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. 2007 Fall;12(3):119–124.

The proteasome inhibitor lactacystin attenuates growth and migration of vascular smooth muscle cells and limits the response to arterial injury

Kurt G Barringhaus 1, Martin E Matsumura 2,✉
PMCID: PMC2323756  PMID: 18650992

Abstract

BACKGROUND

There is emerging evidence that the ubiquitin-proteasome system plays a role in vascular proliferative disorders such as restenosis after percutaneous coronary interventions. The present study examined the effect of proteasome inhibition on cultured vascular smooth muscle cell (VSMC) growth and migration, as well as on vascular lesion formation, following balloon arterial injury in the rat.

METHODS

The effect of the proteasome inhibitor clasto-lactacystin beta-lactone (lactacystin) on cultured VSMC proliferation was assessed using cell proliferation assays and immunohistochemical assessment of S-phase entry. To test the effect of proteasome inhibition on lesion formation and to confirm the role of p21Cip1/Waf1 (p21) in this effect in vivo, carotid injury was performed on anesthetized male Sprague-Dawley rats, followed by local treatment with either lactacystin or vehicle.

RESULTS

Treatment of VSMCs with the proteasome inhibitor lactacystin resulted in a 60% and 80% decrease in cell number versus controls at day 3 and day 5 after treatment, respectively. This effect was accompanied by an 86% decrease in S-phase entry and an increased level of the cyclin-dependent kinase inhibitor p21. Additionally, lactacystin significantly inhibited VSMC migration in a modified Boyden chamber assay. Lactacystin resulted in a 59% reduction of neointimal formation at 14 days following balloon injury. This effect was associated with an early increase in p21 protein in the arterial wall.

CONCLUSIONS

Inhibition of the ubiquitin-proteasome system resulted in the attenuation of VSMC growth both in cultured cells and in an animal model of vascular injury, possibly via a mechanism involving upregulation of the p21 cyclin-dependent kinase inhibitor. These data provide support for a role of the proteasome in the vascular response to injury, and suggest an important role for p21 and attenuation of cellular migration in the mechanism of this effect.

Keywords: Proteasome, Restenosis, Smooth muscle cells

Abnormal proliferation of vascular smooth muscle cells (VSMCs) is a major contributor to numerous pathological processes, and it is believed to be a contributor to the initiation and propagation of atherosclerosis. Numerous strategies for limiting pathological VSMC growth have been devised, including targeting of a variety of cellular growth regulatory pathways (1). The ubiquitin-proteasome pathway is a critical regulator of numerous cellular proteins, including multiple cell cycle regulatory factors (2). Proteins destined to be degraded by this pathway are covalently tagged with polyubiquitin chains. Ubiquitin-linked proteins are then recognized and degraded by the 26S proteasome, a large ATP-dependent enzyme that contains multiple enzymatic moieties (3). Our understanding of the ubiquitin-proteasome pathway has benefited from the development of inhibitors of the 26S proteasome as tools for studying its function and regulation. These inhibitors have been created based on either the natural product lactacystin (4) or on synthetic peptidyl derivatives (5). Lactacystin was originally isolated from actinomycete species because of its ability to inhibit neurite outgrowth. When exposed to aqueous solutions, lactacystin is converted to its active form clasto-lactacystin beta-lactone, which covalently binds to and irreversibly inactivates the proteasome (5,6).

Previous studies (7,8) have supported that the ubiquitin-proteasome pathway plays a role in VSMC proliferation and that proteasome inhibition affects VSMC hyperplasia in an animal model of vascular injury. Furthermore, recent studies (9) support the importance of the ubiquitin-proteasome pathway in the conversion of stable to unstable atherosclerotic plaques. Thus, the proteasome appears to be a potential target for modulation of the atherosclerotic process.

In the present study, we investigated the effect of the proteasome inhibitor lactacystin on the growth of VSMCs in culture and on lesion formation following balloon arterial injury in the rat. Results demonstrated that lactacystin effectively blocked S-phase entry and proliferation of cultured rat aortic VSMCs. A possible mechanism for this cell cycle arrest was through an increase in cellular levels of the G1 cyclin-dependent kinase (cdk) inhibitor p21Cip1/Waf1 (p21). Furthermore, lactacystin significantly inhibited VSMC migration in a modified Boyden chamber assay. Finally, local application of lactacystin at the time of balloon injury of the rat common carotid artery resulted in an increase in vessel wall p21, accompanied by a dramatic reduction in neointimal formation at 14 days following balloon injury. These data support the ubiquitin-proteasome pathway as an important regulator of the pathological increase in VSMC growth and migration, which results in neointimal formation following vascular injury, and supports regulation of p21 expression as a possible mechanism by which the ubiquitin-proteasome pathway is involved in vascular proliferative disorders.

METHODS

Reagents

The irreversible proteasome inhibitor clasto-lactacystin beta-lactone (lactacystin) was obtained from Calbiochem Inc, USA. Dulbecco’s modified Eagle’s medium-Ham’s F12 medium (DMEM-F12), penicillin-streptomycin solution and fetal bovine serum (FBS) were obtained from Gibco BRL, USA.

Effect of lactacystin on growth of cultured VSMCs

Primary rat aortic VSMCs were isolated by enzymatic treatment of rat aortas, and these cells were cultured in DMEM-F12 supplemented with 10% FBS and penicillin-streptomycin solution. Cells were maintained in subconfluent culture conditions by passaging as needed. Low-passage cells were used for experiments (passage 5 to passage 10). Before these cells were used, VSMC lineage was confirmed by positive staining using antibodies against smooth muscle alpha-actin and smooth muscle myosin heavy chain. VSMCs were plated onto 96-well plates at a density of 5000 cells/well, and they were allowed to grow in either 20 μM lactacystin or 0.1% DMSO (vehicle control). The concentration of lactacystin was determined from previous experiments assessing the inhibition of VSMC proliferation using 10 μM, 20 μM and 40 μM of lactacystin. In these experiments, the 20 μM concentration had the greatest inhibition of cell growth without obvious proapoptotic effects (data not shown). At 72 h following plating, cells were counted using a colorimetric cell number assay (Celltiter, Promega Corp, USA). A total of three repeats were performed (n=5 for each repeat).

Effect of lactacystin on S-phase entry of VSMCs

VSMCs were plated on coverslips and grown in DMEM-F12 with 10% FBS containing either 20 μM lactacystin or vehicle for 24 h. Cells were then pulsed with bromodeoxyuridine (BrdU) for 8 h and stained for BrdU using a fluorescein isothiocyanate (FITC)-labelled anti-BrdU staining system according to the manufacturer’s protocol (In Situ Cell Proliferation Kit, Roche Diagnostics, USA). Coverslips were mounted on medium containing propidium iodide (PI) (Vectashield, Vector Laboratories, USA). Slides were examined under fluorescent microscopy for BrdU (FITC) and nuclei (PI), and were counted and expressed as a percentage of BrdU-positive nuclei (which appear yellow under fluorescent microscopy due to a combination of both red PI and green FITC fluorescence). The assay was repeated three times, and the results of six high-power field per sample were summed for statistical analysis.

Effect of lactacystin on levels of p21 in VSMCs

VSMCs were plated in six-well plates and grown in DMEM-F12 with 10% FBS containing either 10 μM or 20 μM lactacystin or vehicle, and were harvested after 0 h, 24 h, 48 h and 72 h of treatment. Equivalent amounts of protein lysate were run on polyacrylamide gels and transferred to polyvinylidene difluoride membranes, blocked overnight in 4% milk in phosphate-buffered saline and incubated for 1 h with a 1:200 dilution of a rabbit polyclonal anti-p21 antibody (Santa Cruz Biotechonology Inc, USA). Membranes were then incubated with a 1:1000 dilution of an appropriate horseradish peroxidase-conjugated antimouse secondary antibody (Santa Cruz Biotechnology Inc, USA) for 1 h and Luminol reagent (Santa Cruz Biotechnology Inc, USA) for 1 min, and were then exposed on autoradiographic film.

Effect of lactacystin on VSMC migration

VSMC migration assay has been described previously (10). Briefly, VSMCs were plated onto migration chambers (8.0 μm pore size) (BioCoat, Becton-Dickinson, USA) at a density of 50,000 cells/chamber. Twenty-four hours later, the medium in the upper chamber was changed to serum-free DMEM-F12 with either 20 μM lactacystin or vehicle. To the lower chamber, DMEM-F12 with 10% FBS was added. The chambers were incubated for 24 h, at which time the membranes were washed and fixed in 2% paraformaldehyde. The tops of the membranes were wiped with a cotton swab to remove nonmigrated cells, and the membranes were stained with 4′,6-diamidino-2-phenylindole for 15 min at room temperature. Membranes were mounted under coverslips and analyzed under fluorescence microscopy. A total of six repeats were performed in each group, with five random fields for each repeat counted. Results were reported as the average number of cells per field.

Effect of lactacystin on neointimal formation and vascular expression of p21

Male Sprague-Dawley rats (300 g to 350 g) underwent balloon injury as described previously (11). Briefly, the right common and internal carotids were isolated and clamped, and the external carotid was isolated and an arteriotomy was made. Through the external carotid, a 1.5 by 20 mm angioplasty balloon was passed into the common carotid. The balloon was inflated and withdrawn three times to denude the endothelium. Following balloon injury, a 1 cm length of the injured common carotid was isolated and treated with either 40 μM lactacystin or vehicle, then diluted in normal saline (n=5 for each group) for 20 min. Following this, the external carotid was ligated, and blood flow in the common carotid was restored. Fourteen days following treatment, animals were sacrificed with an intraperitoneal injection of ketamine and xylazine. A midline sternotomy was performed, and the animals were pressure perfused with 4% paraformaldehyde via a cannula in the left ventricle. The treated portion of each carotid was paraffin embedded, and serial 5 μm sections were obtained every 1 mm for a total of 5 mm starting at the carotid bifurcation. The sections were de-paraffinized and stained with hematoxylin and eosin for histomorphometric analysis. The internal elastic lamina, media and neointimal areas were measured using image analysis software (Media Cybernetics Inc, USA).

Additional animals were sacrificed at four days following vessel injury using an intraperitoneal injection of ketamine and xylazine. Vessels were pressure perfused as described above, and tissue was processed for p21 immunostaining. Five-micrometer vessel sections were stained for p21 in the following manner: slides were deparaffinized and treated with antigen unmasking solution (Santa Cruz Biotechnology, USA) as per the manufacturer’s protocol. Slides were then treated with 0.3% hydrogen peroxide for 20 min, washed and incubated overnight with a 1:200 dilution of a rabbit anti-p21 antibody (Santa Cruz Biotechnology, USA) at 4°C. Slides were then washed and incubated with a 1:1000 dilution of a biotinylated antirabbit secondary antibody (Santa Cruz Biotechnology, USA). They were then washed and treated with Vector ABC reagent (Vector Laboratories, USA) for 30 min, washed and incubated with DAB substrate solution for 5 min. Slides were then counterstained with hematoxylin and mounted using Vectashield (Vector Laboratories, USA).

Statistical analysis

Data were compared using Student’s t test to evaluate two-tailed levels of significance.

RESULTS

Treatment of cultured VSMCs with lactacystin reduces cell numbers, inhibits S-phase entry and increases levels of p21

To determine the effect of proteasome inhibition on the growth of cultured VSMCs, 10 μM and 20 μM of lactacystin were added to rat aortic VSMCs in serum. Ten micromolar of lactacystin was chosen as the lowest concentration, because this value is estimated to be the 50% inhibitory concentration for most cultured cell types (6). Vehicle-treated cells resulted in a 121.9% increase in cell number at 72 h after plating. VSMCs treated with 10 μM lactacystin demonstrated an 88.7% increase in cell number. In contrast to the proliferation noted in vehicle-treated cells and those treated with 10 μM lactacystin, VSMCs treated with 20 μM lactacystin demonstrated an 11.9% decrease in cell number after 72 h (Figure 1).

Figure 1.

Figure 1

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Lactacystin inhibited vascular smooth muscle cell (VSMC) proliferation in a dose-dependent manner. Cultured rat aortic VSMCs treated with 20 μM lactacystin demonstrated 11.9% reduction in cell number after 72 h compared with 121.9% increase in control-treated cells (†P<0.001). VSMCs treated with 10 μM lactacsystin demonstrated 88.7% increase in cell number after 72 h. *P not significant versus control

To determine whether the reduction in the number of lactacystin-treated VSMCs was due to the inhibition of cell cycle activity, S-phase entry was measured using BrdU labelling of VSMCs treated with vehicle or 20 μM lactacystin, followed by staining with an FITC-linked anti-BrdU antibody and nuclear staining with PI. The percentage of cells staining positive for BrdU was defined as the number of yellow-stained nuclei divided by the number of yellow plus green nuclei times 100 (Figure 2A). Compared with vehicle-treated cells, treatment of VSMCs with lactacystin resulted in an 86% reduction in S-phase entry, as indicated by cells staining positive for BrdU (Figure 2B).

Figure 2.

Figure 2

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Lactacystin inhibited vascular smooth muscle cell (VSMC) S-phase entry and increased levels of the cyclin-dependent kinase inhibitor protein p21. A,B VSMCs treated with 20 μM lactacystin demonstrated reduced S-phase entry versus vehicle-treated cells, as indicated by reduced bromodeoxyuridine staining (bromodeoxyuridine plus nuclei turn yellow) (49% versus 7%; *P<0.005). C Treatment of VSMCs with 20 μM lactacystin resulted in increased levels of p21 protein by Western blot, peaking at 24 h to 48 h after addition of lactacystin

Cells traversing the G1-S boundary of the cell cycle are, in part, regulated by the balance between G1 cyclins, cdks and cdk inhibitors. Given the significant reduction in S-phase entry in lactacystin-treated cells, the effect of lactacystin on levels of the G1 cdk inhibitor p21 was examined by immunoblotting for p21 in VSMCs grown in serum and treated with either 20 μM lactacystin or vehicle (Figure 2C). Compared with control-treated cells, the p21 protein level was dramatically increased starting at 24 h following treatment with 20 μM lactacystin, and it remained increased compared with the control as late as 48 h following the addition of lactacystin. Thus, an increase of the p21 protein level is a possible mechanism for the inhibition of S-phase entry and resultant VSMC proliferation in response to proteasome inhibition. Of note, p21 levels decreased between 48 h and 72 h after treatment with lactacystin.

Lactacystin treatment inhibits VSMC migration

In addition to enhanced proliferation, the response of VSMCs of the vessel wall to arterial injury results in an increase in cellular migration (12). However, the effect of proteasome inhibition on VSMC migration had not previously been studied. Thus, the effect of lactacystin on the migration of cultured VSMCs to serum were examined in a modified Boyden chamber assay. Treatment of VSMCs with 20 μM of lactacystin resulted in a 46% reduction in cellular migration toward medium containing 10% FBS (Figure 3).

Figure 3.

Figure 3

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Lactacystin treatment inhibited the migration of vascular smooth muscle cells in a modified Boyden chamber assay. Compared with vehicle-treated cells, lactacystin resulted in 46% reduction in vascular smooth muscle cell migration (81 cells/field versus 44 cells/field; *P=0.002)

Local treatment with lactacystin at the time of balloon injury limits neointimal formation and results in an increased level of p21 protein early after injury

Cell culture studies (13) have demonstrated that lactacystin inhibits VSMC growth and migration, two processes that play a critical role in the contribution of VSMCs to neointimal formation following vascular injury. To test the effect of proteasome inhibition on vascular lesion formation in vivo, male Sprague-Dawley rats underwent carotid balloon injury, followed by a 20 min surgical dwell with either 40 μM lactacystin or vehicle control. Fourteen days after injury, animals were sacrificed and vessels were analyzed. Lactacystin-treated arteries demonstrated a 61% reduction of neointimal area versus vehicle-treated vessels (Figure 4A), as well as a 59% reduction in the neointima-to-media ratio (Figure 4B). No differences in media areas were noted between vehicle- and lactacystin-treated vessels, confirming that lactacystin had no effect on vessel remodelling. This finding was expected given the lack of significant vessel remodelling using the present model seen in previous studies (14).

Figure 4.

Figure 4

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Lactacystin treatment at the time of balloon injury inhibited neointimal formation in the rat carotid. A Neointimal area of animals sacrificed 14 days after injury or treatment. Compared with vehicle-treated animals, lactacystin-treated vessels demonstrated 61% reduction in neointimal area (0.029mm2 versus 0.075mm2; *P<0.05). B Neointima-to-media ratio of animals 14 days after injury or treatment. Lactacystin-treated animals demonstrated 59% reduction in neointima-to-media ratio (0.229 versus 0.569; *P<0.05). C Representative vessel cross-sections from lactacystin- and vehicle-treated groups (arrow indicates internal elastic lamina)

Data on cultured VSMCs have demonstrated that lactacystin treatment results in increased p21 protein levels. Thus, the effect of lactacystin on levels of p21 was examined at four days following balloon injury. This time point was chosen based on previous studies (15) that demonstrated an induction of endogenous p21 starting at three to five days after balloon injury in the present model. Compared with vehicle-treated vessels (Figure 5A), lactacystin-treated vessels demonstrated increased medial levels of p21 protein staining at four days after balloon vessel injury (Figure 5B).

Figure 5.

Figure 5

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Lactacystin treatment at the time of balloon injury increased p21 protein level expression four days after injury or treatment. Representative section of immunostaining for p21 in the control (A) versus lactacystin-treated (B) rats. Inset High-powered view of the vessel wall

DISCUSSION

Control of the cellular degradative apparatus has emerged as a major mechanism for efficiently and rapidly regulating protein levels (2). In particular, it has become clear that protein degradation is a significant means by which the cell tightly regulates levels of cell cycle factors in actively dividing cells (16). Recent data (5,17) support the ubiquitin-proteasome system as a major pathway for protein degradation, and as such, there is growing interest in the proteasome as a target for modulating abnormal cellular growth in a variety of human diseases. In the present study, we demonstrated that inhibition of the proteasome regulates both the growth and the migration of cultured VSMCs. Additionally, we demonstrated that local inhibition of the proteasome at the time of balloon injury in the rat carotid artery reduced the degree of subsequent intimal hyperplasia, the dominant mechanism resulting in luminal narrowing in the present model.

The ubiquitin-proteasome system is responsible for the degradation and turnover of up to 80% of cellular proteins (5). Thus, the cellular changes that occur in response to proteasome inhibition are complex. A previous study (7) examined the effect of proteasome inhibitors on the ultrastructure of cultured VSMCs. Treatment of VSMCs with lactacystin resulted in an increased level of contractile elements as detected by electron microscopy, suggesting that the ubiquitin-proteasome system was involved in the regulation of VSMC phenotype. Taken together with the present study, these data suggest that the proteasome may play a key role in linking VSMC phenotype and proliferative activity.

Our finding of the upregulation of the cdk inhibitor p21 by lactacystin is intriguing, because this represents a possible mechanism for the lactacystin-induced cell cycle arrest of VSMCs. This confirms the findings of a previous study (8) that examined the upregulation of p21 in response to treatment of cultured VSMCs with the reversible proteasome inhibitor MG132 (Cbz-leucyl-leucyl-leucinal), and extends this relationship to an animal model of vascular injury (8). In support of a direct role of p21 in mediating the effect of proteasome inhibition on VSMC growth, we have demonstrated that cells treated with lactacystin are blocked from entry into S-phase, consistent with inhibition of G1 progression by p21. Furthermore, previous studies (18) have demonstrated that direct adenoviral-mediated overexpression of p21 is capable of limiting neointimal formation in several animal models of vascular injury. The apparent discordance in the temporal expression pattern of p21 in cultured VSMCs (which was notable for a peak in p21 expression at 48 h after lactacystin treatment and a decline thereafter) versus those in the vessel wall (which was characterized by strong p21 upregulation at four days after vessel injury) deserves comment. Given that p21 expression is complex and certainly involves mechanisms independent of the regulation of protein degradation, it is possible that the decrease in p21 expression after 48 h was due to differential regulation of genes involved in p21 transcription in cultured VSMCs versus those of the carotid wall, and this regulatory mechanism was sufficient to overcome the effect of proteasome inhibition on p21 protein levels (19). Given that our cultured VSMCs were not primary lines, but rather cells that had been passaged multiple times, it is highly likely that they were phenotypically modulated with respect to VSMCs in vivo and may have altered cell cycle regulation.

Our finding that lactacystin inhibits VSMC migration may also represent an effect of proteasome inhibition mediated through regulation of p21. Previous work (20) has demonstrated that direct overexpression of p21 in cultured rabbit arterial VSMCs leads to a reduction in the migration of these cells. Thus, p21 may represent a common pathway by which lactacystin inhibits both VSMC proliferation and migration after arterial injury.

Admittedly, the finding of increased p21 levels following lactacystin treatment does not conclusively implicate this factor in the mechanism of lactacystin-induced growth inhibition. In particular, recent work has demonstrated that the inhibition of the proteasome alters multiple cellular pathways that could potentially result in growth arrest, including induction of the heat shock response (21), regulation of the nuclear accumulation of p53 (22) and activation of endothelial nitric oxide synthase (23). A better understanding of the mechanisms of lactacystin-induced VSMC growth arrest may uncover ways to target specific components of the ubiquitin-proteasome pathway to inhibit cell growth with maximal efficacy and minimal cellular toxicity.

To the best of our knowledge, the present study is the first to provide evidence of a role for the proteasome in the regulation of VSMC migration. Recent studies (24) have demonstrated a relationship between the proteasome and the migration of neoplastic cells. The proteasome inhibitor bortezomib was shown to inhibit the migration of multiple myeloma cells via inibition of caveolin-1. The specific mechanism by which lactacystin results in attenuated VSMC chemo-taxis is unknown. Of note, a previous study (25) demonstrated that integrin-mediated attachment of both NIH3T3 fibroblasts and human umbilical endothelial cells to the extracellular matrix resulted in proteasomal degradation of p21, suggesting that cellular adhesion, a critical component of cellular migration, may be regulated, in part, by the proteasome regulation of p21 in the vessel wall. Additionally, previous studies (26) have demonstrated that ubiquitin-mediated degradation of the platelet-derived growth factor-beta receptor is critical for the modulation of the cellular response to platelet-derived growth factor-beta, a factor that is known to induce VSMC migration. Further studies are needed to define the mechanisms by which proteasome inhibition leads to decreased cell motility.

CONCLUSIONS

We have shown that inhibition of the ubiquitin-proteasome pathway by the 26S proteasome inhibitor lactacystin results in a significant reduction of VSMC proliferation in vivo and vascular lesion formation in vivo. Possible mechanisms of this effect are via inhibition of proteasome-mediated destruction of the cell cycle inhibitor p21 and via inhibition of cellular migration, an effect known to be mediated, at least in part, by regulation of p21. The present study adds to our growing understanding of the role of protein degradation pathways in response to vascular injury, functions that may represent pharmacological targets for attenuating pathological VSMC growth contributing to vascular proliferative disorders.

ACKNOWLEDGEMENTS

This study was supported by National Institutes of Health/National Heart, Lung, and Blood Institute award K08 HL-04431 (Dr Martin E Matsumura) and National Institutes of Health/National Heart, Lung, and Blood Institute Training Grant T32 HL-07355 (Dr Kurt G Barringhaus).

Footnotes

CONFLICT OF INTEREST: The authors report no competing financial or nonfinancial interests associated with this study.

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