This Article Abstract Full Text (PDF) All Versions of this Article: 33/12/2858    most recent 01.STR.0000038098.04291.F6v1 Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Permissions Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Axisa, B. Articles by Napoli, C. Search for Related Content PubMed PubMed Citation Articles by Axisa, B. Articles by Napoli, C. Pubmed/NCBI databases Compound via MeSH Substance via MeSH Medline Plus Health Information Carotid Artery Disease Hazardous Substances DB DOXYCYCLINE Related Collections Cell biology/structural biology Carotid Stenosis Other Stroke Treatment - Medical Carotid endarterectomy Mechanism of atherosclerosis/growth factors

(Stroke. 2002;33:2858.)
© 2002 American Heart Association, Inc.

Original Contributions

Prospective, Randomized, Double-Blind Trial Investigating the Effect of Doxycycline on Matrix Metalloproteinase Expression Within Atherosclerotic Carotid Plaques

Benedict Axisa, FRCS; Ian M. Loftus, MD, FRCS; A. Ross Naylor, MD, FRCS; Steven Goodall, BSc; Louise Jones, PhD, FRCPath; Peter R.F. Bell, MD, FRCS Matthew M. Thompson, MD, FRCS

From the Departments of Surgery and Pathology (L.J.), University of Leicester, Leicester, UK.

Correspondence to Professor M.M. Thompson, Professor of Vascular Surgery, Department of Vascular Surgery, 4th Floor St James Wing, St George’s Hospital, Blackshaw Rd, London SW17 0QT, UK. E-mail mattT11{at}aol.com

	Abstract

Top Abstract Introduction Methods Results Discussion References MMP Inhibition and the... References

 
Background and Purpose— Elevated levels of matrix metalloproteinases (MMPs), particularly MMP-1 and MMP-9, have been implicated in plaque rupture. It has been suggested that inhibition of MMPs may stabilize vulnerable atherosclerotic plaques and improve clinical outcome. The aim of the study was to investigate the ability of doxycycline, a nonspecific MMP inhibitor, to reduce MMP concentration in carotid atheroma.

Methods— The study design was a prospective, double-blind randomized trial. One hundred patients requiring carotid endarterectomy were randomized to receive 200 mg/d doxycycline or placebo for 2 to 8 weeks before surgery. During endarterectomy, carotid plaques were retrieved. The concentrations of MMPs and doxycycline were determined in the atherosclerotic tissue by enzyme-linked immunosorbent assay and high-performance liquid chromatography, respectively. Clinical events were recorded, as was the rate of preoperative embolization (transcranial Doppler).

Results— Analysis of endarterectomized specimens demonstrated a mean doxycycline concentration of 6.0 µg/g wet weight in treated patients. Administration of doxycycline significantly reduced the concentration of MMP-1 in carotid plaques from a mean of 14.8 to 10.3 ng/100g wet weight (P=0.038). This difference was due to decreased MMP-1 transcript (P<0.001). There was no difference in any other MMP (MMP-2, -3, or -9) or tissue inhibitor of matrix metalloproteinases–1 or –2.

Conclusions— Doxycycline penetrated atherosclerotic plaques with acceptable tissue levels. This resulted in a reduction in MMP-1 concentration because of decreased expression.

Key Words: atherosclerosis • drug therapy • metalloproteinases

	Introduction

Top Abstract Introduction Methods Results Discussion References MMP Inhibition and the... References

 
Acute disruption of an atherosclerotic plaque is a prelude to the onset of clinical ischemic events, including stroke and myocardial infarction. Matrix metalloproteinases (MMPs) are a family of zinc-dependent endopeptidases that act as the physiological regulators of the extracellular matrix. A review of the available evidence suggests that these enzymes play a pivotal role in the evolution and development of atherosclerotic plaques.¹ In recent years, the role of MMPs in acute plaque disruption has received increasing attention because proteolytic disruption of the fibrous cap overlying an atheromatous plaque may be a final common pathway in many clinical events.

Previous studies have identified the presence of several MMPs within atherosclerotic plaques, of which MMP-1 and MMP-9 appear to be the most important. Galis and colleagues² described a localized increase in the expression of MMP-1, MMP-3, and MMP-9 in the vulnerable shoulder and cap of the plaque, in tandem with an increase in overall proteolytic activity. Several investigators have now described increased expression of MMP-1 in vulnerable regions of atherosclerotic lesions^3,4 and suggested that this enzyme may be partly responsible for plaque rupture.

See Editorial Comment, page 2864

More recent work has clearly demonstrated an increase in the level and expression of MMPs in unstable carotid plaques.^5–7 Experimental and clinical evidence has demonstrated that vulnerable atherosclerotic plaques may be characterized by an imbalance between MMPs and their inhibitors and that artificially enhancing antiproteolytic mechanisms may stabilize these plaques.^8,9

Pharmacotherapy to prevent plaque disruption is an attractive option. Hypothetically, inhibition of MMPs within the atherosclerotic plaque may prevent disruption of the shoulders and fibrous cap of the plaque and thus inhibit plaque rupture. Doxycycline is a member of the tetracycline antibiotic family that, apart from its antibacterial action, is also a potent nonselective MMP inhibitor. Several investigations have demonstrated that doxycycline may inhibit MMP expression and activity in a number of vascular disorders.^10–12 The aim of the present study was to determine the effect of doxycycline on MMP concentration within carotid plaques.

	Methods

Top Abstract Introduction Methods Results Discussion References MMP Inhibition and the... References

 
Protocol
The study was designed as a double-blind, randomized, controlled trial to evaluate the effects of doxycycline on MMP production within carotid plaques of patients undergoing carotid endarterectomy. The Leicestershire Research Ethics Committee approved the study.

Patients were recruited from a "single visit" carotid clinic, where patients with carotid disease underwent a vascular consultation and subsequent duplex evaluation. All patients who were considered to require an elective carotid endarterectomy were eligible for the study. Patients with hypersensitivity to tetracycline or systemic lupus erythematosus or who required urgent surgery were excluded from the study. Patients were selected for carotid endarterectomy on the basis of a duplex scan that demonstrated a carotid stenosis >70% (peak systolic velocity >230 cm/s). Indications for carotid endarterectomy in the 100 randomized patients were stroke in 34, transient ischemic attacks in 27, amaurosis fugax in 26, and central retinal artery occlusion in 3; 7 patients were asymptomatic.

Randomization was performed after written informed consent was obtained. Patients were randomized to doxycycline 100 mg twice daily or placebo. After randomization, patients were given a provisional date for surgery 2 to 8 weeks from their initial consultation. Some patients required further medical evaluation or specialized investigations that were organized at this stage. Patients remained on the same medication until surgery. The day before surgery, patients were admitted, and a plasma sample was collected for determination of doxycycline concentration. A carotid duplex scan was repeated at this time, and patients were monitored for preoperative embolization (transcranial Doppler). Carotid endarterectomy was performed in a standardized manner, and the carotid plaque was retained for estimation of MMP concentration, doxycycline penetration, histological analyses, and MMP expression.

The primary outcome measure for the study was the concentration of MMPs and tissue inhibitor of MMPs (TIMPs) in the endarterectomized carotid plaque (with particular reference to MMP-1 and -9). Overall, 19 patients were withdrawn from the trial because they did not undergo carotid endarterectomy within the specified time period and thus plaque MMP concentrations could not be determined.

Secondary outcomes were preoperative cerebral embolization, histological plaque analysis, and doxycycline concentration in serum and plaque. Clinical events were also recorded and reported on an intention-to-treat basis (preoperative transient ischemic attack, stroke, and death) but were not considered an end point of the trial.

The sample size for the study was calculated to detect a difference in MMP-1 concentration between the groups with an effect size of 0.75. At a significance level of 0.05 and a power of 0.9, 70 patients would be needed to refute the null hypothesis. An anticipated dropout rate of 30% necessitated that 100 patients be recruited.

Assignment and Masking
The unit of randomization was the patient. Patients were assigned to placebo or doxycycline 100 mg twice daily by a randomly generated numerical sequence. Placebo and doxycycline were packaged in identical capsules and supplied in receptacles containing a 6-week supply. These were renewed with identical drug if necessary. The investigators were blinded as to treatment allocation until all samples had been analyzed.

Sample Collection
A blood sample was obtained from patients the day before surgery, and plasma was collected for determination of serum doxycycline concentration. Plasma samples were stored at -85°C until use.

Carotid endarterectomy was performed with a standard technique and with minimal manipulation of the specimen. After endarterectomy, the carotid plaque was divided longitudinally through the point of maximum stenosis. An attempt was made to divide the plaque equally with reference to areas of ulceration, thrombosis, or hemorrhage. One half was snap-frozen for later MMP extraction. The other half was divided longitudinally again, with the specimens prepared for histological analysis and doxycycline quantification.

Doxycycline Concentration
Carotid plaques were snap-frozen in liquid nitrogen and stored at -80°C. Doxycycline concentrations in plasma samples and carotid plaques were quantified with a high-performance liquid chromatography–based methodology designed for this study.¹³

MMP Extraction and Quantification
MMP and TIMP levels were quantified by gelatin zymography and enzyme-linked immunosorbent assay (ELISA) as previously been described.⁵ Briefly, plaques were stored at -70°C until MMPs were extracted. During the extraction process, samples were dialyzed to remove molecules with a molecular weight <12 000 Da (doxycycline, 512 Da). The active and latent forms of MMP-2 and MMP-9 were analyzed with substrate gel zymography. The total concentrations of MMP-1, -2, -3, and -9, TIMP-1, and TIMP-2 were quantified with the Biotrack assay system (Amersham, UK).⁵

MMP-1 Expression
Reverse-transcription polymerase chain reaction (RT-PCR) was performed to confirm and compare the expression of MMP-1 within the plaque. In selected samples, total RNA was extracted from the tissue with Trizol reagent (Life Technologies), and RT-PCR was performed using sequences detailed below, according to established methodology.⁵ MMP-1 expression was analyzed in only 9 plaques because most plaques had insufficient tissue to allow for MMP and doxycycline analysis, histological examination, and mRNA extraction. Molecular analysis was possible only in very bulky plaques. Samples were therefore selected for expression studies on the basis of weight and were not consecutive.

Primers were designed according to GeneBank data. The 3-antisense primer was complementary to bases 1496 through 1518 in the 3-untranslated region (5-GGACTCACACCATGTGTTTTCC-3), and the 5-sense primer was defined by bases 1114 through 1135 of the hemopexinlike domain (5-GGGCTGTTCAGG-GACAGAATGT-3) of MMP-1. Amplification of cDNA was performed with Taq DNA polymerase with 40 cycles of denaturation, annealing, and extension at 94°C, 59°C, and 72°C, respectively, for 1 minute. Negative controls included samples without the primers and polymerase. MMP-1 expression was compared with ß-actin by densitometric analysis of ethidium bromide–stained gels.

Histological Analysis
Immediately after endarterectomy, samples were placed in fresh 4% paraformaldehyde. After decalcification, samples were paraffin embedded and sectioned at 4-mm intervals. Sections were stained with hematoxylin and eosin, elastic van Gieson, and monoclonal antibodies for MMP-1 and -9 (R&D Systems).¹⁴ An experienced histopathologist (L.J.) who was blinded to patient identity and randomization then evaluated sections. Plaques were characterized with reference to the presence or absence of cap foam cells, cap thinning, intraplaque hemorrhage, plaque necrosis, ulceration, and calcification.

Preoperative Embolization
Patients were monitored with transcranial Doppler insonation of their ipsilateral middle cerebral artery for 30 minutes the day before surgery. The number of emboli was recorded and characterized as a marker of plaque instability.^5,6

Statistical Analysis
Distribution of the data sets was analyzed with the Kolmogorov-Smirnov test. Because data were normally distributed, results were expressed as mean values with SD. Differences in continuous variables were analyzed with the unpaired t test. Discrete variables were compared with Fisher’s exact test.

	Results

Top Abstract Introduction Methods Results Discussion References MMP Inhibition and the... References

 
Participant Flow and Follow-Up
Patient demographics at randomization are illustrated in Table 1. Overall, 100 patients were randomized. The trial profile is summarized in Figure 1. There were 19 patients who were withdrawn from the trial before carotid endarterectomy. Most were patients who required specialists’ opinions or investigation of comorbid medical conditions. These patients had their surgical procedure deferred for >6 months after randomization and were therefore withdrawn from the primary study. Two patients had a discrepancy between their duplex stenosis performed at randomization and the day before surgery. Repeat examinations determined that the degree of carotid stenosis was <70% and operative intervention was considered inappropriate (Table 2).

View this table: [in this window] [in a new window]   TABLE 1. Patient Characterization at Randomization

View larger version (15K): [in this window] [in a new window]   Figure 1. Trial flow diagram. Detailed reasons for patient withdrawal are given in Table 2.

View this table: [in this window] [in a new window]   TABLE 2. Characteristics of Patients Who Were Withdrawn From the Trial

Interestingly, 3 patients in the placebo group developed carotid occlusion between randomization and carotid surgery compared with 1 patient in the doxycycline-treated group.

Doxycycline Concentration
No patients in the placebo group demonstrated evidence of doxycycline within plasma or carotid plaque. Patients receiving doxycycline had a mean±SD plasma doxycycline concentration of 3.5±1.3 µg/mL 3 hours after ingestion.

In the cohort of patients receiving active drug, the mean±SD concentration of doxycycline within the excised endarterectomy tissue was 6.0±2.6 µg/g wet weight.

MMP Concentration
The concentrations of MMP-1, -2, -3, and -9 and TIMP-1 and -2 were quantified in the carotid plaques with ELISA. The results are illustrated in Table 3. The main finding of the study was a significant reduction in MMP-1 within the cohort treated with doxycycline. Interestingly, the ratio of TIMP-1 to MMP-1 was increased in the treated group, which suggested a change in enzyme balance toward inhibition of collagenolysis. This difference, however, failed to reach statistical significance. Gelatin zymography demonstrated no significant differences between the groups with respect to MMP-2 or -9 in pro or active form (data not shown).

View this table: [in this window] [in a new window]   TABLE 3. Concentrations in the Carotid Plaques of Patients Receiving Doxycycline 200 mg/d or Placebo

In the patients randomized to doxycycline, there was no relationship between the duration of therapy and MMP-1 levels (Pearson’s correlation coefficient, -0.003; P=0.98) (Figure 2).

View larger version (16K): [in this window] [in a new window]   Figure 2. MMP-1 concentration in patients treated with doxycycline as a function of duration of therapy. Best-fit linear regression line with 95% CIs is demonstrated. There was no relationship between concentration and time (Pearson’s correlation coefficient, -0.14, P=0.41).

MMP-1 Expression
MMP-1 expression was analyzed in a subset of 9 plaques (Figure 3). Expression was measured with semiquantitative RT-PCR and was compared with ß-actin. RT-PCR demonstrated a significant reduction in the ratios of MMP-1 to ß-actin in the plaques derived from patients receiving doxycycline compared with control subjects (P=0.0002; 95% CI, 0.26 to 0.13).

View larger version (54K): [in this window] [in a new window]   Figure 3. Ethidium bromide gel comparing MMP-1 expression in patients receiving either doxycycline or placebo. ß-Actin controls were used to standardize samples. Patients receiving doxycycline demonstrated a significant reduction in MMP-1 expression in their carotid plaques (P<0.001).

MMP-1 Immunocytochemistry
Immunocytochemistry of untreated plaques with monoclonal antibodies to MMP-1 demonstrated intense staining in areas of macrophage infiltration in the shoulders of the carotid plaques. MMP-1 was predominantly identified in the cytoplasm of macrophages. Plaques treated with doxycycline demonstrated a similar pattern of staining as control tissues, with MMP-1 demonstrated predominantly in the shoulder regions. The intensity of staining was reduced compared with control tissue. No MMP-1 was identified in smooth muscle cells (Figure 4).

View larger version (57K): [in this window] [in a new window]   Figure 4 . Photomicrograph demonstrating a transverse section through a carotid plaque from the control group and doxycycline-treated patients. a, Negative control without primary antibody to MMP-1. Magnification x200. b, Carotid plaque from the control group. Section stained with monoclonal antibody to MMP-1. Dense macrophage infiltration can be visualized at the shoulder region of the plaque that is heavily stained for MMP-1. Magnification x200. c and d, Carotid plaques from the placebo (c) and doxycycline-treated (d) groups. Section was stained with monoclonal antibody to MMP-1. Intensity of staining in the doxycycline-treated patients is reduced compared with that of control subjects. Magnification x200.

Histological Analysis
The histological characterization of the carotid plaques is illustrated in Table 4. There were no significant differences in plaque morphology between the 2 groups.

View this table: [in this window] [in a new window]   TABLE 4. Morphological Characteristics of Carotid Plaques

Preoperative Embolization
In this study, 1 patient receiving doxycycline (2.2%) and 4 patients on placebo (10.8%) had preoperative evidence of cerebral embolization (P=0.17, Fisher’s exact test).

	Discussion

Top Abstract Introduction Methods Results Discussion References MMP Inhibition and the... References

 
The present study has demonstrated that the nonspecific metalloproteinase inhibitor doxycycline was concentrated in atherosclerotic tissue and caused significant inhibition of MMP-1 in carotid plaques. The tissue concentrations of the other potentially important MMPs or their physiological inhibitors were not affected. Doxycycline has been advocated as a possible therapeutic agent in several vascular disorders.^11,15 Doxycycline has direct actions on MMPs through reversible inhibition of the enzyme and reduction of gene transcription.¹⁶ In addition to these effects, doxycycline decreases the levels of interleukin (IL-1, IL-1ß, IL-6)¹⁷ and nitric oxide synthase,¹⁸ which may act to reduce proteolytic activity, as well as having effects on arterial diameter and vessel wall fragmentation.¹⁹

To be effective in reducing MMP activity in vascular lesions, doxycycline must be efficiently delivered to the tissues. Previous studies have demonstrated that tetracyclines localize to atherosclerotic plaques. Franklin et al²⁰ revealed that tetracycline rapidly penetrated the aortic wall and achieved a median concentration of 2.9 µg/g tissue wet weight, which was sufficient for biological activity. In the present study, 200 mg/d of orally administered doxycycline resulted in a mean concentration of 6.1 µg doxycycline/g wet weight of carotid plaque and a serum level of 3.5 µg/mL. Recent observations from Prall et al²¹have suggested that doxycycline concentrations >2 µg/mL are likely to be effective in reducing MMP concentrations in clinical studies.

This reduction in MMP-1 observed in the present study has confirmed previous reports that have demonstrated inhibition of MMP-1 by relatively low concentrations of doxycycline in other tissues.²² The inhibition observed in human carotid plaques may have significant clinical consequences. Recent experimental studies have suggested that MMP-1 may play a significant role in atherogenesis and plaque rupture. Chen et al²³ demonstrated that losartan reduced lipid deposition within a rabbit model and that this effect may be linked to a reduction in MMP-1 expression. Similarly, other groups have observed that some HMG CoA reductase inhibitors alter smooth muscle accumulation and collagen deposition in a hyperlipidemic model and have postulated that MMP-1 may play a pivotal role in this process.^24,25

The function that MMP-1 plays in human plaque rupture has not been completely defined, although evidence suggests a pivotal role. Elevated levels of MMP-1 have been demonstrated in unstable atheromatous carotid plaques,⁴ and the enzyme has been localized to activated white cells.²⁶ Interestingly, oxidized low-density lipoprotein is known to stimulate MMP-1 expression in human endothelial cells.²⁷ As well as its direct proteolytic capacity, MMP-1 plays an integral role in the cascade of MMP production and activation that occur in the tissues. MMP-1 has the ability to cleave type I, II, and III collagen and plays a central role in the activation of MMP-2 and -9.²⁸ The pleiotropic effects of MMP-1 make this enzyme an attractive target for pharmacotherapy to reduce proteolysis in unstable carotid plaques.

The present study failed to detect any differences in the concentrations of other MMPs. Previous studies have demonstrated a reduction in MMP-9 levels in vascular tissue. Curci et al investigated MMP-9 production and expression in patients with abdominal aortic aneurysms.²⁹ A significant reduction of MMP-9 was demonstrated in the doxycycline treated patients by immunoblotting and RT-PCR. Previous studies from our group demonstrated that highly symptomatic carotid plaques were characterized by elevated MMP-9 levels.⁵ Unpublished observations on cultured human monocytes suggested that serum levels of doxycycline >2 µg/mL doxycycline reduced MMP-9 secretion by 20%. In the present investigation, the MMP-9 concentration in carotid plaques from the doxycycline-treated group was reduced by a similar amount but failed to reach statistical significance.

One criticism of the present study was that MMP protein concentration, rather than MMP activity, was used as the primary end point. Ideally, it would have been preferable to quantify the extent of MMP-dependent elastolytic and collagenolytic action through the use of activity assays. However, these assays have been technically difficult and unreliable in the past²⁹ and have not been successfully implemented in our laboratory. Despite these limitations, doxycycline reduced the concentration of MMP-1, and RT-PCR suggested that this action was mediated by reduced gene transcription.

In the long term, inhibition of MMP-1 in high-risk carotid or coronary plaques might be expected to reduce the incidence of plaque rupture or progression. The present study was powered to investigate MMP concentrations in atherosclerotic tissue, so a reduction in clinical events was not expected. However, several clinical findings warrant comment, although they were not considered end points of the clinical trial. Overall, 4 patients in the trial developed a carotid occlusion between randomization and surgery: 3 in the placebo group and 1 in the doxycycline cohort. This was an unexpectedly high number (the usual rate in our center is <1.5%) but did represent a tendency toward a lower occlusion rate in patients treated with doxycycline. Clearly, a larger cohort would be required to investigate the clinical effects of MMP inhibition on clinical outcome, but these initial data show some promise.

There are a number of limitations in the trial. The primary concern was the number of patients who were withdrawn before carotid endarterectomy. Some had a clinical event that served as a definitive end point, but most were withdrawn because they required specialist medical treatment before endarterectomy. In retrospect, it may have been more appropriate not to randomize these patients until their medical comorbidity had been evaluated and treated. Patients also received doxycycline for variable times before surgery, but there was no relationship between MMP levels and duration of treatment.

The trial did not address the role of the antimicrobial effect of doxycycline on carotid atheroma. Numerous studies have demonstrated an association between carotid atherosclerosis and Chlamydia pneumoniae.³⁰ It has been suggested that eradication of chlamydia may improve clinical events in patients with coronary atherosclerosis, although definitive results are awaited.³¹ Doxycycline has activity against chlamydia in atherosclerotic plaques,³² and experimental evidence has suggested that antichlamydial therapy may reduce plaque volume.³³

Despite these limitations, the present study suggests that doxycycline administration to patients with unstable atherosclerotic plaques was associated with inhibition of MMP-1. Further studies are required to determine the role of MMP inhibition in modulating adverse clinical events in patients with widespread atheroma.

	Acknowledgments

 
We wish to acknowledge the support of the Stroke Association and the Royal College of Surgeons of England.

Received April 12, 2002; revision received June 25, 2002; accepted July 2, 2002.

	References

Top Abstract Introduction Methods Results Discussion References MMP Inhibition and the... References

 

1. Loftus IM, Naylor AR, Bell PR, Thompson MM. Matrix metalloproteinases and atherosclerotic plaque instability. Br J Surg. 2002; 89: 680–694.[CrossRef][Medline] [Order article via Infotrieve]
2. Galis ZS, Sukhova GK, Lark MW, Libby P. Increased expression of matrix metalloproteinases and matrix degrading activity in vulnerable regions of human atherosclerotic plaques. J Clin Invest. 1994; 94: 2493–2503.[Medline] [Order article via Infotrieve]
3. Nikkari ST, O’Brien KD, Ferguson M, Hatsukami T, Welgus HG, Alpers CE, et al. Interstitial collagenase (MMP-1) expression in human carotid atherosclerosis. Circulation. 1995; 92: 1393–1398.[Abstract/Free Full Text]
4. Sukhova GK, Schonbeck U, Rabkin E, Schoen FJ, Poole AR, Billinghurst RC, et al. Evidence for increased collagenolysis by interstitial collagenases-1 and -3 in vulnerable human atheromatous plaques. Circulation. 1999; 99: 2503–2509.[Abstract/Free Full Text]
5. Loftus IM, Naylor AR, Goodall S, Crowther M, Jones L, Bell PR, et al. Increased matrix metalloproteinase-9 activity in unstable carotid plaques: a potential role in acute plaque disruption. Stroke. 2000; 31: 40–47.[Abstract/Free Full Text]
6. Loftus IM, Naylor AR, Bell PR, Thompson MM. Plasma MMP-9: a marker of carotid plaque instability. Eur J Vasc Endovasc Surg. 2001; 21: 17–21.[CrossRef][Medline] [Order article via Infotrieve]
7. D’Armiento FP, Bianchi A, de Nigris F, Capuzzi DM, D’Armiento MR, Crimi G, et al. Age-related effects on atherogenesis and scavenger enzymes of intracranial and extracranial arteries in men without classic risk factors for atherosclerosis. Stroke. 2001; 32: 2472–2480.[Abstract/Free Full Text]
8. Rouis M, Adamy C, Duverger N, Lesnik P, Horellou P, Moreau M, et al. Adenovirus-mediated overexpression of tissue inhibitor of metalloproteinase-1 reduces atherosclerotic lesions in apolipoprotein E–deficient mice. Circulation. 1999; 100: 533–540.[Abstract/Free Full Text]
9. Knox JB, Sukhova GK, Whittemore AD, Libby P. Evidence for altered balance between matrix metalloproteinases and their inhibitors in human aortic diseases. Circulation. 1997; 95: 205–212.[Abstract/Free Full Text]
10. Boyle JR, McDermott E, Crowther M, Wills AD, Bell PR, Thompson MM. Doxycycline inhibits elastin degradation and reduces metalloproteinase activity in a model of aneurysmal disease. J Vasc Surg. 1998; 27: 354–361.[CrossRef][Medline] [Order article via Infotrieve]
11. Thompson RW, Baxter BT. MMP inhibition in abdominal aortic aneurysms: rationale for a prospective randomized clinical trial. Ann N Y Acad Sci. 1999; 878: 159–178.[Abstract/Free Full Text]
12. Treharne GD, Boyle JR, Goodall S, Loftus IM, Bell PR, Thompson MM. Marimastat inhibits elastin degradation and matrix metalloproteinase 2 activity in a model of aneurysm disease. Br J Surg. 1999; 86: 1053–1058.[CrossRef][Medline] [Order article via Infotrieve]
13. Axisa B, Naylor AR, Bell PR, Thompson MM. Simple and reliable method of doxycycline determination in human plasma and biological tissues. J Chromatogr B Biomed Sci Appl. 2000; 744: 359–365.[CrossRef][Medline] [Order article via Infotrieve]
14. Crowther M, Goodall S, Jones JL, Bell PR, Thompson MM. Localization of matrix metalloproteinase 2 within the aneurysmal and normal aortic wall. Br J Surg. 2000; 87: 1391–1400.[CrossRef][Medline] [Order article via Infotrieve]
15. Porter KE, Thompson MM, Loftus IM, McDermott E, Jones L, Crowther M, et al. Production and inhibition of the gelatinolytic matrix metalloproteinases in a human model of vein graft stenosis. Eur J Vasc Endovasc Surg. 1999; 17: 404–412.[CrossRef][Medline] [Order article via Infotrieve]
16. Hanemaaijer R, Visser H, Koolwijk P, Sorsa T, Salo T, Golub LM, et al. Inhibition of MMP synthesis by doxycycline and chemically modified tetracyclines (CMTs) in human endothelial cells. Adv Dent Res. 1998; 12: 114–118.[Abstract/Free Full Text]
17. Solomon A, Rosenblatt M, Li D, Monroy D, Ji Z, Lokeshwar BL, et al. Doxycycline inhibition of interleukin-1 in the corneal epithelium. Invest Ophthalmol Vis Sci. 2000; 41: 2544–2557.[Abstract/Free Full Text]
18. Amin AR, Patel RN, Thakker GD, Lowenstein CJ, Attur MG, Abramson SB. Post-transcriptional regulation of inducible nitric oxide synthase mRNA in murine macrophages by doxycycline and chemically modified tetracyclines. FEBS Lett. 1997; 410: 259–264.[CrossRef][Medline] [Order article via Infotrieve]
19. Tronc F, Mallat Z, Lehoux S, Wassef M, Esposito B, Tedgui A. Role of matrix metalloproteinases in blood flow-induced arterial enlargement: interaction with NO. Arterioscler Thromb Vasc Biol. 2000; 20: E120–E126.
20. Franklin IJ, Harley SL, Greenhalgh RM, Powell JT. Uptake of tetracycline by aortic aneurysm wall and its effect on inflammation and proteolysis. Br J Surg. 1999; 86: 771–775.[CrossRef][Medline] [Order article via Infotrieve]
21. Prall AK, Longo GM, Mayhan WG, Waltke EA, Fleckten B, Thompson RW, et al. Doxycycline in patients with abdominal aortic aneurysms and in mice: comparison of serum levels and effect on aneurysm growth in mice. J Vasc Surg. 2002; 35: 923–929.[CrossRef][Medline] [Order article via Infotrieve]
22. Shlopov BV, Stuart JM, Gumanovskaya ML, Hasty KA. Regulation of cartilage collagenase by doxycycline. J Rheumatol. 2001; 28: 835–842.[Medline] [Order article via Infotrieve]
23. Chen H, Li D, Mehta JL. Modulation of matrix metalloproteinase-1, its tissue inhibitor, and nuclear factor-B by losartan in hypercholesterolemic rabbits. J Cardiovasc Pharmacol. 2002; 39: 332–339.[CrossRef][Medline] [Order article via Infotrieve]
24. Fukumoto Y, Libby P, Rabkin E, Hill CC, Enomoto M, Hirouchi Y, et al. Statins alter smooth muscle cell accumulation and collagen content in established atheroma of Watanabe heritable hyperlipidemic rabbits. Circulation. 2001; 103: 993–999.[Abstract/Free Full Text]
25. Ikeda U, Shimpo M, Ohki R, Inaba H, Takahashi M, Yamamoto K, et al. Fluvastatin inhibits matrix metalloproteinase-1 expression in human vascular endothelial cells. Hypertension. 2000; 36: 325–329.[Abstract/Free Full Text]
26. Schonbeck U, Mach F, Sukhova GK, Murphy C, Bonnefoy JY, Fabunmi RP, et al. Regulation of matrix metalloproteinase expression in human vascular smooth muscle cells by T lymphocytes: a role for CD40 signaling in plaque rupture? Circ Res. 1997; 81: 448–454.[Abstract/Free Full Text]
27. Huang Y, Mironova M, Lopes-Virella MF. Oxidized LDL stimulates matrix metalloproteinase-1 expression in human vascular endothelial cells. Arterioscler Thromb Vasc Biol. 1999; 19: 2640–2647.[Abstract/Free Full Text]
28. Nagase H. Activation mechanisms of matrix metalloproteinases. Biol Chem. 1997; 378: 151–160.[Medline] [Order article via Infotrieve]
29. Curci JA, Mao D, Bohner DG, Allen BT, Rubin BG, Reilly JM, et al. Preoperative treatment with doxycycline reduces aortic wall expression and activation of matrix metalloproteinases in patients with abdominal aortic aneurysms. J Vasc Surg. 2000; 31: 325–342.[CrossRef][Medline] [Order article via Infotrieve]
30. Mosorin M, Surcel HM, Laurila A, Lehtinen M, Karttunen R, Juvonen J, et al. Detection of Chlamydia pneumoniae–reactive T lymphocytes in human atherosclerotic plaques of carotid artery. Arterioscler Thromb Vasc Biol. 2000; 20: 1061–1067.[Abstract/Free Full Text]
31. Dunne MW. Rationale and design of a secondary prevention trial of antibiotic use in patients after myocardial infarction: the WIZARD (Weekly Intervention with Zithromax [Azithromycin] for Atherosclerosis and Its Related Disorders) trial. J Infect Dis. 2000; 181 (suppl 3): S572–S578.[CrossRef][Medline] [Order article via Infotrieve]
32. Gieffers J, Solbach W, Maass M. In vitro susceptibilities of Chlamydia pneumoniae strains recovered from atherosclerotic coronary arteries. Antimicrob Agents Chemother. 1998; 42: 2762–2764.[Abstract/Free Full Text]
33. Fong IW. Antibiotics effects in a rabbit model of Chlamydia pneumoniae-induced atherosclerosis. J Infect Dis. 2000; 181 (suppl 3): S514–518.[CrossRef][Medline] [Order article via Infotrieve]

Editorial Comment

Claudio Napoli, MD, PhD, Guest Editor

	MMP Inhibition and the Development of Cerebrovascular Atherosclerosis: The Road Ahead

Top Abstract Introduction Methods Results Discussion References MMP Inhibition and the... References

 
It has been 40 years since the first member the matrix metalloproteinase (MMP) family of specialized enzymes was described.¹ Structural, molecular, and biochemical approaches have subsequently contributed to piecing together the puzzle of how MMPs work and how they contribute to the pathophysiology of various disease processes.¹ Because degradation of the extracellular matrix scaffold enables reshaping of tissue, participation of MMPs has become the object of intense recent scientific interest in relation to pathogenic events involved in vascular remodeling.

MMPs are hypothesized to play a pivotal role in the pathogenesis of several central nervous system disorders (reviewed in Reference ²). Indeed, increased levels of expression of MMP-9 (gelatinase B) and MMP-2 (gelatinase A) have been observed in Alzheimer’s disease, stroke, multiple sclerosis, and amyotrophic lateral sclerosis.² Aberrant activity of MMPs was also observed during human brain focal ischemia,³ cardioembolic stroke,⁴ and atherogenesis of intracranial arteries.⁵ There is now convincing evidence of increased MMP activity during acute plaque disruption.^1,2,6 The hypothesis that MMPs contribute to weakening of atherosclerotic plaques is especially attractive for the potential development of therapeutic interventions aimed at preventing plaque disruption and its clinical sequelae. Accordingly, intervention experimental studies demonstrated that the MMP inhibitor KB-R7785 has a protective efficacy against focal cerebral ischemia in mice.⁷ Moreover, MMP inhibition (with the inhibitor batimastat, BB-94) attenuates mechanisms involved in experimental thrombolysis-induced hemorrhage.⁸ Furthermore, an elegant study demonstrates that there were no differences in systemic hemodynamic parameters and gross cerebrovascular anatomy between wild-type mice and mutant mice with a targeted knockout of the MMP-9 gene.⁹ After induction of focal ischemia, similar reductions in cerebral blood flow were obtained. In the MMP-9 knockout mice, ischemic lesion volumes were significantly reduced compared with wild-type littermates in male and female mice.⁹ In normal wild-type mice, the MMP inhibitor batimastat also significantly reduced ischemic lesion size.⁹ However, batimastat had no detectable protective effect when administered to MMP-9 knockout mice subjected to cerebral ischemia. These data further corroborate the concept that MMP-9 plays a deleterious role in the development of brain injury after brain ischemia. In another study, wild-type and MMP-2 knockout mice were subjected to permanent and transient occlusions of the middle cerebral artery.¹⁰ MMP-9 levels were increased in all brains after ischemia. MMP-2 levels did not show a significant increase in wild-type mice and were not detectable in knockout mice.¹⁰ Equivalent ischemic reductions in perfusion in wild-type and knockout mice were observed. These data indicate that MMP-2 does not contribute to acute tissue damage in this model of focal cerebral ischemia. Therefore, further studies are needed to investigate specific pathophysiological mechanisms involving MMP (and its isoforms) in the development of brain ischemic damage. In this light, the use of the broad MMP inhibitor KB-R7785 or batimastat (or newly developed drugs) should be developed preclinically, promoting the design of more focused studies in humans.

On the other hand, doxycycline has well-described anticollagenase properties and is used in the treatment of 1-antitrypsin deficiency panniculitis.¹¹ Moreover, this drug was proposed to suppress the growth of abdominal aortic aneurysms¹² and was used in the treatment of recalcitrant recurrent corneal erosions, which is associated with increased MMP activity.¹³ These studies provided the "soil" for a new study appearing in this issue of Stroke.¹⁴ This study, done in patients elected for carotid endarterectomy, demonstrates that 200 mg/d doxycycline penetrated atherosclerotic carotid plaques with acceptable tissue levels.¹⁴ This intervention study also induced a reduction in MMP-1 tissue expression in carotid specimens.¹⁴ MMP-2 and MMP-9 activities were not affected by the drug, and there was no clinical difference in patient outcome.¹⁴ No causal relationship can be postulated in a correlation study, but future studies should evaluate MMP activities rather that MMP protein levels, and a large cohort of patients is needed for evaluating differences in outcome. Because arterial MMP levels increase with age,⁵ patients should be also age stratified, and doxycycline must be administered following reproducible protocols in terms of doses and days of treatment before to investigate clinical end points. Indeed, the natural history of atherosclerosis is different in intracranial arteries compared with extracranial arteries.^5,15,16 Until adult age, increased MMP activities are associated with increased endogenous resistance to oxidative stress in intracranial but not extracranial arteries.⁵ This balance is lost in elderly patients.⁵ Thus, the beneficial effects of doxycycline seen in this issue of Stroke¹⁴ can decrease with increasing age.

Department of Medicine

University of California, San Diego, and

Department of Medicine

University of Naples

Naples, Italy

	References

Top Abstract Introduction Methods Results Discussion References MMP Inhibition and the... References

 

1. Brinckerhoff CE, Matrisian LM. Matrix metalloproteinases: a tail of a frog that became a prince. Nat Rev Mol Cell Biol. 2002; 3: 207–214.[CrossRef][Medline] [Order article via Infotrieve]
2. Yong VW, Power C, Forsyth P, Edwards DR. Metalloproteinases in biology and pathology of the nervous system. Nat Rev Neurosci. 2001; 2: 502–511.[CrossRef][Medline] [Order article via Infotrieve]
3. Clark AW, Krekoski CA, Bou SS, Chapman KR, Edwards DR. Increased gelatinase A (MMP-2) and gelatinase B (MMP-9) activities in human brain after focal ischemia. Neurosci Lett. 1997; 238: 53–56.[CrossRef][Medline] [Order article via Infotrieve]
4. Montaner J, Alvarez-Sabin J, Molina C, Angles A, Abilleira S, Arenillas J, Gonzalez MA, Monasterio J. Matrix metalloproteinase expression after human cardioembolic stroke: temporal profile and relation to neurological impairment. Stroke. 2001; 32: 1759–1766.[Abstract/Free Full Text]
5. D’Armiento FP, Bianchi A, de Nigris F, Capuzzi DM, D’Armiento MR, Abete P, Crimi G, Palinski W, Condorelli M, Napoli C. Age-related effects on atherogenesis and scavenger enzymes of intracranial and extracranial arteries in men without classical risk factors for atherosclerosis. Stroke. 2001; 32: 2472–2480.[Abstract/Free Full Text]
6. Loftus IM, Naylor AR, Bell PR, Thompson MM. Matrix metalloproteinases and atherosclerotic plaque instability. Br J Surg. 2002; 89: 680–694.[CrossRef][Medline] [Order article via Infotrieve]
7. Jiang X, Namura S, Nagata I. Matrix metalloproteinase inhibitor KB-R7785 attenuates brain damage resulting from permanent focal cerebral ischemia in mice. Neurosci Lett. 2001; 305: 41–44.[CrossRef][Medline] [Order article via Infotrieve]
8. Lapchak PA, Chapman DF, Zivin JA. Metalloproteinase inhibition reduces thrombolytic (tissue plasminogen activator)–induced hemorrhage after thromboembolic stroke. Stroke. 2000; 31: 3034–3040.[Abstract/Free Full Text]
9. Asahi M, Asahi K, Jung JC, del Zoppo GJ, Fini ME, Lo EH. Role for matrix metalloproteinase 9 after focal cerebral ischemia: effects of gene knockout and enzyme inhibition with BB-94. J Cereb Blood Flow Metab. 2000; 20: 1681–1689.[CrossRef][Medline] [Order article via Infotrieve]
10. Asahi M, Sumii T, Fini ME, Itohara S, Lo EH. Matrix metalloproteinase 2 gene knockout has no effect on acute brain injury after focal ischemia. Neuroreport. 2001; 12: 3003–3037.[CrossRef][Medline] [Order article via Infotrieve]
11. Humbert P, Faivre B, Gibey R, Agache P. Use of anti-collagenase properties of doxycycline in treatment of alpha 1-antitrypsin deficiency panniculitis. Acta Derm Venereol. 1991; 71: 189–194.[Medline] [Order article via Infotrieve]
12. Thompson RW, Liao S, Curci JA. Therapeutic potential of tetracycline derivatives to suppress the growth of abdominal aortic aneurysms. Adv Dent Res. 1998; 12: 159–165.[Abstract/Free Full Text]
13. Dursun D, Kim MC, Solomon A, Pflugfelder SC. Treatment of recalcitrant recurrent corneal erosions with inhibitors of matrix metalloproteinase-9, doxycycline and corticosteroids. Am J Ophthalmol. 2001; 132: 8–13.[CrossRef][Medline] [Order article via Infotrieve]
14. Axisa B, Loftus IM, Naylor AR, Goodall S, Jones L, Bell PRF, Thompson MM. A prospective, randomized, double-blind trial investigating the effect of doxycycline on matrix metalloproteinase expression within atherosclerotic carotic plaques. Stroke. 2002; 33: 2858–2865.[Abstract/Free Full Text]
15. Napoli C, Paternò R, Faraci FM, Taguchi H, Postiglione A, Heistad DD. Mildly oxidized low-density lipoprotein impairs responses of carotid but not basilar artery in rabbits. Stroke. 1997; 28: 2266–2272.[Abstract/Free Full Text]
16. Napoli C, Witztum JL, de Nigris F, Palumbo G, D’Armiento FP, Palinski W. Intracranial arteries of human fetuses are more resistant to hypercholesterolemia-induced fatty streak formation than extracranial arteries. Circulation. 1999; 99: 2003–2010.[Abstract/Free Full Text]

This article has been cited by other articles:

				J. L. Johnson, R. Fritsche-Danielson, M. Behrendt, A. Westin-Eriksson, H. Wennbo, M. Herslof, M. Elebring, S. J. George, W. L. McPheat, and C. L. Jackson Effect of broad-spectrum matrix metalloproteinase inhibition on atherosclerotic plaque stability Cardiovasc Res, August 1, 2006; 71(3): 586 - 595. [Abstract] [Full Text] [PDF]

				G. Stoll and M. Bendszus Inflammation and Atherosclerosis: Novel Insights Into Plaque Formation and Destabilization Stroke, July 1, 2006; 37(7): 1923 - 1932. [Abstract] [Full Text] [PDF]

				C. Franco, B. Ho, D. Mulholland, G. Hou, M. Islam, K. Donaldson, and M. P. Bendeck Doxycycline Alters Vascular Smooth Muscle Cell Adhesion, Migration, and Reorganization of Fibrillar Collagen Matrices Am. J. Pathol., May 1, 2006; 168(5): 1697 - 1709. [Abstract] [Full Text] [PDF]

				J. P.G. Sluijter, D. P.V. de Kleijn, and G. Pasterkamp Vascular remodeling and protease inhibition-bench to bedside Cardiovasc Res, February 15, 2006; 69(3): 595 - 603. [Abstract] [Full Text] [PDF]

				C. M. Dollery and P. Libby Atherosclerosis and proteinase activation Cardiovasc Res, February 15, 2006; 69(3): 625 - 635. [Abstract] [Full Text] [PDF]

				B. Galobardes, G. D. Smith, M. Jeffreys, S. Kinra, and P. McCarron Acne in Adolescence and Cause-specific Mortality: Lower Coronary Heart Disease but Higher Prostate Cancer Mortality: The Glasgow Alumni Cohort Study Am. J. Epidemiol., June 15, 2005; 161(12): 1094 - 1101. [Abstract] [Full Text] [PDF]

				K.J. Molloy, M.M. Thompson, J.L. Jones, E.C. Schwalbe, P.R.F. Bell, A.R. Naylor, and I.M. Loftus Unstable Carotid Plaques Exhibit Raised Matrix Metalloproteinase-8 Activity Circulation, July 20, 2004; 110(3): 337 - 343. [Abstract] [Full Text] [PDF]

				A. R. Morgan, K. Rerkasem, P. J. Gallagher, B. Zhang, G. E. Morris, P. C. Calder, R. F. Grimble, P. Eriksson, W. L. McPheat, C. P. Shearman, et al. Differences in Matrix Metalloproteinase-1 and Matrix Metalloproteinase-12 Transcript Levels Among Carotid Atherosclerotic Plaques With Different Histopathological Characteristics Stroke, June 1, 2004; 35(6): 1310 - 1315. [Abstract] [Full Text] [PDF]