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. 2000 Jul;130(5):1076–1082. doi: 10.1038/sj.bjp.0703388

Participation of kinins in the captopril-induced inhibition of intimal hyperplasia caused by interruption of carotid blood flow in the mouse

Costanza Emanueli 1,2, Maria Bonaria Salis 1,2, Carlos Figueroa 4, Julie Chao 5, Lee Chao 5, Leonardo Gaspa 1, Maurizio C Capogrossi 2, Paolo Madeddu 1,3,*
PMCID: PMC1572154  PMID: 10882392

Abstract

  1. In the rat balloon injury model, angiotensin-converting enzyme (ACE) inhibitors prevent vascular remodelling by inhibiting angiotensin II generation and kinin breakdown. We investigated if ACE inhibition also prevents the structural vascular responses to disruption of carotid artery blood flow and if kinin potentiation plays a role in such a protection.

  2. Morphometric analysis of the structural alterations caused by ligation of the left carotid artery was performed 14 days after surgery in J129Sv wild-type mice (B2+/+) drinking normal tap water or water containing captopril (120 mg kg−1 per day). In addition, the effect of captopril on vascular remodelling was tested in B2+/+ given the bradykinin (BK) B1 receptor antagonist des-Arg9-[Leu8]-BK (DALBK, 50 nmol kg−1 per day, intraperitoneally) or the BK B2 receptor antagonist D-Arg,[Hyp3,Thi5D-Tic7,Oic8]-BK (icatibant, 1 μmol kg−1 per day, intraperitoneally), and in B2 receptor gene knockout mice (B2−/−).

  3. Interruption of blood flow resulted in carotid artery intimal hyperplasia and media thickening in untreated B2+/+, these responses being partially suppressed by captopril. The inhibition of intimal thickening exerted by captopril was reduced in B2+/+ given DALBK or icatibant (P<0.05 for both comparisons) as well as in B2−/− (P<0.05). Neither antagonism of kinin receptors nor disruption of the B2 receptor gene altered the suppressive effect of captopril on media thickening. The protection of vascular wall structure was independent of the reduction in blood pressure by captopril.

  4. These results demonstrate that kinins participate in the inhibitory effect of captopril on intimal hyperplasia via B1 and B2 receptor signalling. Our findings may have important implications in treating vascular remodelling evoked by altered shear stress conditions.

Keywords: Intimal hyperplasia, angiotensin converting enzyme inhibitors, kinins, receptor antagonists, nitric oxide, knockout mice

Introduction

Intimal hyperplasia due to neointima formation often causes clinically significant restenosis of arteries that have undergone therapeutic percutaneous transluminar angioplasty (Glagov, 1994). The potential of angiotensin-converting enzyme (ACE) inhibitors in prevention of neointima formation was first addressed in a rat model of carotid artery balloon injury (Powell et al., 1989). This protective effect was mainly attributed to inhibition of the ACE-induced generation of angiotensin II a potent vasopressor implicated in vascular smooth muscle cell (SMC) growth (Itoh et al., 1993), proliferation (Huckle & Earp, 1994), and migration (Farhy et al., 1997). In addition, as ACE is also responsible for kinin breakdown, the possible contribution of kinins in the favourable cardiovascular effects exerted by ACE inhibitors needs to be considered (see Linz et al., 1995).

The presence of a local kallikrein-kinin system in the vasculature has been firmly established (Madeddu et al., 1993; 1994). Kinins are vasoactive peptides released by kallikreins from the precursor kininogens. Binding of kinins to specific 7 trans-membrane domain, G-protein-coupled B1 and B2 receptors expressed on vascular endothelial cells (ECs) stimulates the formation of nitric oxide (NO) and prostacyclin, thus leading to activation of cyclic GMP and cyclic AMP pathways (Blaukatt et al., 1996; D'Orleans-Juste et al., 1989). Differently from the constitutively expressed B2 receptors, B1 are inducible receptors, i.e. their expression is activated only under particular pathophysiologic conditions, such as inflammation (see Ahluwalia & Perretti, 1999), hypertension (Emanueli et al., 1999), and diabetes (Zuccolo et al., 1996). The relevance of B2 receptors in prevention of vascular remodelling by ACE inhibitors has been demonstrated in the rat carotid balloon injury model by the use of the selective receptor antagonist icatibant (deBlois et al., 1992; Farhy et al., 1993). Whether the B1 receptor subtype is involved in these processes remains unknown.

Recently, a mouse model has been introduced in which vascular wall remodelling is caused by permanent changes in shear stress conditions, as obtained by interruption of carotid artery blood flow (Kumar et al., 1997; Kumar & Lindner, 1997; Bryant et al., 1999). In this case, vascular endothelium is not removed, thus allowing addressing the importance of substances that are released or targeted to ECs (Langille & O'Donnell, 1986). Furthermore, availability of a murine model of vascular remodelling is essential to exploit the informative potential of gene knockout strategy that is generally carried out in mice.

The aim of the present study was 2 fold: (1) to evaluate if ACE inhibition prevents vascular remodelling induced by disruption of carotid artery blood flow in the mouse and (2) to determine if kinins play a role in this phenomenon via activation of B1 and/or B2 receptors.

To this purpose, mice underwent ligature of the left carotid artery and were given captopril alone or in combination with B1 or B2 receptor antagonists. The preventive effect of captopril on vascular remodelling was also evaluated in mice in which the gene encoding for the B2 receptor was knocked-out by gene targeting and homologous recombination (B2−/−) (Borkowski et al., 1995; Madeddu et al., 1997).

Methods

Experiments were conducted using a protocol that was approved by the Institutional Animal Care and Use Committee. All procedures complied with the standards for care and use of animal subjects as stated in the Guide for the Care and the Use of Laboratory Animals (Institute of Laboratory Animal Resources, National Academy of Sciences, Bethesda, MD, U.S.A.). Male (2–3 months of age) J129 Sv wild-type mice (B2+/+) were obtained from Jackson Laboratory (Bar Harbor, MN, U.S.A.). B2−/−, generated by gene targeting and homologous recombination on a J129 Sv genetic background (Borkowski et al., 1995), were kindly provided by Dr Fred Hess (Merck Laboratories, West Point, PA, U.S.A.).

Surgical procedures

With mice under 2,2,2-tribromoethanol anaesthesia (880 mmol kg−1, intraperitoneally), the left common carotid artery was exposed through a small midline incision of the neck and ligated with 6-0 silk just proximal the bifurcation of the vessel to disrupt blood flow. At the same occasion, osmotic minipumps (Alzet, Alza Co., Palo Alto, CA, U.S.A.) were implanted into the abdomen for continuous intraperitoneal (i.p.) drug delivery. In additional groups of B2+/+ and B2−/−, the carotid artery was exposed, but not ligated (sham-operation).

Experimental protocols

Sham-operated animals were used for reference. Carotid artery-ligated B2+/+ or B2−/− were given regular tap water to drink or water containing captopril (120 mg kg−1 per day for 14 days). In addition, B2+/+ received an i.p. infusion (12 μl per day) of the bradykinin (BK) B1 receptor antagonist des-Arg9-[Leu8]-BK (DALBK, 50 nmol kg−1 per day), the BK B2 receptor antagonist D-Arg, [Hyp3,Thi5D-Tic7,Oic8]-BK (icatibant, 1 μmol kg−1 per day), DALBK and icatibant combined (at the doses stated above), or their vehicle (sterile saline). Each group consisted of at least six mice. Selectivity of icatibant for kinin B2 receptors has been previously reported in the mouse (Madeddu et al., 1997; Nsa Allogho et al., 1997). DALBK reportedly acts as a selective B1 receptor antagonist in rat vasculature (Morbidelli et al., 1998). Although partial agonistic effect was recognized in the mouse stomach (Nsa Allogho et al., 1997), we have found that DALBK, at the dose indicated above, is able to antagonize the hypotensive effect of des-Arg9-BK in the mouse and is devoid of residual agonistic activity (Paolo Madeddu, unpublished observations).

Additional groups of carotid artery-ligated B2+/+ received the NO synthase inhibitor N-nitro-arginine-L-methyl-ester (L-NAME, 1.4 mmol kg−1 per day, orally), or captopril in combination with L-NAME (same dose and route as indicated above). The doses of captopril and L-NAME were chosen according to previous studies (Emanueli et al., 1997; Madeddu et al., 1997). Each group consisted of at least six mice.

In a separate set of experiments, carotid artery-ligated B2+/+ and B2−/− (n=9 and 7, respectively) were chronically given the Angiotensin II AT1 receptor antagonist losartan (15 mg kg−1 per day, orally).

Haemodynamic measurements

Systolic blood pressure (SBP) was measured in unanaesthetized mice by tail-cuff plethysmography (Visitech System, U.S.A.) before and 14 days after carotid ligation in B2−/− treated with captopril or vehicle and in B2+/+ treated with vehicle, captopril, or captopril in combination with DALBK, icatibant, or L-NAME. Body weight was recorded at the same occasions.

Morphometric analysis

Fourteen days after carotid artery ligation or sham-operation, mice were anaesthetized and perfusion fixed at 100 mmHg with 4% paraformaldehyde in 0.1 mol l−1 phosphate buffer (pH 7.3). The whole left common carotid artery was excised and placed for 24 h in 4% paraformaldehyde. Vessels were then processed for paraffin embedding. Five serial sections (200 μm apart) of 3 μm thickness were cut starting from 1 mm below the carotid artery bifurcation and proceeding to the aortic arch. Sections were stained with haematoxylin-eosin. Morphometric analysis was performed using a dedicated software package (KS300, Zeiss). The area of tunica intima (I) was recognized as the area delimited by the luminal circumference and the internal elastic lamina (IEL). The area of tunica media (M) was recognized as the area delimited by IEL and the external elastic lamina (EEL). In addition to I and M areas, the length of IEL and EEL were measured. IEL was taken as an index of inward remodelling, and EEL as a measure of vascular constriction. The total cell number per I and M cross section was evaluated at a magnification of 1000×using a calibrated grid. Then, I and M cellular density was calculated by dividing total cell count by the respective area. For each carotid artery, the values obtained from the five sections were averaged.

Drugs

Captopril, L-NAME, and DALBK were purchased from Sigma (Milan, Italy) and losartan from Merck (Milan, Italy). Icatibant was a generous gift of Hoechst Pharmaceutical Co. (Frankfurt, Germany).

Statistical analysis

Data are expressed as mean±s.e.mean. Multivariate repeated-measures ANOVA was performed to test for interaction between time and grouping factor. In multiple comparisons among independent groups in which ANOVA and P test indicated significant differences, the statistical value was determined according to Bonferroni's method. Differences within and between groups were determined using paired or unpaired Student's t-test, respectively. A probability (P) value less 0.05 was considered significant.

Results

Haemodynamic measurements

Captopril did not affect the surviving rate or the gain in body weight of B2+/+ or B2−/− (data not shown).

As shown in Table 1, basal SBP was higher in B2−/− compared to B2+/+ (P<0.05). This difference between strains persisted after carotid artery ligation in vehicle-treated mice. Captopril significantly decreased SBP levels in carotid artery-ligated B2−/− and B2+/+, so that after 14 days of ACE inhibitor treatment no significant difference was observed between the two strains. In B2+/+, the hypotensive effect of captopril was not altered by the administration of kinin receptor antagonists or L-NAME.

Table 1.

Tail-cuff systolic blood pressure of B2+/+ or B2−/− under basal conditions (Basal SBP) and after 14 days from cartoid ligation (Final SBP)

graphic file with name 130-0703388t1.jpg

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Morphometric analysis

In vehicle-treated B2+/+, disruption of carotid artery blood flow determined a reduction in vessel lumen area by a combination of I hyperplasia (I area: 32,685±7648 μm2 vs not measurable at the same magnification in sham-operated mice), M thickening (M area: 32,891±4361 vs 21,520±5368 μm2 in sham-operated mice, P<0.05), and reduction in IEL and EEL lengths (975±143 vs 1281±141 μm and 1102±75 vs 1380±171 μm in sham-operated mice, respectively, P<0.05 for both comparisons).

As shown in Figure 1, in B2+/+ captopril inhibited I (P<0.001) and M (P<0.05) hyperplasia, leading to reduction in I/M ratio (0.13±0.01 vs 0.97±0.22 in vehicle-treated mice, P<0.01). Captopril did not alter the effect of carotid artery ligature on IEL or EEL length (data not shown).

Figure 1.

Figure 1

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Bar graphs show the areas of tunica intima (I, A) and media (M, B) of the left common carotid artery and the ratio between I and M areas (C) at 14 days from disruption of blood flow in wild-type J129 Sv mice (B2+/+) given vehicle (normal drinking water; n=12), captopril (120 mg kg−1 per day in the drinking water; n=7), captopril plus the B1 receptor antagonist des-Arg9-[Leu8]-bradykinin (DALBK) (50 nmol kg−1 per day, i.p.; n=7), captopril plus the B2 receptor antagonist D-Arg,[Hyp3,Thi5,D-Tic7,Oic8]-bradykinin (icatibant, 1 μmol kg−1 per day, i.p.; n=7), or captopril plus the nitric oxide synthase inhibitor, N-nitro-L-arginine-methyl-ester (L-NAME, 1.4 mmol kg−1 per day in the drinking water, n=7). Values are means±s.e.mean.

In vehicle-treated B2+/+, chronic administration of DALBK or icatibant alone did not change the vascular remodelling response to carotid artery ligation (data not shown). However, as shown in Figure 1A, either B1 or B2 receptor antagonism partially prevented the inhibition of I thickening exerted by captopril (P<0.05 for both comparisons). The combined administration of DALBK and icatibant did not further reduce the captopril-induced inhibition of I hyperplasia (I area: 25,985±6040, P=N.S. vs captopril plus DALBK or captopril plus icatibant).

In contrast, the captopril-induced-suppression of M thickening was not altered by DALBK, icatibant (Figure 1B), or the two antagonists in combination (data not shown).

L-NAME alone did not affect the vascular response to carotid artery ligation in vehicle-treated B2+/+ (data not shown), but it reduced the inhibition of I thickening exerted by captopril (Figure 1A). L-NAME did not change captopril-induced effect on M hyperplasia (Figure 1B).

In carotid artery-ligated B2+/+, captopril reduced total cell count per I cross section (35±3 vs 224±65 cells in vehicle-treated mice, P<0.001, Figure 2A), and increased I cell density (15±6 vs 5±1 cells/mm2 in vehicle-treated mice, P<0.05, Figure 2B). In contrast, as shown in Figure 2C, D, captopril produced borderline changes in total cell count per M cross section (P=0.07) or cell density (P=0.09). Either DALBK or icatibant or L-NAME antagonized captopril-induced effect on I cell number (P<0.05 for all comparisons, Figure 2A), but only icatibant and L-NAME antagonized the effect on I cell density (P<0.05, Figure 2B).

Figure 2.

Figure 2

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Bar graphs show the total cell count per cross section and the cell density in tunica intima (I, A,B) and media (C,D) at 14 days from disruption of carotid artery blood flow in B2+/+ given vehicle (n=12), captopril (n=7), captopril plus DALBK (n=7), captopril plus icatibant (n=7), or captopril plus L-NAME (n=7). Values are means±s.e.mean.

Figure 3 shows the typical remodelling response to interruption of blood flow and the preventive action of captopril against I and M hyperplasia. In addition, the effects of B1 or B2 antagonism on captopril-induced protection against I thickening are shown in the same figure.

Figure 3.

Figure 3

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Pictures show representative haematoxilin and eosin-stained left common carotid artery transverse sections of B2+/+ treated with vehicle (A), captopril (B), captopril in combination with the B1 receptor antagonist DALBK (C), or captopril in combination with the B2 receptor antagonist icatibant (D). Vessels were taken 14 days after blood flow interruption. Sections were obtained at 1 mm from carotid artery bifurcation. Scale bar corresponds to 10 μm.

Next, we examined the characteristics of carotid artery structure in mice with genetic disruption of the B2 receptor. No morphologic difference was seen when comparing sham-operated carotid arteries of B2−/− with those of B2+/+ (data not shown). In addition, as shown in Figure 4, the absence of the B2 receptor gene did not affect the vascular wall response to carotid artery ligation in vehicle-treated animals (Figure 4). However, as shown in Figure 4A, the null mutation was associated with a significant reduction in captopril-induced preventive effect on I hyperplasia (I area: 8381±593 vs 2459±250 μm2 in B2+/+, P<0.05). By contrast, inhibition of M thickening by captopril was similar in B2−/− and B2+/+ (Figure 4B). As a consequence, I/M ratio was higher in B2−/− compared to B2+/+ (P<0.05).

Figure 4.

Figure 4

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Bar graphs show the areas of left common carotid artery tunica intima (I, A) and media (M, B), and the ratio between I and M areas (C) at 14 days from disruption of blood flow in wild-type (B2+/+, left) and B2 receptor gene knockout mice (B2−/−, right) given vehicle (B2+/+: n=12, B2−/−: n=10) or captopril (B2+/+: n=7, B2−/−: n=10). Values are means±s.e.mean.

In carotid artery-ligated B2−/−, captopril reduced total cell count per I cross section (95±3 vs 179±16 cells in vehicle-treated mice, P<0.01) and increased I cell density (12±1 vs 7±1 cells/mm2 in vehicle-treated mice, P<0.05).

Finally, we evaluated the effect of AT1 receptor antagonism on vascular remodelling. Losartan inhibited I and M hyperplasia similarly in B2+/+ and B2−/− (data not shown). In addition, the antagonist decreased I cell number per cross section and increased I cell density with no difference between strains (data not shown). Cell number per M cross section did not change, while M cell density decreased following losartan treatment (data not shown) with no differences between strains.

Discussion

At the best of our knowledge, this is the first study demonstrating a preventive action of ACE inhibition in a mouse model of I hyperplasia. The importance of B2 receptor signalling in this phenomenon has been revealed by pharmacological blockade or genetic disruption of the receptor. Another novel discovery is that also B1 receptors participate in the suppressive effect of ACE inhibition on I hyperplasia.

Implication of kinins in the preventive effect of ACE inhibitors against neointima formation has been previously reported in the rat (deBlois et al., 1992; Farhy et al., 1993). In the above studies, arterial remodelling was triggered by endothelium removal using a balloon catheter. However, development of intimal lesions in atherosclerosis models and in human vascular disease occurs in the absence of noticeable endothelial denudation at sites of altered haemodynamics associated with low shear stress. Therefore, we thought it would be worthwhile to address the role of kinin receptors in a murine model of vascular remodelling attributable to altered shear stress conditions (Kumar & Lindner, 1997). Attention was focused on the B1 receptor in consideration of the mounting importance of this subtype receptor in inflammatory-like conditions (see Ahluwalia & Perretti, 1999). Induction of B1 receptor-dependent vasoconstriction following balloon catheter injury reportedly occurs in the rabbit carotid artery (Pruneau et al., 1994). However, the overall relevance of these changes to vascular remodelling remains unclear. We have found that B1 receptor antagonism does not alter the remodelling response to interruption of carotid artery blood flow, but prevents in part the inhibitory effect of captopril against I hyperplasia. These results indicate a role for the B1 receptor signalling in the preventive effect of ACE inhibitors against I thickening. The functional importance of B1 receptors might have been enhanced by ACE inhibition because of a shift of kinin degradation through kininase I in the direction of des-Arg9-derivatives (Israel & Saavedra, 1987; Lamontagne et al., 1995). Immunohistochemistry studies have clearly demonstrated the presence of inflammatory cells (macrophages and monocytes) in the adventitia, developing intima, and near the luminar surface of carotids that undergo blood flow disruption (Kumar & Lindner, 1997). In this inflammatory milieu, the B1 receptor may be up-regulated, thus enabling cells expressing this receptor subtype to act as magnets for kinin peptides augmented by ACE inhibition.

While knockout of the B1 receptor is still awaited, availability of null mutant B2−/− has allowed to determine the impact of the absence of B2 receptors on the development of cardiovascular phenotype (Madeddu et al., 1997). We found that this genetic defect does not result in abnormalities of the structure of carotid artery under basal conditions or in altered vascular remodelling responses to carotid artery ligature. However, according to our results a functional B2 receptor signalling participates in the inhibition of I hyperplasia induced by captopril. Recently, the hypothesis that ACE inhibitors exert beneficial cardiovascular effects by blocking B2 receptor desensitization has been advanced (Minshall et al., 1997). Whether such an intriguing mechanism is relevant in the protection against I thickening remains to be determined.

The near-stasis conditions in more distal segment of the ligated carotid artery may result in activation of extrinsic coagulation pathway. Therefore, we cannot exclude that kinins released by the plasma kallikrein from high molecular weight kininogen might contribute together with vascular tissue kinins to the protective effect of captopril. However, as previously shown by Kumar & Lindner (1997), preservation of endothelium maintains a non-thrombotic surface in this model.

Consistent with previous results (Emanueli et al., 1997), we found that neither disruption nor pharmacological blockade of the B2 receptor altered the blood pressure fall in response to ACE inhibition in mice. Therefore, it appears unlikely that systemic haemodynamic changes are responsible for the different structural vascular responses observed among groups given captopril. Furthermore, in our model the driving factor for vascular remodelling is constituted by the permanent alterations in shear stress resulting from cessation of blood flow, and not by systemic blood pressure.

Participation of kinins in captopril-induced protective effects appears to be restricted to inhibition of I hyperplasia, since the suppressive action of ACE inhibition on medial thickening was not antagonized by manipulation of kinin receptors. Moreover, kinin blockade inhibits only in part captopril-induced protection against I hyperplasia. These findings are in line with the fundamental role of Angiotensin II in vascular remodelling as indicated by our results in losartan-treated animals.

ACE inhibition decreased I total cell count with the participation of both B1 and B2 receptors in this phenomenon. By contrast, M total cell count was not affected by captopril either alone or in combination with receptor antagonists. Taken together these results suggest that increased kinin levels by captopril could inhibit SMC proliferation. This is in accordance with recent findings from our group showing that kinins released by tissue kallikrein inhibit SMC proliferation (Murakami et al., 1999). In vitro experiments indicate that binding of kinins to aortic SMC receptors stimulates prostacyclin formation, thus leading to increased cyclic AMP levels and subsequent inhibition of SMC proliferation (Dixon et al., 1990). The mechanism by which increased kinin levels inhibit vascular growth in vivo may also involve the induction and/or activation of NO synthase, and in fact L-NAME diminished the inhibition of I hyperplasia exerted by captopril in our experimental setting as well as in the rat balloon injury model (Farhy et al., 1993). Binding of kinins to vascular endothelial B2 receptors increases NO release (Zhang et al., 1997). Once released by ECs, NO reaches SMC where it activates guanilate cyclase, thus increasing the production of cyclic GMP, a potent inhibitor of SMC proliferation and migration (Cornwell et al., 1994; Grag & Hassid, 1989; Sarkar et al., 1996). Accordingly, Farhy et al. (1997) showed that kinin-stimulated NO release contributes to the beneficial effect of ACE inhibitors in the rat balloon injury model by reducing SMC migration from the tunica media to the I. As already stated above, we found that M cell count was unchanged by captopril. We would expect that if ACE inhibition blocked SMC migration to I without affecting proliferation rate, the total number of cell would be increased in the tunica media. Since this was not the case, it is likely that both SMC migration and proliferation were inhibited by captopril. Kinins have been demonstrated to attenuate programmed cell death in ischaemic tissue (Yoshida et al., 2000). Since hypoxia-induced apoptosis occurrs in the early phases of vascular remodelling after carotid artery ligation (Kumar & Lidner, 1997), it is possible that an anti-apoptotic effect of kinins may in part balance the kinin-induced inhibitory effect on SMC proliferation. Further studies using markers of cellular turnover (i.e. cell proliferation and apoptosis) may help address this issue.

The finding that I cell density was augmented in captopril-treated group, with this effect being antagonized by icatibant, favours a suppressive role of kinins, via the B2 receptor, against matrix production and/or deposition. This is in accordance with in vitro experiments demonstrating that BK down-regulates extracellular matrix protein production via NO and cyclic GMP (Kim et al., 1999) and with a report showing that kinin B2 receptor antagonism enhances the spontaneous interstitial deposition of collagen in response to myocardial infarction in the rat (Wollert et al., 1997). An alternative explanation for kinin-mediated effects of captopril on cell density would be a decrease in cell size.

In conclusion, we have demonstrated that endogenous kinins acting on both their receptor subtypes play an important role in the preventive effect of ACE inhibition against I hyperplasia in a mouse carotid artery model in which vascular remodelling is induced by cessation of blood flow. These findings underline the importance of the kallikrein-kinin system in vascular biology and may have important implications in treating I hyperplasia evoked by altered shear stress conditions.

Acknowledgments

The financial support of Telethon-Olnus (grant A.105) is gratefully acknowledged. This study was supported in part by National Institutes of Health (Grants HL29397 and HL52196) Biomed 96-1160 and Regione Autonoma Della Sardegna (assessorato Della Pubblica Istruzione). In addition, we would like to thank Dr Elena Cigola from the University of Parma for the expert advices in histologic procedures and Dr Renzo Filippetti, Mr Vittorio Lelii, and Mr Leandro Travaglini from the Universtià Cattolica del Sacro Cuore (Rome, Italy) for their assistance in the animal care.

Abbreviations

ACE

angiotensin converting enzyme

B1

BK B1 receptor

B2

BK B2 receptor

B2−/−

B2 receptor gene knockout mice

B2+/+

wild-type mice

BK

bradykinin

DALBK

des-Arg9-[Leu8]-BK

EC

vascular endothelial cell

EEL

external elastic lamina

I

tunica intima

icatibant

D-Arg,[Hyp3,Thi5,D-Tic7,Oic8]-BK

IEL

internal elastic lamina

L-NAME

N-nitro-arginine-L-methyl-ester

M

tunica media

NO

nitric oxide

SBP

systolic blood pressure

SMC

vascular smooth muscle cell

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