Restenosis occurs in 30-50% of cases after percutaneous coronary angioplasty (PTCA), and no pharmacologic intervention has conclusively been shown to reduce the restenosis rate (1,2). Lumen compromise in atherosclerotic and restenotic arteries was previously thought to be exclusively dependent on vascular smooth-muscle cell (VSMC) proliferation and migration, but more recently, importance has been attributed to the role of constrictive vascular remodeling (vessel shrinkage) (3,4). The mechanisms underlying remodeling are unknown, but it has been suggested that inflammatory reactions in response to vessel injury (e.g., cytokine-stimulated disturbances in VSMC synthesis and degradation of extracellular matrix) can contribute to the remodeling process (3-7).
Pentoxifylline (PTX) is a xanthine derivative used in the treatment of peripheral vascular disease (8,9), and this agent is known for its favorable effects in experimental models of inflammatory vascular injury (10-12). By acting as a nonselective phosphodiesterase inhibitor that increases intracellular levels of cyclic nucleotides [cyclic adenosine monophosphate (cAMP) and cyclic guanosine monophosphate (cGMP)], PTX has been shown to suppress a variety of cellular processes implicated in vascular lesion formation [i.e., platelet aggregation (13), VSMC proliferation (14,15), apoptosis (programmed cell death) (16), activation of NF-κB transcription factors (15), tumor necrosis factor-α (TNFα) synthesis (17,18), and fibroblast collagen production (19-21)]. We therefore used the well-characterized and reproducible rat model to examine the effects of PTX on the arterial response to balloon injury, and we also assessed the influence of PTX on VSMC migration and collagen production in vitro.
MATERIALS AND METHODS
Vascular injury and PTX treatment
Endothelial denudation of the left common carotid artery in male Sprague-Dawley rats (average weight, 300 g) was performed with a Fogarty 2F balloon catheter, as previously described (22). Rats were anesthetized with intraperitoneal sodium barbital (50 mg/kg), and the left external and distal common carotid arteries were exposed by a midline incision. The balloon catheter was advanced to the aortic arch through an external carotid artery arteriotomy and pulled back 3 times with the balloon sufficiently distended with saline to generate slight resistance. After removal of the balloon catheter, the external carotid artery was ligated. Starting 3 days before balloon injury, animals were randomly assigned to treatment with intraperitoneal PTX (75 mg/kg/day; PTX group) or intraperitoneal saline (control group). This dose of PTX has previously been shown to abolish cyclosporine-induced vascular toxicity in rats (23). The femoral artery blood pressure was determined before PTX administration and at 14 days, and rats were killed by cervical dislocation under sodium barbital anesthesia at 24 h or 14 days after injury. The investigation conformed with the Guide for the Care and Use of Laboratory Animals published by the U.S. National Institutes of Health (NIH publication No. 85-23, revised 1996).
After killing on postoperative day 14, the rats were perfusion-fixed under constant pressure (100 mm Hg) with 4% (wt/vol) buffered formaldehyde. The common carotid arteries were harvested and cut into four segments, which were embedded in paraffin, and 3- to 5-μm sections were stained with van Gieson Orcein. The luminal area (area circumscribed by the intima border), neointimal area (area between the lumen and the internal elastic lamina), medial area (area between the internal and the external elastic lamina), total vessel area (EEL; area circumscribed by the external elastic lamina), and intimal-to-medial ratio (I/M ratio; neointimal area divided by medial area) were calculated by computerized digital planimetry by using a videomicroscope with dedicated image-analyzing software (Leitz Texture analyzing system). The measurements from the four sections of each vessel were averaged for statistical analysis.
Proliferating cell nuclear antigen (PCNA) is an essential co-factor for DNA polymerase δ, and in the rat model, the presence of PCNA in VSMCs is considered to be a specific indicator of cell replication (24). Expression of PCNA was evaluated by immunohistochemical analysis of arterial sections at 24 h or 14 days after injury with use of a monoclonal anti-PCNA antibody (clone PC10, Dako) at a dilution of 1:100, essentially as previously described (24). Negative controls were incubated in the absence of the primary antibody. The number of PCNA-positive nuclei and the total number of nuclei within each vessel layer were counted, and proliferation was expressed as the PCNA labeling index [100% × (number of PCNA-positive nuclei/total number of nuclei)]. In addition, the neointimal cell density (nuclei/mm2) in vessels removed 14 days after balloon injury was determined by dividing the number of intimal nuclei with the intimal cross-sectional area.
Apoptosis was detected in situ by using terminal deoxynucleotidyl transferase (TdT)-mediated dUTP-biotin nick-end labeling (TUNEL) of fragmented DNA, as previously described (25). One section of each artery was processed as a positive control by pretreatment with DNAase I (100 U/ml; Worthington Biochemical Corporation), and a negative control was incubated in the absence of TdT. Sections of mouse small intestine also served as positive controls (25). Cells were counted under a light microscope, and the cells with a clear nuclear labeling were defined as TUNEL positive. The number of TUNEL-positive nuclei and the total number of nuclei were counted, and apoptosis was expressed as the TUNEL index [100% × (number of TUNEL-positive nuclei/total number of nuclei)].
VSMC collagen production and migration in vitro
We used a solid-phase enzyme-linked immunosorbent assay (ELISA)to assess the amount of soluble (nonfibrillar) collagen types I, IV, and VI released into the growth medium by cultured human VSMCs (26). Human VSMCs were grown for 24 h in Dulbecco's modified Eagle's medium (DMEM; Gibco) containing 0-1,000 μM PTX. Cell viability was assessed by the trypan blue exclusion method. Purified human collagen type I, IV, or VI (Sigma) was prepared in 0.5 M CH3COOH. Serial dilutions of the collagen standards or incubation medium were made in DMEM with 0.5% human serum albumin, and 100 μl was added to the wells of 96-well polystyrene microtiter plates (Nunc, Roskilde, Denmark). The fluid phase was evaporated by incubation in a hood for 24 h at room temperature. Wells were washed 3 times with buffer (25 mM Na2PO4, 150 mM NaCl, 0.05% Tween 20, and 0.1% bovine serum albumin, pH 7.2), and incubated with biotin-labeled polyclonal goat antibodies against human collagen type I, IV, or VI (dilution, 1:4,000; Southern Biotechnology Associates Inc., Birmingham, AL, U.S.A.). Streptavidin-labeled horseradish peroxidase was added (dilution, 1:6,000), and a color reaction was developed with a tyramine-based ELISA amplification system (Renaissance; New England Nuclear Life Science Products, Boston, MA, U.S.A.). The plates were read in a spectrophotometer, and linear standard curves were obtained from wells with the dilutions of purified collagen. All experiments were performed in triplicate, and the absorbance in wells from VSMCs grown in the absence of PTX was determined as the control value, with which the absorbance in wells from VSMCs grown in the presence of PTX was compared.
For assessment of VSMC migration, cultures of rat aortic VSMCs were prepared by the explant technique and examined in a modified Boyden chamber assay (27) by using nitrocellulose filters (8 μm thick, 8-μm pores) coated with type-I collagen solution at 100 μg/ml (Vitrogen 100; Collagen Corp., Fremont, CA, U.S.A.). VSMCs were trypsinized and resuspended at a concentration of 1 × 106 cells/ml in serum-free medium containing 0.2% bovine serum albumin (BSA). The cell suspension was placed in the upper chamber, and serum-free medium containing 0.2% BSA with or without 5 ng/ml platelet-derived growth factor (PDGF) and PTX (0-1,000 μM) was placed in the lower chamber. After 4 h at 37°C, the medium was removed, and VSMCs sticking to the filter were fixed in 99% ethanol and stained with Giemsa solution. The number of cells that had migrated through the filter was counted, and all experiments were performed in triplicate.
Data are expressed as mean ± SEM. Statistical analysis was performed by using a two-sided, two-sample t test, paired t test, or analysis of variance (ANOVA) as appropriate. A value of p < 0.05 was considered to be statistically significant.
Morphometric analysis and blood pressure measurements
In the first series of experiments, 19 rats underwent the balloon injury procedure and received PTX (n = 10) or saline (control group, n = 9) from 3 days before to 14 days after injury. As shown in Figs. 1 and 2, there was a marked neointimal thickening of injured arteries in control rats 14 days after balloon injury, and the neointimal area was significantly reduced by treatment with PTX (0.074 ± 0.001 vs. 0.172 ± 0.003 mm2; p < 0.001). The medial area (0.143 ± 0.001 vs. 0.176 ± 0.001 mm2; p < 0.01) and I/M ratio (0.50 ± 0.02 vs. 0.990 ± 0.12; p < 0.001) were also reduced by PTX. However, administration of PTX was associated with constrictive remodeling [i.e., a decrease in EEL (0.601 ± 0.010 vs. 0.744 ± 0.011 mm2; p < 0.01)], and as a consequence of these processes, the luminal area did not differ between the two treatment groups (0.340 ± 0.007 vs. 0.319 ± 0.011 mm2; p = 0.62). The mean arterial blood pressure before balloon injury and after 14 days was not different in rats treated with PTX (102 ± 4 vs. 99 ± 3 mm Hg; p = 0.45) or controls (104 ± 6 vs. 97 ± 3 mm Hg; p = 0.38).
Proliferative activity, apoptosis, and neointimal cell density
As shown in Figs. 3-5, substantial replication and apoptosis of medial VSMCs were detected by PCNA immunochemistry and TUNEL in carotid arteries from 14 rats (n = 7 in each treatment group) killed 24 h after balloon injury, and these responses were not significantly altered by treatment with PTX. At 14 days after injury, PCNA expression and TUNEL were virtually limited to neointimal cells, and differences were not observed between the two treatment groups (n = 10 in PTX group; n = 9 in control group). In arteries removed 14 days after injury, administration of PTX significantly increased the cell density in the neointima (3,476 ± 504 cells/mm2 vs. 2,215 ± 232 cells/mm2; p < 0.05).
VSMC collagen production and migration in vitro
Incubation with PTX resulted in a concentration-dependent inhibition of collagen type I production from cultured human VSMCs (Fig. 6), whereas significant effects of the agent on VSMC synthesis of collage type IV or VI were not observed (data not shown). PTX (10-1,000 μM) did not influence VSMC viability as determined by trypan blue exclusion.
PDGF produced a concentration-dependent increase in the migration of rat VSMCs (not shown). As demonstrated in Fig. 7, PTX failed to influence VSMC migration toward PDGF, and the agent did not modify random VSMC movement (chemokinesis) in the absence of PDGF.
In this study, PTX was found to inhibit neointimal thickening and induce constrictive vascular remodeling after balloon injury in the rat model. These effects were not associated with apparent alterations of VSMC proliferative activity or apoptosis, but they were paralleled by increased neointimal cell density, indicating that the decrease in total vessel and neointimal cross-sectional areas after PTX administration were primarily dependent on a reduction of the lesional extracellular matrix content. In agreement with this contention, PTX had the capacity to inhibit VSMC collagen type I synthesis in vitro, whereas the agent failed significantly to affect PDGF-induced VSMC migration.
Neointimal formation represents the typical response of the arterial wall to a wide range of injuries (e.g., hypercholesterolemia, hypertension, or trauma to any layer of the vessel wall), and is characterized by a sequence of proliferation of medial VSMCs, migration of these cells to the intima, and subsequent proliferation of intimal cells (1,2). The cyclic nucleotides cAMP and cGMP have well-established roles in inhibiting VSMC mitogenic signaling pathways (28,29), and phosphodiesterase activity is induced in proliferating VSMCs (30). Relatively limited knowledge is available regarding the regulation and sensitivity to pharmacologic inhibition of the seven families (i.e., >25 different isoenzymes) of phosphodiesterases that currently compose this class of enzymes, and the therapeutic potential of clinically available phosphodiesterase inhibitors in suppressing neointimal thickening has not been exploited previously. Recently, however, Indolfi and co-workers (31) demonstrated that local delivery of aminophylline in a gel surrounding the injured rat carotid artery markedly reduced neointimal formation, and this effect was reversed by concomitant local application of an inhibitor of cAMP-dependent protein kinase. In that study, systemic treatment with aminophylline failed to inhibit neointimal thickening, and potential effects on vessel remodeling, VSMC proliferation, apoptosis, migration, and collagen production were not addressed (31).
PTX has beneficial effects in models of inflammatory vessel injury when administered in a dosage comparable to that used in the present study (10-12), and the agent may suppress various mechanisms thought to be involved in neointimal formation and vascular remodeling (13-20). For example, PTX is known to inhibit VSMC proliferation in vitro (14,15,32) (and own unpublished results). However, we were unable to demonstrate any significant effect of PTX on the proliferative activity as detected by PCNA immunostaining in balloon-injured arteries removed 24 h or 14 days after injury (Fig. 4), and this observation underscores the multiplicity and redundancy of mitogenic signals in the injured vessel wall (1,2). Apoptosis is a distinct mode of programmed cell death characterized by cytoplasmic shrinkage and DNA cleavage into 180 bp fragments, which are readily detected by TUNEL (25). Extensive apoptosis of VSMCs was recently shown to occur in animal models of balloon injury (33-35) and in human atherosclerotic and restenotic lesions (36). As PTX can inhibit apoptosis in vitro (16), and VSMCs undergo apoptosis when exposed to stimulators of cAMP-dependent protein kinase (19), we hypothesized that interference with apoptosis could be a mechanism underlying the effect of PTX on vascular remodeling. However, treatment with PTX failed significantly to alter the apoptotic response at the two time points investigated (i.e., 24 h and 14 days after injury), as indicated by TUNEL (Fig. 5). The data therefore suggest that VSMC replication and death are not critical determinants of constrictive vascular remodeling in this model of arterial injury.
Reports of vessel remodeling after balloon injury have previously been limited to humans and large animal models (e.g., rabbits and pigs), and increasing evidence has suggested that disturbances in the regulation of VSMC extracellular matrix synthesis and degradation contribute importantly to this phenomenon (3-7). Because of the nature of immunohistochemical staining techniques, there is at present no established reliable way to measure the extracellular matrix content by these methods, but in our study, a reduction of lesional matrix was clearly suggested in PTX-treated rats by the increase in neointimal cell density in the face of unchanged markers of proliferation and apoptosis. This finding was unexpected, because PTX was thought to have the potential to increase extracellular matrix formation [e.g., by inhibition of TNFα-induced VSMC synthesis of matrix metalloproteases (6,17,18,20)]. The in vivo results were, however, corroborated by the demonstration that PTX exerted a concentration-dependent inhibitory effect on VSMC production of collagen type I in vitro (Fig. 6). This finding is in agreement with reports that PTX can inhibit fibroblast synthesis of collagen, glycosaminoglycan, and fibronectin (19,20), and PTX also has been shown to inhibit experimental liver fibrosis (21). Whether the inhibition of collagen type I production observed in vitro is sufficient to explain the effect of PTX after arterial injury in vivo remains to be determined, but it is notable that collagen type I is the predominant collagen in vascular lesions (37), and the notion that PTX-induced constrictive remodeling by inhibition of VSMC collagen I production is supported by recent results indicating a diminished collagen content in vessels with constrictive remodeling (7). Interestingly, previous studies suggested that the extent of neointimal formation after arterial injury may be an important determinant of adaptive remodeling (vessel enlargement) (38,39). It is therefore possible that the failure of many pharmacologic agents to inhibit lesion formation after arterial injury can be explained, in part, by a mechanism similar to the one we observed with PTX [i.e., inhibition of neointimal formation and concomitant induction of constrictive vessel remodeling or suppression of vessel enlargement (40)].
Alterations of VSMC migratory activity are considered to be an important determinant in the arterial response to injury (41). In the rat model, the intimal cell number 4 days after injury has previously been used as a marker of VSMC migration, because VSMC migration first occur between days 3 and 4 after injury [i.e., when intimal VSMCs were thought to have undergone only limited proliferation (42)]. However, the validity of this method is questionable, given our current understanding of the nonlinearity of events during neointimal formation (1,2), and in our hands, the small but highly variable number of intimal cells detected at day 4 after balloon injury usually displayed PCNA expression, indicating that proliferation had already added to the neointimal cell number (data not shown). We therefore examined the effect of PTX on VSMC migration in vitro, and the finding that the agent was devoid of effect (Fig. 7), combined with the in vivo results, makes it highly unlikely that PTX exerted significant effects on VSMC migration in this model of arterial injury.
The rat model of carotid balloon injury provides a reproducible and manipulable model, in which agents can be screened before testing in other animal models and before human trials. It does not, however, mirror all aspects of human restenosis, and limitations apply to the interpretation of our results. For example, we did not use stereologic methods for determination of cell numbers, and the specificity of TUNEL for apoptosis is not absolute (43,44). Furthermore, the assessment of PCNA expression and TUNEL represents a static snapshot at two time points, and the complete kinetic picture of VSMC proliferation and apoptosis in the balloon-injured vessel wall has not been defined. We conclude that PTX inhibits neointimal formation and induces constrictive vascular remodeling in the rat model of balloon injury by mechanisms involving inhibition of VSMC collagen type I production.
Acknowledgment: This work was supported by The Danish Heart Foundation, Karen Margrethe Torp-Pedersen and Ambassador Emil Torp-Pedersen Foundation, Grosserer Sigurd Abrahamson and wife Addie Abrahamson Memorial Foundation, Eva and Robert Voss Hansen Foundation, and the AP Møller and wife Chastine McKinney Møller Foundation. Lena Classon-Welsh and Stig Haunsø are acknowledged for support.
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Keywords:© 1999 Lippincott Williams & Wilkins, Inc.
Pentoxifylline; Remodeling; Neointima; Vascular smooth-muscle cell; Apoptosis; Collagen