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From the Department of Laboratory Medicine and Pathobiology and theDepartment of Medicine,*
University of Toronto, Toronto; and theTerrence Donnelly Cardiovascular ResearchLaboratories,
St. Michael’s Hospital,Toronto, Ontario, Canada
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Abstract |
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 Top Abstract Materials and MethodsResultsDiscussionReferences |
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In vitro the tetracyclines influence various cellular
functions. They inhibit the proliferation of endothelial and
osteosarcoma cells;13,17,18
induce apoptosis of
macrophages, osteosarcoma, and breast carcinoma
cells;19,20
inhibit tumor cell
migration;13,14
and reduce matrix synthesis by
chondrocytes.21,22
Tetracyclines also decrease the
expression of and destabilize the mRNA for inducible nitric oxide
synthase in macrophages and mesangial cells,23-25
decrease the production of tumor necrosis factor-
,2
and
scavenge reactive oxygen species.26
Most of this work has
been done in cell culture and little is known about the diverse effects
of tetracycline on these cellular processes in vivo.
Currently doxycycline is under investigation in the treatment of atherosclerosis, based on the hypothesis that microorganisms such as Chlamydia pneumoniae are important pathogenic factors.27 However MMP production, cell proliferation, cell migration, apoptosis, and matrix synthesis have all been implicated in the development of intimal thickening during atherosclerosis and restenosis, and given the evidence cited above it is likely that doxycycline will impact many of these processes, independent of its antibiotic properties. Recent studies have shown that doxycycline inhibits outward vessel remodeling in response to increased blood flow,28 and improves function in isolated rat hearts injured by ischemia-reperfusion.29 Intriguingly, a retrospective study of patients presenting with acute myocardial infarction showed decreased incidence of infarction in patients correlated with usage of tetracycline or quinolone antibiotics during the preceding 3 years.30 This study was far from conclusive, because the time course of antibiotic treatment, and molecular and morphological endpoints were not considered.
Despite the effects on many critical cellular processes and promising results in cardiovascular studies, little is known about the potential for doxycycline in modulating intimal thickening. In the current study we examine the effects of doxycycline treatment after balloon catheter denudation of the rat carotid artery, a model in which intimal thickening occurs in four well-defined sequential phases. The first phase of response, medial smooth muscle cell (SMC) replication, peaks at 2 days after injury and declines thereafter.31 The second phase, migration of SMCs into the intima, can be measured at 4 days after injury as the first cells arrive in the intima, and before the cells have time to replicate in this layer.32 We have also measured MMP activity at 4 days, because both MMP-2 and MMP-9 activities are elevated at this time.32 The third phase, intimal SMC replication peaks at 7 days.31 The fourth phase, matrix protein synthesis and accumulation, occurs between 1 week and 4 weeks after injury. The mRNAs for tropoelastin and procollagen are increased at 1 week,33-35 and at 4 weeks maximum neointimal thickening is achieved.31 Our objectives in this study were two-fold, first to characterize the pluripotent effects of doxycycline on cells of the vessel wall using an in vivo model, and second to elucidate the effects of doxycycline treatment on the phases of neointimal lesion development.
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Materials and Methods |
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TopAbstractMaterials and MethodsResultsDiscussionReferences |
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Male Sprague-Dawley rats (3 to 4 months old) (Charles River, Constant, QB) were used in all experiments. Animal experiments were performed in accordance with the guidelines of the Canada Council on Animal Care. Rats were anesthetized by intraperitoneal injection of xylazine (4.6 mg/kg body weight, Rompum; Bayer Inc., Etobicoke, ON) and ketamine (70 mg/kg body weight, Ketaset; Ayerst Veterinarian Laboratories, Guelph, ON), and balloon catheter injury of the left common carotid artery was performed as previously described.32 Doxycycline was administered in the drinking water at a dose of 30 mg/kg/day starting 24 hours before surgery. This dose was used previously to inhibit MMPs in studies of abdominal aortic aneurysm formation in the rat.5 Control rats drank water alone. Rats were sacrificed at various time points after injury, chosen as follows based on previous studies elucidating the kinetics of the injury response. SMC proliferation was measured in the media (2, 4, 7, and 14 days) and intima (7 and 14 days).31 Migration of cells from media to the intima and MMP activity were measured at 4 days.32 Development of the neointima was assessed by measuring intimal area and the ratio of intimal:medial area at 14 and 28 days. Collagen and elastin synthesis were measured at 7 days, and collagen and elastin content of the vessels was measured at 7 and 21 days.31
To label cells entering S phase in the 2, 4, 7, and 14 day groups, a 50-mg pellet of 5-bromo-2'-deoxyuridine (BrdU; Boehringer Mannheim Corp., Montreal, PQ) was implanted subcutaneously at the nape of the neck 24 hours before sacrifice. Rats were killed by an intravenous injection of T-61 (Hoechst Roussel Veterinarian, Regina, SA). Rats were infused with Lactated Ringer’s (Baxter, Toronto, ON) via a catheter placed in the abdominal aorta, followed by perfusion with 0.1 mol/L phosphate-buffered 4% paraformaldehyde at a pressure of 110 mmHg. Vessels were excised and immersed in 4% paraformaldehyde for 1 hour, and then transferred to Ringer’s solution. Samples for histological and morphometric analysis were taken 1 cm and 2 cm downstream of the origin of the common carotid, these were embedded in paraffin blocks and sectioned.
Morphometry
SMC replication rates in the media were measured 2, 4, 7, and 14
days after injury, and replication rates in the intima were measured at
7 and 14 days after injury by immunostaining carotid cross-sections for
BrdU and determining the percentage of BrdU-labeled cells present as
previously described.36
Images of the cross-sections were
obtained using a Nikon E600 microscope (Nikon, Mississauga, ON),
digitized using a digital camera (model C4742-95-12NRB, Hamamatsu,
Inc., Nikon) and analyzed using a computer-assisted morphometric
analysis system (Simple C Imaging Systems, Mars, PA). Measurements of
intimal and medial areas (14 and 28 days after injury) were made as
follows. Lumen area was determined by tracing around the inside edge of
the vessel and quantitating the area inside. Intimal area was measured
as the area encompassed by the internal elastic lamina minus the lumen
area. Medial area was measured as the area encompassed by the external
elastic lamina minus the area encompassed by the internal elastic
lamina (including lumen area). At the 4-day time point, a 1-cm length
was excised from the middle of the common carotid artery and used for
assay of SMC migration into the intima as previously
described.32
Briefly, intimal cells on the surface of
fixed common carotid artery segments were immunostained with an
antibody against histone H1. The number of intimal cell nuclei per
square mm of surface area was counted by light microscopy. The
migration assay takes advantage of the fact that the first SMCs appear
in the intima 3 to 4 days after injury; it takes 
24 hours for the
cells to progress through the cell cycle, so the cells are counted
before going through a round of replication.
Zymograms
Six rats (n = 3 per group for control and doxycycline) were used to measure MMP-2 and MMP-9 activity by gelatin zymography as previously described.32 Extracts of individual carotid arteries were subject to electrophoresis on sodium dodecyl sulfate-polyacrylamide gel electrophoresis gels that contained 0.1% gelatin as a substrate for MMP digestion. After electrophoresis, the gels were incubated 16 hours, then stained with Coomassie Blue, and MMP activity was evident as cleared bands of substrate lysis. The MMPs were identified by their molecular weights and inhibition by ethylenediaminetetraacetic acid or phenanthroline. Activity was quantitated by scanning densitometric analysis using a Bio-Rad gel 700 documentation system and Molecular Analyst software (Bio-Rad Laboratories, Hercules, CA).
Collagen and Elastin Synthesis and Content
Collagen and elastin synthesis was measured at 7 days, and total collagen and elastin content was measured at 7 and 21 days after arterial injury. Briefly, the entire left common carotid artery was excised between its origin at the aorta and the carotid bifurcation, then carefully stripped of adventitia, weighed, cut open, and incubated in Dulbecco’s modified Eagle medium containing 1% fetal calf serum (Invitrogen Canada, Burlington, ON), [14C]-proline (0.5 µCi/ml medium, Amersham Pharmacia Biotech, Baie d’Urfé, PQ), and ascorbic acid (50 µg/ml) for 6 hours. Collagen and elastin content and synthesis were determined according to Strauss and colleagues.37 Results of collagen and elastin synthesis were expressed as cpm [14C]-proline per mg tissue wet weight. Collagen content was measured as hydroxyproline content, and total collagen was calculated assuming that collagen contains 12.77% hydroxyproline by weight.38 Total collagen and elastin content were expressed as mg per carotid segment.
Statistical Analysis
Values are expressed as mean ± SEM. Group means were compared by the two-tailed Student’s t-test for independent samples, or by analysis of variance followed by Fisher’s paired least significant difference (PLSD) to determine differences between groups.
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Results |
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TopAbstractMaterials and MethodsResultsDiscussionReferences |
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SMC Replication and Migration
Medial SMC replication was measured as the percentage of
BrdU-labeled cells in the media after balloon injury. In control rats,
medial replication peaked at 2 days after injury, then progressively
declined between 4 and 14 days (Figure 1A)
. There were no significant
differences in medial replication between control and
doxycycline-treated rats at any time point examined. Intimal SMC
replication in control rats was measured at 7 and 14 days after injury,
was highest at 7 days, then declined by 14 days (Figure 1B)
.
Doxycycline treatment significantly reduced intimal SMC replication;
the replication of SMCs in the intima at 7 days after injury was
reduced from 28.3 ± 2.5% in controls to 17.0 ± 2.8% in
doxycycline-treated rats. Intimal SMC replication was also decreased at
14 days in the doxycycline-treated rats compared to control rats, but
the difference was not statistically significant.
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. The number of intimal SMCs was 212.6 ±
27.5 cells/mm2
of intimal surface area in control
rats compared to 48.3 ± 28.5 cells/mm2
in
doxycycline-treated rats. In the doxycycline-treated rats there was a
slight increase in SMC number in the media; 379 ± 24 cells
compared to 367 ± 19 cells in controls (P
= 0.7). MMP Activity
Previous work from our lab and others has shown that MMPs play an
important role in regulating SMC migration, therefore gelatin zymograms
were used to assess MMP activity in arterial extracts from control and
doxycycline-treated rats. Three prominent bands with molecular weights
of 88, 70, and 62 kd were noted in extracts from the injured carotids
of control rats (Figure 2A)
. We have
previously shown that these lytic bands correspond to active MMP-9,
latent MMP-2, and active MMP-2, respectively.32
Activity
of all three bands was decreased in arterial extracts from the
doxycycline-treated rats (Figure 2A)
. Densitometric analysis of the
zymograms revealed that after doxycycline treatment, activity was
reduced to 65%, 42%, and 7% of control levels for active
MMP-9, latent MMP-2, and active MMP-2, respectively (Figure 2B)
.
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Collagen and elastin are the most abundant matrix proteins in the
vessel wall and increased synthesis and accumulation of these molecules
contributes to the intimal thickening that follows arterial injury. At
7 days after carotid artery injury there were no significant
differences in collagen or elastin synthesis between control and
doxycycline-treated rats (Figure 3)
.
There were no significant differences in collagen or elastin content
between control and doxycycline-treated rats at 7 days after injury
(Figure 4, A and B)
. There was no
difference in collagen content between the two groups at 21 days
(Figure 4A)
. However, there was a decrease in elastin content in the
doxycycline-treated rats at 21 days (Figure 4B)
, although this was not
statistically significant. We compared the accumulation of matrix
proteins between 7 and 21 days, and found that there was a
significant increase in collagen content in both control and
doxycycline-treated rats throughout this time period (Figure 4A)
. By
contrast, there was a significant increase in elastin content in the
control carotids, but not in the doxycycline-treated carotids between 7
and 21 days (Figure 4B)
. These data suggest that elastin accumulation
was impaired after doxycycline treatment. As a result, the relative
proportions of collagen and elastin in the vessel wall were changed
after doxycycline treatment. The ratio of collagen:elastin in control
vessels at 21 days after injury was 1.0 ± 0.2, whereas the ratio
in doxycycline-treated carotids was 1.3 ± 0.1
(P = 0.4).
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The ratio of intima:media area was significantly decreased at 28
days after injury in the doxycycline-treated rats compared to controls
(Figure 5A)
. Intimal area at 14 and 28
days after injury was decreased by doxycycline treatment, however these
differences were not statistically significant (Figure 5B)
. There were
no significant differences in medial area between the treatment groups
at either time point (Figure 5C)
.
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. We also calculated
the density of SMC nuclei per unit of cross-sectional area as an index
of matrix deposition relative to cell number, and found that SMC
density did not differ between the control and doxycycline-treated rats
(Table 1)
.
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Discussion |
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TopAbstractMaterials and MethodsResultsDiscussionReferences |
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Medial SMC replication was not affected by doxycycline treatment. However, doxycycline treatment did attenuate SMC migration from the media to the intima. Furthermore, MMP-2 and MMP-9 activities were reduced in the injured carotid arteries of rats treated with doxycycline. This effect on cell migration was consistent with data we obtained using a different MMP inhibitor, the peptide hydroxamic acid GM 6001.32 We infer therefore that the decrease in migration after doxycycline treatment was secondary to the MMP inhibition. Our finding of decreased MMP activity was in agreement with previous studies showing decreased MMP activity after doxycycline treatment in a rat model of abdominal aortic aneurysm,4,5 and in tissue samples taken from patients treated with doxycycline before aneurysm repair.6
The mechanisms by which doxycycline inhibits the MMPs are not completely understood. Doxycycline binds directly to Zn2+ or Ca2+ associated with the enzyme, blocking the active site or inducing conformational changes that render the proenzyme susceptible to fragmentation during activation.2,39 In our experiments, it is likely that doxycycline dissociated from the MMP enzyme during gel electrophoresis, therefore a reduction in activity on the zymogram gel could indicate that less enzyme was actually present in the arterial extract. This strongly suggests that the mechanisms leading to lowered MMP activity may be related to inhibition of the transcription of MMP mRNAs,40-44 or degradation of the pro-MMP zymogen during extracellular activation.39 We saw decreases in the amount of the active forms of MMP-2 and MMP-9, therefore it is also possible that doxycycline prevented the in vivo activation of pro-MMP zymogens by reactive oxygen species,26 or by the MT1-MMP.45
We found that the third phase of intimal thickening, intimal SMC replication, was reduced after doxycycline treatment. We do not know why doxycycline selectively inhibited the proliferation of intimal and not medial SMCs, however there are important phenotypic differences between these cell types.46 Interestingly, in tissue culture we found that doxycycline inhibited the growth of SMCs derived from the neointima more effectively than SMCs derived from the media (unpublished data), suggesting that there were stable phenotypic differences in doxycycline responsiveness between the SMCs in these two layers of the vessel wall. Another possible explanation for the differential effect on growth is that intimal SMCs are not firmly attached to the matrix because a new immature matrix is being deposited and remodeled around the cells. Reductions in matrix attachment leave cells more susceptible to cell death by apoptosis, and there is in vitro evidence that doxycycline may induce apoptosis.19,20
In our previous work, the MMP inhibitor GM 6001 did not reduce cell proliferation in the vessel wall after injury, and in fact high rates of intimal SMC replication were prolonged from 7 to 10 days after injury in the GM 6001-treated rats compared to controls.47 Similar results were reported by Zempo and colleagues48 using another MMP inhibitor BB-94 (batimastat). Thus the effects of doxycycline seemed to differ from these MMP inhibitors, suggesting that the cell growth inhibition by doxycycline was independent of the anti-MMP activity. In contrast to the in vivo results, other investigators have reported that GM6001 and BB-94 inhibit the proliferation of SMCs in tissue culture.49,50 Tissue culture studies aimed at distinguishing whether the effects of tetracyclines on cell growth are dependent on anti-MMP activity are contradictory. Gilbertson-Beadling and colleagues18 showed that doxycycline inhibited endothelial cell sprouting in an angiogenesis assay whereas GM 6001 did not. Another study comparing several chemically modified tetracyclines either with or without anti-MMP activity showed that the ability of these compounds to inhibit the proliferation of endothelial cells was linked to the anti-MMP activity.17 It seems that the mechanism of the doxycycline effect on cell growth many be different in vivo versus in vitro, and further studies to elucidate the mechanisms of growth control in vivo are needed.
We studied the fourth phase, matrix protein synthesis and accumulation in the injured arterial wall, and found that doxycycline did not inhibit matrix synthesis rates measured at 1 week after injury. However doxycycline did inhibit the accumulation of elastin, but not collagen, in the injured vessel between 1 and 3 weeks. MMPs and serine elastases are both up-regulated after injury; doxycycline inhibits MMP but not serine elastase activity. Therefore, it seems that doxycycline selectively spared the degradation of collagen but not elastin after injury, resulting in an increase in the proportion of collagen to elastin in the artery. This is important because it indicates that doxycycline may have a selective collagen-sparing effect, and therefore it may be a useful treatment to prevent plaque rupture.
The intima:media area ratio was reduced in the doxycycline-treated rats, however there was only a small reduction in intimal size in the doxycycline-treated rats. The most likely explanation for this catch-up effect is that SMC migration was prolonged in the doxycycline-treated rats longer than controls, with SMCs from the media eventually replenishing the intima throughout a longer time course. Migration can only be directly measured at the 4-day time point before cells undergo the first round of replication in the intima, so we cannot measure the full time course of migration. Decreased cell turnover could also result in intimal cell accumulation, and we cannot rule out the possibility that doxycycline enhances the survival of SMCs in the injured vessel wall. However, we think this unlikely because the literature suggests that doxycycline is proapoptotic.19,20
Taken together, our data shows that doxycycline inhibits several phases of the intimal thickening response, and alters the matrix composition of the neointima. One limitation of the rat carotid injury model, as reflected in this study and our previous work,47 is that there is a catch-up phenomenon, which resulted in little change in intimal area after doxycycline treatment. However, our findings of decreased intimal:medial ratio, and the protection against collagen degradation suggested some beneficial long-term effects of doxycycline. This should be pursued in larger animal models of restenosis that do not demonstrate this catch-up phenomenon, and are suitable for the study of plaque rupture.
Finally, it must be remembered that the nonantibiotic effects of doxycycline on arterial remodeling, as shown in this study, may complicate the mechanistic interpretation of studies in which doxycycline is used for the treatment of vascular diseases based on its antibiotic activity. Thus the MMP inhibiting effects of tetracyclines need to be taken into account in the design of future investigations.
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Acknowledgements |
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Footnotes |
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Supported by the Heart and Stroke Foundation of Ontario (grant NA4800 to M. B.) and a Premier’s Research Excellence Award (to M. B. and S. F.). M. B. was a Research Scholar of the Heart and Stroke Foundation of Ontario.
Accepted for publication November 14, 2001.
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