activated downstream of Mst1, and a p38-MAPK inhibitor partially inhibited staurosporine-induced Mst1 and caspase 3 cleavage. These data suggest that p38-MAPK, at least in part, mediates apoptotic response through Mst1 in VSMCs. p38-MAPK is activated both upstream and downstream of Mst1 and may be involved in the positive feedback loop formed by Mst1 and caspase 3. However, the effect of SB203580 was partial, suggesting that some pathway other than p38-MAPK is also involved in apoptosis induced by staurosporine, thus requiring further investigation.
Recently, it has been reported that Hippo, the Drosophila homologue of Mst1/2, promotes apoptosis and proper termination of cell proliferation during development, together with Salvador, the Drosophila homologue of hWW45, and Warts, the Drosophila homologue of large tumor suppressor (LATS), in a signal module complex.28,29 Although mutations in the hWW45 gene have been reported in three cancer cell lines,30 its function has not been examined in detail. The roles of hWW45 or LATS in VSMCs and the relationship between Mst1/2 and hWW45 or LATS in blood vessel require further investigation.
Many environmental factors and endogenous proteins regulate the struggle between apoptosis and proliferation in atherosclerotic lesion. Several reports suggest that apoptosis in atherosclerotic plaque may decrease its stability and induce plaque rupture.31,32 A recent report showed that proapoptotic genes were highly expressed in atherosclerotic plaques from patients with acute coronary syndrome compared with those from patients with stable angina.33 However, induction of apoptosis may be useful for the treatment of in-stent restenosis that is rich in cellular component such as VSMCs.34,35
The data presented in the present study suggest that Mst1 plays a critical role in the vascular remodeling process and could be a therapeutic target for prevention of restenosis after vascular injury.
Acknowledgments
This study was supported in part by grants from Takeda Science Foundation and a Grant-in-aid for Scientific Research from the Ministry of Education, Culture, Sports, Science, and Technology of Japan (17590742). We thank Daniela Zablocki for critical reading of the manuscript.
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Correspondence to Toshihiro Ichiki, MD, Department of Cardiovascular Medicine, Kyushu University Graduate School of Medical Sciences, 3-1-1 Maidashi, Higashi-ku, 812-8582 Fukuoka, Japan. E-mail ichiki@cardiol.med.kyushu-u.ac.jp
Abstract
Objective— Apoptosis of vascular smooth muscle cells (VSMCs) is observed in chronic vascular lesions such as atherosclerotic plaques and is believed to contribute to the vascular remodeling process. Mst1 is a ubiquitously expressed serine/threonine kinase known to be activated in response to a wide variety of nonphysiological apoptotic stimuli. However, little is known of the physiological function of Mst1, and its role in VSMCs has never been examined.
Methods and Results— Treatment of VSMCs with staurosporine induced apoptosis and cleavage of Mst1, which is a marker of its activation, as well as activation of caspase 3. Adenovirus-mediated overexpression of wild-type Mst1 (AdMst1) in VSMCs increased apoptotic cells with activation of caspase 3. Mst1 was induced and activated in the balloon-injured rat carotid artery. Infection with AdMst1 in balloon-injured rat carotid artery suppressed neointimal formation compared with infection with AdLacZ. Infection with AdMst1 significantly increased the apoptotic cell number in the neointima compared with infection with AdLacZ without affecting BrdU incorporation.
Conclusion— Our results suggest that Mst1 plays an important role in the induction of apoptosis of VSMCs, mediating the vascular remodeling process, and may be a potential therapeutic target for vascular proliferative diseases.
Apoptosis of vascular smooth muscle cells (VSMCs) is believed to contribute to the vascular remodeling process. Mst1 mediated VSMC apoptosis in vitro and vivo and suppressed neointimal formation in balloon-injured artery, suggesting that Mst1 mediates the vascular remodeling process and may be a potential therapeutic target for vascular proliferative diseases.
Key Words: Mst1 ? caspase 3 ? apoptosis ? vascular smooth muscle cell ? balloon injury
Introduction
Apoptosis, or programmed cell death, is an active process fundamental to the development and homeostasis of multicellular organisms. Many studies have documented apoptosis of vascular smooth muscle cells (VSMCs) in human advanced atherosclerotic plaques.1–3 Apoptosis of VSMCs has also been documented in various animal models of acute vascular injury.4–6 These studies suggest that the balance between proliferation and apoptosis of VSMCs is one of the critical determinants of atherosclerotic lesion formation and the vascular remodeling process.7–9 Therefore, knowledge of the key regulators of apoptosis may offer novel therapeutic targets in both the prevention and treatment of atherosclerosis.
Mammalian sterile 20-like kinase 1 (Mst1) is a ubiquitously expressed serine/threonine kinase10 which belongs to a mammalian sterile 20-like kinase (Ste20) family.11 Several Ste20 group kinases, including Mst1, have been reported to be involved in apoptotic response and cytoskeletal regulation.11 Mst1 is cleaved by caspase 3 and this cleavage increases kinase activities of Mst1 by removal of the regulatory C-terminal region, which in turn activates caspase 3.12,13 Thus, Mst1 contributes to a positive feedback loop that amplifies apoptotic responses. Most Ste20 group kinases activate the mitogen-activated protein kinase (MAPK) cascades and act as MAP kinase kinase kinase kinase (MAP4K).11 It has been reported that Mst1 activates the p38-MAPK and the c-jun N-terminal kinase (JNK) pathways.12 Mst1 is known to be stimulated in response to a wide variety of nonphysiological apoptotic stimuli such as staurosporine, UV, etoposide, and serum starvation;12–16 however, few physiological activators of Mst1 are known other than engagement of Fas ligand. Recently it was reported that overexpression of Mst1 in cardiac myocytes caused dilated cardiomyopathy by stimulating apoptosis.17 However the role of Mst1 in VSMCs or blood vessel has never been examined.
We report in the present study that overexpression of Mst1 induces VSMC apoptosis both in vitro and in vivo and suppressed neointimal formation in balloon-injured artery.
Materials and Methods
Materials
Dulbecco’s modified Eagle’s medium (DMEM) and fetal bovine serum (FBS) were purchased from GIBCO BRL. Bovine serum albumin (BSA), staurosporine, and SP600125 were purchased from Sigma Chemical Co. PD98059 was purchased from BIOMOL Research Laboratories Inc. SB203580 was a generous gift from GlaxoSmithKline (Middlesex, UK). Horseradish peroxidase conjugated secondary antibodies (anti-rabbit or anti-mouse IgG) were purchased from VECTOR Laboratories Inc. Other antibodies used in the experiments were obtained from Cell Signaling Technology. Ac-DEVD-CHO was purchased from BD Biosciences. Other chemical reagents were purchased from Wako Pure chemicals unless specifically mentioned.
Cell Culture
VSMCs were isolated from the thoracic aorta of Sprague–Dawley rats and grown in a humidified atmosphere of 95% air/5% CO2 at 37°C in DMEM. Cells were grown to confluence and growth-arrested in DMEM with 0.1% BSA for 2 days before use. Passages between 5 and 14 were used for the experiments.
Adenovirus Vector Expressing Wild-Type Mst1 and LacZ
A recombinant adenovirus vector expressing wild-type Mst1 (AdMst1) was reported previously.17 Confluent VSMCs were washed 2 times with PBS and incubated with AdMst1 or adenovirus vector expressing LacZ (AdLacZ) under gentle agitation for 2 hours at room temperature. Then the cells were washed 3 times, cultured in DMEM with 0.1% BSA for 2 days, and used for the experiments. Multiplicity of infection (moi) indicates the number of virus per cell added to a culture dish.
Detection of Apoptosis
After floating cells in the supernatant were collected, attached cells were harvested through trypsinization. Collected cells were stained with Hoechst 33258. The number of apoptotic cells (cell shrinkage, chromatin condensation, and nuclear fragmentation) was counted from 500 cells under fluorescence microscopy. Cells were incubated with cold 70% ethanol and stained with propidium iodine (PI) in the presence of RNase. The fluorescence of PI from 10 000 cells was measured by flow cytometry (EPICS ALTRA MultiCOMP, Beckman Coulter) as described previously.18
Western Blot Analysis
VSMCs were lysed in a sample buffer (5 mmol/L EDTA, 10 mmol/L Tris-HCl, pH 7.6, 1% Triton X-100, 50 mmol/L NaCl, 30 mmol/L sodium phosphate, 50 mmol/L NaF, 1% aprotinin, 0.5% pepstatin A, 2 mmol/L phenylmethylsulfonyl fluoride and 5 mmol/L leupeptin). Western blot analyses of Mst1, cleaved caspase 3, extracellular signal-regulated protein kinase (ERK), p38-MAPK, or JNK were performed as described previously.19
Balloon Injury Model and Infection With Adenovirus
All procedures were approved by the institutional animal use and care committee and were conducted in conformity with institutional guideline. Balloon injury and infection with adenovirus were performed as described previously.18 A male Wistar rat (300 to 350 g) was anesthetized by intraperitoneal administration of pentobarbital sodium. The left common carotid artery was denuded of the endothelium with a 2F Fogarty balloon catheter (Baxter) that was introduced through the external carotid artery. Inflation and retraction of the balloon catheter were repeated 5 times. AdMst1 or AdLacZ was introduced into the lumen, and the carotid artery was incubated for 20 minutes without blood flow. Then viral solution was removed, and blood flow was restored. ?-galactosidase activity was observed in both the neointima and media in the AdLacZ-infected artery (data not shown).
Morphometry and Immunohistochemistry
Morphometry was performed as described previously.18 Immunohistochemistry for Mst1 was performed in frozen cross sections. In brief, after the rats were killed, the carotid artery was embedded in Optimal Cutting Temperature compound and quick-frozen in liquid nitrogen. The samples were sectioned serially at 4 μm thickness, fixed in acetone, and stained immunohistochemically with Mst1 antibody.
Detection of Apoptosis and DNA Synthesis In Vivo
Apoptotic cells were detected by the terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick end-labeling (TUNEL) method with an apoptosis in situ detection kit (Wako Pure Chemicals) as described previously.18 The counterstain was hematoxylin. In vivo labeling with BrdU (0.5 mg/kg), a thymidine analogue that was injected intraperitoneally 3 hours before preparation of the artery, was performed to identify replicating cells by detection of DNA synthesis. Incorporated BrdU was detected immunohistochemically with an anti-BrdU antibody (cell proliferation kit, Amersham Pharmacia Biotech). Quantitative analysis was performed from 500 cells in independent sections from each rat (n=5). The ratio of TUNEL- or BrdU-positive cells to total nucleated cells was expressed as the TUNEL index or BrdU labeling index, respectively.
Statistical Analysis
Statistical analysis was performed with 1-way ANOVA and Fisher test if appropriate. P<0.05 was considered to be statistically significant. Data are shown as mean±SEM.
Results
Mst1 Was Activated by Apoptotic Stimuli in VSMCs
Staurosporine is a potent inducer of apoptosis in many cells. We examined whether staurosporine induced apoptosis in VSMCs. Staining with Hoechst 33258 showed that staurosporine increased the number of apoptotic cells, characterized by chromatin condensation and nuclear fragmentation (Figure 1a). Flow cytometric analysis of DNA content by PI staining showed that staurosporine increased hypodiploid cells, indicating an increase in DNA fragmentation (Figure I, available online at http://atvb.ahajournals.org). Increases in the number of hypodiploid cells by staurosporine were time- and dose-dependent (Figure I). Staurosporine induced cleavage of caspase 3 with a peak at 12 hours (Figure 1b), which indicates activation of caspase 3. These results suggest that staurosporine induced VSMC apoptosis. Mst1 cleavage was induced by staurosporine with a peak at 12 hours, which is the same peak as cleavage of caspase 3 (Figure 1b; Figure II, available online at http://atvb.ahajournals.org). Pretreatment with Ac-DEVD-CHO, a specific inhibitor of caspase 3,20,21 inhibited Mst1 cleavage (Figure 1c). Although Ac-DEVD-CHO almost completely inhibited Mst1 activation, it partially suppressed staurosporine-induced apoptosis (Figure 1c). These data suggest that staurosporine-induced apoptosis is partially dependent on Mst1 pathway. Previous report demonstrated that H2O2 causes VSMC apoptosis22. H2O2 induced Mst1 cleavage with the same peak as cleavage of caspase 3 (Figure 1d). Mst1 cleavage induced by H2O2 was also inhibited by Ac-DEVD-CHO (data not shown). These data suggest that Mst1 was activated by apoptotic stimuli downstream from caspase 3 in VSMCs.
Figure 1. Mst1 was activated by apoptotic stimuli. a, VSMCs were stimulated with staurosporine (1 μmol/L) for 24 hours and stained with Hoechst 33258, which stains nuclei. Proportion of apoptotic cells indicated by nuclear fragmentation (indicated by arrows) is shown as bar graph (n=4). **P<0.01 vs control. b, VSMCs were stimulated with staurosporine (1 μmol/L) for varying periods as indicated in the figure, and expression of cleaved and uncleaved Mst1 and cleaved caspase 3 was detected by Western blot analysis. Density of the specific band was scanned and quantified with an imaging analyzer. The statistical analysis of densitometric measurements of uncleaved and cleaved Mst1 expression is shown in Figure II (n=5). c, VSMCs were preincubated with Ac-DEVD-CHO (100 μmol/L) for 6 hours then stimulated with staurosporine (1 μmol/L) for 12 hours. Expression of Mst1 and cleaved caspase 3 were detected by Western blot analysis. The same results were obtained in other independent experiments and a representative autoradiogram is shown (n=4). Proportion of apoptotic cells is shown in the right panel (n=4). **P<0.01 vs control. d, VSMCs were simulated with H2O2 (1 mmol/L) for varying periods as indicated in the figure, and expression of Mst1 and cleaved caspase 3 was detected by Western blot analysis. The same results were obtained in other independent experiments, and a representative autoradiogram is shown (n=4).
Mst1 Induced VSMC Apoptosis
To clarify the role of Mst1 in VSMCs, we overexpressed Mst1, which induces activation of Mst1 by unknown mechanism.12,23 Infection with AdMst1 dose-dependently increased expression of both uncleaved and cleaved Mst1 (Figure 2a). AdMst1 dose-dependently induced cleavage of caspase 3 (Figure 2a), suggesting that Mst1 activated the caspase pathway. Staining with Hoechst 33258 and PI showed that AdMst1 dose-dependently increased apoptotic cells (Figure 2b). The magnitude of the apoptotic effect of Mst1 overexpression is 29% to 44% of the apoptosis induced by staurosporine. Although infection of AdLacZ slightly increased hypodiploid cells (Figure 2b), it did not activate Mst1 or caspase 3 (Figure 2a) suggesting that the effect of AdLacZ may be nonspecific.
Figure 2. Overexpression of Mst1 induced VSMC apoptosis. a, VSMCs were infected with AdMst1 (1 to 10 moi) or AdLacZ (10 moi) for 48 hours, and expression of Mst1 and cleaved caspase 3 was detected by Western blot analysis. The same results were obtained in other independent experiments, and a representative autoradiogram is shown (n=4). b, VSMCs were infected with AdMst1 (1 to 10 moi) or AdLacZ (10 moi) for 96 hours, and apoptotic cells were detected with Hoechst 33258 and PI staining as described in the legend to Figure 1. Proportion of apoptotic cells is shown as a bar graph (n=4). **P<0.01 vs control and AdLacZ. *P<0.01 vs control
Role of MAPKs
Many mammalian Ste20 homologs have been shown to function as MAP4K.11 Overexpression of Mst1 induced phosphorylation of p38-MAPK and JNK; however, it did not affect phosphorylation of ERK in VSMCs (Figure IIIa, available online at http://atvb.ahajournals.org). This suggests that p38-MAPK and JNK were activated downstream from Mst1. Staurosporine induced phosphorylation of ERK, p38-MAPK, and JNK (Figure IIIb). We therefore examined which pathway was responsible for Mst1 and caspase 3 activation by staurosporine. SB203580, a p38-MAPK inhibitor, partially decreased staurosporine-induced Mst1 and caspase 3 cleavage. However, PD98059, an ERK kinase inhibitor, and SP600125, a JNK inhibitor, did not affect them (Figure IIIc). These data suggest that p38-MAPK is, at least in part, involved in staurosporine-induced Mst1 activation and apoptosis.
Activation of Mst1 in Balloon-Injured Artery
It was reported that both proliferation and apoptosis were increased in the neointima of the balloon-injured rat carotid artery, and that the number of apoptotic cells in neointima peaked after 7 to 14 days.24 We therefore examined whether Mst1 was activated in balloon-injured rat carotid artery. Immunohistochemistry revealed that immunoreactive Mst1 was hardly detectable in intact artery. At 14 days after balloon injury, expression of Mst1 was observed in the nuclei and, to a lesser extent, in the cytoplasm of the neointimal cells (Figure 3a), most of which are positive for -SM actin.18 Western blot analysis showed that expression of both uncleaved and cleaved Mst1 was significantly increased at 14 days after balloon injury, along with cleavage of caspase 3 (Figure 3b). This suggests that Mst1 was increased and activated in the process of vascular remodeling after balloon injury. We overexpressed Mst1 in balloon-injured artery by infection with AdMst1. Infection with AdMst1 suppressed neointimal formation (I/M ratio) compared with infection with AdLacZ 14 days after balloon injury (Figure 4a and 4b). The intimal area was suppressed in AdMst1-infected artery compared with AdLacZ-infected one without affecting the medial area (Figure 4b). It was previously reported that the TUNEL index peaked after 7 to 14 days of vascular injury, and the BrdU labeling index peaked after 7 days.24 TUNEL index in the neointima of AdMst1-infected arteries was significantly increased compared with that of AdLacZ-infected arteries, whereas AdMst1 did not affect the TUNEL index in medial cells at 14 days after balloon injury (Figure 4c). Infection with AdMst1 did not affect the BrdU labeling index in either the neointima or media at 7 days after injury (Figure 4c).
Figure 3. Expression of Mst1 in balloon-injured artery. a, Representative microphotographs of immunohistochemistry for Mst1 in intact and injured carotid artery at 14 days after injury (n=5). b, Western blot analysis of Mst1 and cleaved caspase 3 in balloon-injured rat carotid artery at 14 days after injury is shown. The ratio of uncleaved Mst1 (white bars) or cleaved Mst1 (black bars) to -tubulin is shown in the right panel (n=6). The values are expressed as mean±SEM, **P<0.01 vs control, *P<0.05 vs control.
Figure 4. Overexpression of Mst1 in balloon-injured artery. a, Representative microphotographs of hematoxylin-eosin staining of cross sections of carotid arteries 14 days after balloon injury. The same results were obtained in other independent experiments (n=5). b, The intima/media area ratio (I/M ratio) and cross-section area (CSA) of intima, media, lumen, and total artery in injured artery are shown as bar graphs (n=5). The values are expressed as mean±SEM, *P<0.05 vs control or AdLacZ. c, TUNEL or BrdU labeling were performed in cross sections of carotid arteries 14 or 7 days after balloon injury. TUNEL index of intima (black bars) or media (white bars) is indicated in a left bar graph (n=5). BrdU labeling index of intima (white bars) or media (black bars) is indicated in a right bar graph (n=5). The values are expressed as mean±SEM, **P<0.01 vs control or AdLacZ; N.S.=not significant.
Discussion
In the present study, we showed that overexpression of Mst1 induced VSMC apoptosis in vitro and in vivo and suppressed neointimal formation in balloon-injured artery. This is the first report examining the function of Mst1 in VSMCs and in balloon-injured artery. A recent study reported that cardiac-specific overexpression of Mst1 causes dilated cardiomyopathy,17 suggesting that apoptosis of cardiac myocytes impairs cardiac function. However, overexpression of Mst1 in injured artery has anti-proliferative effect by stimulating apoptosis, which may be used as a therapeutic tool for vascular proliferative lesion such as in-stent restenosis.
Numerous animal models of acute balloon injury of artery have documented apoptotic VSMC death. Several studies demonstrated that balloon injury of vessels induces two waves of VSMC apoptosis. The first wave of apoptosis occurred in the media within hours of the injury.5 The second wave occurs at much later times after injury (days to weeks). VSMC accumulation in the neointima of injured rat carotid arteries reached a maximal level at 2 weeks after injury; however, cellular proliferation continues for up to 12 weeks.25 Therefore, it is thought that this second wave of apoptosis limits lesion growth. The rates of neointimal VSMC death and proliferation are in equilibrium at 2 weeks, thereby preventing further increase in lesion size. Expression of both the uncleaved and cleaved forms of Mst1 was increased after balloon injury. Upregulation of uncleaved and cleaved Mst1 indicated that Mst1 kinase activity was increased and may contribute to the delayed enhancement of apoptosis in the neointimal formation. Although genotoxic agents are known to activate Mst1, this is the first report showing that clinically relevant stimuli such as balloon injury activate Mst1 in blood vessel. An in vitro study suggested that reactive oxygen species may be involved in the activation of Mst1 in injured artery. Regulation of Mst1 expression has not been examined in detail, and the mechanism of Mst1 upregulation and activation in injured rat carotid artery is not clear at this point. Further studies are necessary to determine the mechanism of Mst1 upregulation and activation in injured blood vessel.
We showed in the present study that Mst1 was activated in apoptotic response by staurosporine and that overexpression of Mst1 induced apoptosis in VSMCs. Several studies have demonstrated that Mst1 is necessary for the induction of apoptosis. A kinase-negative mutant of Mst1 in which Lys-59 is changed to Arg inhibited staurosporine-induced chromatin condensation26 and chelerythrine-induced DNA fragmentation.17 Suppression of endogenous Mst1 by cardiac-specific overexpression of the kinase-negative mutant of Mst1 in transgenic mice prevented myocyte death induced by ischemia/reperfusion.17 These data suggest that Mst1 plays an essential role in mediating apoptotic response, which may be true in blood vessel as well.
Most Ste20 group kinases act as MAP4K, and it has been reported that Mst1 activates the p38-MAPK and JNK pathways.12 Mst1 has been shown to function upstream from MEKK1 and MKK7 in the JNK pathway12,27 and upstream from MKK6 in the p38-MAPK pathway.12 These results are consistent with Mst1 function as a MAP4K. However, there are conflicting evidences as to which pathway mediates apoptosis downstream of Mst1. Graves et al reported that the p38-MAPK pathway may mediate Mst1-induced apoptosis because SB203580 inhibited Mst1-induced morphological changes in 293T cells.12 Another group reported that a dominant-negative mutant of JNK inhibited Mst1-induced morphological changes as well as caspase activation, while a dominant-negative p38-MAPK did not.23 In VSMCs, p38-MAPK and JNK were both activated downstream of Mst1, and a p38-MAPK inhibitor partially inhibited staurosporine-induced Mst1 and caspase 3 cleavage. These data suggest that p38-MAPK, at least in part, mediates apoptotic response through Mst1 in VSMCs. p38-MAPK is activated both upstream and downstream of Mst1 and may be involved in the positive feedback loop formed by Mst1 and caspase 3. However, the effect of SB203580 was partial, suggesting that some pathway other than p38-MAPK is also involved in apoptosis induced by staurosporine, thus requiring further investigation.
Recently, it has been reported that Hippo, the Drosophila homologue of Mst1/2, promotes apoptosis and proper termination of cell proliferation during development, together with Salvador, the Drosophila homologue of hWW45, and Warts, the Drosophila homologue of large tumor suppressor (LATS), in a signal module complex.28,29 Although mutations in the hWW45 gene have been reported in three cancer cell lines,30 its function has not been examined in detail. The roles of hWW45 or LATS in VSMCs and the relationship between Mst1/2 and hWW45 or LATS in blood vessel require further investigation.
Many environmental factors and endogenous proteins regulate the struggle between apoptosis and proliferation in atherosclerotic lesion. Several reports suggest that apoptosis in atherosclerotic plaque may decrease its stability and induce plaque rupture.31,32 A recent report showed that proapoptotic genes were highly expressed in atherosclerotic plaques from patients with acute coronary syndrome compared with those from patients with stable angina.33 However, induction of apoptosis may be useful for the treatment of in-stent restenosis that is rich in cellular component such as VSMCs.34,35
The data presented in the present study suggest that Mst1 plays a critical role in the vascular remodeling process and could be a therapeutic target for prevention of restenosis after vascular injury.
Acknowledgments
This study was supported in part by grants from Takeda Science Foundation and a Grant-in-aid for Scientific Research from the Ministry of Education, Culture, Sports, Science, and Technology of Japan (17590742). We thank Daniela Zablocki for critical reading of the manuscript.
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