Angiotensin-Converting Enzyme Inhibition Abolishes Medial Smooth Muscle PDGF-AB Biosynthesis and Attenuates Cell Proliferation in Injured Carotid Arteries
Relationships to Neointima Formation
Background ACE inhibitors can attenuate the development of intimal fibrocellular lesions after balloon catheter vessel injury, but the mechanisms responsible are unknown.
Methods and Results To evaluate how basic fibroblast growth factor (FGF-2) and the platelet-derived growth factor (PDGF) isoforms are affected by ACE inhibition in injured rat carotid arteries in relation to smooth muscle cell (SMC) proliferation, we examined the effects of oral perindopril on FGF-2 and PDGF isoform levels in carotid arteries 2 days after balloon catheter injury. [3H]Thymidine incorporation into medial and intimal SMCs was also assessed. Uninjured vessels contained two forms of FGF-2, with molecular weights of 18 and 22 kD, and PDGF-AA. Two days after injury, FGF-2 and PDGF-AA levels were markedly reduced, but high levels of PDGF-AB became apparent when the SMCs were proliferating. Perindopril completely abolished the biosynthesis of PDGF-AB but had little effect on residual FGF-2. This was accompanied by a 25% reduction in medial SMC proliferation. Neointimal cell proliferation 10 days after injury was unaffected by perindopril, although neointima size was reduced by 30%. Commencing perindopril treatment 4 days after the injury confirmed that early events associated with effects on medial SMCs were the major contributors to the attenuated neointimal lesions.
Conclusions The ability of ACE inhibitors such as perindopril to attenuate neointima formation and growth in balloon catheter–injured rat carotid arteries is dependent on early events in the media, the inhibition of SMC PDGF-AB biosynthesis and attenuation of proliferation. Neointima formation in similarly injured vessels containing SMCs that are either unresponsive to PDGF-AB or exhibit an ACE-independent profile of growth factor biosynthesis responses may account for the ineffectiveness of ACE inhibition in some species.
Restenosis after percutaneous transluminal balloon angioplasty frequently limits the long-term success of the procedure in patients with coronary and/or peripheral vascular disease1 and is often due to the formation of large occlusive neointimal fibrocellular proliferative lesions.2 The processes contributing to lesion formation include platelet activation, with the release of a variety of growth factors, increased expression of growth factors associated with medial SMC replication, cell migration through the internal elastic lamina, neointimal cell proliferation, and matrix biosynthesis.2 3 4
Recent evidence indicates that inhibitors of ACE are effective under some circumstances in attenuating the development of neointimal fibrocellular lesions in injured vessels. In rats, for example, ACE inhibitors attenuate neointimal fibrocellular lesions in the carotid artery after balloon catheter injury,5 vein graft hyperplasia,6 and allograft hyperplasia.7 In rabbits, swine, and nonhuman primates, however, ACE inhibitors are less efficacious.8 9 Similarly, in humans, their ability to attenuate the incidence of restenosis after angioplasty appears to be insignificant.10 These different responses to ACE inhibitors suggest a relatively specific mechanism of growth inhibition and prompted us to investigate further the mechanisms by which they reduce neointimal growth after injury to a vessel wall.
Theoretically, many of the effects of ACE inhibitors in vivo can be attributed to inhibition of angiotensin II production. Angiotensin receptor antagonists attenuate neointima formation in rat carotid arteries after balloon catheter injury.11 Other in vivo findings also implicate angiotensin II in SMC proliferation.12 In cultured vascular SMCs, the mechanisms causing proliferation are dependent on the production of growth factors, and angiotensin II has been reported to elevate mRNAs encoding PDGF-A, transforming growth factor-β1, c-myc, c-fos, and FGF-2.13 It is therefore likely that the ability of ACE inhibitors to attenuate neointimal formation after vessel injury is dependent on their reducing the production of tyrosine kinase–activating growth factors, such as FGF-2 and/or the PDGF isoforms. After balloon catheter injury of the carotid artery, mRNAs encoding the PDGF-A peptide are elevated, whereas PDGF-B mRNA levels appear unaltered.14 FGF-2 mRNA expression also is increased immediately after balloon injury.15
In the present study, we examined how various PDGF isoforms and FGF-2 peptides are affected in relation to cell proliferation in the injured rat carotid artery during ACE inhibition with perindopril. We demonstrate that 2 days after balloon catheter injury, when medial cell proliferation is high, PDGF-AA levels are reduced, whereas PDGF-AB levels are greatly elevated. PDGF isoform peptide levels are abolished during ACE inhibition by perindopril, but FGF-2 peptide levels are not affected. These effects of perindopril on growth factor expression were associated with reductions in medial SMC proliferation and neointimal growth. Neointimal cell proliferation is unaffected by the ACE inhibitor.
Animals and Study Design
Adult male Wistar Kyoto rats weighing 300 to 400 g were obtained from the Baker Medical Research Institute, Melbourne, Australia. Their left common carotid arteries were subjected to balloon catheter injury by surgical procedures approved by the Baker Medical Research Institute and Alfred Hospital Animal Experimentation Committee. Before surgery, the rats were divided into seven groups (see Fig 1⇓), according to whether they were to be given perindopril (1 or 3 mg·kg−1·d−1) or vehicle (water) by daily oral gavage. Therapy was begun 6 days before the operation and ceased on day 2 in groups 1 and 2 and on day 10 in groups 3, 4, and 5 after the operation. Vessel structure, indices of SMC proliferation, and platelet adhesion to the injured vessel wall were then assessed. The effects on PDGF isoforms and FGF-2 were assessed 2 days after vessel injury. These times for examining the effects of perindopril were optimal with respect to SMC proliferation.3 A sixth group of rats began therapy on the high dose of perindopril 4 days after the operation, a time when SMC proliferation and SMC migration from media to intima has already taken place.3 These rats received vehicle for 10 days by gavage before perindopril treatment was begun. The seventh group was given vehicle, commencing 6 days before the angioplasty and finishing 14 days later, to confirm expected high rates of neointimal cell proliferation.3
Balloon Catheter–Induced Vessel Injury
Vessel injury was achieved in the left common carotid artery as described by Clowes et al.3 Briefly, the rats were placed under general anesthesia (ketamine 80 mg/kg and xylazine 10 mg/kg), and a midline neck incision was made to expose the left carotid arteries at the bifurcation. A 2F Fogarty balloon catheter (Edwards Laboratories) was inserted into the common carotid artery and inflated with normal saline to produce a slight resistance upon withdrawal. The inflated balloon catheter was then dragged four times along the length of the common carotid artery. After wound closure, the animals were allowed to recover with parenteral rehydration. Endothelial removal was confirmed in all vessels by vital staining with Evans blue dye (60 mg/kg IV).3
Tissue Collection and Processing
Rats were anesthetized with sodium pentobarbitone, 100 mg/kg body wt (Boehringer Ingelheim), and then perfused via the left ventricle with saline for ≈2 minutes until cleared of blood, then with 2.5% glutaraldehyde in 0.1 mol/L phosphate buffer (PB, pH 7.4) at 120 mm Hg pressure for 7 minutes. Fixed arteries were dissected free of surrounding tissue, and then blocks of tissue 3 to 4 mm long were postfixed in osmium tetroxide (1% for 2 hours), dehydrated in ethanol, and embedded in Epon 812 resin. Cross sections 1 μm thick were cut, stained with 1% toluidine blue in 1% borax, and mounted on glass slides. Carotid vessels for immunohistochemistry or electrophoretic analysis were not perfusion fixed. A small segment of each vessel for immunohistochemistry was mounted in OCT and frozen in isopentane over liquid nitrogen; the remainder was rapidly frozen in liquid nitrogen and stored at −80°C for electrophoretic analysis of growth factors (see below).
[3H]Thymidine Autoradiography and Labeling Frequency
The ability of SMCs in injured vessels to enter the S phase of the mitotic cell cycle was assessed in the different groups after injection of [3H]thymidine (0.5 mCi/kg IP; ICN Radiochemicals) at 17, 9, and then 1 hour before the animals were killed. This procedure labels all cells that enter the S phase of the cell cycle over the 24-hour period before death.3 16 Blood vessels were sectioned as described above, then slides were dipped into autoradiographic emulsion at 42°C (LM1, Amersham) and left in the dark for 14 days at 4°C. The slides were developed in Ilford Phenisol developer, fixed in sodium thiosulfate, and counterstained with toluidine blue. Cell nuclei with three or more overlying silver grains were considered to be labeled above background levels and were counted in three regions of each carotid artery, ie, distal, mid, and proximal, then expressed as a proportion of total nuclei counted in each vessel (thymidine index). Values for the three regions of each injured and noninjured carotid artery were averaged.16
The size of neointimal lesions was estimated for distal, mid, and proximal regions. Sections of the vessels were projected onto a digitizing tablet (Complot Series 7000, Bausch and Lomb), and the intimal and medial perimeters were traced. Areas were calculated by planimetry with proprietary software.
Cross sections 8 μm thick of frozen vessels were cut, air-dried onto gelatinized glass slides, and fixed in acetone at −20°C. After fixation, sections were washed in PBS (pH 7.4) and treated with 0.3% H2O2 in PBS to block endogenous peroxidase activity. After a wash in PBS, sections were incubated with 10% horse serum in PBS for 30 minutes before application of the DG2 monoclonal mouse anti-human FGF-2 antibody with high specificity for FGF-2 compared with FGF-117 (du Pont de Nemours and Co Inc). After 1 hour at room temperature, the sections were washed in PBS. Control sections were incubated with antibody diluent solution (PBS 2% horse serum) instead of the primary antibody. Tissue-bound primary antibody was detected by the ABC method (Vector Laboratories Inc) with diaminobenzidine tetrahydrochloride (Sigma Chemical Co) as the chromogen. Sections were counterstained with hematoxylin, dehydrated in alcohol, and mounted in Depex.
Scanning Electron Microscopy
Perfusion-fixed vessels obtained from animals 4 and 48 hours after balloon catheter injury were rinsed three times in 0.1 mol/L PB (pH 7.4) and placed into 2% osmium tetroxide for 1 hour. Then, after three rinses in 0.1 mol/L PB (pH 7.4), they were dehydrated in a graded ethanol series and stored overnight in acetone. After critical-point drying, the specimens were cut longitudinally to expose the luminal surface and mounted on stubs with carbon dag and sputter-coated with gold. The vessel surface was examined with a scanning electron microscope (Phillips PSEM 515).
Electrophoresis and Western Blotting
FGF-2 and PDGF-AA, -AB, and -BB contents were compared in extracts of vessels after electrophoresis and Western blotting. Soluble protein was obtained by pulverizing three vessels under liquid nitrogen and adding 200 μL buffer (pH 7.4) (0.5% SDS, 50 mmol/L Tris HCl, 100 μmol/L PMSF, and 100 μg/mL leupeptin). After centrifugation at 13 500g for 2 minutes, an aliquot of the supernatant was reserved for protein estimation and the remainder boiled for 3 minutes in sample buffer (0.0625 mol/L Tris [pH 6.8], 10% glycerol, 2% SDS, and 0.0012% bromophenol blue) in the presence or absence of 5% β-mercaptoethanol. For analysis of PDGF isoforms, β-mercaptoethanol was omitted from the sample buffer. Proteins (20 μg for FGF-2 or 50 μg for PDGF) were loaded onto 15% polyacrylamide gels, electrophoresed at 200 V for 1 hour, and then transferred to nitrocellulose at 30 V (overnight at 4°C). Standards, FGF-2 (Bachem, 20 ng), PDGF-AA, PDGF-BB homodimer, or PDGF-AB heterodimer (Upstate Biotechnology Inc, 10 ng) were also electrophoresed and subjected to Western analysis. Nonspecific binding sites on the nitrocellulose were blocked by incubation in skim milk (5 mg/100 mL) in TBS-T. Blot immunostaining for FGF-2 peptides was performed with a high-affinity monoclonal mouse anti-human FGF-2 ( DE6; du Pont de Nemours and Co)17 (1 μg/mL diluted to 1:1000 in TBS-T). Immunostaining for the different PDGF peptides was done with an anti-human polyclonal PDGF-AA IgG prepared by immunizing rabbits with recombinant human PDGF-AA or an anti-human PDGF-BB monoclonal IgG with recombinant human PDGF-BB as the immunogen, both diluted to 1:250 in TBS-T (Upstate Biotechnology Inc). Blots were incubated with secondary biotinylated antibodies diluted to 1:10 000 in TBS-T; for FGF-2 and PDGF-B peptides, a horse anti-mouse antibody was used, whereas rat absorbed IgG anti-rabbit antibody was used to detect PDGF-A peptides. For immunodetection, the enhanced chemiluminescence Western blotting system was used (Amersham). Proteins were quantified with the “Coomassie plus” protein assay kit (Pierce), with BSA as a standard.
Immunoprecipitation and Reductive Alkylation of PDGF Peptides
The PDGF-A peptides were immunoprecipitated with PDGF-AA antiserum by incubation of soluble vessel extracts at 4°C for 2 hours.18 Resulting immunoprecipitates were removed by centrifugation either with or without prior incubation with a 50% protein A–Sepharose slurry in PBS (pH 7.3), and the supernatant was subjected to electrophoresis and Western blotting with the PDGF-BB antiserum (see above). Reductive alkylations were carried out on tissue extracts by inclusion of 50 mmol/L dithiothreitol in the sample buffer and heating of the samples at 90°C for 3 minutes before addition of 100 mmol/L iodoacetamide. After incubation for 15 minutes at 20°C, the samples were electrophoresed.
Semiquantitative Estimation of PDGF-A and PDGF-B mRNA by Reverse Transcription Polymerase Chain Reaction
Total RNA was extracted from carotid arteries by the single-step acid guanidinium thiocyanate–phenol-chloroform method.19 For cDNA production, the concentration of extracted RNA was adjusted to 67 ng/mL and reverse transcribed with random-hexamer priming (Perkin-Elmer RNA-PCR kit) in a mixture of 50 ng RNA, 10 mmol/L Tris-HCl (pH 8.3), 50 mmol/L KCl, 5 mmol/L MgCl2, 1 mmol/L each of dATP, dTTP, dGTP, and dCTP, 5 U ribonuclease inhibitor, 2.5 μmol/L random hexamers, and 12.5 U MuLV reverse transcriptase. First-strand cDNA synthesis was achieved with one cycle of 10 minutes at 25°C, 15 minutes at 42°C, and 5 minutes at 99°C, then the reaction was rapidly cooled to 25°C and placed on ice. This mixture was then used for amplification of PDGF-A, PDGF-B, and ribosomal protein L7 cDNA fragments with isoform-specific oligonucleotide primers. For PCR, the mixture contained 10 mmol/L Tris-HCl (pH 8.3), 50 mmol/L KCl, 1.5 mmol/L MgCl2, 200 μmol/L each of dATP, dTTP, dGTP, and dCTP, 0.8 μmol/L oligonucleotide PCR primers, 0.625 U Amplitaq DNA polymerase, and a 1:200 dilution of anti-Taq DNA polymerase monoclonal antibody (Clontech). The amplification parameters were the appropriate numbers of cycles of 94°C for 1 minute, 63°C for 1 minute, and 72°C for 2 minutes to obtain semiquantitative expression data for each of the cDNA fragments. For these experiments, 50 ng RNA was reverse transcribed, then the optimal PCR cycling length was used for each of the primer pairs, such that the product amplification–RNA relationship was always kept in the log-linear phase. The last cycle consisted of a final extension step at 72°C for 8 minutes before cooling to 25°C. Confirmation that similar amounts of RNA were used in each RT-PCR was carried out by also amplifying the cDNA for L7, a ribosomal protein that is encoded by a noninducible cell cycle–independent gene.20
Samples were electrophoresed on 3% agarose gels containing ethidium bromide and photographed. Verification that the correct fragments were amplified was based on size estimations and diagnostic restriction endonuclease digestion analysis.
Primers specific for PDGF-A chain and PDGF-B chain were designed by use of the published rat cDNA sequence for PDGF-A21 and the partial rat cDNA sequence for PDGF-B (Genbank accession No. L41623) and the L7 rat cDNA sequence.22 To avoid design of primers that had significant homology between isoforms, the PDGF-A and PDGF-B cDNA sequences were compared with each other by the Megalign program of DNAstar, and regions displaying the highest amounts of nucleotide divergence were used for choosing the primers. Primers were designed with the Primer Detective program (TMJ Lowe, Clontech Laboratories). Their characteristics and the expected target sizes of the amplified cDNA fragments are as follows: PDGF-A (190 bases), 5′-GAGTTGATCGAGCGACTGGC-3′ (forward, nucleotides 82 to 101 of cDNA) and 5′-CTTCCTCAATACTTCTCTTCCTGCGAATGG-3′ (reverse, nucleotides 242 to 271 of cDNA); PDGF-B (220 bases), 5′-TGCACAGACTCCGTAGACGAAGATGGGG-3′ (forward, nucleotides 90 to 119 of partial cDNA) and 5′-CACACCAGGAAGTTGGCATTGG-3′ (reverse, nucleotides 288 to 309 of partial cDNA); and L7 (286 bases), 5′-CCTGAGGAAGAAGTTTGCCC-3′ (forward, nucleotides 143 to 162 of cDNA) and 5′-CTTGTTGAGCTTCACAAAGGTGCC-3′ (reverse, nucleotides 405 to 428 of cDNA).
All data are presented as mean±SEM. Differences between groups were tested by one-way ANOVA, and if found significant, the post hoc tests were unpaired t tests. Differences between treatment groups were considered significant when P<.05.
Animal weights were well maintained throughout the study, except for a small reduction (≤5% of body weight) in all treatment groups shortly after surgery. Because mild dehydration was suspected, a consequence of the neck surgery, parenteral fluid supplementation was given during the perioperative period, and all rats gradually regained their weight during the study. There were no significant differences between treatment groups throughout the study, and after 16 days of drug treatment the body weights for vehicle, low-dose, and high-dose perindopril groups were 338±14, 331±5, and 337±11 g, respectively (P>.05).
ACE Inhibition and SMC Proliferation
Because maximum [3H]thymidine labeling of SMC nuclei in injured vessels is known to occur ≈48 hours after injury,3 we initially assessed how perindopril affected the number of SMCs entering the S phase at this time. In vehicle-treated rats, the proportion of SMC nuclei incorporating [3H]thymidine at this time was ≈30 times greater (32.8% versus <1%) than in undamaged carotid vessels (P<.05; Fig 2⇓). In rats treated with perindopril 3 mg·kg−1·d−1, the number of SMCs incorporating [3H]thymidine was 24% lower than in vehicle-treated animals, averaging a labeling frequency of 25.1% (P<.05; see Fig 2⇓). Ten days after injury, the frequency of [3H]thymidine labeling in the vessel media of vehicle-administered animals averaged 5.3±1.8% (Fig 2⇓; P<.05 from uninjured vessels); in perindopril 3 mg·kg−1·d−1–treated rats, it averaged 1.7±0.5%. In the neointima of vehicle-administered rats, the frequency of [3H]thymidine labeling in the injured vessels was high, averaging 21.8±3.9% (P<.05 from uninjured vessels; Fig 2⇓). It was unaffected by the chronic perindopril administration (3 mg·kg−1·d−1), averaging 28±5% (P>.05). The effects of the low dose of perindopril (1 mg·kg−1·d−1) were also not statistically significant (P>.05). In the neointima, SMC [3H]thymidine incorporation 10 days after balloon injury averaged 24.5±1.8%.
ACE Inhibition and Vascular FGF-2
Because the antiproliferative effect of perindopril was detected only within the media of the injured vessel, we examined whether such an effect could be attributed to reductions in the content of FGF-2 within the vessels. Initially, we examined by immunohistochemistry whether immunoreactive FGF-2 peptides had been altered by the perindopril 3 mg·kg−1·d−1 treatment. Two days after the injury, immunostaining for FGF-2 peptides was localized predominantly to cellular elements of the vessel wall, the cytoplasm of SMCs, and some adventitial cells. A similar distribution was seen in vessels of the perindopril-treated animals (Fig 3⇓). Because this immunoreactivity could represent FGF-2 plus its degraded fragments, we also evaluated the effects of perindopril on FGF-2 content by Western blotting (Fig 4⇓). In uninjured vessels, the predominant FGF-2 peptide species possessed a molecular weight of 22 kD and most probably represents nuclear FGF-2 in the SMCs23 (Fig 4⇓). A less intense band of 18 kD comigrated with recombinant FGF-2 and is consistent with cytoplasmic FGF-2. In the injured vessels, 2 days after the balloon catheter procedure, these FGF-2 peptide species represented only a small portion (<5%) of the immunoreactivity detected by Western blotting in the uninjured vessels (Fig 4⇓). The two major FGF-2 immunoreactive peptides in the injured vessels exhibited molecular weights of 12 and 14 kD and probably represent degradation products of both nuclear and cytoplasmic FGF-2. The 14-kD FGF-2 peptide has previously been associated with a cytoplasmic localization, particularly in the late S and G2 phases of the mitotic cell cycle.24 Some higher-molecular-weight proteins exhibiting FGF immunoreactivity could also be observed in extracts of the injured vessels (Fig 4⇓). We did not investigate their origin or identity. These peptide patterns in the injured vessels were unaffected by perindopril (Fig 4⇓).
ACE Inhibition and Vessel PDGF Isoform Content
Because PDGF-A mRNA is known to be elevated after balloon catheter injury of the rat carotid artery,25 we considered the possibility that ACE inhibition affects the expression of PDGF-A peptides in the injured vessels.13 We initially examined by Western blotting the effects of injury on PDGF-A immunoreactive peptides and then evaluated how their distribution pattern was affected by ACE inhibition with perindopril 3 mg·kg−1·d−1. Uninjured carotid vessels of vehicle-treated animals contained two major PDGF-A immunoreactive peptides with apparent molecular weights of 32 and 35 kD (Fig 5⇓). The lower-molecular-weight species comigrated with recombinant PDGF-AA; the higher-molecular-weight peptide could represent long-chain PDGF-AA.26 Other less intense immunoreactive PDGF-A peptides of higher molecular weight could represent precursor peptides. Forty-eight hours after vessel injury, this pattern of expression was dramatically altered. In the injured vessels, a new PDGF-A immunoreactive species with an apparent molecular weight of 29 kD was present, which comigrated with recombinant PDGF-AB, and PDGF-AA was greatly reduced (Fig 5⇓). Lower-molecular-weight PDGF-A peptides were also apparent, probably representing partially degraded PDGFs. Perindopril treatment completely suppressed this pattern of PDGF-A peptide expression in the injured vessels. Because these experiments suggested that PDGF-B peptides were contributing to the elevation in PDGF-A peptides, we then examined the effects of vessel injury and ACE inhibition on PDGF-B peptides. They were not detectable in uninjured carotid arteries (Fig 6⇓). However, 48 hours after injury, the vessels contained at least three significant PDGF-B peptides. The 29-kD peptide comigrated with recombinant PDGF-AB and PDGF-BB. The other significant peptides of ≈48 and 12 kD probably represent precursor and degraded PDGF-B immunoreactive peptide products, respectively. Perindopril 3 mg·kg−1·d−1 treatment completely prevented their induction. It is also possible that the PDGF-B peptides comigrating with PDGF-AB and -BB represent a small amount of PDGF-BB.27
To investigate this possibility, PDGF-A peptides in the vessel extracts were precipitated with the PDGF-AA polyclonal IgG, specific for PDGF-A peptides. After removal of PDGF-A peptides–IgG complexes, the 29-kD peptide previously detectable with the anti–PDGF-BB IgG was no longer present in the supernatant (Fig 7⇓). Other PDGF-B peptides were also removed, indicating that they too were either precursors or degradation products of PDGF-AB. To confirm that these PDGF-B peptides in the injured vessels were disulfide-bonded dimers, reductive alkylation was also performed on the supernatant extracts. In such supernatants, disulfide-bonded dimeric PDGF-B peptides were no longer detectable with the anti–PDGF-BB IgG; the antibody does not interact with reduced and alkylated forms of PDGF-B. Because only peptide levels for PDGF-AB were measured and not biosynthesis, we also evaluated whether perindopril affected platelet adhesion and degranulation at sites of vessel injury. Comparison of the luminal surface of injured vessels both 4 and 48 hours after balloon catheterization and inflation indicated no apparent difference in the extent to which platelets had adhered and flattened to injured vessels of vehicle- and perindopril 3 mg·kg−1·d−1–treated animals (Fig 8⇓).
Effects of Perindopril on PDGF-A and PDGF-B mRNA Levels
Because PDGF-AB biosynthesis in injured vessels was attenuated by perindopril, we also assessed its effects on PDGF-A and PDGF-B mRNA levels in vessels 48 hours after injury by RT-PCR. Amplified cDNA fragments for PDGF-A and PDGF-B were detected in the injured vessels by use of specific oligonucleotide primers (Fig 9⇓), consistent with previous studies indicating the presence of PDGF-A and PDGF-B mRNA expression in injured vessels (Fig 9⇓). In contrast, only PDGF-B mRNA could be consistently detected in injured vessels of the perindopril-treated animals (Fig 9⇓). PDGF-A transcripts were either absent or greatly reduced.
Temporal Dependence of ACE Inhibition on Neointimal Growth
To confirm the significance of the effects of perindopril on medial SMC proliferation and the expression of the various PDGF isoforms in relation to neointima formation, we compared the effect of the “early” and “late” perindopril treatment on neointimal growth. When treatment was commenced early, 6 days before vessel injury, and continued through to day 10 after the injury, neointimal size, defined as the area of tissue bounded inwardly by the internal elastic lamina and outwardly by the luminal edge of the vessel wall, was reduced by 30% (P<.05 from vehicle-treated; Table⇓). Commencing treatment later, 4 days after the injury was inflicted, when neointima formation had already begun,3 caused only a small and not statistically significant reduction of neointimal size (≈8% reduction, P>.05) when continued through to day 10 (Table⇓). At this time, the frequency of [3H]thymidine incorporation into neointimal SMCs averaged 28±4%, similar to labeling frequencies observed in the neointima of animals in which treatment was commenced early. A low (1-mg·kg−1·d−1) dose of perindopril begun early did not affect neointimal SMC [3H]thymidine incorporation and caused a small (≈12%), insignificant reduction in intimal size (P>.05; Table⇓).
This study demonstrates that perindopril abolishes the large, early expression of PDGF-AB in injured rat carotid arteries and that this is associated with attenuation of medial SMC proliferation. FGF-2 peptide levels are unaffected. Our studies on inhibition of neointimal growth by the ACE inhibitor indicate that these and possibly other early events that occur predominantly in the media account for much of its ability to attenuate neointima formation.
The present findings on PDGF-A and PDGF-B peptides in the carotid artery early after injury indicate a somewhat unexpected profile of growth factor expression. Within hours after injury, large transient increases in PDGF-A mRNA levels have been reported, whereas PDGF-B mRNA levels are apparently unchanged.25 The large increases in PDGF-A peptides we observed are consistent with these earlier observations. However, PDGF-AB rather than PDGF-AA accounts for the increase in PDGF levels. Because PDGF-B mRNA levels apparently are not elevated at this early stage after injury,25 the increase in PDGF-B peptides is best accounted for by an elevation in the translation efficiency of its mRNA. Coordinated with the increase in PDGF-AB is a reduction in PDGF-AA, probably because of a switch in biosynthesis from PDGF-AA to PDGF-AB. It is unclear whether this change to the other PDGF isoform is a consequence of an alteration in SMC phenotype, which occurs close to the onset of medial cell proliferation.4 In vitro PDGF-like mitogenic activity is produced by SMCs after modulation to the synthetic phenotype and during proliferation.28
The large increase in PDGF-AB in the injured vessels appears to be dependent on ACE activity and possibly angiotensin II production, because perindopril completely abolishes PDGF-A and PDGF-B peptides as well as dramatically reducing PDGF-A mRNA levels. Angiotensin II receptor antagonists also reduce elevated levels of PDGF-B during development of hypertrophy and sclerosis in a severe partial nephrectomy model.29 This effect was not a consequence of any lowering of blood pressure. Angiotensin II receptor antagonists also attenuate neointimal growth in the balloon catheter–damaged carotid artery,11 and in vivo angiotensin II enhances SMC proliferation in the injured vessel wall.12 In vitro, it stimulates SMCs to produce PDGF-A mRNA and FGF-2 mRNA.13 Despite this latter finding, FGF-2 peptides in the injured vessels were not reduced by the ACE inhibitor.
Our findings demonstrating the release of most of the FGF-2 from the injured carotid artery, together with a large switch in PDGF production from the AA to the AB isoform, are consistent with earlier reports implicating both FGF-2 and PDGFs in the early vessel healing responses.15 30 31 The prevention by perindopril of this PDGF biosynthesis, together with the 25% reduction in medial SMC proliferation after the injury, is consistent with reports on the limited mitogenic potential of PDGF in this vessel.32 We did not examine whether perindopril would affect PDGFs produced by neointimal SMCs because the ACE inhibitor did not reduce their proliferation; these cells apparently do not proliferate in response to PDGF.30 The differential expression of PDGF-AA and PDGF-AB in uninjured and injured vessels has important implications for cell migration within the vessel wall. PDGF-AA is not only a poor mitogen for SMCs14 but is also an inhibitor of their migration; in its presence, the migration initiated by PDGF-AB, PDGF-BB, or fibronectin is prevented.33 Thus, the reduction in PDGF-AA early after injury provides the most favorable conditions for PDGF-AB to stimulate SMC migration. Our observation that perindopril abolishes PDGF expression in the injured carotid artery may explain why the ACE inhibitors and AT1 antagonists can attenuate medial SMC migration into the neointima under these conditions.34
A large component of the early mitogenic activity and cell migration that occurs in the injured vessel despite ACE inhibition by perindopril is probably due to release of FGF-2. Two FGF-2 species were released by the injury, a 22-kD form frequently found in the nucleus24 and a less abundant cytoplasmic form. This released FGF-2 can stimulate mitogenesis by interacting with high-affinity FGF receptors present on the “synthetic” phenotype SMCs.35 In this study, we observed no significant difference in FGF-2 content between injured vessels of the perindopril and control groups. Most of the FGF-2 immunoreactivity in vessels 2 days after injury represents degraded peptides, indicative of high proteolytic activity in the injured vessels; this may also be limiting any increase in FGF-2. The significance of the small residual amount of FGF-2 remains to be determined.
The ability of perindopril to inhibit neointima formation only when administered early is consistent with many of its actions being confined to medial SMCs. Neointimal SMC proliferation was unaffected by the different perindopril dose regimens. Similar conclusions have recently been made by Fingerle et al,36 using the ACE inhibitor cilazapril. Thus, it appears that ACE inhibitors exert their effects on neointimal growth predominantly by attenuating the activities of medial SMCs in injured vessels. Although not proven, it is likely that these effects depend on abolishing PDGF-AB biosynthesis.
Despite many studies confirming the inhibitory effects of ACE inhibitors on neointima formation after injury of the rat carotid artery, these agents are not as effective in other vascular injuries and are apparently ineffective in preventing restenosis of human arteries.16 Differences in study design, drug dosage, vessel structure (including SMC composition), and the types of growth factors involved in the injuries probably account for the discrepancies. In contrast to the rat carotid artery, which is composed primarily of medial SMCs and intimal endothelial cells, the human atherosclerotic lesion is located predominantly in the intimal smooth muscle layer. Despite this difference, several histopathological studies on restenotic lesions after percutaneous transluminal angioplasties indicate that early responses to vessel wall injury can involve medial SMCs. As early as 1 to 6 days after angioplasty, SMCs begin to migrate. Other studies indicate that extensive medial SMC damage is associated with fibrocellular tissue occluding the lumen, and in some instances, two distinct intimal layers have been observed in restenotic lesions.37 Possibly an important factor in the inability of ACE inhibitors to prevent such lesions is more related to regulation of certain growth factors. It may be that the growth factors expressed after vessel injury are not dependent on ACE or that growth factor biosynthesis dependent on ACE does not affect SMC proliferation or migration.
To summarize, our results suggest that the early effects of ACE inhibitors on PDGF-AB expression are likely to account for their ability to attenuate neointimal growth in balloon catheter–injured carotid arteries via effects on medial SMC proliferation and migration.
Selected Abbreviations and Acronyms
|FGF-2||=||basic fibroblast growth factor|
|PCR||=||polymerase chain reaction|
|PDGF||=||platelet-derived growth factor|
|SMC||=||smooth muscle cell|
These studies were supported by a postgraduate research scholarship from the National Heart Foundation of Australia (Dr Wong) and a grant-in-aid from Servier Laboratories, Paris, France. Antibodies against FGF-2 were kindly provided by Dr T.M. Reilly (Wilmington, Del). The technical assistance of Dr C. Neylon and M. Larsen was greatly appreciated.
- Received August 22, 1996.
- Revision received March 3, 1997.
- Accepted March 5, 1997.
- Copyright © 1997 by American Heart Association
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