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(Arteriosclerosis, Thrombosis, and Vascular Biology. 1997;17:2500-2506.)
© 1997 American Heart Association, Inc.

Articles

Short-term Exposure to Thapsigargin Inhibits Neointima Formation in Human Saphenous Vein

S. J. George; J. L. Johnson; G. D. Angelini; ; J. Y. Jeremy

From the Bristol Heart Institute, University of Bristol, Bristol Royal Infirmary, Bristol, UK.

Correspondence to Dr Sarah Jane George, Bristol Heart Institute, University of Bristol, Bristol, BS2 8HW, UK. E-mail s.j.george{at}bristol.ac.uk

	Abstract

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Abstract Vascular smooth muscle cell (VSMC) migration and proliferation are involved in the intimal thickening responsible for late vein graft failure. In addition to growth and chemotactic factors, VSMCs require expression of matrix-degrading enzymes, eg, metalloproteinases (MMP), to relieve the antiproliferative and antimigratory constraints of the extracellular matrix. Thapsigargin irreversibly inhibits Ca²⁺- ATPase, eliciting an increase in intracellular Ca²⁺ and depletion of the intracellular calcium pools that are thought to be involved in the control of VSMC migration, VSMC proliferation, and MMP activity. We therefore studied the effect of thapsigargin on VSMC migration, VSMC proliferation, and MMP expression in human saphenous vein organ cultures. Vein segments were cultured for 14 days, and VSMC proliferation and migration were determined by autoradiography. Cell death was assessed using in situ end-labeling and lactate dehydrogenase release. Using Western blotting, we examined MMP-2 and MMP-9 and tissue inhibitor of metalloproteinases (TIMP)-1 and TIMP-2 expression. Exposure to thapsigargin at 10 nmol/L for 60 minutes before culture significantly inhibited neointimal thickening (60%, P<.05), intimal and medial VSMC proliferation (32%, P<.05 and 37%, P<.05, respectively), and VSMC migration (36%, P<.05). Thapsigargin at 10 nmol/L did not significantly increase cell death or MMP-2, MMP-9, TIMP-1, and TIMP-2 expression. These results suggest that blockade of Ca²⁺-ATPase by thapsigargin inhibits VSMC migration and proliferation involved in neointimal formation without affecting MMP-2 and MMP-9 expression. Because short-term exposure to thapsigargin was sufficient to inhibit neointima formation, this drug may prove useful in the treatment of intimal thickening after arterial bypass graft surgery.

Key Words: metalloproteinases • neointima • calcium • human saphenous vein • smooth muscle cells

	Introduction

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Autologous saphenous vein is the most commonly used conduit for coronary artery bypass graft surgery.¹ However, the long-term clinical success of coronary artery bypass graft with saphenous vein is limited due to medial and intimal thickening, which may lead to narrowing of the graft lumen and ultimately to thrombotic occlusion (50% remain patent after 10 years).¹ Although the etiology of vein graft failure is multifactorial, evidence from animal models and in vitro systems indicates that intimal thickening involves the migration of VSMCs of medial origin to the intima of the graft where they continue to proliferate and secrete extracellular matrix proteins.^2–4 A variety of growth factors and chemotactic agents including platelet-derived growth factor have been shown to be key determinants of VSMC migration and VSMC proliferation.⁵ In addition, the expression of matrix-degrading enzymes such as the MMP family of enzymes is also essential.⁶ It is hypothesized that these enzymes allow the VSMCs to free themselves of the antiproliferative and antimigratory constraints of the extracellular matrix.^6–9

The intracellular mechanisms controlling VSMC replication have not been fully clarified, but an increase in cytosolic Ca²⁺ may be a key component of mitogen-stimulated replication and migration.^10–13 Ca²⁺ mobilization is mediated by inositol trisphosphate, which elicits the release of Ca²⁺ from intracellular stores.^10,11 Furthermore, Ca²⁺ channel blockers have been shown to inhibit VSMC migration and VSMC proliferation in cultured cells and after experimental arterial injury.^14–18 There is paradoxical evidence, however, that substances that mobilize intracellular Ca²⁺ pools, eg, thapsigargin, potently inhibit VSMC proliferation.¹⁹ Thapsigargin causes an increase in cytosolic Ca²⁺ by inhibiting Ca²⁺-ATPase, which catalyzes the resequestration of cytosolic Ca²⁺ into intracellular storage sites.²⁰ The effect of thapsigargin is apparently irreversible and a 30-minute exposure to the drug is sufficient to inhibit replication of isolated VSMCs.¹⁹ In the same study, other Ca²⁺ mobilizers (ionomycin, calcimycin, and cyclopiozonic acid) were much less potent and did not inhibit VSMC replication after short-term exposure.

In this study we have examined the effect of short-term (60 minutes) exposure to thapsigargin on neointima formation, VSMC migration, and VSMC proliferation in the human saphenous vein organ culture model. In this system, VSMCs are in contact with their native extracellular matrix. A neointima containing VSMCs is formed during 14 days in culture, as a result of both VSMC migration and VSMC proliferation.^21,22 Because MMP activity is an obligatory event in VSMC migration and VSMC proliferation,^8,9,23 we have also examined the effect of thapsigargin on the expression of MMP-2 and MMP-9 and TIMP-1 and TIMP-2 using Western blotting and immunocytochemistry. Finally, the impact of thapsigargin on cell injury and death was determined by in situ end-labeling of fragmented DNA and by assay of LDH release.

	Methods

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Materials
All reagents were purchased from Sigma Chemical Co except for those listed below. EM-1 emulsion, ECL reagents, Hybond C nitrocellulose membrane, and [6-³H]thymidine were obtained from Amersham International. Biotin-16-UTP and the LDH assay kit were purchased from Boehringer Mannheim. Sodium heparin was from CP Pharmaceuticals. L-Glutamine, FCS, penicillin, and streptomycin were purchased from Gibco BRL. Horseradish peroxidase-conjugated swine anti-rabbit and rabbit anti-sheep immunoglobulins were from Dako. RPMI-1640 culture media and amphotericin B were obtained from ICN Flow. Papaverine hydrochloride was supplied by McCarthy Medical. QB-end-10 monoclonal antibody was supplied by Oxoid Unipath Ltd. DNA polymerase I (Klenow) large fragment was from Promega. Gentamicin was from Roussel. Purified MMP-2 and MMP-9 proteins were from TCS Biologicals. Sheep anti-TIMP-1 and anti-TIMP-2 polyclonal antibodies were generous gifts of Dr Gillian Murphy (Strangeways Research Laboratories, Cambridge, UK). Purified TIMP-1 and TIMP-2 proteins were kind gifts of Dr Andrew Baker (Bristol Heart Institute, Bristol, UK).

Vein Collection
Human saphenous vein segments were obtained and cultured by a modification of the method of Pedersen and Bowyer²⁴ as described previously by Soyombo et al.²¹ Human saphenous vein segments were obtained from 24 patients undergoing coronary artery bypass grafting. Briefly, "surgically prepared" segments were obtained from each patient from the ankle region after completion of the last proximal anastomosis. These veins had been subjected to adventitial stripping, side branch ligation, gentle manual distention, and storage in heparinized blood. Twenty-two patients were men and 2 were women; the mean age of the patients was 58.6 years (range 40 to 76 years). Ethical permission was obtained from the United Bristol Hospital Trust Ethics Committee (ref. E2847). Vein segments were collected in sterile 20 mmol/L HEPES-buffered RPMI-1640 tissue culture medium containing 0.225 mg/ml papaverine hydrochloride, 5 µg/ml amphotericin B, and 20 IU/ml sodium heparin.

Short-term Exposure to Thapsigargin
Segments were placed in wash medium consisting of 20 mmol/L HEPES-buffered RPMI-1640 tissue culture medium supplemented with 2 mmol/L L-glutamine, 8 µg/ml gentamicin, 100 IU/ml penicillin, and 100 µg/ml streptomycin. The adventitia was removed, and then the vein was cut longitudinally, and divided into 10-mm segments. One segment was fixed in 10% buffered formal saline as a day 0 control. Vein segments were pinned down with minuten pins, endothelial surface on top, on polyester mesh resting on Sylgard resin in glass Petri dishes. The vein segments were incubated at 37°C under 95% air/5% CO₂ in incubating medium (2 g/L bicarbonate buffered RPMI-1640 tissue culture medium supplemented with 2 mmol/L L-glutamine, 8 µg/ml gentamicin, 100 IU/ml penicillin, and 100 µg/ml streptomycin) supplemented with 10 nmol/L thapsigargin for 30, 60, or 120 minutes. Control vein segments were incubated at 37°C under 95% air/5% CO₂ in incubating medium supplemented with the vehicle (ethanol) alone for 120 minutes. The segments were washed three times with wash medium and then cultured for 14 days in incubating medium supplemented with 30% (vol/vol) FCS, 1 µCi/mL [6-³H]thymidine at 37°C under 95% air/5% CO₂, changing the medium every 2 days. Vein segments were then washed twice with phosphate-buffered saline (0.15 mol/L NaCl, 7.5 mmol/L Na₂HPO₄, and 1.9 mmol/L NaH₂PO₄, pH 7.4), fixed in 10% buffered formal saline, processed, and paraffin wax-embedded.

Continuous Exposure to Thapsigargin
In the continuous exposure organ experiments, organ cultures were set up as described for the short exposure organ cultures. Vein segments were cultured for 14 days in incubating medium supplemented with 30% (vol/vol) FCS, 1 µCi/ml [6-³H]thymidine, and thapsigargin at 1, 5, 10, 100, and 1000 nmol/L at 37°C under 95% air/5% CO₂. Control vein segments were cultured for 14 days in incubating medium supplemented with 30% (vol/vol) FCS, 1 µCi/ml [6-³H]thymidine and the vehicle (ethanol) alone. The medium was changed every 2 days. After culture, the vein segment was washed three times with wash medium and then placed in serum-free incubation medium for 24 hours at 37°C under 95% air/5% CO₂. The conditioned medium was removed and stored at -20°C until analysis, and the vein segments were fixed in 10% buffered formal saline, processed, and paraffin wax-embedded at right angles to the original direction of blood flow.

Histological Analysis
Transverse 3-µm-thick sections were cut and mounted on 3-aminopropyl triethoxy silane coated slides. Serial sections were examined by both Miller's elastic van Gieson staining, Mayer's hematoxylin and eosin staining, and modified Alcian blue van Gieson staining.²⁵ VSMCs were identified by immunocytochemical analysis using monoclonal anti--smooth muscle actin antibody (clone 1A4) as described previously.²¹ Endothelial cells were identified by immunocytochemical analysis using QB-end-10 antibody.²⁶

Assessment of Intimal Thickening, VSMC Proliferation, and VSMC Migration
Proliferating cells were detected by autoradiography as described previously.^21,22 The number of labeled and unlabeled neointimal VSMCs were counted, and the latter was used as an estimate of VSMC migration. The neointimal thymidine labeling index was calculated by dividing the number of labeled neointimal VSMCs by the total number of neointimal VSMCs and expressing the result as a percentage. The percentage of labeled medial VSMCs was determined in three 0.25-mm² areas, and the mean was calculated to give the medial thymidine labeling index. The mean neointima thickness was determined by measuring the thickness of the neointima at 10 points on the vein segment with an image analysis system (Microscale TC) and calculating the average. The vein section length (ie, circumference) was measured with a calibrated microscope eyepiece graticule, and the total number of neointimal cells was expressed per unit length, to normalize for variation in the size of veins between patients.

Cell Viability
To assess the effect of culturing in the presence of 1 µCi/ml [³H]thymidine on cell viability, paired segments of vein were cultured as described above in the presence or absence of 1 µCi/ml [³H]thymidine for 7 and 14 days (n=6). Cell viability was examined by determining the tissue concentrations of ATP and DNA and ISEL as described previously.^21,22 The number of ISEL-positive neointimal cells was determined and expressed as a percentage of the total number of neointimal cells. In addition, the percentage of positive medial cells in three 0.25-mm² fields was calculated and the average was determined.

The effect of thapsigargin on cell viability was examined by ISEL and by measuring LDH release. Conditioned medium were diluted to the equivalent of 1 mg wet weight of vein segment and assayed for LDH activity using the LDH assay kit according to the manufacturer's protocol. Results were calculated as international units per liter. The total tissue LDH activity and DNA concentration per milligram wet weight were determined in 12 segments of vein segments, and the LDH activity per cell and the number of cells per milligram wet weight were calculated. With these figures the LDH activity in the conditioned medium were expressed as the percentage of cells that must have died to release this enzyme activity.

Western Blotting
Previous studies in our laboratory using human saphenous vein (unpublished data, 1996) and pig carotid artery²⁷ have demonstrated that expression of MMP-2 and MMP-9 in tissue levels closely resembles secretion of these proteins into conditioned medium. Collection of conditioned medium is both easy and allows the tissue to be analyzed histologically. Therefore, MMP-2 and MMP-9 levels in conditioned medium collected from the organ cultures (n=6) were examined by Western blotting, as described previously.²⁸ Culture with 0.1 mmol/L cycloheximide demonstrated that gelatinase release was due to de novo protein synthesis.²⁸ TIMP-1 and TIMP-2 levels were also determined using Western blotting of the conditioned medium. Conditioned medium collected from one patient were always compared on the same gel. The conditioned medium was concentrated 40-fold using Amicon 10 centrifugal concentrators and then diluted with serum-free incubation medium to according to the wet weight of the sample, in order to control for variations in vein segment size. Conditioned medium was subjected to electrophoresis on 7.5% and 10% (wt/vol) polyacrylamide gels for MMP and TIMP proteins, respectively. All gels were calibrated with high molecular mass protein standards and 150 ng of purified MMP-2 and MMP-9 proteins or 50 ng of purified TIMP-1 and TIMP-2 proteins. Proteins were transferred to Hybond C nitrocellulose membrane. Transferred MMP-2 and MMP-9 proteins were detected using rabbit anti-human MMP-2 and rabbit anti-human MMP-9 antisera diluted 1:1000, horseradish peroxidase swine anti-rabbit immunoglobulins diluted 1:2000 and ECL reagents. Transferred TIMP-1 and TIMP-2 proteins were detected using sheep anti-human TIMP-1 and sheep anti-human TIMP-2 polyclonal antibodies at 50 µg/ml, horseradish peroxidase rabbit anti-sheep immunoglobulins diluted 1:2000, and ECL reagents. The mean gelatinase and TIMP levels (n=6) were determined by densitometric scanning of the autoradiographs using a Bio-Rad GS-690 imaging densitometer. The optical density (ODx millimeters squared) of each band was corrected for variation between Western blots by expressing it as a percentage of the value obtained for the purified standard on the same gel. Bovine MMP-2 and MMP-9 and TIMP-1 and TIMP-2 proteins in the FCS used for culturing were not detected by Western blotting (data not shown).

Immunocytochemical Analysis for MMP-2 and MMP-9
Cells expressing MMP-2 and MMP-9 protein were identified by immunocytochemical analysis using rabbit anti-human MMP-2 and MP-9 antisera, as described previously.²⁸

Statistical Analysis
Data were analyzed using ANOVA for multiple comparisons. Paired analysis between two groups was performed using paired Student's ttest where ANOVA indicated significance for the multiple comparison. Statistical significance was accepted when P<.05.

	Results

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Organ Culture
As observed previously,^21,22 histological staining revealed that before culture (day 0) the veins had an incomplete endothelial monolayer and a fenestrated elastic lamina (Fig. 1a) above a media composed of VSMCs (Fig. 1b). After 14 days in organ culture, a highly cellular, elastin-free intima developed (Fig. 1c). The intimal cells were identified as VSMCs (Fig. 1d) beneath a single layer of endothelial cells (data not shown). No significant difference in cell viability was detected in the presence and absence of 1 µCi/ml [³H]thymidine using ISEL and the concentrations of ATP and DNA in tissue extracts (data not shown).

View larger version (144K): [in this window] [in a new window]   Figure 1. Histological appearance of veins before and after culture. A, One of six veins before culture stained with Miller's elastic van Gieson stain. B, The same vein stained for smooth muscle cells with a monoclonal antibody against -smooth muscle actin. C, A paired vein segment after 14 days in culture stained with Miller's elastic van Gieson stain. D, The same vein after 14 days in culture stained with monoclonal antibody against -smooth muscle actin. Arrows in all panels indicate the boundary between the intima and the media. The scale bar in a applies to all panels and represents 25 µm. The brown color in b and d indicates positive cells.

Effect of Short-term Exposure to Thapsigargin on Neointima Formation, VSMC Proliferation, and VSMC Migration
Neointima formation, determined by neointimal thickness and the total number of neointimal cells per millimeter, was significantly inhibited by a 60-minute exposure to 10 nmol/L thapsigargin before culture for 14 days (Table 1 and Fig. 2d and 2e). The neointimal and medial VSMC proliferation indices were also significantly reduced by a 60-minute pretreatment with thapsigargin (Table 1 and Fig. 2e). Furthermore, VSMC migration estimated by the number of unlabeled neointimal VSMCs was significantly reduced by a 60-minute exposure to thapsigargin (Table 1). Although a 30-minute exposure to thapsigargin significantly reduced neointimal thickness and intimal and medial proliferation, it was insufficient to reduce the total number of neointimal cells per millimeter and the number of unlabeled neointimal VSMCs (Table 1). Exposure to thapsigargin for 120 minutes did not cause increased inhibition of neointima formation, VSMC migration, or VSMC proliferation compared with a 60-minute exposure (Table 1). Modified Alcian blue staining demonstrated the presence of mucopolysaccharide-containing extracellular matrix surrounding the neointimal VSMCs after 14 days in culture (Fig. 2c), confirming previous findings.²⁹ The presence of mucopolysaccharide-containing extracellular matrix around neointimal cells was not reduced in the neointima of vein segments exposed to thapsigargin before culture (Fig. 2f).

View this table: [in this window] [in a new window]   Table 1. Effect of Short-term Exposure to 10 nmol/L Thapsigargin on Neointima Formation and VSMC Proliferation, Migration, and Death

View larger version (152K): [in this window] [in a new window]   Figure 2. Effect of short-term exposure to thapsigargin on neointima formation. a, One of six veins cultured for 14 days in the absence of thapsigargin (control) and stained with Miller's elastic van Gieson stain. b, Hematoxylin and eosin-stained autoradiograph of the same section, c, The same vein segment stained with modified Alcian blue van Gieson stain. Many silver-stained nuclei (ie, proliferating cells) are present in the neointima and media. d, A paired vein segment exposed to 10 nmol/L thapsigargin before 14 days in culture and stained with Miller's elastic van Gieson stain. e, Hematoxylin and eosin-stained autoradiograph of the same section. f, The same vein segment stained with modified Alcian blue van Gieson stain. Fewer silver-stained nuclei (ie, proliferating cells) are present in the neointima and media. Arrows in all panels indicate the boundary between the intima and the media. The scale bar in a applies to all panels and represents 50 µm.

Effect of Continuous Exposure to Thapsigargin Neointima Formation, VSMC Proliferation, and VSMC Migration
Continuous exposure to 10 nmol/L thapsigargin significantly reduced neointima formation, as measured by neointimal thickness and the total number of neointimal cells per millimeter, to a similar extent as 60 minutes of exposure (Table 2). Neointimal and medial cell proliferation and the number of unlabeled neointimal cells were also significantly (P<.05) inhibited by continuous exposure to thapsigargin to the same degree as short-term exposure (Table 2). Continuous exposure to 1 and 5 nmol/L thapsigargin did not significantly inhibit neointima formation (P<.05), whereas concentrations >10 nmol/L (100 and 1000 nmol/L) did (Table 2).

View this table: [in this window] [in a new window]   Table 2. Effect of Continuous Exposure to Thapsigargin on VSMC Proliferation and Death and Neointima Formation

Cell Viability
The percentage of cells with fragmented DNA in the neointima and the media was not affected by short-term or continuous exposure to 10 nmol/L thapsigargin, but was significantly greater (P<.05) than the control in the presence of 100 and 1000 nmol/L thapsigargin (Tables 1 and 2). The LDH activity released into the conditioned medium was not increased by culturing in the presence of 10 nmol/L thapsigargin (Table 2), indicating that thapsigargin is not toxic at the lowest effective concentration (10 nmol/L). In view of the toxicity observed at higher concentrations (100 and 1000 nmol/L) subsequent studies on MMPs were carried out only at 1, 5, or 10 nmol/L thapsigargin.

Expression of MMP-2 and MMP-9 and TIMP-1 and TIMP-2 Proteins
After 14 days in culture in the absence of thapsigargin, high levels of MMP-2 protein were detected in all cells of the vein segment, while MMP-9 expression was detected at high levels in the neointima and in some of the medial VSMCs. Expression of these MMPs was not affected by culturing in the presence of 1, 5, or 10 nmol/L thapsigargin (data not shown).

Western blotting using rabbit anti-human MMP-9 antisera detected a 95-kD protein (Fig. 3A), equivalent to the electrophoretic mobility of the pro-MMP-9 standard, and rabbit anti-human MMP-2 antisera detected 72- and 68-kD proteins (Fig. 3B), equivalent to the electrophoretic mobility of the pro-MMP-2 and active MMP-2 standards, respectively. Densitometric scanning of autoradiographs (n=6) revealed that thapsigargin had no significant effect on the amount of MMP-2 or MMP-9 released into the conditioned medium (Table 3).

View larger version (41K): [in this window] [in a new window]   Figure 3. Detection of MMP-2 and MMP-9 proteins in conditioned medium collected after 14 days in culture using Western blotting. Representative Western blots (n=6) of conditioned medium collected between days 14 and 15 from organ cultures supplemented with 0, 1, 5, or 10 nmol/L thapsigargin. Conditioned medium was concentrated 40-fold, and proteins were detected using antibodies to (A) MMP-9 and (B) MMP-2. Lanes 1, control; lanes 2, 1 nmol/L thapsigargin; lanes 3, 5 nmol/L thapsigargin; lanes 4, 10 nmol/L thapsigargin; and lanes 5, purified protein.

View this table: [in this window] [in a new window]   Table 3. Expression of MMP-2 and MMP-9 and TIMP-1 and TIMP-2

Western blotting for TIMP-1 and TIMP-2 proteins revealed 29- and 22-kD proteins, equivalent to the electrophoretic mobility of the TIMP-1 and TIMP-2 standards, respectively (Fig. 4A and 4B). Densitometric scanning of autoradiographs (n=6) revealed that thapsigargin had no significant effect on the amount of TIMP-1 or TIMP-2 released into the conditioned medium (Table 3).

View larger version (105K): [in this window] [in a new window]   Figure 4. Detection of TIMP-1 and TIMP-2 proteins in conditioned medium collected after 14 days in culture using Western blotting. Representative Western blots (n=6) of conditioned medium collected between days 14 and 15 from organ cultures supplemented with 0, 1, 5, or 10 nmol/L thapsigargin. Conditioned medium was concentrated 40-fold, and proteins were detected using antibodies to (A) TIMP-1 and (B) TIMP-2. Lanes 1, control; lanes 2, 1 nmol/L thapsigargin; lanes 3, 5 nmol/L thapsigargin; lanes 4, 10 nmol/L thapsigargin; and lanes 5, purified proteins.

	Discussion

Top Abstract Introduction Methods Results Discussion References

 
This study demonstrates that thapsigargin is a potent inhibitor of neointima formation in organ culture of the human saphenous vein. The mechanism by which 60 minutes of exposure to 10 nmol/L thapsigargin before 14 days in culture inhibits neointima formation by approximately 60% is unknown. Thapsigargin may reduce neointima formation by irreversible inhibition of the normal function of certain intracellular Ca²⁺ storage pools. However, these findings may be due to the presence of a "time window" that is sensitive to thapsigargin. It is possible that key events critical for subsequent neointima formation occur very early after human saphenous veins are placed in culture and that these are thapsigargin sensitive. This speculation is supported by previous studies in the rat balloon injury model, which demonstrated the existence of a time window for inhibition of intimal thickening and VSMC proliferation using calcium antagonists³⁰ and heparin.³¹ Whether such a time window exists for thapsigargin in the human saphenous vein organ culture model could be established by delaying the exposure to thapsigargin for 24 hours and examining the effect on neointima formation. Furthermore, neointima formation may be inhibited by a combination of both of these mechanisms.

With the aim of gaining more information on the mechanism of action of thapsigargin, we examined the critical exposure time. We exposed vein segments for 30 and 120 minutes and compared the effect on neointima formation with 60 minutes of exposure. Exposure for 120 minutes significantly inhibited neointima formation, but there was no additional effect compared with 60 minutes of exposure. This demonstrates that 60 minutes of exposure to 10 nmol/L thapsigargin gives the maximal inhibitory effect. However, 30 minutes of exposure significantly reduced neointimal thickness and VSMC proliferation but was insufficient to inhibit the number of neointimal cells and the number of unlabeled neointimal cells. This suggests that 30 minutes is insufficient to significantly inhibit VSMC migration.

To understand how short-term exposure to thapsigargin is effective in inhibiting neointima formation, it is necessary to examine the effect of thapsigargin on the processes that are involved in neointima formation: VSMC migration, VSMC proliferation, extracellular matrix synthesis, and apoptosis. It has previously been established that neointima formation in the human saphenous vein organ culture model involves migration of medial VSMCs to the intima and VSMC proliferation.^21,22 The number of neointimal cells unlabeled with tritiated thymidine in this model is an estimate of VSMC migration since these cells can only arise by migration. The significant reduction in the number of unlabeled neointimal cells indicates that VSMC migration is inhibited by treatment with 10 nmol/L thapsigargin for 60 minutes before culture. We have also recently found that thapsigargin at 10 nmol/L inhibits VSMC migration in vitro (unpublished data, 1997). The reduction in the number of labeled neointimal cells in both the intima and media demonstrates that thapsigargin also potently inhibits VSMC proliferation. After 14 days in culture, modified Alcian blue van Gieson stains neointima cells yellow indicating the presence of muscle and surrounding extracellular matrix stains green indicating the presence of mucopolysaccharides (Fig. 1c), confirming previous findings.²⁹ The amount of extracellular matrix-containing mucopolysaccharides surrounding each neointimal cell was not affected by pretreatment with thapsigargin, suggesting that neointima formation is not reduced by inhibition of extracellular matrix synthesis. We examined whether thapsigargin inhibited neointima formation by stimulating apoptosis using ISEL. We did not detect higher levels of apoptosis in thapsigargin-treated cultures. From these results we can therefore conclude that thapsigargin inhibits neointima formation in the human saphenous vein organ culture model by blocking VSMC migration and VSMC proliferation.

In healthy blood vessels, the extracellular matrix maintains VSMCs in a state of relative quiescence.³² After injury to the vessel, however, MMPs are rapidly expressed and activated;; this may allow extracellular matrix degradation, and promote VSMC migration and VSMC proliferation and remodeling of the vessel.⁶ Furthermore, the involvement of MMPs in VSMC migration^7,8,9,33 and VSMC proliferation^9,33 has been demonstrated directly using synthetic MMP inhibitors. Therefore, we examined whether thapsigargin inhibits neointima formation by reducing MMP expression. The expression and secretion of the pro- and active forms of MMP-2 and the pro-form of MMP-9, which are markedly increased after 14 days in culture,²³ were unaffected by the concentrations of thapsigargin that inhibited neointima formation, VSMC migration, and VSMC proliferation. This demonstrates that the reduction in neointima formation by thapsigargin is not mediated by inhibition of MMP-2 and MMP-9 expression. Because the activity of MMPs is dependent on the level of TIMP expression, we also examined TIMP-1 and TIMP-2 protein expression. Similarly, there was no effect on TIMP-1 and TIMP-2 protein levels, suggesting that MMP-2 and MMP-9 activity is not affected by thapsigargin. However, we have previously shown using a synthetic MMP inhibitor (Ro 31-9790) that MMP activity is essential for neointima formation.³³ This demonstrates that MMP-2 and MMP-9 are necessary but not sufficient to mediate neointimal thickening, because thapsigargin inhibits neointimal formation in the presence of these enzymes. Thapsigargin must therefore interfere with another part of the migratory and proliferative machinery, for example, other proteases or a chemotactic signal.

These findings suggest that thapsigargin may be clinically useful for preventing neointima formation in coronary artery vein grafts. Thapsigargin cannot be administered systemically for reasons of toxicity, but the short-term exposure results suggest that it could be used in coronary artery bypass grafting to treat veins ex vivo before implantation. Before pursuing this in animal models, however, it is necessary to examine in more detail the effect of thapsigargin on cell viability. We determined the effect of long-term (14-day) exposure to a concentration gradient (1, 5, 10, 100, and 1000 nmol/L) of thapsigargin on neointima formation and cell death. Thapsigargin caused a dose-dependent effect on neointima formation and VSMC migration and proliferation. At 100 and 1000 nmol/L, the general histological and the ISEL results demonstrated that thapsigargin was toxic. Therefore, the most effective nontoxic dose, determined by ISEL and LDH release, was 10 nmol/L. These findings question the value of previous studies, which have used higher doses of thapsigargin. For example, a recent study showed that thapsigargin inhibits MMP-2 and MMP-9 activity in HT-1080 cells.³⁴ Thapsigargin was ineffective at 5 nmol/L, but was effective at 50 and 500 nmol/L. However, we have demonstrated that concentrations of thapsigargin >10 nmol/L cause an increase in cell death, assessed by ISEL of fragmented DNA. It is possible that the inhibitory effects of thapsigargin 50 nmol/L on MMP activity are the result of cytotoxicity.

One very important consideration concerning these results is the apparent irreversibility of the effect of thapsigargin on intracellular Ca²⁺ stores. Further studies are required to determine whether thapsigargin binds irreversibly to Ca²⁺-ATPase and irreversibly inhibits resequestration of Ca²⁺ to intracellular pools in the vein segments since conflicting data exist concerning its irreversibility in isolated cells.^11,35 Because human saphenous vein segments can only be maintained in organ culture for 14 days, longer term studies to investigate the irreversibility of thapsigargin could be carried out in animal models of vein grafting, eg, the pig arteriovenous graft model. If thapsigargin does bind irreversibly, this may cause problems in the vein graft, such as its ability to contract or the normal rate of VSMC proliferation to maintain vessel integrity. Furthermore, thapsigargin may cause problems by leaching out of the grafted vein and entering the bloodstream.

In conclusion, this study demonstrates that short-term exposure to thapsigargin potently inhibits neointima formation and VSMC migration and VSMC proliferation in the human saphenous vein. Ex vivo treatment of human saphenous veins could inhibit neointimal formation after coronary artery bypass grafting and increase the long-term success rate of this procedure.

	Selected Abbreviations and Acronyms

 

FCS = fetal calf serum ISEL = in situ end labeling LDH = lactate dehydrogenase MMP = matrix metalloproteinase TIMP = tissue inhibitor of metalloproteinase VSMC = vascular smooth muscle cell

	Acknowledgments

 
This work was supported by the Garfield Weston Trust, The Wellcome Trust, and the British Heart Foundation. We thank Melanie Smith for her excellent technical help. We also thank Dr Chris Jackson for useful discussions.

Received March 1, 1997; accepted August 26, 1997.

	References

Top Abstract Introduction Methods Results Discussion References

 
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