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Cardiovascular Research 1999 42(3):761-772; doi:10.1016/S0008-6363(98)00340-X
© 1999 by European Society of Cardiology
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Copyright © 1999, European Society of Cardiology

Angiotensin II receptor antagonists prevent neointimal proliferation in a porcine coronary artery organ culture model

David P. Wilsona,c, Laura Sawarda,c, Peter Zahradkaa,c,* and Po Kee Cheungb

aDepartment of Physiology, University of Manitoba, Manitoba, Canada
bDepartment of Medicine, University of Manitoba, Manitoba, Canada
cInstitute of Cardiovascular Sciences, Rm 3040, St. Boniface General Hospital Research Centre, Winnipeg, Manitoba, Canada R2H 2A6

* Corresponding author. Tel.: +1-204-237-2289; fax: +1-204-233-6723. E-mail address: peterz@sbrc.umanitoba.ca (P. Zahradka)

Received 19 August 1998; accepted 4 November 1998


   Abstract
Top
 Abstract
1 Introduction
 2 Materials
 3 Results
 4 Discussion
 5 Conclusion
 References
 
Objectives: Angiotensin II (AngII) generation in response to vascular injury has long been assumed to influence neointimal proliferation contributing to restenosis. This concept has been supported by evidence that ACE inhibition and AT1 receptor blockade effectively limits restenosis in the rat. On the other hand, ACE inhibition has proven ineffective in clinical trails. The present study examines the response of the porcine coronary artery after angioplasty in vitro and compares the actions of an ACE inhibitor to AngII receptor antagonists. Methods and results: Captopril, an ACE inhibitor, and the AngII receptor antagonists, losartan and PD123319, were evaluated for their ability to attenuate neointimal proliferation in a porcine organ culture model of coronary restenosis. The neointima was significantly increased by 300% after angioplasty compared to non-angioplasty controls. The AT1 receptor antagonist, losartan, produced a significant reduction in neointimal index at 10–5 mol/l, while its in vivo metabolite, EXP3174, reduced neointimal proliferation at 10–6 mol/l. PD123319, a selective antagonist of the AT2 receptor, also restricted neointimal proliferation at 10–5 mol/l. Treatment with captopril (10–6 mol/l) increased the neointimal proliferation by approximately 200% after angioplasty. Conclusions: Direct blockade of AngII receptors effectively inhibits cell proliferation and restenosis post-angioplasty in vitro. ACE inhibition, exclusive of flow, does not attenuate proliferative restenosis. These data suggest that AngII contributes to neointimal proliferation and validates the concept that receptor antagonists could contribute to the therapeutic management of restenosis.

KEYWORDS Restenosis; Angiotensin II; Angioplasty; Smooth muscle, porcine


   1 Introduction
Top
 Abstract
 1 Introduction
2 Materials
 3 Results
 4 Discussion
 5 Conclusion
 References
 
Coronary heart disease is a leading cause of death in the industrialized world. While pharmacological agents such as lipid lowering drugs, antiplatelet/antithrombotic agents and vasodilators have provided some therapeutic benefits for patients with vascular disease, mechanical revascularization remains the most effective approach for dealing with advanced lesions. Procedures such as coronary bypass or angioplasty (with or without stents), however, are costly. Although initially less expensive, angioplasty may double or triple in cost as 30–50% of patients experience symptomatic restenosis of the coronary artery and may therefore require a second or third treatment.

Restenosis is the primary reason for the failure of angioplasty intervention and its development may be attributed to four phenomena: (1) platelet aggregation and thrombus formation, (2) arterial recoil, (3) vascular remodelling and (4) neointimal proliferative response. Currently, a number of therapies have been designed to deal with platelet aggregation, including treatment with heparin, aspirin and other antiplatelet factors [1–4]. Although arterial recoil can be managed reasonably well by inserting stents, the proliferative response is less well understood and, hence, has proved more difficult to treat.

The proliferative response may be divided into four phases: (1) medial smooth muscle cell (SMC) proliferation, which begins 24 h post injury, is associated with SMC death as well as the release of basic fibroblast growth factor (FGF-2), angiotensin II (AngII) and {alpha}-adrenergic hormones, (2) migration of SMCs across the internal elastic lamina (IEL) to form a neointima involves platelet derived growth factor (PDGF), AngII, transforming growth factor (TGF)-β and FGF-2, (3) replication of SMCs in the neointima is stimulated by PDGF, AngII, TGF-β and insulin-like growth factor (IGF)-1 and (4) the vasculature has an increased and prolonged responsiveness to growth factors such as TGF-β, FGF-2, AngII and PDGF [5].

Substantial interest in the role of AngII in neointimal proliferation was fostered by the finding that ACE inhibitors could attenuate neointimal development following angioplasty in the rat [6]. Application of ACE inhibitors or AngII receptor antagonists in rabbit [7], baboon [8] or porcine [9, 10] systems, on the other hand, has provided conflicting data pertaining to the efficacy of either ACE inhibitors or receptor antagonists such as losartan (AT1 specific) and PD123319 (AT2 specific) in blocking restenosis [6, 10–14]. Two explanations have been proposed to account for these species differences. It has been observed that kinin receptor antagonists interfere with the protective effects obtained with ACE inhibitors in rodents [15, 16]. Given that ACE inhibition leads to an increase in circulating kinin concentration, it has been suggested that ACE inhibitors function by promoting kinin accumulation in addition to decreasing AngII levels. Alternatively, tissue AngII synthesis may occur via an ACE-independent mechanism mediated by peptidases such as chymase [17, 18]. Regardless of the source, it seems unequivocal that AngII does indeed play a role in promoting both hypertrophic and hyperplastic vascular SMC growth, and local RAS activation has been implicated in the vascular response to injury. With respect to AngII receptors, the AT1 subtype is by far the best characterized and it is responsible for the modulation of AngII-mediated vasoconstriction. The second major AngII receptor subtype (AT2) has a less well defined role, although a diverse array of functions, such as development [19], apoptosis [20] and proliferation [21], have been attributed to it. While transgenic knockouts of the AT2 receptor have been developed [22], a physiological function for this receptor subtype could not be discerned during fetal developmental.

The objective of this study was to determine whether an ACE inhibitor and/or AngII receptor antagonists were able to directly reduce neointimal proliferation associated with re/stenosis, independent of hemodynamic reduction of vascular wall stress. This aim was accomplished by using a modified in vitro porcine model of coronary stenosis after balloon angioplasty [23] to examine the effects of AngII receptor antagonists and ACE inhibition on neointimal formation. Our findings suggest that the ability of AngII receptor antagonists to inhibit AngII mediated smooth muscle proliferation correlated closely with their ability to attenuate neointimal formation.


   2 Materials
Top
 Abstract
 1 Introduction
 2 Materials
3 Results
 4 Discussion
 5 Conclusion
 References
 
Tissue culture medium (DMEM-high glucose), antibiotics, trypsin and multiwell culture dishes (Linbro) were obtained from ICN-Flow (Costa Mesa, CA, USA). Fetal bovine serum (FBS), antibiotic–antimycotic (100x10 000 U/ml penicillin G, 10 000 µg/ml streptomycin sulfate, 25 µg/ml amphotericin) and culture plates were purchased from Gibco (Grand Island, NY, USA). Superfrost Plus glass microscope slides were supplied by Fisher Scientific (Nepean, ON, Canada). Insulin, pyruvate, transferrin, ascorbic acid, selenium, captopril, AngII and Cy3-conjugated anti-mouse antibody were purchased from Sigma (St. Louis, MO, USA). [Methyl-3H]thymidine was obtained from Dupont-NEN (Mississauga, ON, Canada). Bromodeoxyuridine (BrdU), anti-BrdU and anti-vonWillebrand factor monoclonal antibodies were purchased from Boehringer-Mannheim (Laval, PQ, Canada). Angioplasty catheters (20x3.5 mm) were obtained from Scimed Life Systems (Maple Grove, MN, USA). Losartan and PD123319 were provided by Dupont–Merck and Parke–Davis, respectively.

2.1 Dissection and in vitro coronary preparation
Pig hearts (size matched, 340–440 g, with the exception of the experiment shown in Fig. 3b where the heart weight was 490–660 g) were obtained from the local abattoir and transferred to the laboratory (on ice) within 30 min of excision. Hearts were placed on ice and the LADCA (left anterior descending coronary artery) flushed with PBS containing 10xantibiotic–antimycotic (AB–AM). A standard angioplasty catheter (20x3.5 mm) was inserted into the coronary artery and passed along the LADCA distal to the first major bifurcation. The catheter was inflated to five atmospheres for 1 min. Control vessels were treated in an identical manner with the exception of catheter insertion and inflation, and a similar 20-mm region of the LADCA just distal to the first major branch was used. Each vessel was cut into four equal 5-mm segments, and one control or catheter treated segment was randomly placed per culture dish. Dishes were incubated at 37°C, 5% CO2 in 20% FBS–DMEM–10xAB–AM. Media and drugs were changed every second day with the concentration of AB–AM reduced to 1x over days 6–14.


Figure 3
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  		Fig. 3 Contribution of angioplasty and AngII receptor subtype AT1 antagonist administration to neointimal proliferation 14 days following treatment. Neointimal proliferation in LADCA is expressed as the neointimal index and is relative to "without angioplasty" control (set to 100). (a) Statistically significant differences were observed in comparisons between angioplasty and non-angioplasty groups (+), non-angioplasty and losartan (10–6, 10–7 mol/l) treatment after angioplasty (+), and angioplasty with or without losartan (10–5 mol/l) treatment (*). (b) With EXP3174, the active metabolite of losartan, significant differences were realized between non-angioplasty control, and angioplasty plus EXP3174 treatment (*), as well as between angioplasty and angioplasty plus EXP3174 treated samples (+). Symbols: +, without angioplasty control versus treatment (P<0.05); *, angioplasty control versus treatment (P<0.05). Differences in control groups between the experiment illustrated in (a) and (b) reflect variations in heart weight and number of vessels per treatment (a) n=6–8, mean heart weight 340–440 g, (b) n=4 per group, mean heart weight 490–660 g.

 
2.2 Morphometric determinations
Vessel segments were removed from culture on the fourteenth day, placed in formalin or Streck fixative (Streck, Omaha, NE, USA) and passed through a graded ethanol series prior to embedding in JB-4 (Polysciences, Warrington, PA, USA). Embedding in resin is necessary because the delicate neointima is often lost in standard paraffin or cryosectioned material. The tissue blocks were faced to remove 1.5 mm of the cut surface of the vessel to avoid artifacts generated by the cut site. Sections of 1–2 µm were stained with Lee’s methylene blue, and photomicrographs taken using an Olympus BH2 microscope. Photographs were scanned with an HP Scan Jet at 300 dpi, and imported into SIGMASCAN/IMAGE software (Jandel Scientific) for morphometric determination of intimal and medial areas. The neointimal index represents a ratio of neointimal area to medial area transformed to a percent of non-angioplasty treated control [(treated group neointimal/medial area)/(control group intimal/medial area)]x100 (n=6–8 per treatment).

2.3 Smooth muscle cell culture
Primary cultures of VSMC were prepared by migration from free-floating explants of porcine coronary artery segments [24]. Following 5–10 days in culture, cells began to migrate from explants and attach to the culture dish (migration day 1). The migrating population from day 7–14 was used for all cell studies. Following trypsinization (0.5% trypsin), these cells (at passage 2) were seeded in 24-well culture dishes at 5·103 cells/well and grown in DMEM containing 20% FBS until approximately 75% confluent (1–2 days). The growth medium was removed and replaced with DMEM supplemented with 5 µg/ml transferrin, 1·10–9 mol/l selenium, 2·10–4 mol/l ascorbate, 1·10–8 mol/l insulin for 96 h. Triplicate sets of cells were treated with AngII±receptor antagonists in the presence of 2 µCi [3H]thymidine for 48 h. The cells were subsequently lysed with 1.0 ml of a solution containing 10 mmol/l Tris–HCl, pH 7.4, 100 mmol/l NaCl, 1 mmol/l EDTA and 0.5% SDS, and the nucleic acids, precipitated with an equal volume of cold 20% trichloroacetic acid (TCA), were collected on Whatman GF/A glass fibre filters. The filters were washed four times with 5% TCA, once with ethanol and the radioactivity determined by liquid scintillation counting. In a parallel experiment, cells were treated in the absence of radiolabeled thymidine for 96 h, harvested by trypsinization and total cell number measured using a haemocytometer. A third triplicate set of cells was treated with AngII±receptor antagonists in the presence of 1 µmol/l BrdU. Cells were fixed with methanol 96 h post-treatment and processed for immunocytochemical staining as outlined by the manufacturer.

2.4 Data and statistical analysis
Morphometric, scintillation counts and cell number data were quantified and graphically represented as means±S.E.M. Data were analyzed using ANOVA while the Bonferroni (versus control) method was used to compare treatment means versus non-angioplasty and angioplasty controls (significance was set at P<0.05).


   3 Results
Top
 Abstract
 1 Introduction
 2 Materials
 3 Results
4 Discussion
 5 Conclusion
 References
 
3.1 Neointimal proliferation in response to balloon angioplasty in vitro
Incubation of porcine coronary artery segments in vitro resulted in the development of a neointima consisting of 2–3 cell layers associated with a compact extracellular matrix and few disruptions in the IEL (Fig. 1A and A'). This progression occurred in the presence of a single layer of endothelial cells, although irregularity of staining for von Willebrand’s factor suggested that the process of culture either resulted in some endothelial cell loss or caused a functional change in these cells (data not shown).


Figure 1
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  		Fig. 1 Effect of angioplasty on the left anterior descending coronary vascular morphology by organ culture at 14 days post-treatment. An emphasis has been placed on demonstrating the range of intimal proliferation, the presence of breaks in the IEL and the degree of cell loss and fibrosis in the tunica media. Photomicrographs of LADCA after staining with Lee’s methylene blue, representing the range of response to treatment; (A, A') control without angioplasty or drug treatment, (B, B') following angioplasty, without drug treatment, (C, C') following angioplasty plus 10–5 mol/l losartan, (D, D') following angioplasty plus 10–5 mol/l PD123319. In each pair of micrographs, the first is the minimum response observed over 14 days (A, B, C, D) and the second is the maximum response obtained (A', B', C', D'). Although overlap in the individual response of vessels exists, examination of the entire sample population resolves significant differences between groups (P<0.05). Arrow indicates internal elastic lamina; m=media, n=neointima. Magnification 141x.

 
A 14-day culture period following balloon catheter inflation within the vessels produced a significantly greater neointima compared to non-catheter treated vessels (Fig. 1A and A' vs. B and B'). An important characteristic of this model is the variability in neointimal formation that is observed. For this reason, two panels (minimum response A, B, C, D and maximum response A', B', C', D') are provided for each treatment condition depicted in Fig. 1. Quantification of the neointimal index indicated there was an increase of >300% relative to non-injured control vessels (Fig. 2). Distinguishing features of this neointima include numerous breaks in the IEL, extensive extracellular matrix (ECM) deposition and an increase in cell number with subadjacent medial cell loss. It is important to recognize that the region is not uniform with respect to cell arrangement and composition, since the injury is often eccentric and irregular. Focal areas that exhibit neointimal mass commonly have a pronounced loss of nuclear staining in the subadjacent medial smooth muscle layer (Fig. 1).


Figure 2
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  		Fig. 2 Effect of the ACE inhibitor captopril on proliferative stenotic response. Neointimal proliferation was significantly different between control and angioplasty treated group+ (P<0.05). Administration of captopril (10–6 mol/l) enhanced the index of proliferation by >200% compared with the angioplasty treated group *(P=0.05). The neointimal index of the untreated control group was set to 100 as indicated in Section 2. +, without angioplasty control versus treatment (P<0.05), *, angioplasty control versus treatment (P<0.05) n=6–8.

 
Although preparation of the vessel segments for culture causes a cut site injury, the neointima produced during the healing process, which can be seen in longitudinal section (data not shown), does not extend more than 1 mm from the edge. Furthermore, this region was carefully removed prior to morphometric analysis.

3.2 Effect of ACE inhibition on neointimal proliferation
Administration of the ACE inhibitor captopril at doses of 10–6 mol/l in rats has proven effective in reducing neointimal proliferation post angioplasty [6], however, ACE inhibitors have proven less effective in reducing neointimal proliferation in non-rodent systems [25, 26]. In order to measure the efficacy of captopril on inhibiting neointimal proliferation in the in vitro porcine model we chose a dose of 10–6 mol/l. It was observed that, rather than reducing neointimal area, captopril increased the magnitude of neointimal proliferation 200% beyond balloon injured vessels (Fig. 2, Table 1). The elevation in neointimal index obtained with captopril treatment may represent either an increase in intimal area or a reduction in medial area. Table 1 demonstrates, however, that captopril treatment leads to a profound increase in intimal area with little effect on medial area. Since this inhibitor was ineffective in reducing neointimal proliferation induced by balloon catheter inflation, the contribution of AngII, or at least its mode of generation, was brought into question. Therefore, to circumvent the question of mechanism of AngII generation we opted to target an AngII mediated proliferative response via AT1 and AT2 specific receptor blockade.


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  		Table 1 Effect of angioplasty and angioplasty plus administration of captopril on intimal and medial area

 
3.3 Actions of AngII AT1 and AT2 receptor blockade on neointimal proliferation
LADCA culture in the presence of the AT1 receptor antagonist losartan over the concentration range 10–7 to 10–5 mol/l produced a dose-dependent reduction in neointimal proliferation (Fig. 3a). The concentration of losartan required to reduce neointimal proliferation to control levels was high, 10–5 mol/l. In fact, losartan treatment at 10–5 mol/l produced a 69% reduction in the neointimal index compared with angioplasty controls. At more pharmacological doses (10–6 and 10–7 mol/l), losartan reduced intimal proliferation by 49 and 41%, respectively, compared to angioplasty alone; however, the almost 50% reductions in neointimal index were not statistically significant. The lack of statistical significance is, in part, a consequence of the variability that exists in response to angioplasty (see Fig. 1C and C') which reduces the statistical power and thereby the ability to resolve differences between group means. Nevertheless, visual inspection demonstrates that differences between the treatments can be readily distinguished. The significant reduction in neointimal index observed with high doses of losartan warranted examination of the effective concentration of EXP3174, the active metabolite of losartan in vivo, since it was uncertain if losartan is metabolized effectively in vitro [27]. The reduction in neointimal index obtained with 10–6 mol/l EXP3174 (Fig. 3b) was not significantly different from losartan at 10–5 mol/l, confirming that indeed EXP3174 is an order of magnitude more potent than losartan. Interestingly, higher concentrations of EXP3174 (e.g. 10–5 mol/l) produced a neointima not significantly different from angioplasty treatment alone (Fig. 3b), suggesting a possible agonist effect or cross-reaction with other receptors at high doses.

Blockade of the AT2 receptor using PD123319 over the range 10–7 to 10–5 mol/l produced a dose-dependent reduction in neointimal proliferation (Fig. 1D and D', Fig. 4). Analysis of data obtained with PD123319 treatment revealed a response that was similar to that obtained with the AT1 receptor antagonist. Treatment of vessels with high concentrations of PD123319 (10–5 mol/l) produced a neointimal index that was not significantly different from control and represented a 67% reduction in neointimal index compared with angioplasty treatment alone. Administration of PD123319 at lower concentrations (10–6, 10–7 mol/l) produced a 43% reduction and 2% increase in neointimal index, respectively (P=NS). It is interesting to compare the response of 10–7 mol/l losartan which produced a 41% reduction in neointimal index, whereas PD123319 over the same concentration range produced no intimal reduction. Losartan and PD123319, however, performed equally well in the range 10–5–10–6 mol/l, although there was a apparent reduction in both neointimal and medial proliferation produced by these drugs (Table 2).


Figure 4
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  		Fig. 4 Contribution of angioplasty and AngII receptor subtype AT2 antagonist administration to neointimal proliferation 14 days following treatment. Neointimal proliferation in the left anterior descending coronary artery is expressed as the neointimal index (n=6–8, mean heart weight 340–440 g). Non-injured vessels have a significantly lower index of proliferation (+) compared to angioplasty-treated coronary explants. Angioplasty plus the administration of 10–5 mol/l PD123319 produced a neointimal index that was significantly lower than the angioplasty treated control group (*). Lower concentrations of PD123319 (10–6 mol/l, 10–7 mol/l) resulted in neointimal proliferation significantly greater than non-angioplasty control levels (+). Symbols: +, without angioplasty control versus treatment (P<0.05); *, angioplasty control versus treatment (P<0.05).

 

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  		Table 2 Effect of angioplasty and angioplasty plus administration of Losartan and PD123319 on intimal and medial area

 
3.4 Effect of AngII receptor blockade on smooth muscle cell proliferation
To establish whether the AngII receptor antagonists could directly inhibit porcine vascular SMC proliferation, losartan and PD123319 were added to explant-derived cultures of quiescent porcine coronary artery SMCs which had been treated with 10–6 mol/l AngII. Either losartan or PD123319 blocked the stimulation of bromodeoxyuridine incorporation by AngII when added individually to the cultures (Fig. 5). Confirmation that this effect was coupled to an inhibition of DNA synthesis was obtained by measuring [3H]thymidine incorporation in the presence of losartan or PD123319 (Fig. 6). These data indicate that losartan or PD123319 (10–5 mol/l) interfere with the action of AngII on SMC proliferation resulting in a 35 and 30% reduction in DNA synthesis, respectively, compared to AngII treated controls. These values represent an inhibition of the stimulation produced by AngII of ~100% for losartan and PD123319. Furthermore, in order to determine if AngII treatment resulted in an increase in cell number, total cell counts were made following AngII and receptor antagonist administration. Treatment with AngII plus either losartan or PD123319 (10–5 mol/l) reduced cell number by 29% compared to AngII alone (Fig. 7). This value represents a 71% inhibition of the increase in cell number attributed to AngII.


Figure 5
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  		Fig. 5 Bromodeoxyuridine (BrdU) incorporation by explant-derived coronary artery smooth muscle cells. Experimental treatments were (A) quiescent cells, (B) following 10–6 mol/l AngII, (C) following AngII plus losartan 10–6 mol/l, and (D) AngII plus PD123319 10–6 mol/l. BrdU incorporation was visualized using anti-BrdU/Cy3 and epifluorescence microscopy. Magnification: 43x.

 

Figure 6
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  		Fig. 6 Effect of AngII receptor antagonists on thymidine incorporation by explant-derived porcine coronary artery SMCs. Thymidine incorporation was measured following administration of AngII (10–6 mol/l), AngII plus losartan (10–6 mol/l), and AngII plus PD123319 (10–6 mol/l) to quiescent SMCs. Statistically significant differences are indicated for control versus AngII treatment (+, P<0.05), and AngII treatment alone versus AngII treated with antagonist (*, P<0.05). Control=quiescent cells without AngII stimulation.

 

Figure 7
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  		Fig. 7 Influence of receptor antagonists on AngII-stimulated cell division. Effects of exogenously added AngII (10–6 mol/l) in the presence or absence of losartan or PD123319 on cell number were conducted with explant-derived coronary artery smooth muscle cells. Statistically significant differences (P<0.05) between AngII treated and unstimulated (quiescent) controls (+), and AngII control versus AngII treated with agonist (*) are indicated. Total cell number was determined 96 h after stimulation.

 

   4 Discussion
Top
 Abstract
 1 Introduction
 2 Materials
 3 Results
 4 Discussion
5 Conclusion
 References
 
To address the issue of whether local production of AngII by SMCs is sufficient to mediate restenosis, we employed an organ culture model of stenosis following balloon angioplasty. The vascular responses observed in this system reflect the reaction of SMCs in the absence of infiltrating cells, pressure, flow, and recolonization by endothelial cells. The organ culture model thus provides a unique opportunity to examine SMC-derived factors.

Proliferation leading to an additional two to three layers of intimal cells was observed in the non-injured control vessel (Fig. 1A and A'). As reported by Voisard et al. [23], who used a coronary artery organ culture model similar to the one employed in this investigation, as well as Koo and Gotlieb [28] using a porcine aortic organ culture system, formation of a limited neointima appears to be a normal response of vessels placed into culture even in the presence of an apparently intact endothelial layer. Nevertheless, significantly greater neointimal proliferation was observed after balloon injury (Fig. 1B and B', Fig. 2). The ability to reproducibly detect changes in neointimal size indicated that this model was a suitable tool for evaluating the role of AngII as a mediator of smooth muscle cell proliferation in response to injury. In addition, several vascular changes that were detectable in the vessel segments after injury were useful for full characterization of the response. These included: (1) increased intimal thickening, (2) disruption of the internal elastic lamina, (3) abundant ECM deposition, (4) accumulation of SMC in the intimal region and (5) focal subintimal/medial cell loss and associated fibrosis (Fig. 1).

Systemic and local vascular AngII has two biological functions, since it operates to both maintain and increase blood pressure as well as to stimulate VSMC growth [29–31]. It has become evident that these functions are independent but may be indirectly cooperative, since both hypertrophic and hyperplastic growth occur under conditions where blood pressure has either been maintained at normal levels or elevated with AngII administration [30, 31]. Independent of blood pressure change, AngII apparently functions by promoting the rapid synthesis and release of growth factors such as PDGF and TGF-β [5, 32–35], as well as hypertrophic agents such as endothelin [36]. A direct role for AngII in neointimal proliferation was indicated by Laport and Escher [37] who demonstrated that [Sar1]AngII, a peptide antagonist, prevented neointimal formation. Blockade of AngII synthesis with ACE inhibitors has been shown to attenuate hypertension and injury-induced proliferation in rat [6]. Contrary to the rat model, experiments conducted in rabbit [7], baboon [8], porcine [9, 10] and human [24, 25] systems have not found ACE inhibitors effective in reducing neointimal proliferation. Using the present model, which eliminated the variables of vascular wall tension and blood flow (among others), we found that the ACE inhibitor captopril in fact increased neointimal area by >200%. A comparable effect has not been observed in vivo, suggesting other factors not active in the organ culture system are important elements of the vascular response exclusive of the smooth muscle component. In order to reconcile this conflict, one may consider the possibility that the vasodilatory effects (reduction in wall tension) of ACE inhibitors in vivo may offset a wall stress-dependent proliferative effect. A second consideration is that clinical studies, such as the recently completed MERCATOR [24] and MARCATOR [25] trials, examining ACE inhibitor efficacy in reducing restenosis utilized angiography as an index of restenosis. Although angiography provides anatomical evidence for the development of a lesion, it cannot identify the mechanism responsible for its formation. Only a complete histological examination could distinguish, for example, between reduced neointimal proliferation and compensatory vasodilatation. Indeed, a number of recent reports [15, 16, 38, 39] examining the mechanism of ACE inhibitor action in congestive heart failure support the hypothesis that ACE inhibitor action may be largely mediated by an accumulation of bradykinin as much as or more than reductions in AngII. In fact, bradykinin has been shown to induce proliferation in a number of cell types [40–42], however, this has yet to be demonstrated specifically in SMCs. A further alternative is that there exist species-dependent variation in the pathways responsible for generating tissue-derived AngII. Okonishi et al. [43] has put forth the hypothesis that AngII may be generated locally via a chymase-dependent mechanism. If this is true, ACE inhibitors would have less effect on local AngII concentrations yet still reduce systemic AngII and elevate systemic bradykinin levels. Danser et al. [44], however, have recently shown using isolated perfused porcine coronary and carotid arteries (a preparation similar to ours) that the bulk of AngII generation is via a mechanism that is abrogated by administration of the ACE inhibitor captopril. These data do not take into account the contributions of blood cells which infiltrate injured tissue almost immediately and are known to be a potent source of proteolytic enzymes, including chymases [45]. The current literature suggests that the majority of AngII production in rat occurs via an ACE-dependent mechanism [43, 46, 47]. In other species such as dog, there appears to be a 60:40 split between ACE and ACE-independent mechanisms [17, 47, 48]. With respect to humans, the literature suggests local AngII production may involve a chymase-dependent pathway [47, 49, 50]. It is important to realize, however, that consensus on this point has not been reached. For example, the data of Zisman et al. [51] supports the existence of a local ACE-dependent pathway, while the data of Wolny et al. [50] as well as recent work from Balcells et al. [47], point to a serine protease, perhaps chymase, as important in local AngII production in humans. Interestingly, it is possible that the presence of infiltrating cells, which are capable of converting AngI to AngII in tissue preparations, or methodological differences as outlined by Balcells and coworkers [47, 52] could explain some of these apparently contradictory results. It is therefore possible that AngII produced in response to vascular injury could be derived from both ACE-dependent and ACE-independent pathways.

Once the effect of ACE inhibition was realized, it became important to establish if direct blockade of AngII receptors, independent of the effects on wall stress reduction, is able to attenuate neointimal proliferation in response to injury. As indicated in the results, there was a concentration-dependent reduction in neointimal index brought about by AT1 or AT2 receptor blockade (Figs. 3 and 4Go). Indeed, our results with an AT1 receptor antagonist at concentrations of 10–6 mol/l are in agreement with the results of Huckle et al. [10] who reported a non-significant 12% reduction in intimal thickness was realized using the AT1 receptor antagonist L-158809. Our experiments, which examined the vascular response to a broader concentration range of receptor antagonist (one major advantage of the in vitro culture model), demonstrated that high concentrations of losartan (10–5 mol/l) significantly attenuated neointimal proliferation. Is this a real effect or is losartan at 10–5 mol/l toxic? Data derived from treatment with the more active losartan metabolite EXP3174 revealed that lower concentrations are required to attenuate proliferation, and even higher concentrations (10–5 mol/l) restore intimal proliferation to angioplasty-treated control levels. These results suggest either a partial agonist effect or cross reactivity with other receptors at high concentrations, and clearly indicate the absence of toxicity.

It is interesting that many groups [10, 30, 31], in an effort to attenuate vascular proliferation, have administered AT1 receptor antagonists at concentrations that are effective in normalizing or reducing blood pressure. This practice assumes that all effects mediated by the AT1 receptor are pressure dependent, and precludes the following alternatives: (1) trophic effects may be mediated by AT1 pressure-dependent and pressure-independent mechanisms, where pressure-independent mechanisms may require greater receptor occupancy than would be required to normalized blood pressure and (2) changes in receptor density at a local/regional injury site may cause these areas to become more or less sensitive to administration of a receptor antagonist than the non-injured vasculature. The latter point is supported by evidence showing vascular tissue that has been injured does not have the same receptor profile as uninjured tissue. Specifically, it was demonstrated that, although AT1 receptor mRNA expression is elevated upon administration of losartan, receptor binding is actually reduced in the kidney [53]. As such, drug dosages should be tailored to effect a change in action at the site of injury rather than systemic effects. If this precludes the use of systemic administration due to systemic toxicity, then local delivery of drugs with a shorter half-life should be sought.

The response to injury in the model is not uniform, as demonstrated in Fig. 1 and by the variability in neointimal index. This is very similar to the clinical setting where only 30–50% of patients develop restenosis as defined by angiography. Nevertheless, it is realized that all patients undergo restenosis to some degree. In the statistical analysis of our data, we have chosen not to partition high responders from low responders since we are at present unable to do this clinically prior to treatment. This limitation in the paradigm may actually be an asset in that it closely represents the clinical situation of variable restenosis in both rate and extent. Our cell culture studies, in which the experimental conditions are more uniform, provide supportive evidence that AngII receptor antagonists may be effective at lower concentrations in a selected and less heterogenous culture system of migrating SMCs.

Reduction in neointimal proliferation post-angioplasty [13], as well as in medial arterial wall hypertrophy and fibrosis in hypertensive rats [30], has been obtained with both local administration (at 1 mg/kg/day) and high systemic doses (30 mg/kg/day) of AT2 receptor antagonist, respectively. While Huckle et al. [10] reported AT2 receptor antagonism to be ineffective in reducing neointimal proliferation, their systemic route of administration provided significantly lower early concentrations of drug (24 nmol/l) than we (Fig. 4) or others have found successful [12, 21]. Our cell culture data with AT2 receptor antagonism support a number of other studies over a range of cell types, including smooth muscle, that indicate the presence of an AT2 receptor antagonist can attenuate cell proliferation [53–57]. In contrast, an elegant study by Nakajima et al. [58], in which AT2 receptors were over-expressed in vascular SMCs and injured carotid arteries, showed the presence of excess AT2 receptors actually inhibited cell growth and neointimal formation, possibly through the induction of apoptosis. The involvement of AT2 receptors in these processes was confirmed by abrogation of this response through administration of an AT2 receptor antagonist. Interestingly, one of the commonly used AT2 receptor antagonists, CGP42112A, has been shown to act as both an antagonist and agonist, depending on the concentration used [59, 60]. This finding may account for some of the discrepancies in proliferative versus anti-proliferative effects attributed to AT2 receptor activation. Clarification of these seemingly opposite observations should provide insight for understanding the biological contribution of the AT2 receptor to RAS function.


   5 Conclusion
Top
 Abstract
 1 Introduction
 2 Materials
 3 Results
 4 Discussion
 5 Conclusion
References
 
We have provided two lines of evidence at the cell and organ level that AngII receptor blockade of both AT1 and AT2 receptors at high concentrations is effective in attenuating cell and neointimal proliferation. In contrast, the ACE inhibitor captopril, independent of its blood pressure lowering effect, does not have a significant effect in attenuating proliferative restenosis and may actually cause increases in neointimal proliferation. Based on these results, we anticipate that local delivery of high concentrations of AngII receptor antagonists in vivo, as shown by Taguchi et al. [12], does indeed result in a reduction in the proliferative element of stenosis following angioplasty. Considering the multitude of co-factors involved in restenosis, it is unlikely that blockade of the RAS in vivo will completely abrogate proliferative restenosis, as we have shown in vitro. Furthermore, the results we have obtained reflect only the responses of the vascular smooth muscle component and do not address the possible influence of other factors released from other sources. On the other hand, combinatorial treatment with an AngII receptor antagonist and an agent which influences an unrelated cellular process may be more successful, as has recently been demonstrated by Matsuno et al. [61] using losartan and a fibrinogen receptor antagonist.

Time for primary review 22 days.


   Acknowledgements
 
The authors would like to thank Dr. E.K. Kardami for a critical review of the manuscript. This study was supported by grants from the Paul H.T. Thorlakson Foundation Fund and the St. Boniface Research Foundation (PKC) and from the Medical Research Council Group in Experimental Cardiology (PZ). DW is the recipient of a University of Manitoba Graduate Fellowship and LS was supported by a studentship from the Manitoba Health Research Council.


   References
Top
 Abstract
 1 Introduction
 2 Materials
 3 Results
 4 Discussion
 5 Conclusion
 References
 

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