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INFLAMMATION AND IMMUNOPHARMACOLOGY
Faculté de Pharmacie (M.E.M., G.M., A.A.), Faculté des Arts et des Sciences, Département de Mathématiques et de Statistique (M.C., Y.L.) and Faculté de Médecine Vétérinaire (P.D.), Université de Montréal, Montréal, Québec, Canada; Centre Hospitalier Universitaire de Québec, Centre de Recherche, Québec, Canada (F.M.); and Institute of Biochemistry II, Johann Wolfgang Goethe University School of Medicine, Frankfurt, Germany (W.M.-E.)
Received April 14, 2005; accepted September 13, 2005.
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). Moreover, two recent studies have shown AE incidence higher than previously appreciated, up to 0.68% of enalapril-treated hypertensive patients (Packer et al., 2002; Kostis et al., 2004). Also, a 3-fold incidence increase was reported in African-American patients, and it was nominally higher in smokers (Coats, 2002). AE affects notably the extremities, abdominal cavity, face, larynx, and tongue. However, a laryngeal localization is at high risk. Although the clinical symptoms of AE have been attributed to the vasoactive peptide bradykinin, no definitive experimental evidence supports an obligatory role for this agent.
The nonapeptide bradykinin is the prototype of kinins, a family of powerful bioactive autacoids, involved in a series of physiological and pathological cardiovascular responses, mainly vasodilatation, increased capillary permeability, and pain processes (Leeb-Lundberg et al., 2005). Bradykinin and kallidin (Lys-bradykinin) exert their pharmacological activities by binding to their constitutively expressed kinin B2 receptor before being metabolized by multiple peptidases (Leeb-Lundberg et al., 2005). The identity of the metallopeptidases involved in bradykinin metabolism in vitro and their relative importance vary according to the biological medium considered. In various cell types and tissues, such as kidney, endothelial cells, and cardiomyocytes, neutral endopeptidase 24.11 (NEP, neprilysin) plays an important role in the degradation of bradykinin (Raut et al., 1999). In human plasma, bradykinin is metabolized mostly by three metallopeptidases (Decarie et al., 1996; Cyr et al., 2001). Angiotensin I-converting enzyme (ACE) constitutes the main degradation pathway. Carboxypeptidase N transforms bradykinin and kallidin into their active metabolites, des-arginine9-bradykinin (des-Arg9-BK) and Lys-des-Arg9-BK (des-Arg10-kallidin), respectively (a minor metabolic pathway in plasma unless ACE is inhibited). We have also shown that aminopeptidase P (X-prolyl aminopeptidase, APP) plays an important role in the metabolism of kinins in plasma, mostly for des-Arg9-BK (Cyr et al., 2001). Although the carboxytruncated metabolites of bradykinin and kallidin are largely inactive under normal conditions, they are the agonists of the strongly regulated B1 receptors, of which receptor synthesis is increased in experimental models of inflammation under the control of cytokines, MAP kinases, and specific transcription factors (Leeb-Lundberg et al., 2005).
We have previously reported a decreased degradation of endogenous des-Arg9-BK in the plasma of hypertensive patients who, while treated with an ACEi, experienced an AE (Molinaro et al., 2002). This anomalous breakdown was linked to a decreased aminopeptidase P plasma activity, supporting a pathogenic mechanism relying on kinin catabolism (Adam et al., 2002; Molinaro et al., 2002). Human aminopeptidase P exists in both cytosolic and membrane-bound forms, the latter being most likely responsible for plasma activity (Molinaro et al., 2005). Moreover, a recent report describes a single nucleotide polymorphism within the membrane-bound aminopeptidase P (mAPP) gene XPNPEP2 linked to low plasma aminopeptidase P activity (Duan et al., 2005). Although plasma aminopeptidase P deficiency states could predispose to AE in some ACEi-treated patients, little is known about the physiological roles of this metallopeptidase in tissues. These observations based on plasma can hardly be extrapolated to a localized AE affecting the oropharyngeal tissues.
The first aim of this paper was to quantify metallopeptidases and kinin receptor mRNAs in oropharyngeal tissues in swine using novel real-time PCR analysis. As a second step, we have defined the effect of inflammation and of acute and chronic ACE inhibition on the expression of these biochemical entities that limit and mediate, respectively, the pharmacological activity of kinins during an episode of AE. As a sequel to our preceding in vitro investigations, we have defined the effect of these in vivo treatments on the metabolism of endogenous kinins in plasma.
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), K(Dnp)PPGK(Abz) for mAPP (Molinaro et al., 2005), and (Abz)YRK(Dnp)P for ACE (Araujo et al., 2000) were gifts from Prof. A. Carmona (Department of Biophysics, Escola Paulista de Medicina, Universidade Federal de São Paulo, São Paulo, Brazil). PCR primers were synthesized by Biocorp Inc. (Montreal, QC, Canada). Animals. Twenty-four healthy male cross-bred barrows (Yorkshire x Landrace) approximately 2 months old and weighing 16.1 ± 1.6 kg were purchased from a specific pathogen-free farm (Faculté de Médecine Vétérinaire, Université de Montréal, Montréal, Canada) and were kept in an isolated room. Within the room, the animals were housed in separate pens. Food and water were provided ad libitum throughout the experiment.
Experimental Protocol. The experimental protocol was approved by the Faculty of Veterinary Medicine ethics committee (Université de Montréal) in accordance to the Canadian Council on Animal Care Guidelines. The 24 animals were randomly allocated in four different treatment groups: 1) the one-time ACEi treatment group (enalaprilat 100 µg/kg i.v. injection once), 2) the 7-day ACEi treatment group (enalapril maleate tablet 20 mg p.o.) 12 h apart for 7 consecutive days, 3) the LPS group [5 µg/kg dissolved in 1 ml of NaCl (0.9%) i.v. once], and 4) untreated animals (control group). At the end of the experiments, all of the animals were euthanized with an i.v. lethal dose of sodium pentobarbital (540 mg/ml). The one-time ACEi group was euthanized 4 h postenalaprilat i.v. injection, the 7-day ACEi group was euthanized on the morning of the eighth day, and the LPS animals were sacrificed 6 h post-LPS i.v. injection. Sacrifice immediately followed the last blood samplings. Two control pigs were sacrificed with each group of treated animals.
Venous blood samples were collected in 1) the one-time ACEi group before injection, 45 min post-ACEi injection, and 4 h post-ACEi injection; 2) the 7-day ACEi group on the first (before the first oral dose of ACEi), third, and eighth days of treatment; and 3) the LPS group before and 6 h after LPS injection. Control group samples were taken at all time points on six animals. Blood samples were collected into sodium citrate and EDTA-treated tubes (Vacutainer; BD Biosciences, Franklin Lakes, NJ). The citrated blood samples were immediately centrifuged (2400g for 10 min), and the plasma from each animal was aliquoted in polypropylene tube and immediately stored at –80°C until further analysis. Tissues from the oropharyngeal zone (parotid gland, tongue, and laryngeal tissue) and a piece of kidney were quickly excised upon euthanasia, washed in ice-cold saline solution, and flash-frozen in liquid nitrogen and/or conserved in RNAlater for protein and RNA extraction.
Assessment of the Systemic Inflammatory Response. C-reactive protein, an indicator of a systemic inflammatory response, was quantified in plasma with a commercial solid-phase sandwich immunoassay kit (PHASE RANGE porcine C-reactive protein assay kit; Tri-Delta Diagnostics, Inc., Morris Plains, NJ) according to the manufacturer's instructions. Total and differential leukocytes counts were routinely obtained with a Coulter counter (Beckman Coulter, Fullerton, CA), and these systemic parameters were complemented by rectal temperature assessment in LPS-treated and control animals.
Biochemical Investigations in Plasma. The activity of ACE, aminopeptidase P, and carboxypeptidase N was measured using the methods described previously. ACE activity was determined by Bühlmann ACE radioenzymatic assay (ALPCO: American Laboratory Products Company, Windham, NH) according to the manufacturer's instructions. The activity of aminopeptidase P and carboxypeptidase N was assessed by fluorimetric assay as described previously (Cyr et al., 2001). The metabolism of the endogenous kinins, bradykinin and its active metabolite des-Arg9-BK, was studied through in vitro plasma contact system activation as described extensively elsewhere (Cyr et al., 2001; Molinaro et al., 2002), using two specific competitive chemiluminescent enzyme immunoassays, as previously described (Decarie et al., 1994; Raymond et al., 1995).
Total RNA Isolation from Tissues. Total RNA was isolated using the TRIzol reagent and RNAqueous-4PCR kit (Ambion) according to the manufacturer's instructions with modification. In brief, 
50 mg of each tissue was first homogenized in the TRIzol reagent, and chloroform was added. After centrifugation, the aqueous phase was mixed with the lysis/binding solution and the RNAqueous-4PCR protocol was followed subsequently. All RNA samples were finally DNase-treated to remove traces of genomic DNA, quantified using RiboGreen fluorescent nucleic acid stain (RNA quantification kit; Invitrogen, Carlsbad, CA), and stored at –80°C until use.
Reverse Transcription and Real-Time Polymerase Chain Reaction. The 0.5-µg amount of total RNA was transcribed into cDNA using 50 units of Moloney murine leukemia virus reverse transcriptase (Applied Biosystems, Foster City, CA) and 5 µM oligo(dT)16 primer. Quantification of the RNAs was performed by real-time PCR using the LightCycler 2.0 apparatus (Roche Diagnostics, Mannheim, Germany). Two microliters of cDNA were brought to a final volume of 20 µl containing 2 mM MgCl2, 2 µl of LightCycler-FastStart DNA SYBRGreen I Mix (Roche Diagnostics), and 0.5 or 0.7 µM primers (Table 1) in water. After initial activation of the DNA polymerase at 95°C for 10 min, the amplification conditions were as follows: 47 cycles consisting of denaturation at 95°C for 15 s, annealing for 12 s at 62°C [glyceraldehydes 3-phosphate dehydrogenase (GAPDH), B1 receptor, B2 receptor, and neprilysin] or 15 s at 60°C (mAPP), and extension at 72°C. The extension times were calculated from the amplicon size (base pairs/25). Fluorescence data were acquired at the end of each extension phase. After amplification, a melting curve analysis from 65 to 98°C with a heating rate of 0.1°C/s with a continuous fluorescence acquisition was made. Standard curves were created from specific PCR products. The corresponding real-time PCR efficiency (E) of one cycle in the exponential phase was calculated from the slopes calculated by the LightCycler software according to eq. 1 (Pfaffl, 2001).
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Quantification was done by using a mathematical model in eq. 2 to determine the relative quantification of a target gene compared with a reference gene. The relative quantification of a target transcript is based on the PCR efficiency of the individual transcripts and crossing point (Cp) deviation (calculated by the second derivative maximum method) of a control and an unknown sample normalized by a reference transcript. This relative expression ratio can be calculated as shown in eq. 2,
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Measurement of ACE, mAPP, and Neprilysin Enzymatic Activities in Tissues. Tissues from the oropharyngeal zone, namely from the parotid gland, tongue, and laryngeal tissue, and a piece of kidney weighing 500 mg were homogenized in Tris buffer [50 mM Tris, 100 mM NaCl, pH 7.4, and 1x Complete protease inhibitor solution (protease inhibitor cocktail tablets; Roche Diagnostics)] with a Polytron homogenizer (Brinkmann Instruments, Rexdale, ON, Canada) and sonicated for 10 min. A solution of CHAPS (Sigma-Aldrich) was added to each homogenate to final concentration of 8 mM, incubated on ice for 2 h, and centrifuged (1750g, 4°C), and the supernatant containing total membrane proteins was collected (Raut et al., 1999). Final protein concentrations were determined using the bicinchoninic acid method (Pierce Chemical, Rockford, IL).
Metallopeptidase (ACE, mAPP, and neprilysin) activities were measured in tissue extracts using quenched fluorescent substrates. Reactions were run in duplicate in 96-well flat bottom plates (Costar UV plate 3635; Corning Life Sciences, Acton, MA) in a final volume of 150 µl. Ten milligrams of membrane protein extracts in a 50 mM Tris-Cl buffer, pH 7.4, 100 mM NaCl, and 10 µM ZnCl2 for ACE and neprilysin or 100 mM Hepes buffer, pH 7.4, for mAPP were incubated with a 10 µM final concentration of (Abz)YRK(Dnp)P for ACE, (Abz)RGL(EDDnp) for neprilysin, or K(Dnp)PPGK(Abz) for mAPP. Plates were preincubated at 37°C for 15 min before substrate addition. Fluorescence (excitation wavelength of 340 nm, emission wave-length of 420 nm) was assessed kinetically in a FL600 microplate fluorescence plate reader (BioTek, Winooski, VT). Fluorescent units were converted into picomoles of hydrolyzed substrate based on a standard fluorescence curve.
Immunohistochemistry. Lingual tissue samples from all pigs were obtained at necropsy and fixed using 10% buffered formalin phosphate for histological examination. Paraffin-embedded tissue sections (5-µm thick) were prepared, submitted to an antigen-retrieving procedure (Marceau et al., 1999), and immunostained at 37°C for 1.5 h with one of the following antibodies: monoclonal anti-
-actin (clone 1A4, dilution 1:100; Sigma-Aldrich), monoclonal anti-human-B2 receptor (Blaukat et al., 2003), polyclonal anti-von Willebrand factor (vWF, dilution 1:100; DakoCytomation California Inc., Carpinteria, CA), or a mixture of polyclonal antibodies raised against six distinct peptides from the B1 receptor human sequence (Mazenot et al., 2001). The antibody staining was revealed using horseradish peroxidase-coupled goat anti-mouse IgG (dilution 1:100; Sigma-Aldrich) for primary monoclonal antibodies or monoclonal anti-rabbit IgG (dilution 1:200; Sigma-Aldrich) for polyclonal antibodies (30-min reactions, 37°C). The secondary antibodies were allowed to react for 5 to 15 min at 25°C with the Immunopure metal-enhanced diaminobenzidine substrate (Pierce Chemical). Endogenous peroxidase was initially inhibited in tissue sections using 3% H2O2 (5 min).
Statistical Analysis. The systemic inflammatory response and the enzymatic activities in plasma are analyzed using a two-way analysis of variance with repeated measures on one of the factors. The factors are the groups with four levels (one-time ACEi treatment, 7-day ACEi treatment, LPS, and control) and the time with two levels [before experimental protocol (T0) and at sacrifice (TS)], the latter being the repeated factor. In case of interaction, separate analyses of variance with the factor group are performed at each time. Significant differences with the control groups were further assessed using Dunnett's test (p < 0.05).
For the normalized ratio of the examined target gene/GAPDH generated by the real-time PCR analysis, a logarithmic transformation was used to bring the data closer to normality. The statistical significance was established using the mean values of the log normalized ratio calculated for each animal for a given tissue. The biochemical investigations in tissues are analyzed separately for each tissue. For the log normalized ratio and the biochemical parameters, a one-way analysis of variance with a factor group with four levels (one-time ACEi treatment, 7-day ACEi treatment, LPS, and control) was used followed by Dunnett's test when a significant difference was observed (p < 0.05).
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Biochemical Analysis in Plasma. Enzymatic activities measured in blood plasma for ACE, aminopeptidase P, and carboxypeptidase N are presented in Table 2. For ACE, the four groups progressed differently and a significant interaction was observed between the factors time and group [F(3,20) = 43.417, p < 0.001]. At T0, a difference was obtained between the groups [F (3,20) = 4.77, p = 0.011]; the one-time ACEi treatment group was significantly different from the control group (p = 0.025; an unexplained variation observed before drug dosing). At TS, a significant difference was observed [F(3,20) = 105.81, p < 0.001]; as expected, the one-time and 7-day ACEi treatments lead to a significantly lower ACE activity (p < 0.001) when compared with LPS and control groups. Both ACEi and LPS treatments do not affect significantly plasma activities of aminopeptidase P at TS. Indeed, no interaction between the factors time and group and no time effect were noted [F (3,20) = 0.452, p = 0.719; and F(1,20) = 0.286, p = 0.599, respectively] (data not shown). For carboxypeptidase N, there is a significant interaction between the factors time and group [F(3,20) = 5.088, p = 0.009]. No group effect at each time was observed, but a time effect was significant only for LPS group where carboxypeptidase N activity was 67 ± 10 nmol · min–1 · ml–1 at T0 and increased to 86 ± 6 nmol · min–1 · ml–1 at euthanasia (p < 0.001).
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Endogenous Bradykinin and Des-Arg9-BK Metabolism. Fig. 2 illustrates the calculated area under the curve (AUC), representing the accumulation and subsequent catabolism of immunoreactive bradykinin and des-Arg9-BK measured during the in vitro activation of the contact system using glass beads in 1 ml of plasma sampled from each animal at TS. For bradykinin, a significant interaction was observed between the factors time and group [F(3,20) = 8.440, p = 0.001]. No significant difference was noted for data at T0 [F(3,20) = 1.205, p = 0.333], but at TS, the difference was significant [F(3,20) = 60.94, p < 0.001] and obtained for the one-time and 7-day ACEi treatment groups when compared with the control group (p < 0.001, respectively). For des-Arg9-BK, the same outcome was observed [F(3,20) = 7.069, p = 0.002]. No difference was noted at T0 [F(3,20) = 1.745, p = 0.190], but one occurred at euthanasia [F(3,20) = 11.475, p < 0.001]; in fact, the one-time ACEi (p = 0.006) and 7-day ACEi (p < 0.001) treatment groups were statistically higher from the control group. For the LPS group, the AUC was not statistically different from the control group, neither for bradykinin nor for des-Arg9-BK. There were no differences among groups from samples collected at every other time (data not shown).
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Metallopeptidase Activities in Tissues. Metallopeptidase activities measured in tissue extracts from control animals using quenched fluorescent substrates are given in Table 5. Neither acute nor 7-day ACEi treatments, nor the single LPS injection, significantly affected ACE activities in the kidney, tongue, parotid gland, or laryngeal tissue when compared with the control group (data not shown). However, a significant treatment effect was noted for the neprilysin activity in the kidney and for the mAPP activity in parotid gland [F(3,20) = 19.567, p < 0.001; and F(3,20) = 3.440, p = 0.036, respectively] when compared with the control group. Indeed, the one-time and 7-day ACEi treatment significantly increased neprilysin activity in the kidney, 20461 ± 841 (p < 0.001), and mAPP activity in the parotid gland, 294 ± 151 (p = 0.027), respectively.
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Immunohistochemistry Applied to Lingual Tissue. The tongue, examined below the epithelial surface, contains connective tissue and striated muscle along with blood vessels of various sizes (Fig. 5, hematoxylin and eosin stain). The blood vessels are further identified by immunoreactivity for the endothelial cell marker vWF (one-cell thick lines) and by the smooth muscle cell marker -actin (thicker labeling in arteriole and venule walls). Unexpectedly, tissues from all animals treated for 7 days with the ACEi exhibit only faint vWF vascular staining (Fig. 5). The staining for the B2 receptor is present in endothelial cells of identifiable blood vessels in all groups, but the comparatively weaker B1 receptor signal is only found after treatment with LPS or 7-day ACEi.
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). Here we have set out to analyze in more detail the pathogenesis of ACEi-induced AE. To this end, we used an animal model (i.e., the pig) that is large enough to allow repeated blood sampling and harvesting of oropharyngeal tissues to support multiple analyses.
Clinical and experimental lines of evidence plead for a multifactorial mechanism of ACEi-associated AE. Angioedema could be the result of a coincidence of distinct pharmacological, metabolic, and pathophysiological factors. Previously, we have shown that AE patients have an anomaly of the metabolism of des-Arg9-BK because of a defect of plasma aminopeptidase P activity, evidenced only in the presence of ACEi (Adam et al., 2002; Molinaro et al., 2002). We have shown recently that this low aminopeptidase P depends largely on genetic factors (Duan et al., 2005). Because the incidence of AE is higher in hypertensive smokers treated with an ACEi (Coats, 2002), we analyzed the effect of inflammation (a pathophysiological factor) on the tissue expression of metallopeptidases and on the plasma metabolism of kinins. Inflammation was mimicked by a sublethal dose of LPS. We chose a dose substantially less than that typically used in rats and mice, because, like human and nonhuman primates, pigs are extremely sensitive to LPS (Warren et al., 1997). Since AE episodes have been reported as early as on the first day (Wood et al., 1987) or later during ACEi treatment (pharmacological factor) (Hedner et al., 1992; Shionoiri et al., 1996), two groups of pigs were treated, acutely or chronically. The dosages of a single injection of 100 µg/kg enalaprilat i.v. and the per oral application of 20 mg of enalapril twice daily were chosen in preliminary studies, because they both allowed consistent inhibition of plasma ACE activity, as extensive as in our in vitro studies (Cyr et al., 1999; Molinaro et al., 2002).
Methods previously developed in our laboratory to define the metabolism of endogenous kinins (Decarie et al., 1994; Raymond et al., 1995; Cyr et al., 2001) were applied to the plasma of the experimental animals. A significant in vitro accumulation of kinins (as assessed by the AUC; Fig. 2) has been monitored in the one-time and 7-day ACEi treatment groups when compared with controls. Because aminopeptidase P activities in plasma were similar in both ACEi-treated groups, the observed increased bradykinin and des-Arg9-BK concentrations are exclusively a consequence of ACE blockade and not of a nonspecific inhibition of the activity of aminopeptidase P by ACEi. These observations are important as Hooper et al. (1992) reported a nonspecific inhibition of purified pig aminopeptidase P by some ACEi.
We provide evidence for the expression of kininases and kinin receptors in the oropharyngeal tissues of swine. For this purpose, we developed the relative quantification of our target gene transcripts XPNPEP2 (membrane aminopeptidase P), MME (neprilysin), BDKRB1 (B1 receptor), and BDKRB2 (B2 receptor) in comparison with the reference GAPDH gene transcript. After an analytical validation, a mathematical approach for data analysis was presented to calculate the relative expression of normalized logarithmic ratios.
We show that gene expression of the metallopeptidases neprilysin and mAPP in the oropharyngeal tissues is lower than that measured in the kidney when expressed as a ratio to GAPDH (Table 3 and data not shown), and the enzyme activity measurements fully support this observation. The kidney is a valuable positive expression control that contains all components of the kallikrein-kinin system. A 7-day treatment with an ACEi increased mAPP mRNA expression in the tongue and laryngeal tissue and also increased neprilysin mRNA expression in kidney, tongue, and parotid gland tissues; paradoxically, neprilysin expression was decreased after a single administration of enalaprilat, as well as in LPS-treated animals, and this only in the kidney. As in the control animals, the corresponding enzyme activities were rather stable, with occasional changes that did not match the mRNA variation. Although these observations suggest that other factors regulate the activity of the peptidases, a high variation of the low activities did not reach statistical significance, even when the activities were higher or lower than those measured in the control animals (Table 5 and data not shown). The mechanisms by which ACEi leads to changes in metallopeptidase mRNA expression in the present experimental model remain to be elucidated, although other laboratories have reported that ACEi increase ACE mRNA and activity in cell systems and in some tissues (Chai et al., 1992; King and Oparil, 1992). We were unable to assess the effect of ACEi on ACE expression itself, because obtaining valid and specific primers for the porcine gene was not possible and attempts to synthesize primers based on homologous mammalian sequences remained unsuccessful. To circumvent these problems, the quantification of enzyme activities in tissue homogenates using highly sensitive and specific internally quenched fluorescent substrates for ACE was done, as well as for neprilysin and mAPP (Molinaro et al., 2005). In fact, the enzyme activities in oropharyngeal tissues could not be measured using traditional endpoint methods (Blais et al., 1999). The effects of ACEi treatments on tissue ACE activities contrast with those observed in plasma; no significant difference was seen in ACE catalytic activity in any tissue for any treatment. These results parallel others in this laboratory for rat and human heart, and they could be explained by a washout of the ACEi during the tissue homogenization for membrane preparation (Kinoshita et al., 1993; Blais et al., 1997, 2000a).
Local tissue inflammation in ACEi-treated pigs took the form of a strong up-regulation of the mRNA levels for the kinin B1 receptor in all oropharyngeal and renal tissues after the 7-day drug treatment only. This contrasts with B2 receptor regulation modulated in a tissue-specific manner by 7-day ACEi treatment (mRNA up-regulated in the tongue and laryngeal tissue) and in an apparently inconsistent manner with other tissues or treatments. B1 and B2 receptor genes are well documented to be differently regulated in vivo (Leeb-Lundberg et al., 2005). B1 receptor mRNA was expected to be low in healthy tissues but up-regulated during inflammatory conditions. In our hands, LPS administration at a mid-range level that does not overtly stress the animals (Warren et al., 1997) did not substantially alter kinin receptor mRNA expression in any of the tested tissues despite a marked systemic inflammatory reaction (Naess et al., 1989). The observed increased plasma carboxypeptidase N in the LPS group supports the idea that the synthesis of this protein is increased by inflammation and can be considered to be an acute-phase reactant protein, as previously observed in patients with inflammatory arthritis (Chercuitte et al., 1987). The lack of B1 receptor mRNA up-regulation in this experimental group could be explained by the low dose used and short duration of the endogenous cytokine action (as monitored by the febrile reaction). However, the lingual immunohistochemistry study suggests that some endothelial B1 receptor protein was still present 6 h post-LPS after a hypothetic rise and fall of the corresponding mRNA; this induction reproduces published functional results for this species (Siebeck et al., 1996). ACEi-induced B1 receptor expression has been observed in chronically treated rats (Marin-Castano et al., 2002), although 48-h treatment with enalaprilat did not induce B1 receptor expression in the healthy rabbit despite an effective ACE blockade that should theoretically potentiate endogenous kinins (Marceau et al., 1999). A species- and physiological state-dependent toxic reaction to ACEi could explain the up-regulation of B1 receptor without overt systemic inflammatory reaction (as C-reactive protein remained low and leukocyte counts were normal in ACEi-treated pigs). Vasopressin or epinephrine administration to human subjects depletes endothelial vWF in biopsies from the oral mucosa (Takeuchi et al., 1988). Such depletion was observed in the lingual vasculature in the 7-day ACEi group and may result from a slow onset hemodynamic adaptation that includes the secretion of these hormones and a form of endothelial stimulation that favors B1 receptor expression.
In conclusion, here we provide a sensitive real-time PCR analyses that permit gene expression of metallopeptidases and kinin receptors in the oropharyngeal tissues in the pig. We have detected subtle changes after ACEi treatment. The low activity of kininases, the presence of specific receptors, mainly B1 receptor, and the modulatory effect of inflammation and ACEi support a plausible mechanism whereby kinins could initiate a vasogenic inflammatory process (AE) via the sustained stimulation of either or both kinin receptor subtypes. These results obtained using ACEi could be the basis of future investigations on the pathophysiology of AE associated with more "modern" cardiovascular drugs; e.g., AT1 receptor antagonists and vasopeptidase inhibitors. Moreover, encouraging results in the clinical trial of the B2 receptor antagonist Icatibant support that kinin receptor blockade is of therapeutic interest in hereditary AE (Rosenkranz et al., 2004). It remains to be seen whether B1 receptor blockade could be more effective in this and other forms of AE.
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ABBREVIATIONS: ACEi, angiotensin I-converting enzyme inhibitor; AE, angioedema; ACE, angiotensin I-converting enzyme; NEP, neprilysin; AUC, area under curve; des-Arg9-BK, des-arginine9-bradykinin; mAPP, membrane-bound aminopeptidase P; PCR, polymerase chain reaction; LPS, lipopolysaccharide; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; vWF, von Willebrand factor; (Abz)RGL(EDDnp), (o-aminobenzoic acid)-Arg-Gly-Leu-(ethylenediamine 2,4-dinitrophenyl); K(Dnp)PPGK(Abz), Lys-(2,4-dinitrophenyl)-Pro-Pro-Gly-Lys-(o-aminobenzoic acid); (Abz)YRK(Dnp)P, (o-aminobenzoic acid)-Tyr-Arg-Lys-(2,4-dinitrophenyl)-Pro; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid.
Address correspondence to: Dr. Albert Adam, Université de Montréal, Faculté de Pharmacie, Room 3190, 2900 Blvd.Édouard-Montpetit, C.P. 6128, succ Centre-ville, Montréal, Québec H3C 3J7, Canada. E-mail: albert.adam{at}umontreal.ca
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