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Am J Physiol Heart Circ Physiol 281: H1648-H1656, 2001;
0363-6135/01 $5.00
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Vol. 281, Issue 4, H1648-H1656, October 2001

Bradykinin receptor antagonists attenuate neointimal proliferation postangioplasty

Lorraine Yau, David P. Wilson, Jeffery P. Werner, and Peter Zahradka

Department of Physiology, University of Manitoba and Institute of Cardiovascular Sciences, St. Boniface General Hospital Research Centre, Winnipeg, Manitoba, Canada R2H 2A6


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Bradykinin has been linked to the development of restenosis in response to vascular injury. We therefore examined the effect of bradykinin on vascular smooth muscle cell growth and neointimal formation in organ culture. Bradykinin stimulated both RNA and DNA synthesis (by 175%) in smooth muscle cells from either porcine or human coronary arteries and increased cell number in a concentration-dependent manner. Both p42/44 mitogen-activated protein kinase (MAPK) and p38 kinase were also activated. Treatment with [Hyp3,Tyr(Me)8]bradykinin, a B2 receptor agonist, stimulated thymidine incorporation by 146%, whereas B1-selective Lys-des-Arg9-bradykinin had no effect. Addition of the B2 antagonist HOE-140 reduced the stimulation by 56%, whereas B1-selective des-Arg-HOE-140 had no significant effect. Similarly, HOE-140 attenuated angioplasty-induced neointimal formation in organ culture with an efficacy approaching 100% inhibition. These experiments suggest that bradykinin promotes smooth muscle proliferation after vascular injury, presumably via B2 receptor-dependent activation of MAPK family pathways, and may explain the negative outcome of angiotensin converting enzyme inhibitor therapy on restenosis in nonrodent models.

restenosis; vascular smooth muscle; angioplasty; porcine; human; HOE-140


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

BALLOON ANGIOPLASTY has become one of the most common interventions for the treatment of coronary artery disease. However, the incidence of failure due to restenosis remains relatively high (30-50%), often necessitating a second revascularization procedure. Restenosis is the result of compromised vascular integrity, and its development is characterized by the following four events: 1) platelet aggregation and thrombus formation, 2) arterial hyperresponsiveness and recoil, 3) vascular remodeling, and 4) neointimal proliferation of smooth muscle cells (SMC) (30). Although a number of treatments have been designed to counter some of these responses, including deployment of stents and antiplatelet glycoprotein therapy, significant reductions in restenosis rates have not been achieved (21).

Neointimal formation is the culmination of a number of cellular events occurring in the vessel wall. It has been clearly established that the neointima develops as a result of medial SMC proliferation and subsequent migration across the internal elastic lamina (30). A variety of growth factors, cytokines, and vasoactive substances influence SMC migration and proliferation both in culture and in animal models (5, 20, 30). Despite intensive study to determine the efficacy of contractile antagonists in restricting neointimal formation, less effort has been directed toward vasodilatory agents such as bradykinin (BK).

The beneficial effects of angiotensin-converting enzyme (ACE) inhibition in heart failure and restenosis have been attributed to the regulation of both angiotensin II (ANG II) synthesis and BK degradation by ACE (11). BK contributes to wound healing (both exudative and inflammatory phases), and elevated levels of BK have been found after vascular injury (10, 17). As such, BK becomes a logical target for study in the process of restenosis. Although BK may be expected to influence the vasomotor response under these conditions to oppose recoil, it may also contribute to the remodeling process. The failure of ACE inhibitor therapy to reduce clinical rates of restenosis, for example, suggests that BK has a more prominent role in damaged and diseased arteries than originally anticipated from animal models.

The objective of this study was to determine whether BK influences SMC growth independent of the endothelium and to define the contribution of BK type 1 (B1) and type 2 (B2) receptors. This goal was accomplished using primary cultures of both porcine and human coronary artery SMCs to measure specific parameters of cell proliferation and monitor activation of intracellular signaling. Additionally, an in vitro porcine model of coronary stenosis (36) was employed to examine the significance of BK to neointimal formation. Our results suggest that stimulation of SMC growth by BK may promote neointimal proliferation postangioplasty in the absence of endothelium.


    METHODS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Cell culture. Primary cultures of SMCs were prepared by migration from free-floating explants of porcine coronary artery segments isolated from fresh porcine hearts (29). Alternatively, this approach was adapted for the selective isolation of SMCs from human coronary artery atherectomy samples obtained in accordance with a protocol approved by the University of Manitoba. Cells were propagated in DMEM (GIBCO-BRL) containing 20% fetal bovine serum (GIBCO-BRL). Cells were used after the second passage only to ensure consistency between preparations. When the cells were 75% confluent, the growth medium was replaced with DMEM supplemented with 5 µg/ml transferrin, 1 nmol/l selenium, 20 mmol/l ascorbate, 11 µg/ml pyruvate, and 10 nmol/l insulin for 96 h. Quiescent cells were used for all growth assays.

RNA and DNA synthesis. Triplicate sets of cells were treated with BK ± receptor antagonists [HOE-140 and des-Arg-HOE-140 (dA-HOE), Peninsula Laboratories] in the presence of 2 µCi [3H]thymidine (New England Nuclear) for 96 h. Alternatively, cells were stimulated with receptor-specific agonists {Lys-des-Arg9-BK; Sigma and [Hyp3,Tyr(Me)8]BK; Calbiochem} instead of BK. Parallel treatments were conducted for 6 h in the presence of 2 µCi [3H]uridine (New England Nuclear). Incorporation of radiolabel into DNA or RNA was monitored by trichloroacetic acid precipitation as previously described (37).

Cell number. Triplicate sets of cells were treated with BK ± receptor antagonists for 96 h and harvested by trypsinization, and the total cell number was quantified with a Coulter counter.

Bromodeoxyuridine incorporation. Cells were treated in triplicate with BK ± receptor antagonists in the presence of 1 µmol/l bromodeoxyuridine (BrdU), fixed with methanol 96 h posttreatment, and processed for immunocytochemical staining with anti-BrdU antibody as outlined by the supplier (Roche).

Organ culture. Porcine hearts were transferred on ice from the abattoir. A standard angioplasty catheter (3.5 mm ± 20 mm; Scimed Life Systems) was inserted into the left anterior descending coronary artery distal to the first major bifurcation. The catheter was inflated to 6 atmospheres for 1 min. After dissection from the heart, each vessel was cut into four equal 5-mm segments, and individual segments (from control or injured vessels) were randomly placed into culture wells (12-well dishes) containing DMEM with 20% fetal bovine serum and ×10 antibiotic/antimycotic (GIBCO-BRL). Media and treatments were changed every second day with the concentration of antibiotic/antimycotic reduced to ×1 over days 6-14. Details of this model have been published previously (36).

Morphometry. Vessel segments were removed from culture on the 14th day and embedded in JB-4 resin (Polysciences). 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 digital photographs captured with a DAGE 330 camera were imported into SigmaScan/Image software (Jandel Scientific) for morphometric determination of intimal and medial areas (36). The neointimal index represents a ratio of neointimal area to medial area.

Immunoblot analysis. Cells prepared in 12-well culture dishes were treated with BK ± receptor antagonists and lysed with SDS/gel loading buffer (37). Equal amounts of each lysate (according to protein content) were then loaded onto a 10% polyacrylamide gel, transferred to polyvinylidine difluoride membrane, and immunostained with the following specific antibodies: PY20 (1:5,000 dilution; Transduction Laboratories); phospho/nonphospho-mitogen-activated protein kinase (MAPK, 1:1,000 dilution; New England Biolabs); and phospho/nonphospho-p38 kinase (1:1,000 dilution; New England Biolabs).

Data and statistical analysis. Morphometric, radiotracer incorporation, and cell number data were quantified and graphically presented as means ± SE. Densitometric analysis of autoradiograms was conducted with a Bio-Rad G670 densitometer under nonsaturating conditions as previously described (37). Data were analyzed with either the Student's t-test or the Fisher's least significant difference ANOVA to compare treatment means versus controls (significance was set at P < 0.05).


    RESULTS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Effect of BK on SMC growth and proliferation. The administration of BK (10-9 to 10-5 mol/l) to porcine SMC in culture resulted in a concentration-dependent increase in SMC growth. Ninety-six hours after treatment, only the highest BK concentration produced a significant increase in DNA synthesis as measured by [3H]thymidine incorporation (Fig. 1A). In parallel with the [3H]thymidine incorporation study, levels of RNA synthesis were also determined. Six hours after BK administration, there was a significant concentration-dependent increase in [3H]uridine incorporation, with 10-6 and 10-5 mol/l being the most effective (Fig. 1B). Although [3H]thymidine incorporation is a reliable index of DNA synthesis, it was unclear whether exogenously applied BK led to SMC hypertrophy or hyperplasia. Consequently, cell number was quantified directly using a Coulter counter. Both 10-6 and 10-5 mol/l BK significantly increased SMC cell number compared with cells without BK treatment (Fig. 1C). Although it may be argued that these concentrations of BK are nonphysiological, previous studies in our lab documented the effect of proteolytic enzymes on the efficacy of ANG II and showed there is a 100-fold change in the effective concentration (Litchie and Zahradka, unpublished observations). Furthermore, the conditions used to prepare the cells for experiments (specifically no media change after a 96-h incubation to ensure cell quiescence before BK addition) lead to high levels of secreted proteases capable of rapidly degrading this peptide and, thus, influence the concentration response. However, attempts to compensate by addition of a protease inhibitor cocktail resulted in complete inhibition of cell proliferation (tested with a nonpeptide mitogen), presumably due to nonspecific inhibition of an essential secreted proteinase (Wilson and Zahradka, unpublished observations). Therefore, on the basis of the response to BK at 10-5 mol/l (Fig. 1), this concentration was used in all subsequent studies with BK receptor antagonists.


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  		Fig. 1.   Effect of bradykinin (BK) concentration on smooth muscle cell (SMC) proliferation. Thymidine incorporation (A) and uridine incorporation (B) in response to various BK concentrations (10-9 to 10-5 mol/l) was used to assess DNA and RNA synthesis, respectively. Data are expressed as percent control with control (untreated, quiescent SMCs) set to 100%. Cell number (C) was quantified with a Coulter counter 96 h after stimulation of quiescent cells with BK (10-7 to 10-5 mol/l). *Statistically significant differences for control vs. BK treatment, P < 0.05. Data are presented as means ± SE (n = 3 experiments).

Effect of BK receptor blockade on SMC growth. Once it was established that BK stimulated SMC growth, the effect of nonpeptide B1 and B2 BK receptor antagonists on SMC growth was examined. Both HOE-140 (B2-selective) and dA-HOE (B1-selective) significantly attenuated BK-mediated [3H]thymidine incorporation at 10-6 mol/l (Fig. 2A). More specifically, B1 and B2 receptor blockade reduced [3H]thymidine incorporation by 82% and 56%, respectively. When the receptor antagonists were combined, DNA synthesis was reduced to control levels (Fig. 2A). BK receptor antagonists were then assayed for their effect on BK-mediated cell division. Interestingly, whereas either 10-6 mol/l HOE-140 or 10-6 mol/l dA-HOE significantly attenuated BK-mediated [3H]thymidine incorporation (Fig. 2A), neither produced a statistically significant decrease in cell number when added individually (Fig. 2B). Nevertheless, the values of the mean for these treatments were lower than in the absence of the antagonists. In contrast, combined administration of the receptor antagonists prevented the increase in cell number obtained with BK treatment (Fig. 2B).


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  		Fig. 2.   Effect of BK receptor antagonists on SMC proliferation. Thymidine incorporation (A) was measured after the administration of BK ± receptor antagonists to quiescent SMCs. Data are expressed as percent control with control (untreated, quiescent SMCs) set to 100%. Total cell number (B) was determined with a Coulter counter 96 h after stimulation. dA-HOE, des-Arg-HOE-140. *Statistically significant differences for control vs. BK treatment, P < 0.05; **BK vs. BK plus antagonists, P < 0.05. Data are presented as means ± SE of individual experiments conducted in triplicate.

In agreement with the thymidine incorporation experiments, BK (10-5 mol/l) increased the number of cells staining positive for BrdU from 1 to 22%, as determined by immunochemical staining after 96 h (Fig. 3). Attenuation of BrdU incorporation to near basal levels was achieved on the addition of either HOE-140 (10-6 mol/l) or dA-HOE (10-6 mol/l) individually.


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  		Fig. 3.   Modulation of bromodeoxyuridine (BrdU) incorporation by BK-stimulated SMCs with BK receptor antagonists. Quiescent SMCs were stimulated with BK (10-5 mol/l) for 96 h in the presence or absence of HOE-140 (10-6 mol/l) or dA-HOE (10-6 mol/l). Incorporation of BrdU was visualized by anti-BrdU/Cy3 epifluorescence microscopy (magnification ×40). Untreated cells (A), BK-treated cells (B), and BK-treated cells coincubated with either HOE-140 (C) or dA-HOE-140 (D). Quantitative analysis was performed by counting positive cells in 4 distinct fields for each slide. E: means ± SE for three independent experiments are presented graphically. *Statistically significant differences vs. untreated control cells, P < 0.05.

Because receptor specificity of antagonists can be of concern when they are employed at high concentrations, we validated the results described in Fig. 2A by monitoring [3H]thymidine incorporation after treating the cells with the receptor selective agonists Lys-des-Arg9-BK (B1 specific) and [Hyp3,Tyr(Me)8]BK (B2 specific). It was observed that only [Hyp3,Tyr(Me)8]BK was stimulatory and, in contrast, Lys-des-Arg9-BK had no effect, even at a concentration of 10-4 mol/l (Fig. 4).


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  		Fig. 4.   Influence of BK receptor agonists on thymidine incorporation by quiescent SMCs. Thymidine incorporation was measured after treatment of quiescent SMCs with either Lys-des-Arg9-BK (LdABK) or [Hyp3,Tyr(Me)8]BK (HTBK) at various concentrations as indicated. Control, quiescent cells without agonist stimulation. Data are presented as means ± SE (n = 3 experiments) expressed as a percent relative to control (set to 100%). *Statistically significant differences for control vs. treatment, P < 0.05.

Effect of BK and BK receptor blockade on human SMC growth. Although porcine SMCs provide an excellent model for studying SMC growth, the relevance to human physiology is unclear. To address the possibility that species variation may exist, this study was extended to include primary cultures of SMC obtained from atherectomy samples of human coronary artery. Addition of BK increased DNA synthesis as measured by [3H]thymidine incorporation in a concentration-dependent manner (data not shown). As was observed with the porcine SMCs, 10-6 and 10-5 mol/l BK were the most effective concentrations. The administration of BK (10-5 mol/l) plus BK receptor antagonists to human SMC produced a reduction in DNA synthesis by 85% and 74% for the B2 and B1 receptor blockers, respectively (Fig. 5). Combined addition of B2 and B1 receptor antagonists did not influence DNA synthesis further (Fig. 5). Direct quantification of cell number in one experiment revealed 8,800 ± 320 cells per well (24-well dish, n = 3) after 96 h in the absence of BK. BK (10-5 mol/l) stimulation resulted in 13,580 ± 980 cells per well, whereas inclusion of both BK receptor antagonists (HOE-140 + dA-HOE, both at 10-6 mol/l) reduced this number to 8,380 ± 220 cells per well. When added individually, no significant effect was observed, as seen in Fig. 2B. Similar results were obtained in two independent experiments.


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  		Fig. 5.   Effect of BK and BK receptor antagonists on thymidine incorporation by human coronary artery SMCs. Thymidine incorporation was measured after the administration of BK (10-5 mol/l), BK plus 10-6 mol/l HOE-140, BK plus 10-6 mol/l dA-HOE-140, and BK plus both receptor antagonists (10-6 mol/l each) to quiescent SMCs. Data are expressed as percent control with control set to 100%. Control, quiescent cells without BK stimulation. *Statistically significant differences for control vs. BK treatment, P < 0.05; **BK vs. BK plus treatment with antagonists, P < 0.05. Data are presented as means ± SE (n = 3 experiments).

Effect of BK and BK receptor antagonists on neointimal proliferation. Previous studies have shown that after injury, tissue BK levels become elevated (10, 17). After we established that high concentrations of BK are capable of enhancing SMC growth and that the BK receptor antagonists attenuate growth, it seemed logical to determine whether high concentrations of BK and BK receptor antagonists also affected neointimal proliferation postangioplasty. We have previously shown with an in vitro no-flow model of stenosis that injury due to balloon inflation results in an increase in the neointimal index (36). Inclusion of BK at 10-5 mol/l in the culture media neither reduced nor augmented neointimal proliferation postangioplasty (Fig. 6). Similar negative results were obtained in the absence of angioplasty (data not shown). On the other hand, addition of HOE-140 to BK-treated vessels postangioplasty significantly reduced the neointimal index compared with either angioplasty or angioplasty plus BK treatment (Fig. 6). In comparison, the B1 receptor antagonist dA-HOE, did not reduce the neointimal index significantly. Simultaneous addition of both receptor antagonists was no more effective than HOE-140 alone (Fig. 6).


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  		Fig. 6.   Effect of BK and BK receptor antagonists on angioplasty-induced neointimal (n) formation in organ culture. Contribution of angioplasty, BK (10-5 mol/l), and BK receptor antagonist (10-6 mol/l HOE-140, 10-6 mol/l des-Arg-HOE-140). Administration to neointimal formation is presented at 14 days after treatment. A-C: micrographs of vessel sections prepared and stained with Lee's methylene blue after 14 days of culture. m, Medial region. The specific conditions are noninjured (A), balloon injured and treated with BK (B), and balloon injured and treated with BK, HOE-140, and dA-HOE (C). Medial and intimal areas were calculated as described in METHODS (D). Neointimal formation is expressed as the neointimal index (n/m area) and is relative to "without angioplasty" control. *Statistically significant differences for noninjured control vs. balloon-angioplasty treatment, P < 0.05; **balloon-angioplasty vs. balloon-angioplasty plus antagonists, P < 0.05. Results are presented as means ± SE (n = 8 each treatment set), with BK (10-5 mol/l), HOE-140 (10-6 mol/l), and dA-HOE (10-6 mol/l) maintained for the entire culture period at the indicated concentrations.

Effect of BK receptor antagonists on activation of cell signaling pathways. Protein tyrosine phosphorylation and p42/44 MAPK are activated by BK (34), and MAPK is activated by balloon injury (39). We therefore used MAPK activation to examine the mechanism coupling specific BK receptors and neointimal proliferation. Transient and sustained increases in protein tyrosine phosphorylation were obtained on treatment with 10-6 mol/l BK in both porcine (Fig. 7A) and human (not shown) SMCs. Prominent changes were observed in proteins of 71 and 103 kDa, which remained phosphorylated over the entire 1-h period that was examined. Short-term increases were noted with proteins of 32, 46, and 56 kDa. Western blot analysis with phosphorylation-specific antibodies demonstrated a transient increase (peak at 5-15 min) in phosphorylation of p42/44 MAPK (Fig. 7B) and p38 kinase (not shown) after stimulation with BK. On the basis of this information, subsequent experiments were terminated at 6 min. Treatment with BK stimulated the phosphorylation of both MAPK and p38 kinase (Fig. 8, A and B). These changes were not due to an increase in the amount of protein as indicated by control anti-MAPK and anti-p38 kinase antibodies (Fig. 8). A similar analysis of c-Jun NH2-terminal protein kinase (JNK) was not conducted because changes in JNK phosphorylation were not observed (data not shown). Pretreatment with both BK receptor antagonists, HOE-140 and dA-HOE, prevented MAPK and p38 kinase phosphorylation (Fig. 9). Individually, HOE-140 reduced phosphorylation to basal unstimulated levels, whereas dA-HOE decreased MAPK and p38 kinase phosphorylation to a lesser extent. Lane 1 (DMSO) of Fig. 9 served as both the vehicle control for the BK receptor antagonists and the positive control for BK stimulation of both MAPK and p38 kinase. Interestingly, the inhibition of MAPK phosphorylation by these antagonists corresponded to the effects observed in the organ culture system, presumably indicating a correlation between these processes.


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  		Fig. 7.   Effect of BK on protein tyrosine phosphorylation and mitogen-activating protein kinase (MAPK) activation in SMCs. Immunoblot analysis of BK (10-6 mol/l) treated SMCs after administration over a time course of 1 h was conducted with phosphotyrosine-specific PY20 (A) and anti-phospho-MAPK (B) antibodies. A, left: the positions of protein bands that show changes in intensity; right, molecular mass markers. One of three separate blots is depicted for both A and B.



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  		Fig. 8.   Effect of BK concentration on the phosphorylation of MAPK and p38 kinase. Immunoblot analysis was conducted after harvesting SMCs treated for 6 min with varying concentrations of BK (10-7 to 10-5 mol/l). Antibodies specific for the phosphorylated and unphosphorylated forms of MAPK (pMAPK/MAPK) (A) and p38 kinase (pp38/p38) (B) were used. One of three separate blots is depicted for each panel. Densitometric analysis of band intensity was conducted as described in METHODS. The mean ± SE was calculated for each condition (n = 3), and these are compared graphically in C. *Statistically significant differences vs. untreated control, P < 0.05.



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  		Fig. 9.   Effect of BK receptor antagonists on the phosphorylation of MAPK and p38 kinase. Immunoblot analysis was conducted after harvesting SMCs treated for 6 min with BK (10-5 mol/l) after pretreatment (10 min) with BK receptor antagonists. Antibodies specific for pMAPK/MAPK (A) and pp38/p38 (B) were used. BK receptor antagonists HOE-140 (HOE) and dA-HOE (dAH) were employed at 10-6 mol/l. One of three separate blots is depicted for each panel. Densitometric analysis of band intensity was conducted as described in METHODS. Means ± SE were calculated for each condition (n = 3) and these are compared (C). *Statistically significant differences vs. untreated control, P < 0.05.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

SMC proliferation, a critical process in the formation of a neointimal lesion, is promoted by factors such as ANG II, platelet-derived growth factor, and fibroblast growth factor (5, 20). It has also been reported that BK modulates SMC growth (19, 34), and testing whether this peptide can influence proliferative vascular disease was the primary objective of our study. Although we expected BK would inhibit cell growth based on reports that BK antagonizes the effects of various growth-promoting vasoconstrictors (9, 16, 35), our evidence shows BK increases SMC growth in the absence of an endothelium, a result that is sensitive to BK receptor antagonists.

Studies examining the effect of BK on cell growth have provided contradictory results. For instance, BK inhibits the growth of platelet-derived growth factor-induced SMCs and gingival fibroblasts (8, 24). Similarly, Ritchie et al. (28) demonstrated that BK blocks ANG II-mediated hypertrophy of adult and neonatal ventricular myocytes but only in the presence of endothelial cells. In contrast, when endothelial cells were absent, as is the case of our primary SMC cultures or in vessels postangioplasty, BK was found to have a direct hypertrophic effect on ventricular myocytes (28). In agreement with these observations, Levesque et al. (19) reported BK-enhanced thymidine incorporation by a cell line derived from rabbit aortic SMC. Furthermore, studies by LaMorte et al. (18) have demonstrated that BK, like ANG II and endothelin-1, activates a Gq-dependent signaling pathway that typically stimulates cell growth (6), whereas Jaffa and colleagues (13, 34) have speculated that BK functions as a mitogen for rat aortic SMCs based on observations that BK activates MAPK and c-fos. These points support our findings (Figs. 1-3), which show BK stimulates the proliferation of porcine SMC. Furthermore, we have shown that BK has a positive effect on the growth of human SMCs (Fig. 5) in agreement with the observations of Raicu et al. (27).

Although our findings with isolated porcine and human SMCs indicate BK operates as a mitogen, considerable evidence has accumulated to show BK inhibits the proliferation of rat aortic SMCs (22, 33, 38). This point was emphasized by the recent report of Agata et al. (1), who showed that adenoviral-mediated over-expression of kallikrein in rat carotid arteries, presumably resulting in elevated BK synthesis, reduces neointimal formation after balloon angioplasty. A species-specific response to BK may be especially relevant to vascular injury. It has been established that ACE inhibitors can suppress neointimal proliferation postangioplasty in rodents (26), but that these drugs lack efficacy when applied to humans as demonstrated in clinical trials (12, 25). Similar negative outcomes have been reported for rabbits, baboons, and pigs (4, 14, 15). Although there is no rationale for the ineffectiveness of ACE inhibitor therapy in humans with respect to vascular injury, it is known that ACE inhibition is beneficial in the treatment of congestive heart failure (3). Interestingly, increases in BK levels have been postulated to mediate these positive actions of ACE inhibitor therapy. Given that increases in BK have been detected in dogs and humans subsequent to coronary ligation and PTCA, respectively (10, 17), BK is likely to be an important element in vascular pathophysiology as well. However, in nonrodents, it is possible that ACE inhibitor-dependent augmentation of local BK levels would promote neointimal formation and thus negate any advantage associated with a decrease in ANG II synthesis. Furthermore, increased BK levels would influence vascular tissues independent of any contribution by chymase to the production of ANG II (31) although chymase inhibitors might enhance the effectiveness of a BK receptor antagonist in reducing neointimal proliferation (32).

BK receptors are G protein linked, and two pharmacologically distinct subtypes designated B1 and B2 have been cloned (15). This study has shown that a B2 receptor antagonist is capable of decreasing BK-dependent SMC growth (assayed by [3H]thymidine incorporation) (Fig. 2A). Other studies have also indicated the effect of BK on cell growth, whether positive or negative, is mediated by the B2 receptor (7). The partial inhibition obtained in the presence of dA-HOE, a presumed B1 receptor antagonist, can be accounted for by a nonspecific interaction with the B2 receptor, as suggested by Marceau et al. (23). Although the selectivity of HOE-140 could similarly be challenged at the concentrations required for maximal inhibition (2), the data obtained with the receptor-specific agonists Lys-des-Arg9-BK and [Hyp3,Tyr(Me)8]BK (Fig. 4) conclusively establish that BK exerts its effects on SMC growth through the B2 receptor.

The relevance of experimental results produced in animal models to human pathophysiology is often elusive, and the issue of a species-specific response is particularly important with respect to vascular injury. Within this context, it has been established that the ability of ACE inhibitors to suppress neointimal proliferation postangioplasty is restricted to rodents. A rational explanation for the ineffectiveness of ACE inhibitors in humans has not been forthcoming, and, for this reason, no mechanism has been proposed. However, our results show that human coronary artery SMCs, as with their porcine counterparts, are sensitive to exogenously added BK and respond with a positive growth response that can be inhibited with BK receptor antagonists. Our findings, therefore, suggest a role for BK in promoting restenosis postangioplasty and provide evidence that pharmacological intervention via local application of BK receptor antagonists could be a viable option in situations where elevated BK levels are suspected.


    ACKNOWLEDGEMENTS

We thank Dr. PoKee Cheung, Section of Cardiology, St. Boniface General Hospital, for providing the human arterectomy samples, as well as Julieta Werner and Jason Voldeng for expert technical assistance.


    FOOTNOTES

This study was supported by a grant to the Canadian Institutes of Health Research Group in Experimental Cardiology. L. Yau and D. P. Wilson were the recipients of graduate studentships from the St. Boniface Research Foundation and the University of Manitoba, respectively.

Address for reprint requests and other correspondence: P. Zahradka, Institute of Cardiovascular Sciences, St. Boniface General Hospital Research Centre, 351 Tache Ave., Winnipeg, Manitoba, Canada R2H 2A6 (E-mail: peterz{at}sbrc.ca).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received 18 January 2001; accepted in final form 29 May 2001.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

1.   Agata, J, Miao RQ, Yayama K, Chao L, and Chao J. Bradykinin B1 receptor mediates inhibition of neointima formation in rat artery after balloon angioplasty. Hypertension 36: 364-370, 2000[Abstract/Free Full Text].

2.   Aramori, I, Zenkoh J, Morikawa N, O'Donnell N, Asano M, Nakamura K, Iwami M, Kojo H, and Notsu Y. Novel subtype-selective nonpeptide bradykinin receptor antagonists FR167344 and FR173657. Mol Pharmacol 51: 171-176, 1997[Abstract/Free Full Text].

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Am J Physiol Heart Circ Physiol 281(4):H1648-H1656
0363-6135/01 $5.00 Copyright © 2001 the American Physiological Society


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