Minocycline Inhibits Smooth Muscle Cell Proliferation,... : Journal of Cardiovascular Pharmacology (original) (raw)
INTRODUCTION
Restenosis following balloon angioplasty reduces vessel patency and limits long-term procedural success. Migration of vascular smooth muscle cells (SMC) plays an important role in the formation of the neointima seen in restenotic lesions. 1 Under normal circumstances, SMCs are located in the media of the artery where they are enmeshed in an extracellular matrix composed of collagen, fibronectin, proteoglycans and other glycoproteins. To migrate from the media to the intima, SMCs must be capable of degrading this matrix. 2,3
Following injury to the vessel wall, SMCs undergo a phenotypic change from a contractile state to a synthetic one producing proteolytic enzymes that digest and remodel the extracellular matrix. 4–8 Expression of urokinase and tissue-type plasminogen activator occurs shortly after balloon injury. 5 These serine proteases catalyze the conversion of plasminogen to plasmin, an enzyme capable of degrading a broad array of matrix molecules. 5 Vascular SMCs also produce various matrix metalloproteinases (MMP). 9 These proteolytic enzymes facilitate SMC migration and play an integral part in the process of vascular remodeling. 4
The tetracyclines are a class of antimicrobial agents that have been used for years for the treatment of bacterial and rickettsial infections. 10 Recently, these drugs have been found to be inhibitors of MMPs. Specifically, doxycycline and chemically modified tetracycline (CMT) have been shown to have direct inhibitory effects on the expression of both MMP-2 (Gelatinase A) and MMP-9 (Gelatinase B) in human epithelial cells and rat smooth muscle cells. 11–13 Tetracycline, when given to rats in combination with flurbiprofen (a non-steroidal anti-inflammatory drug), completely inhibited collagenase activity and significantly inhibited gelatinase activity in joint tissue. 14
In the present study, we sought to determine whether minocycline, a semisynthetic derivative of chlortetracycline, could inhibit human aortic smooth muscle cell migration and proliferation in vitro. We also examined whether minocycline, when given to rats around the time of balloon injury of the carotid artery, could inhibit neointimal formation. We tested three routes of administration to maximize the potential therapeutic effect and minimize toxicity.
METHODS
Materials
CytoTox 96™ cytotoxicity assay system and CellTiter 96™ AQ proliferation assay kits were purchased from Promega Corp. (Madison, WI). FBS and M199 were purchased from Gibco Laroratories (Grand Island, NY). Minocycline was purchased from Sigma Chemical Co. (St. Louis, MO).
Cell Culture
Human aortic SMC were obtained from Clonetics Corp. (San Diego, CA) and subcultured using smooth muscle growth medium (Clonetics) containing human EGF (10 ng/ml), human fibroblast growth factor (2ng/ml), dexamethasone (0.39 μg/ml), 5% FBS, gentamycin (50 μg/ml), and amphotericin-B (50 ng/ml) at 37°C in a humidified 95% air/5% CO2 atmosphere. The growth medium was changed every other day until confluence was reached. The cells used for experiments were from passages 5–10.
Western Blot Analysis
Western blot analysis was performed by a standard procedure. The monoclonal MMP-2 antibody, recognizing both the active and latent forms of the enzyme, was purchased from Calbiochem (San Diego, CA). Monoclonal anti-beta-actin was purchased from Sigma Chemical (St. Louis, MO). Cells were treated with or without minocycline and harvested 48 hours after treatment. 40 μg of total protein was loaded in each well. MMP-2 protein was detected using the ECL system from Amersham (Arlington Heights, IL) using an antibody dilution of 1:5000. Densitometry was performed using a Molecular Dynamics Personal Densitometer SI from Amersham Biosciences (Piscataway, NJ).
SMC Proliferation Assay
Human aortic SMC were grown to 60%–70% confluence in 12-well tissue culture plates (22.6 mm diameter; Costar Corp., Cambridge, MA). SMC were then washed three times with basal M199 and incubated with M199 supplemented with 0.2% bovine albumin for 48 hours as previously described. 15 Thereafter, cells were incubated with various concentrations of minocycline for 48–96 hours at 37°C in a humidified 95% air/5% CO2 atmosphere. SMC were trypsinized and cell numbers were immediately determined by triplicate counts with a Coulter counter (model Z1; Coulter Electronics, Beds, United Kingdom).
SMC Migration
SMC migration activity was assayed in a modified micro-Boyden chamber 16,17 using a polycarbonate filter of 8.0 μm (diameter) pore size (Costar Corp.) to divide the upper and lower well chambers. Cultured human aortic SMC were trypsinized and suspended at a concentration of 5 × 105 cells/ml in M199 supplemented with 0.2% bovine albumin. A volume of 1 mL of cell suspension was placed in the upper chamber, and 2 mL of the same medium containing vehicle in combination with varying concentrations of minocycline was loaded in the lower chamber of the apparatus. After 48 hours of incubation (37°C, 5% CO2 in air), the cells on the upper and lower sides of the filter were trypsinized and counted using a Coulter counter. Migration activity was determined by the ratio of cell number of triplicate counts in the upper and lower chambers of the apparatus.
Rat Balloon Injury Studies
Male Sprague-Dawley rats (Charles River Breeding Laboratories, Wilmington, MA), weighing 300–350 g, were used. The Columbia University Institutional Animal Care and Use Committee, Health Sciences Division approved all animal procedures. Each animal was anesthetized with an intraperitoneal injection of 100 mg/kg ketamine (Ketalar; Parke Davis, Morris Plains, NJ), and 5 mg/kg xylazine (Rompum; Mobay Corp., Shawnee, KS). The left carotid artery of each animal was isolated by a midline incision, suspended on ties, and stripped of adventitia. A 2F Fogarty catheter was introduced through the external carotid artery of each rat and advanced to the aortic arch; the balloon was inflated in the common carotid artery to produce moderate resistance to catheter movement and then gradually withdrawn to the entry point. The entire procedure was repeated three times for each animal. The wounds were then sutured closed, and the rats were returned to their cages.
Minocycline was administered to male Sprague-Dawley rats in one of three ways: orally by gavage, via intraperitoneal injection or locally applied by means of a pluronic gel. In the latter group, immediately after balloon injury, 200 μL of solution of 25% pluronic gel or 25% pluronic gel containing minocycline was applied to the exposed adventitial surface of the carotid artery at the site of balloon injury. The pluronic solutions were prepared as outlined by the manufacturer (BASF Wyandotte Corporation, Wyandotte, MI) and maintained at 4°C. Prechilled pipettes and tips were used to apply the gel solutions to the common carotid arteries. The treated area constituted about half of the carotid artery and represented the portion that lies within the neck. On contact with tissues at 37°C, pluronic solutions gel instantaneously generating a translucent layer that envelops the treated regions.
Fourteen days after balloon injury, animals were killed with ketamine and xylazine. The carotid arteries were removed, fixed in 4% formalin, and stained with hematoxylin and eosin for light microscopy in a standard manner. The quantitative measurement of rat intima, media and lumen area was done using image microscopy and image measurement software (Optimetric, Bioscan, Edmonds, WA). The borders of the external elastic lamina (EEL), internal elastic lamina (IEL) and lumen were traced freehand. The medial area (EEL area-IEL area), and intimal area (IEL area-lumen area) were determined by area subtraction.
Statistical Analysis
Data are presented as the mean ± SD of the independent experiments. Statistical significance was determined by one-way ANOVA and Fisher's PLSD test (Stat View 4.01; Brain Power, Inc., Calabasas, CA). For data of treated and untreated segments of carotid arteries, a paired t test was used. A P value of < 0.05 was considered statistically significant between the means.
RESULTS
Matrix Metalloproteinase-2 Expression
We found that human aortic smooth muscle cells abundantly express matrix metalloproteinase-2. Western blot analysis revealed that minocycline at doses of 1, 25 and 50 μg/ml blunted MMP-2 expression in a dose-dependent fashion (Fig. 1).
Western blot analysis demonstrates that human aortic smooth muscle cells abundantly express MMP-2 and that minocycline inhibits this expression in a dose-dependent fashion. The two bands represent the 72 kDa zymogen and 66 kDa active form of MMP-2. The production of MMP-2 protein as measured by densitometry for 0, 1, 25 and 50 μg/ml were 125,418; 97,197; 65,128; and 54,832 optical density units, respectively.
SMC Proliferation
We examined the effects of varying concentrations of minocycline on SMC proliferation over time. After 48 hours of incubation with minocycline at doses of 10 and 25 μg/ml, SMC proliferation was significantly inhibited (83.8 ± 8.8% of control values, P < 0.0001 and 70.0 ± 1.4% of control values, P < 0.0001, respectively). By 96 hours, minocycline at concentrations of 1 μg/ml (84.0 ± 13.1%, P = 0.022), 4 μg/ml (68.0 ± 7.8%, P < 0.0001), 10 μg/ml (53.0 ± 2.9%, P < 0.0001) and 25 μg/ml (49.0 ± 7.6%, P < 0.0001) showed a remarkable inhibitory effect on SMC proliferation (Fig. 2). A tetrazolium calorimetric proliferation assay was used to assess cytotoxicity. Minocycline, at concentrations as high as 100 μg/ml, had no noticeable cytotoxic effect on SMCs grown in SMC growth medium for 96 hours. Nor were there any prominent morphologic changes in minocycline treated cells.
Effects of increasing concentrations of minocycline on smooth muscle cell (SMC) proliferation. Cells were incubated with various concentrations of minocycline (0.2–25 μg/ml) for 48–96 hours at 37°C in a humidified 5% CO2 atomsphere. SMCs were trypsinized and cell numbers were immediately determined by triplicate counts with a Coulter counter. Incubating SMCs with increasing doses of minocycline progressively reduced their proliferation at 96 hours as shown here. Values are mean ± SD * P = 0.022; ** P < 0.0001 relative to control.
SMC Migration
The results of SMC migration studies using a modified micro-Boyden chamber are shown in Figure 3. Increasing concentrations of minocycline inhibited SMC migration in a dose-dependent fashion. Drug concentrations of 1 μg/ml had no significant effect (98.8 ± 8.1% of control values, p=NS) whereas 100 μg/ml of minocycline nearly eliminated SMC migration (14.8 ± 8.1% of control values, P < 0.0001).
Effects of increasing concentrations of minocycline on smooth muscle cell (SMC) migration. Migration activity was assessed in a modified micro-Boyden chamber. Cultured human aortic SMCs (5 × 105/ml) were added to the upper chamber and varying concentrations of minocycline (1–100 μg/ml) were added to vehicle and pipetted into the lower chamber. SMC migration was determined using a Coulter counter after a 48-hour incubation at 37°C in a humidified 5% CO2 atomsphere. Migration activity was determined by the ratio of cell number of triplicate counts in the upper and lower chambers of the apparatus. Values are mean ± SD * P = 0.004; ** P < 0.0001 relative to control.
Neointima Formation in Rat Carotid Arteries Following Balloon Injury
Rats (n = 7) were fed minocycline 125 mg/kg daily for 7 days prior to and 14 days following balloon injury. Compared with control rats (n = 6), those treated with oral minocycline had a non-significant reduction in intimal area (0.17 ± 0.06 versus 0.21 ± 0.05, P = 0.21), but no difference in the intima/media area ratio (1.07 ± 0.42 versus 1.08 ± 0.16, P = 0.99).
When delivered by intraperitoneal injection at high doses (250 mg/kg/d), minocycline was uniformly lethal. At intermediate doses (70 or 100 mg/kg/d), one-half of the minocycline treated rats died. Surviving rats (n = 7) received minocycline for 2 days prior to and 14 days following balloon injury, and had a significant reduction in intimal area (Fig. 4). These minocycline-treated animals had gross evidence of liver toxicity manifested by hepatomegaly and bile stasis.
Differing effects of oral versus intraperitoneal (IP) administration of minocycline on rat carotid artery neointimal area (A), medial area (B) and intima/media area ratio (C). Rats (n = 7) were fed minocycline 125 mg/kg daily for 7 days prior to and 14 days following balloon injury and had a 20% reduction in intimal area as compared with control (P = 0.21). Rats (n = 7) who received IP minocycline 70–100 mg/kg daily two days prior to injury and continuing 14 days post-injury had a 76% reduction in intimal area and 77% reduction in intima/media area ratio compared with control (n = 6). There was no significant change in medial area in the treatment groups versus control. Values are mean ± SD * P = 0.21; ** P < 0.0001; †P = 0.99; ††P < 0.0001 relative to control.
In an attempt to limit toxicity, minocycline was given in divided doses for a shorter a period of time. Rats (n = 8) received 35 mg/kg injections twice daily beginning one day before injury and continuing for five days post-procedure. These rats were killed four weeks after balloon injury. Compared with controls (n = 4) a significant reduction was seen in both intimal area (0.11 ± 0.02 versus 0.17 ± 0.01, P = 0.001) and intima/media area ratio (0.72 ± 0.14 versus 1.24 ± 0.13, P < 0.001) without any reduction in hepatotoxicity (Fig. 5). Another group of rats (n = 13) was treated with lower doses of minocycline (20–40 mg/kg/d) for 2 days prior and 14 days following injury. These lower doses produced no obvious hepatotoxicity, however, in spite of receiving the drug for a longer period of time, these animals had no reduction in intimal area or intima/media area ratio (Fig. 6).
Effects of divided dosing and shorter treatment duration on neointimal area (A), medial area (B) and intima/media area ratio (C). Rats (n = 8) received intraperitoneal injections of minocycline 35 mg/kg twice daily for one day prior and 5 days following balloon injury and were killed four weeks after injury. There was a 34% reduction in intimal area (A) and 42% reduction in intima/media area ratio (C). No significant change was observed in measured medial area. Values are mean ± SD * P < 0.01; ** P < 0.001 relative to control.
Effects of intraperitoneal injection of low dose minocycline. Rats received intraperitoneal injections of minocycline 20 mg/kg daily (n = 6) or 40 mg/kg daily (n = 7) for 2 days prior and 14 days following balloon injury. There was no significant reduction in either intimal area (A) or intima/media area ratio (C) versus control (n = 6). Values are mean ± SD.
To maximize the local concentration of drug and eliminate systemic toxicity, we applied minocycline locally to the adventitial surface of the injured segment of carotid artery via pluronic gel. This produced an unexpected increase in intimal proliferation (Fig. 7). The pH of the gel in the treated animals was 3.5, whereas control animals were treated with a pluronic gel with a physiologic pH.
Effects of locally administered minocycline on intimal area (A), medial area (B) and intima/media area ratio (C). Immediately after balloon dilatation, 200 μL of solution of 25% pluronic gel or 25% pluronic gel containing minocycline was applied to the exposed adventitial surface of the carotid artery at the site of injury. Compared with control rats (n = 5) treated with pluronic gel alone, those treated with minocycline (20 or 100 mg/ml, n = 3 and 5, respectively) had an unexpected increase in intimal growth. The pH of the gel was around 3.5 for the treated animals.
DISCUSSION
In the present study we demonstrate that minocycline has potent in vitro anti-proliferative and anti-migratory effects upon vascular smooth muscle cells. We also show that minocycline can limit neointimal hyperplasia following arterial injury in rats. Our findings suggest that by preventing negative arterial remodeling, minocycline could have a role in the treatment of restenosis following percuataneous coronary interventions. Limiting this therapeutic benefit, however, is significant systemic toxicity that is encountered when minocycline is administered in effective doses.
The MMPs are a complex family of enzymes that are capable of digesting all components of the extracellular matrix. 18,19 The rat carotid artery constitutively expresses MMP-2 (Gelatinase A), a 72-kDa gelatinase that degrades type IV collagen and elastin. 4 Following balloon injury, expression of MMP-2 is up-regulated along with expression of MMP-9 (Gelatinase B). MMP-9 mRNA levels and activity peak 6 hours after balloon injury and are still present after 7 days. 20 MMP-2 mRNA levels show a decrease in expression 24 hours after vascular injury, but increase in the 5- to 14-day period after injury. 21 Expression of both MMP-2 and MMP-9 is believed to facilitate SMC migration from the media to the intima. MMP-1, also referred to as interstitial collagenase, is not expressed by rat vascular smooth muscle cells, but is known to be produced by human vascular SMCs. 4,22 It has been suggested that this enzyme plays an important role in the migration of SMCs and the eventual formation of atherosclerotic plaques. 22
Our results demonstrate that minocycline inhibits MMP-2 in a dose dependent fashion. Likewise, we show that increasing concentrations of minocycline progressively abrogated SMC proliferation and migration. When administered to rats orally, however, high doses of minocycline (125 mg/kg/d) produced only a non-significant reduction in neointimal area. The drug was apparently not well tolerated since minocycline-treated rats experienced frequent vomiting and diarrhea, and, as such, may not have absorbed the total dose. Attempts to deliver a similar dose by intraperitoneal injection were limited by the fact that this dose was uniformly lethal to a cohort of rats. Intermediate doses of 70–100 mg/kg/d were as effective as the higher doses at reducing cross-sectional neointimal area, but were similarly lethal to one-half of the animals tested. Among the surviving rats, significant weight loss and hepatotoxicity was noted. This included on a gross level hepatomegaly and the presence of yellow tumors presumably composed of deposits of minocycline. Histologically, bile stasis was clearly identified.
Similar side effects can be seen in humans. Acute hepatitis with fulminant liver failure is a rare and potentially severe side effect that has been reported among patients taking minocycline for treatment of acne vulgaris. 23 Other less frequent, but documented toxicities include a Loffler-like syndrome with pulmonary infiltrates, wheezing, fever, and eosinophilia; exfoliative dermatitis; and a lupus-like syndrome. 23,24
Attempts to minimize these side effects in further animal studies proved unsuccessful or of no benefit. Administering intermediate doses of minocycline in divided daily doses for a shorter period of time achieved a similar level of neointimal inhibition as higher doses, however there was no reduction in hepatic side effects. No obvious hepatic toxicity was seen in animals treated with low dose minocycline (20–40 mg/kg/d). Unfortunately, low doses of minocycline produced no therapeutic benefit.
In the final set of experiments, we locally delivered minocycline to the sight of arterial injury believing that we could achieve a reduction in neointimal hyperplasia and avoid systemic toxicity. Our previous experience in applying phosphorothioate oligodeoxynucleotides to the adventitial surface of carotid arteries by means of a pluronic gel led us to believe that diffusion of drug into the media does indeed occur. 25,26 What we discovered in the current experiments was that our therapy had an opposite effect actually stimulating neointimal hyperplasia beyond that seen with pluronic gel alone.
A possible explanation for this discrepancy may lay in the fact that minocycline is a crystalline base. To get it into solution with the pluronic gel, hydrochloric acid was used in concentrations that resulted in the pH of the final solution being between 3.5 and 4.0. Such application of an acidic solution may have resulted in a sustained injury to the vessel wall in reaction to which SMCs proliferated. In our prior work, as in the control arm of these experiments, the pH of the pluronic solutions was neutral and no difference in neointimal area was observed between balloon injured arteries treated with saline and those treated with pluronic gel alone. Of course, direct toxicity from minocycline and not the HCl cannot be excluded as a possible explanation for these discrepant observations.
We have postulated that one mechanism by which minocycline prevents intimal growth following arterial injury is through the inhibition of matrix metalloproteinase expression. Other possible mechanisms, including the induction of apoptosis, were not examined in this study. Tolomeo et al. have shown that chemically modified tetracyclines, particularly CMT-8, can induce apoptosis in myeloid leukemia cells. 27 Whether minocycline can induce apoptosis in vascular smooth muscle cells remains unanswered. Another limitation of our study is that we measured MMP-2 protein levels by Western blotting but did not use zymography to directly measure metalloproteinase activity. Our results, however, complement those of Bendeck et al. who reported that doxycycline reduced the activity of both latent and active MMP-2 as well as MMP-9. 11
CONCLUSION
In conclusion, we have shown that minocycline is an inhibitor of matrix metalloproteinase-2 expression and displays potent anti-migratory and anti-proliferative effects upon vascular smooth muscle cells in vitro. Minocycline can reduce neointimal hyperplasia following balloon injury of the carotid artery in rats, but its efficacy is limited by its systemic toxicity. If minocycline could be delivered in such a way as to minimize this toxicity, perhaps by incorporating it onto a drug-eluting stent, minocycline may offer a pharmacologic treatment of restenosis.
ACKNOWLEDGMENTS
This work was supported by the Sol and Margaret Berger Foundation, Clifton, NJ (Dr. Rabbani).
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Keywords:
Minocycline; metalloproteinases; vascular smooth muscle; arterial injury; restenosis
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