Bolus Endovascular PDGFR-β Antisense Treatment Suppressed Intimal Hyperplasia in a Rat Carotid Injury Model
Background—Intimal thickening in accelerated arteriopathies relies on the migration of medial vascular smooth muscle cells (VSMCs) and their proliferation within the neointima. Activation of platelet-derived growth factor receptor-β (PDGFR-β) expressed in injured VSMCs is responsible for the migration of medial VSMCs to the intima. In the present study, we wanted to assess whether a single local endovascular delivery of antisense PDGFR-β in injured rat carotid arteries would be sufficient to prevent intimal hyperplasia and how it might contribute to the vascular healing process.
Methods and Results—A bolus of antisense PDGFR-β delivered into injured rat carotid arteries reduced PDGFR-β protein overexpression by >90% from day 3 to 28 after injury. At day 28 after injury, compared with injured untreated carotids, treatment with antisense PDGFR-β reduced intimal hyperplasia by 58% and medial VSMC migration by 49% and improved vascular reendothelialization by 100% and vascular reactivity (EC50) to acetylcholine by 5-fold.
Conclusions—A single-bolus luminal delivery of antisense PDGFR-β to injured rat carotids reduced intimal hyperplasia, improved the reendothelialization process, and led to the recovery of endothelium-dependent regulation of vascular tone.
Restenosis after coronary angioplasty occurs in 20% to 50% of treated patients and remains the major limitation to the long-term effectiveness of this procedure.1 2 3 The expanded catheter removes the endothelium and injures many of the vascular smooth muscle cells (VSMCs) located in the innermost layer of the media of the artery. This is followed by the proliferation of medial VSMCs as early as 1 day after injury, followed by their migration from the media into the intima (4 to 7 days), after which proliferation of intimal VSMCs results in intimal hyperplasia.4 5 6
Reports support the role of platelet-derived growth factor (PDGF) in mediating arterial intimal hyperplasia after an angioplasty and in the formation of atherosclerotic lesions.7 8 9 10 PDGF is secreted as 3 different isoforms: PDGF-AA, -AB, and -BB, and binds to 2 distinct subtype receptors, PDGFR-α and -β, which can form homodimers and heterodimers.11 12 PDGF dimers and both PDGF receptor subtypes are overexpressed in VSMCs from human atherosclerotic plaques and at sites of postangioplasty repair in animal and human arteries.10 13 14 15
In rats, administration of polyclonal antibodies to all forms of PDGF before and after balloon injury (BI) of carotid arteries inhibited intimal hyperplasia but not the first wave of proliferation in the media.16 17 18 Interestingly, antibodies directed against the PDGF-B chain blocked intimal thickening, whereas antibodies to the PDGF-A chain prevented intimal hyperplasia.19 Infusion of PDGF-BB into a carotid BI in rats increased intimal thickening but had little effect on intimal VSMC proliferation, suggesting that the decrease in intimal hyperplasia was due to the inhibition of medial VSMC migration.20
We recently reported that a sustained perivascular delivery for 14 days of an antisense (AS) oligomer complementary to specific PDGFR-β mRNA sequences after a BI in rat carotids prevented PDGFR-β protein overexpression and intimal hyperplasia.15 The aim of the present study was to investigate whether a single endovascular delivery of AS PDGFR-β would be sufficient to reduce intimal hyperplasia by limiting either VSMC migration or proliferation. We also investigated the possibility that inhibition of PDGFR-β overexpression would favor endothelial regrowth and the return of vasomotor activity.
Induction of Intimal Hyperplasia
BI of common carotid arterial endothelium was performed in male Sprague-Dawley rats (325 to 400 g) as described previously.15 Animals were euthanized at different periods of time (0, 3, 7, 14, and 28 days) after injury with an overdose of ketamine and xylazine, exsanguinated, and perfused with 100 mL of Ringer’s lactate solution by the left ventricle. The left (treated) and right (untreated) segments of the common carotid arteries were removed and fixed in 10% formalin PBS. The segments were embedded in paraffin, cut into 6-μm longitudinal sections, and stained with Masson’s trichrome solution. The areas of the intima and media and the intima-to-media (I:M) area ratio were calculated by computerized digital planimetry.
AS Oligonucleotide Therapy
We used an AS oligonucleotide phosphorothioate backbone sequence to the murine PDGFR-β mRNA subunit (AS-PDGFR-β: TATCACTCCTGGAAGCCC). A scrambled (SCR) sequence (SCR-PDGFR-β: GTGATAGTATGCCGAGCA) was used as control. In the present study, we used only 1 AS sequence, because we showed the efficiency of this AS in a previous report.15 After BI of the left common carotid artery, we introduced a 22-gauge infusion cannula into the external carotid arteriotomy and administered 0.2 mL of 0.9% NaCl solution to flush the residual blood-borne elements. The AS or SCR oligonucleotide solution (200 μg/25 μL of PBS 0.01 mol/L) was infused into the temporarily isolated segment of the left common carotid artery for a 30-minute period. Then the arteriotomy was ligated, the left common carotid artery was released, the wounds were closed, and the animals were returned to their cages. The protocol was performed in accordance with the Canadian Council on Animal Care guidelines.
Evaluation of Vascular Reactivity
Carotid arteries were harvested at death and placed in Krebs-Ringer solution. Rings of 4 to 5 mm from the medial portion of the left (treated) and right (untreated) carotids were mounted with 2 triangle 5-0 stainless steel wires. The adjacent segments (distal and proximal) were fixed in formalin for analysis. Experiments were performed in organ chambers filled with 25 mL of Krebs-Ringer solution and indomethacin 0.01 mmol/L and gassed with 95% O2/5% CO2 at 37°C. Vessels were passively stretched (≈1.5 g) while the contraction generated by a depolarizing solution containing physiological KCl (20 mmol/L) was assessed. The organ chamber was rinsed with fresh Krebs-Ringer solution and equilibrated for 45 minutes. Phenylephrine (PE; 10−6 mol/L) was used to achieve a submaximal contraction. An endothelium-dependent vasorelaxation was induced by the addition of cumulative acetylcholine (ACh) concentrations (10−9 to 3.17×10−5 mol/L). Calcium ionophore A23187 (2.5×10−7 mol/L) was added to obtain the maximal endothelium-dependent vasorelaxation. Sodium nitroprusside (10−5 mol/L) was added to mediate a direct VSMC relaxation.
Immunohistochemistry of PDGFR-β, PCNA, and ecNOS Expression
The immunohistochemistry procedures on arterial sections were performed as described previously.15 The primary antibodies used were rabbit polyclonal anti-human PDGFR-β IgG (UBI), monoclonal anti-human proliferative cell nuclear antigen (PCNA) IgG (Zymed Laboratories Inc), and monoclonal anti-human endothelial cell constitutive nitric oxide synthase (ecNOS) IgG (Transduction Laboratories)].
Data are mean±SEM. Statistical comparisons were determined by ANOVA followed by an unpaired Student’s t test with Bonferroni’s correction for multiple comparisons. Data were considered significantly different if a value of P<0.05 was observed. Relaxation is expressed as a percentage of preconstricting tone. EC50 (concentration of ACh producing a half-maximal relaxation) has been calculated for each segment with the Statview program.
Expression of PDGFR-β Protein Subunit
In native arteries, basal expression of the PDGFR-β subunit was observed immunohistochemically on 1.4±0.4% of medial VSMCs (Figures 1A⇓ and 2⇓). PDGFR-β protein increased 8.7-fold in medial VSMCs (P<0.001) by day 3 after injury, reached a plateau at day 7 (12.6-fold increase, P<0.001), and returned to basal levels by day 14 (Figures 1B⇓ and 2⇓). The presence of intimal VSMCs was observed by day 7 after injury, with 18.3±3.7% of intimal VSMCs staining positively for PDGFR-β protein. By day 14, the PDGFR-β protein expression in intimal VSMCs returned to basal level (Figures 1B⇓ and 2⇓).
Treatment with AS-PDGFR-β prevented PDGFR-β protein overexpression in medial VSMCs at days 3 and 7 by 90% and 93%, respectively (P<0.001). Similarly, PDGFR-β protein level was reduced by 60% (P<0.05) in intimal VSMCs at day 7 (Figures 1C and 2) and was at the basal level observed in native medial VSMCs at day 14 (Figure 2⇑). Three days after injury, treatment with an SCR oligomer reduced the PDGFR-β protein expression on medial VSMCs by 42% (P<0.05). This reduction, however, was significantly less (P<0.05) than the reduction mediated by the AS-PDGFR-β (90%) (Figure 2⇑). At day 7, SCR treatment did not reduce PDGFR-β protein expression in medial or intimal VSMCs (Figures 1D⇑ and 2⇑), and by day 14 the PDGFR-β protein expression returned to basal levels (Figure 2⇑).
The intimal and medial areas (mm2) and the I:M area ratio were determined after a vascular injury. The medial areas in BI rat carotid arteries at days 7, 14, and 28 after injury were 0.101±0.007, 0.109±0.005, and 0.105±0.004 mm2, respectively (Figure 3A⇓) and fluctuated by <14% compared with the medial area of native carotid arteries (data not shown). Treatment of the BI carotid arteries with AS-PDGFR-β increased the medial area by 33%, 3%, and 13% at days 7, 14, and 28, respectively (P<0.01 at day 7 and P=NS at days 14 and 28). SCR treatment increased the medial area by 23%, 14%, and 16.5% (P=NS at day 7 and P<0.05 at days 14 and 28) (Figure 3A⇓).
Intimal hyperplasia developed during the first 7 days and was maximal within 14 days. The intimal areas in BI groups at days 7, 14, and 28 were 0.025±0.005, 0.116±0.012, and 0.091±0.011 mm2 (Figure 3B⇑). An AS-PDGFR-β treatment reduced the intimal hyperplasia by 37%, 40%, and 56% (P=0.07 [NS], P<0.05, and P<0.01) at days 7, 14, and 28, respectively, whereas the SCR treatment did not reduce the intimal hyperplasia (Figure 3B⇑). The I:M area ratios in BI carotids were 0.256±0.047, 1.102±0.126, and 0.899±0.099, respectively (Figure 3C⇑). An AS-PDGFR-β treatment reduced these ratios by 50%, 47%, and 58% (P=0.08 [NS], P<0.01, P<0.001), respectively, whereas the SCR treatment did not significantly alter the I:M area ratios compared with BI groups (Figure 3C⇑).
The induction of a carotid BI did not affect the medial VSMC count throughout the first 14 days compared with native vessels (467±38 cells) (Figure 4⇓). At day 28 after injury, however, all groups demonstrated an increased number of medial VSMCs compared with native media. The VSMC count increased by 11% (P=NS) in the untreated BI group, by 32% (P<0.05) in the AS-PDGFR-β–treated group, and by 47% (P<0.01) in the SCR-treated group. The difference between the AS-PDGFR-β and the BI groups was not significant (Figure⇑ 4). At days 7, 14, and 28, the number of intimal VSMCs in BI arteries was 422±67, 1285±100, and 1004±126, respectively. AS-PDGFR-β reduced the number of intimal VSMCs at days 7, 14, and 28 by 47%, 33%, and 50% (P<0.05, P<0.05, P<0.01), respectively, compared with the BI group. The SCR oligomer did not reduce the intimal VSMC count at any time point (Figure 4⇓).
The medial density of VSMCs in native carotid arteries was 4253±160 VSMCs/mm2. The fluctuation density of medial VSMCs at days 3, 7, 14, and 28 after injury in BI or AS-PDGFR-β – or SCR-treated groups was always <20% compared with the VSMC density observed in native medial VSMCs. The variation of medial VSMC density between the BI group and the groups treated either with AS-PDGFR-β or SCR oligomer was also <20% (data not shown). The intimal VSMC densities in the BI group at days 7, 14, and 28 after injury were 14 762±1143, 11 466±496, and 11 939±681 VSMCs/mm2. The AS-PDGFR-β significantly reduced the intimal VSMC density by 29% only at day 7 (data not shown).
SMC Proliferative Activity
In native carotid arteries, the percentage of proliferative medial VSMCs was 1.2±0.4% (Figures 5A⇓ and 6⇓). At days 3 and 7 in the BI group, PCNA expression on medial VSMCs increased to 7.8±2.4% (P<0.01) and 6.8±1.3% (P<0.001) compared with native medial VSMCs and returned to the basal level of PCNA expression observed in uninjured medial VSMCs by day 14 (Figures 5B⇓ and 6⇓). Intimal VSMC PCNA expression was quantified from days 7 to 28 after injury. In the BI group, the percentage of PCNA expression at day 7 was 9.8±2.4%, and it returned to near basal expression by day 14 (Figures 5B and 6). A treatment with AS-PDGFR-β or SCR oligomer did not significantly reduce PCNA overexpression on medial and intimal VSMCs compared with the BI group at any time point (Figures 5C⇓ and 5D⇓ and 6).
To evaluate the extent of reendothelialization, immunohistochemical staining was performed to detect the expression of ecNOS. In native carotid arteries, ecNOS-positive cells covered 96.7±0.5% of the internal elastic lamina (Figures 7A⇓ and 8⇓). Immediately after the passage (3 times) of an inflated balloon, the degree of endothelialization (day 0) was reduced to 2.7±0.3% (Figures 7B⇓ and 8⇓). In the BI group, reendothelialization occurred but remained incomplete (Figures 7C⇓ and 8⇓). Treatment with AS-PDGFR-β increased the extent of reendothelialization at each time point compared with the BI group (Figures 7D⇓ and 8⇓). The application of SCR oligomer did not favor reendothelialization (Figure 8⇓).
Ex Vivo Carotid Vascular Reactivity
Segments of carotid arteries were precontracted to submaximal level with PE (10−6 mol/L). PE-induced contraction in endothelium-intact native arteries (E+; 0.68±0.04 g) was less than in freshly denuded arteries (day 0; 1.38±0.12 g). At 14 and 28 days after injury, PE-induced contraction varied between 0.97±0.11 and 1.28±0.10 g in BI or AS-PDGFR-β – and SCR-treated arteries (data not shown).
On PE-precontracted arteries, ACh induced a complete relaxation of endothelium-intact segments (E+; Figure 9⇓). The relaxant effect of ACh, which was absent in freshly denuded arteries (BI day 0) and maximal on days 14 and 28, produced only 13.4±3.7% (day 14) and 36.1±6.8% (day 28) of vasorelaxation (Figure 9⇓). AS-PDGFR-β but not SCR significantly improved (time-dependently) the efficacy of ACh-induced relaxation compared with the BI group (Figure 9⇓). After the addition of the highest concentration of ACh (3.17×10−5 mol/L), the calcium ionophore A23187 (10−7 mol/L) was added to obtain the maximal endothelium-dependent vasorelaxation. The addition of A23187 to injured carotid arteries either untreated (BI) or treated with the AS-PDGFR-β or SCR oligomers never induced >10% relaxation at 14 and 28 days after injury (Figure⇑ 9). Sodium nitroprusside (10−5 mol/L), which induces a direct VSMC relaxation, produced 100% relaxation in all treated groups (Figure 9⇓).
In the present study, we show that a local endovascular delivery of AS-PDGFR-β at the injured carotid artery site not only reduced the formation of intimal hyperplasia but also enhanced reendothelialization and almost completely restored the endothelium-dependent relaxing function. It is also very interesting to note that such treatment prevented, rather than simply delaying, the overexpression of PDGFR-β protein, which normally peaks 7 days after injury. Finally, we showed that the reduction of intimal hyperplasia mediated by AS-PDGFR-β treatment was not due to a reduction of medial and/or intimal VSMC proliferative activity but rather was attributable to the inhibition of medial VSMC migration into the intima.
After a BI, PDGFR-β protein expression increased in the media and the neointima. This was maximal at day 7 and returned to its baseline level at day 14. These results are in agreement with previous reports that have shown transient PDGFR-β protein overexpression in rat and human injured arteries.10 14 Bilder et al21 reported that a selective PDGFR-β tyrosine kinase inhibitor given orally twice a day for 28 days decreased by 30% the I:M area ratio in injured porcine coronary arteries. Banai et al22 showed that a local intravascular delivery of a PDGF-receptor tyrosine kinase blocker reduced by 40% the I:M area ratio of BI porcine femoral arteries. Finally, Hart et al23 showed that repeated intravenous administration of mouse/human chimeric anti–PDGFR-β antibodies combined with a sustained heparin delivery decreased the I:M area ratio by 40% in BI baboon saphenous arteries. In our study, the single-bolus endovascular application of AS-PDGFR-β was sufficient to prevent the overexpression of PDGFR-β protein throughout the entire 28 days of our experiment, and this might explain why our treatment was more efficient (58%) in reducing the development of intimal hyperplasia than the above-mentioned studies. In our previous report,15 the sustained perivascular application of AS-PDGFR-β reduced the I:M area ratio by 60% to 80%. Our present results suggest that a sustained release of the AS-PDGFR-β is not necessary to achieve its optimal biological effect and reinforce the concept that the blockade of initial events after acute vascular injury might be sufficient to have prolonged benefits.23 24
We calculated the number of medial and intimal VSMCs and their density per square millimeter (VSMCs/mm2), as well as the VSMC proliferative activity in the different groups studied. Although medial VSMC count was increased 28 days after injury in all 3 groups, medial VSMC density at each time point in BI and AS-PDGFR-β – or SCR oligomer–treated groups never fluctuated by >20% compared with VSMC density observed in the media of native carotid arteries. AS-PDGFR-β treatment reduced the number of intimal VSMCs at days 7, 14, and 28 by up to 50% compared with the BI group without altering intimal VSMC density at days 14 and 28. In addition, a treatment with either the AS-PDGFR-β or the SCR oligomer did not significantly reduce PCNA overexpression at any time point in medial and intimal VSMCs as observed in the BI group (Figures 5⇑ and 6⇑). These results demonstrate that the treatment of an injured rat carotid artery with AS-PDGFR-β did not alter the proliferative activity of the medial or intimal VSMCs. Thus, the reduction in intimal VSMC number and the I:M area ratio is attributed to the inhibition of medial VSMC migration into intima.
We observed that the passage of an inflated balloon in rat carotid arteries led to an almost complete denudation of the endothelium. In the untreated BI arteries, a progressive reendothelialization was achieved, but <25% of the luminal area was covered by day 28. The application of AS-PDGFR-β increased the extent of reendothelialization by 2-fold at each time point, such that nearly 50% of the neointima was covered by neoendothelial cells at 28 days. This result, combined with a 58% reduction of the I:M ratio observed in the same carotid arteries treated with AS-PDGFR-β, supports the hypothesis that the inhibition of VSMC migration from the injured media has the double beneficial effects of reducing intimal hyperplasia and improving the vascular healing process.
Finally, our results demonstrate that the contractile (PE) and relaxant (sodium nitroprusside) properties of VSMCs were unaltered by the different treatments. Most importantly, at 14 days and more convincingly at 28 days after injury, AS-PDGFR-β treatment significantly improved endothelium-dependent relaxation. The maximal relaxation produced by ACh more than doubled, and the estimated concentration of ACh needed to induce 50% of its maximal relaxation was reduced by 2- and 5-fold at 14 and 28 days, respectively, compared with injured untreated carotid arteries. Our results suggest that a 50% reendothelialization of injured rat carotid arteries might be sufficient to induce an almost complete endothelium-dependent vasorelaxation as observed in native arteries.
In conclusion, we have shown that the local endovascular delivery of a single bolus of AS-PDGFR-β at the injury site is sufficient to block the initial and delayed PDGFR-β protein overexpression, reduce the formation of intimal hyperplasia, and improve the degree of reendothelialization sufficiently to restore endothelium-dependent relaxant function to the injured carotid arteries. These data demonstrate the clinical potential of AS-PDGFR-β to prevent accelerated arteriopathies and promote vascular healing of injured areas.
This study was supported by grants from the Medical Research Council of Canada (MT-14378) and the Heart and Stroke Foundation of Québec to Dr Sirois. Dr Sirois is a recipient of a scholarship from the Heart and Stroke Foundation of Canada, and C.H. Boucher of a studentship from the Fonds pour la Formation de Chercheurs et l’Aide à la Recherche (FCAR)/Fonds de Recherche en Santé du Québec (FRSQ) foundation. We wish to thank Dominique Lauzier for her technical assistance and Dr Jean-François Tanguay for his scientific input.
- Received February 18, 2000.
- Revision received April 6, 2000.
- Accepted April 7, 2000.
- Copyright © 2000 by American Heart Association
Liu MW, Roubin GS, King SB III. Restenosis after coronary angioplasty: potential biologic determinants and role of intimal hyperplasia. Circulation. 1989;79:1374–1387.
Clowes AW, Schwartz SM. Significance of quiescent smooth muscle migration in the injured rat carotid artery. Circ Res. 1985;56:139–145.
Lindner V, Reidy MA. Proliferation of smooth muscle cells after vascular injury is inhibited by an antibody against basic fibroblast growth factor. Proc Natl Acad Sci U S A. 1991;88:3739–3743.
Williams LT. Signal transduction by the platelet-derived growth factor receptor. Science. 1989;243:1564–1570.
Tanizawa S, Ueda M, van der Loos CM, et al. Expression of platelet derived growth factor B chain and β receptor in human coronary arteries after percutaneous transluminal coronary angioplasty: an immunohistochemical study. Heart. 1996;75:549–556.
Raines EW, Bowen-Pope DF, Ross R. Platelet-derived growth factor. In: Sporn MB, Roberts AB, eds. Handbook of Experimental Pharmacology: Peptide Growth Factor and Their Receptors. Heidelberg, Germany: Springer-Verlag; 1990:73–262.
Seifert RA, Hart CE, Phillips PE, et al. Two different subunits associate to create isoform-specific platelet-derived growth factor receptors. J Biol Chem. 1989;264:8771–8778.
Ross R, Masuda J, Raines EW, et al. Localization of PDGF-B protein in macrophages in all phases of atherogenesis. Science. 1990;248:1009–1012.
Majesky MW, Reidy MA, Bowen-Pope DF, et al. PDGF ligand and receptor gene expression during repair of arterial injury. J Cell Biol. 1990;111:2149–2158.
Sirois MG, Simons M, Edelman ER. Antisense oligonucleotide inhibition of PDGFR-β receptor subunit expression directs suppression of intimal thickening. Circulation. 1997;95:669–676.
Ferns GAA, Raines EW, Sprugel KH, et al. Inhibition of neointimal smooth muscle accumulation after angioplasty by antibody to PDGF. Science. 1991;253:1129–1132.
Jackson CL, Raines EW, Ross R, et al. Role of endogenous platelet-derived growth factor in arterial smooth muscle cell migration after balloon catheter injury. Arterioscler Thromb. 1993;13:1218–1226.
Hart CE, Clowes AW. Platelet-derived growth factor and arterial response to injury. Circulation. 1997;95:555–556.
Jawien A, Bowen-Pope DF, Lindner V, et al. Platelet-derived growth factor promotes smooth muscle migration and intimal thickening in a rat model of balloon angioplasty. J Clin Invest. 1992;89:507–511.
Bilder G, Wentz T, Leadley R, et al. Restenosis following angioplasty in the swine coronary artery is inhibited by an orally active PDGF-receptor tyrosine kinase inhibitor, RPR101511A. Circulation. 1999;99:3292–3299.
Banai S, Wolf Y, Golomg G, et al. PDGF-receptor tyrosine kinase blocker AG1295 selectively attenuates smooth muscle cell growth in vitro and reduces neointimal formation after balloon angioplasty in swine. Circulation. 1998;97:1960–1969.
Hart CE, Kraiss LW, Vergel S, et al. PDGFβ receptor blockade inhibits intimal hyperplasia in the baboon. Circulation. 1999;99:564–569.
Edelman ER, Simons M, Sirois MG, et al. c-myc in vasculoproliferative disease. Circ Res. 1995;76:176–182.