Integrative Physiology

Decreased Flow-Dependent Dilation in Carotid Arteries of Tissue Kallikrein–Knockout Mice

Sonia Bergaya, Pierre Meneton, May Bloch-Faure, Eric Mathieu, François Alhenc-Gelas, Bernard I. Lévy, Chantal M. Boulanger
https://doi.org/10.1161/01.RES.88.6.593
Circulation Research. 2001;88:593-599
Originally published March 30, 2001

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Abstract

Abstract— Flow-dependent dilation is a fundamental mechanism by which large arteries ensure appropriate blood supply to tissues. We investigated whether or not the vascular kallikrein-kinin system, especially tissue kallikrein (TK), contributes to flow-dependent dilation by comparing wild-type and TK-knockout mice in which the presence or absence of TK expression was verified. We examined in vitro changes in the outer diameter of perfused carotid arteries from TK+/+ and TK−/− mice. In both groups, exogenous bradykinin caused a similar dilation that was abolished by the B2 receptor antagonist HOE-140, as well as by the NO synthase inhibitor Nω-nitro-l-arginine methyl ester. However, purified kininogen dilated only TK+/+ arteries, demonstrating the essential role of TK in the vascular formation of kinins. In TK+/+ arteries, increasing intraluminal flow caused a larger endothelium-dependent dilation than that seen in TK−/−. In both strains the flow response was mediated by NO and by endothelium-derived hyperpolarizing factor, whereas in TK−/− vasoconstrictor prostanoids participated as well. HOE-140 impaired flow-dependent dilation in TK+/+ arteries while showing no effect in TK−/−. This compound reduced the flow response in TK+/+ arteries to a level similar to that in TK−/−. After NO synthase inhibition, HOE-140 no longer affected the response of TK+/+. Impaired flow-dependent dilation was also observed in arteries from knockout mice lacking bradykinin B2 receptors as compared with wild-type animals. This study demonstrates the active contribution of the vascular kallikrein-kinin system to one-third of the flow-dependent dilation response via activation of B2 receptors coupled to endothelial NO release.

Large arteries accommodate changes in blood flow by increasing their diameter via the release of endothelial factors such as NO, prostacyclin, and endothelium-derived hyperpolarizing factor (EDHF).1 2 3 4 This flow-dependent dilation (FDD) response represents a fundamental physiological mechanism that acts to oppose vasoconstriction, normalize shear stress, and ensure appropriate blood supply to tissues.

Bradykinin modulates vascular smooth muscle tone by stimulating endothelial B2 receptors and releasing NO and other mediators.5 6 7 Bradykinin is a product of the cleavage of high and low molecular weight kininogens resulting from activation of proteases such as plasma and tissue kallikreins (TKs), cathepsins, or calpains.8 9 10 11 12 The presence in the vessel wall of kininogens and kallikrein-like activities, including TK, suggests the existence of an endogenous vascular kinin-generating system, different from the circulating plasma kallikrein-kinin system.13 14 15 16 However, the physiological role of the vascular kallikrein-kinin system itself has not been fully established because of the possible contribution of preadsorbed plasma kallikrein to vascular kinin formation and the lack of specific inhibitors of TK.17 18 19

Experimental evidence suggests that endogenously formed kinins could participate in the regulation of vascular tone. For instance, cultured endothelial cells may release sufficient amounts of kinins to activate B2 receptors and to stimulate release of NO.20 In addition, exposure of isolated blood vessels to exogenous kallikrein, purified kininogens, or inhibitors of kinin degradation has been observed to cause a relaxation response mediated by B2 receptor activation.21 22 23 24 However, these experiments did not resolve the question of whether or not blood vessels were capable of generating enough vasoactive kinins from their endogenous kallikrein-kinin system to affect their diameter. Observations that a bradykinin B2 receptor antagonist decreases FDD in human arteries suggest that activation of vascular B2 receptors by either circulating or locally formed kinins mediates this physiological response.25 These experiments did not reveal which kinin-generating pathway is involved in FDD.

The purpose of the present study is to elucidate the role of TK in FDD using isolated carotid arteries from mice lacking the TK klk1 gene versus wild-type mice. The results demonstrate the active role of TK in arterial adaptation to changes in luminal flow.

Materials and Methods

Animal Groups

TK-null mice were obtained by targeted disruption of the TK klk1 gene that was accomplished by replacing 100 bp of exon 4 with the neomycin-resistance gene in embryonic stem cells.26 Breeding of heterozygous mice derived from these embryonic stem cells led to wild-type (TK+/+), heterozygous (TK+/−), and homozygous (TK−/−) littermate mice. Experiments were performed on littermate 12-week-old F2 male mice harboring a mixed genetic 129/Sv-C57BL/6 background. Each genotype at the TK locus was identified by extraction of DNA from mouse tail, and the presence of targeted TK alleles was detected by polymerase chain reaction (PCR).

Some experiments were also performed on male 12-week-old littermate bradykinin B2 receptor–deficient mice (rB2 −/−), which have been backcrossed for 7 generations on the C57BL/6 genetic background.27

Blood Pressure Measurement

Blood pressure was measured in trained and conscious mice using a tail-cuff piezoelectric sensor (LETICA-LE5002). The animals were placed in a prewarmed restrainer during the blood pressure recording. Values were derived from an average of 5 measurements per animal at each time point. Systolic and diastolic pressures were then recorded over a 5-day period.

In Vivo Flow Rate Measurement and Artery Preparation

Mice were anesthetized with ketamine (2.5 mg/g IP; Rhône Mérieux). The left carotid artery was carefully and minimally dissected before measuring in situ the intraluminal flow rate using an ultrasonic apparatus (Transonic Systems Inc). The artery was maintained on a piezoelectric sensor, which can detect flow rates in a range from 0 to 2.5 mL/min. This captor connected to a transit time measurement system and provided an immediate measure of flow rate values. Then, a midsternal thoracotomy was performed and a bolus of heparin (50 IU/30 g body weight) was injected in the sus-hepatic vein. Left and right common carotid arteries were carefully exposed and quickly excised. The procedure was in accordance with the European Community guidelines on the care and use of laboratory animals (Ministère de l’Agriculture, France, authorization No. 07430).

Both carotid arteries were placed immediately in ice-cold modified Krebs-Ringer solution (control solution; composition [in mmol/L] as follows: NaCl 118.3, glucose 5.5, KCl 4.7, CaCl2 2.5, KH2PO4 1.2, MgSO4 1.2, NaHCO3 25, and HEPES 5 [pH 7.4]) gassed with a mixture of 95%O2/5%CO2. The left carotid artery was immediately frozen in liquid nitrogen and kept at −80°C for further total RNA extraction and reverse transcriptase (RT)–PCR analysis. The right carotid artery was used for further in vitro experiments.

RNA Extraction and RT-PCR Analysis

For each group of animals, carotids from 10 mice were pooled and total RNA was extracted according to the Trizol reagent protocol (Life Technologies). The quality of the RNA preparation was confirmed by ethidium bromide staining.

The single-strand cDNA synthesis was carried out in 20 μL of reaction buffer, consisting of first-strand buffer 5× (GIBCO-BRL), RNase inhibitor (40 IU/μL), dNTP (25 mmol/L), DTT (100 mmol/L) (Amersham), and reverse Moloney murine leukemia virus (200 IU/μL). The RT reaction was performed by incubating the reaction mixture for 90 minutes at 37°C followed by 10 minutes at 65°C, using a 3′-primer (5′-CACACTGGAGCTCATCTGGGTATTCAT-3′). The PCR included 35 cycles of the 3 steps of denaturation (94°C, 45 seconds), annealing (65°C, 45 seconds), and extension (72°C, 105 seconds), using the above-mentioned 3′-oligonucleotide and the other 5′-oligonucleotide (5′-GCTTCACCAAATATCAATGTGGGGGTATC-3′). These primers were taken respectively from exons 2 and 4 of the kallikrein klk1 gene, thus synthesizing a 0.4-kb fragment of the TK klk1 cDNA.28 29 The cDNA was amplified using 5 IU/μL of Taq DNA polymerase (Life Technologies) and 20 μmol/L of each set of primers in 50 μL of buffer 10× (containing, in mmol/L, Tris-HCl [pH 8.4] 22, KCl 55, MgCl2 1.65, and dNTP 25, as well as 10 μL of loading dye [0.02% of red cresol and 60% of sucrose]).

In addition, expression of GAPDH was evaluated in parallel to that of TK. Briefly, total RNA was reversed transcribed as mentioned above using the 3′-primer (5′-CATGTAGGCCATGAG-GTCCACCAC-3′). Then, the RT product was amplified during 25 cycles as described for TK, using the 3′-primer and the other 5′-oligonucleotide (5′-TGAAGGTCGGTGTGAACGGATTTGGC-3′). The migration of both PCR products (TK and GAPDH) was then followed on a 2% agarose gel, and they were revealed after ethidium bromide staining.

In Vitro Measurement of the Arterial Diameter

The right carotid artery (≈7 mm in length) was placed in a thermostated bath containing control solution (37°C; 10 mL) continuously gassed with a mixture of 95%O2/5%CO2. The artery was cannulated at both extremities and then perfused continuously in vitro in a system in which flow and pressure can be modified independently, as described earlier.30 The control solution (warmed and gassed throughout the experiment) was used for intraluminal and extraluminal perfusions provided by the mean of 2 perfusion pumps. The carotid artery was maintained under a transmural pressure of 70 mm Hg throughout the experiments. The artery outer diameter was determined with a binocular loop (Nachet, Amethyste 0227) connected to a video-camera system (Living System Instruments, Inc). The pressure at both ends of the arterial segment was monitored using pressure transducers. The 2 catheters on which the carotid artery was mounted presented the same resistance to flow. Furthermore, intraluminal pressure upstream and downstream of the blood vessel was controlled by means of a pressure servo-control (Living System Instruments, Inc), so that flow could be increased without change in pressure. Therefore, the average pressure between distal and proximal pressures can be assumed to be representative of lumen pressure. The outer diameter, as well as proximal and distal pressures, was continuously recorded using Acqknowledge 881 (MP100WS) software (Biopac System, Inc).

At the beginning of each experiment, vessels were equilibrated for 45 minutes at 70 mm Hg and perfused with an intraluminal flow rate of 10 μL/min. The presence of the endothelium was evaluated by assessing the relaxation by acetylcholine (1 μmol/L) during phenylephrine-induced (1 μmol/L) contraction. Experiments were not included in further analysis when the relaxation by acetylcholine was <60% of that induced by sodium nitroprusside (0.1 mmol/L), revealing that the endothelial layer was damaged. In some experiments, the endothelium was removed by air injection (for 1 minute) through the vessel, and contractions to acetylcholine (1 μmol/L) were then observed. At the end of each experiment, passive diameter was obtained after incubation of the artery (40 minutes) with a Ca2+-free control solution containing EGTA (2 mmol/L) and sodium nitroprusside (0.1 mmol/L), which abolished the smooth muscle tone. Phenylephrine and all of the inhibitors or antagonists used in this study were delivered both in the intraluminal and extraluminal perfusions. Acetylcholine, bradykinin, and kininogen were infused intraluminally using a syringe-infusion pump connected to the intraluminal circulation upstream of artery (dilution rate factor of 1/10; intraluminal flow rate of 440 μL/min). Sodium nitroprusside was present in both intra- and extraluminal perfusions.

In Vitro Protocols

The response to bradykinin (10 pmol/L, intraluminally) was evaluated during exposure to phenylephrine (1 μmol/L; intraluminal flow rate of 440 μL/min) and in the presence of a cyclooxygenase inhibitor (indomethacin, 3 μmol/L), an angiotensin-converting enzyme inhibitor (perindoprilat, 10 μmol/L), a carboxypeptidase inhibitor (mergepta, 1 μmol/L), an aminopeptidase inhibitor (bestatin, 10 μmol/L), and a neutral endopeptidase inhibitor (thiorphan, 1 μmol/L). The preparations were exposed intraluminally and extraluminally to the inhibitors for 40 minutes before bradykinin stimulation. Pilot experiments revealed that bradykinin concentrations higher than 10 pmol/L caused contraction, whereas those lower than 1 pmol/L did not affect the arterial diameter (data not shown). For each group, the response to bradykinin (10 pmol/L) was examined under control conditions and after exposure to the bradykinin B2 receptor antagonist HOE-140 (0.1 μmol/L)31 or the NO synthase inhibitor Nω-nitro-l-arginine methyl ester (L-NAME; 0.1 mmol/L) in separate experiments. The intraluminal flow rate during exogenous bradykinin infusion was 440 μL/min.

Response to a mixture of high and low molecular weight kininogen semipurified from bovine plasma was assessed using the same protocol as described above for exogenous bradykinin. This semipurified kininogen was devoid of kinase activity and of activatable prekallikrein activity.32 The final concentration of the kininogen preparation used (1.06 mg/L, intraluminally) could generate up to 100 ng of bradykinin per mg (100 pmol/L). In addition, no contaminating free bradykinin was detected by radioimmunoassay in the preparation, indicating that free BK was absent or lower than 0.004 pmol/L under these conditions and could therefore not contribute to the kininogen vasoactive effects. For each group, the response to kininogen was examined under control conditions and after exposure to HOE-140 or L-NAME in separate experiments.

All experiments evaluating the response to increases in intraluminal flow rate were performed in the presence of phenylephrine (1 μmol/L) and in the absence of inhibitors of kinin degradation pathway. When the contraction to phenylephrine was stable for at least 10 minutes, the intraluminal flow rate was increased in a stepwise manner from 10 to 800 μL/min. Each flow rate was applied for ≈3 to 9 minutes, until the diameter reached a plateau, and then was augmented to the next level. Experiments were performed in the presence of either L-NAME (0.1 mmol/L), diclofenac (a cyclooxygenase inhibitor; 1 μmol/L), or HOE-140 (1 μmol/L). When K+ (20 mmol/L) was used, the concentration of phenylephrine was decreased to 10 nmol/L to match the decrease in diameter among all experimental conditions. Each antagonist or inhibitor was incubated for 40 minutes in the intraluminal and extraluminal perfusions. For each group, the response to increase in flow rate was examined under control conditions and after exposure to the different inhibitors and antagonists in separate experiments.

Drugs and Chemical Agents

The compounds used for in vitro studies were acetylcholine chloride, ([2S, 3R]-3-amino-2-hydroxy-4-phenylbutanoyl)-l-leucine (bestatin), bradykinin, diclofenac, indomethacin, L-NAME, l-phenylephrine hydrochloride, and dl-3-mercapto-2-benzylpropanoyl-glycine (thiorphan) (from Sigma). HOE-140 was kindly provided by Drs H.J. Lang and B.A. Schölkens (Hoechst-Marion-Roussel, Frankfurt, Germany). dl-2-Mercaptomethyl-3-guanidinoethylthiopropanoic acid (mergepta) was purchased from Calbiochem. Perindoprilat was kindly supplied by Dr G. Lerebours from the Institut de Recherche Servier (Courbevoie, France).

Data Analysis and Statistics

Data are given as changes in diameter (micrometers) of the artery obtained during contraction with phenylephrine. Results are expressed as mean±SEM for n animals used for each experimental protocol. The contribution of endogenous kinins to FDD was estimated as the percentage difference in area under the flow-dependent response curves (as measured by the trapezoidal rule) for TK+/+ versus TK−/− carotid arteries. Statistical evaluation was performed by use of ANOVA for factorial or repeated measures, followed by a Scheffe t test.33 Values of P<0.05 were considered to be statistically significant.

Results

Expression of TK was examined by RT-PCR in carotid arteries from TK+/+ and TK−/− mice. Results show that expression of TK was prevented after targeted disruption of the klk1 gene in TK−/− mice (Figure 1⇓). Mean arterial blood pressure was not different among the strains (TK+/+, 95±3; TK+/−, 98±3; and TK−/−, 96±2 mm Hg, n=6; P=0.65). Neither was there any significant difference in in vivo carotid artery blood flow (TK+/+, 599±61; TK+/−, 605±67; and TK−/−, 635±50 μL/min, n=10; P=0.12).

Figure 1.
Figure 1.

Expression of the TK gene in the carotid artery of TK+/+ and TK−/− mice. This typical RT-PCR experiment is representative of 3 different experiments obtained on different pools of carotid arteries. Expression of GAPDH was observed in the same samples in parallel experiments.

In perfused TK+/+, TK+/−, and TK−/− carotid arteries, exposure to phenylephrine, bradykinin, acetylcholine, or sodium nitroprusside caused comparable changes in outer diameter (Table⇓). After endothelium removal, bradykinin no longer increased arterial diameters (data not shown). In all groups, L-NAME or the B2 receptor antagonist HOE-140 significantly inhibited the response to bradykinin (Table⇓). Exogenous kininogen caused a significant diameter increase in arteries from TK+/+ mice but not in those from TK+/− or TK−/− mice (Table⇓). In TK+/+ arteries, exposure to L-NAME or HOE-140 prevented the kininogen-induced dilation. Although kininogen did not affect the artery diameter in both TK+/− and TK−/− mice, subsequent exposure to acetylcholine caused a complete dilation in these preparations (Table⇓).

View this table:
Table 1.

Changes in Outer Diameter (μm) of Carotid Arteries from TK+/+, TK+/−, and TK−/− Mice, in Response to Phenylephrine (Phe), Acetylcholine (Ach), Bradykinin (BK), Kininogen, and Sodium Nitroprusside (SNP)

Stepwise increases in intraluminal flow rate affected carotid artery diameter in all 3 groups of mice. In TK+/+ arteries, the increase in flow rates resulted in a diameter increase, whereas this response was significantly smaller in preparations from TK+/− and TK−/− mice (P=0.046 and P=0.0001, respectively) (Figures 2⇓ and 3⇓). Actually, low flow rates (between 0 and 200 μL/min) caused a decrease in diameter in TK−/− (Figures 2⇓ and 3⇓). The contribution of endogenous kinins to the flow response was estimated at 34% as measured by the difference in area under the flow-dependent response curves for the TK+/+ versus TK−/− groups.

Figure 2.
Figure 2.

Representative traces of FDD in perfused carotid arteries in TK+/+ (top) and TK−/− mice (bottom). Traces indicate time course of changes in diameter (μm) after increase in flow rates during contraction to phenylephrine (Phe). Acetylcholine (Ach) was added extraluminally at the end of the experiment to investigate again endothelium-dependent responses. i.l. indicates intraluminally; e.l., extraluminally.

Figure 3.
Figure 3.

Flow-induced changes in diameter (μm) in perfused carotid arteries from TK+/+ (n=10; ▪), TK+/− (n=7; ▴), and TK−/− mice (n=8; •). *Significant difference compared with TK+/+ (P<0.05).

In TK+/+ arteries, FDD was significantly impaired by L-NAME but was not affected by the cyclooxygenase inhibitor diclofenac (P=0.0001 and P=0.16, respectively) (Figure 4⇓). Exposure to the combination of L-NAME and diclofenac did not augment the effect of L-NAME alone (P=0.75). However, exposure to the combination of L-NAME, diclofenac, and K+ (20 mmol/L) abolished FDD in TK+/+ arteries (Figure 4⇓). In TK−/− arteries, the vasoconstrictor response observed at low flow rates (<200 μL/min) was abolished by diclofenac but was unaffected by L-NAME (P=0.013 and P=0.14, respectively; Figure 4⇓). At higher flow rates (>200 μL/min), the dilation observed in TK−/− arteries was not affected by diclofenac but was significantly decreased by L-NAME (P=0.38 and P=0.04, respectively; Figure 4⇓). Diclofenac did not further impair the dilation observed in the presence of L-NAME alone (P=0.19 when comparing L-NAME versus L-NAME+diclofenac). Finally, FDD in TK−/− arteries was abolished by the combination of L-NAME, diclofenac, and K+ (20 mmol/L) (P=0.007; Figure 4⇓).

Figure 4.
Figure 4.

Flow-dependent responses (μm) in carotid arteries from TK+/+ (top) and TK−/− mice (bottom). Top, In TK+/+ mice, experiments were performed under control conditions (n=10; ▪) and in the presence of L-NAME (n=8; ○) or diclofenac (n=5; ▵) or in the presence of L-NAME, diclofenac (DICLO), and potassium (n=3; ×). Bottom, In TK−/− mice, experiments were performed under control conditions (n=8; ▪) and in the presence of L-NAME (n=8; ○) or diclofenac (n=5; ▵), or in the presence of L-NAME, diclofenac, and potassium (n=3; ×). *Significant difference compared with control conditions (P<0.05).

In TK+/+ arteries, the B2 receptor antagonist HOE-140 significantly impaired FDD (P=0.002). The response obtained in the presence of HOE-140 was not different from that observed with L-NAME (P=0.11; Figure 5⇓). Combining L-NAME and HOE-140 did not significantly affect the flow response observed in the presence of L-NAME alone (P=0.33). Unlike what was observed in TK+/+ mice, HOE-140 had no significant effect on FDD in TK−/− mice (P=0.76; Figure 5⇓). Interestingly, the flow-induced response of TK+/+ carotid arteries exposed to HOE-140 was similar to the response obtained in TK−/− under control conditions (P=0.10). Finally, FDD in the presence of L-NAME plus HOE-140 was not significantly different from that observed in the presence of L-NAME alone in TK−/− arteries (P=0.14).

Figure 5.
Figure 5.

Flow-dependent responses (μm) in carotid arteries from TK+/+ (top) and TK−/− mice (bottom). Top, In TK+/+ mice, experiments were performed under control conditions (n=10; ▪) and in the presence of L-NAME (n=8; □) or HOE-140 (n=9; ▿), or in the presence of L-NAME plus HOE-140 (n=3; ×). Bottom, In TK−/− mice, experiments were performed under control conditions (n=8; ▪) and in the presence of L-NAME (n=8; ○) or HOE-140 (n=6; ▿) or in the presence of L-NAME plus HOE-140 (n=3; ×). *Significant difference compared with control conditions (P<0.05).

The response to increases in intraluminal flow rate was also evaluated in carotid arteries from mice lacking bradykinin B2 receptors (rB2−/−) (Figure 6⇓). In rB2+/+ mice, increases in flow rate augmented the diameter, whereas this response was significantly smaller in preparations from rB2−/− mice (P=0.0016). FDD was significantly impaired by HOE-140 in arteries from rB2+/+ mice (P=0.012) but not in preparations from rB2−/− mice (P=0.70).

Figure 6.
Figure 6.

Flow-induced changes in diameter (μm) in carotid arteries from rB2+/+ mice (top; n=5; ▪) and rB2−/− mice (bottom; n=5; •). Experiments were performed under control conditions (▪ and •) and in the presence of HOE-140 (□ and ○). *Significant effect of HOE-140 compared with control conditions (P<0.05).

Discussion

The present study demonstrates the role of TK in the vascular formation of kinins and the contribution of the vascular kinin-kallikrein system to about one-third of the flow-dependent arterial dilation response, through activation of bradykinin B2 receptors coupled to endothelial NO release.

The mouse carotid artery appears to be an appropriate experimental model to investigate the role of the endogenous vascular kinin-generating system. The presence of TK mRNA observed in TK+/+ arteries is in agreement with previous studies performed with blood vessels and cultured vascular endothelial and smooth muscle cells.14 20 34 35 Bradykinin B2 receptors are functional in this blood vessel given that bradykinin is seen to cause an endothelium-dependent increase in diameter, which is blocked by the B2 receptor antagonist HOE-140 in both TK+/+ and TK−/− mice. In addition, the inhibitory effect of L-NAME demonstrates that the dilation by bradykinin is mediated by the release of endothelial NO. The high sensitivity of the mouse carotid artery to bradykinin is similar to that of other isolated blood vessels and is consistent with the circulating levels of kinins measured in rat plasma.36 37

The response to kininogen in wild-type mice most likely results from the metabolism by TK of exogenous kininogen into kinins. This is suggested by the fact that the dilation evoked by exogenous kininogen is blocked by the bradykinin B2 receptor antagonist and by the NO synthase inhibitor. A similar observation was made earlier in the isolated rat kidney.21 The fact that kininogen, unlike exogenous bradykinin, does not cause a relaxation in TK−/− and TK+/− arteries suggests that TK is an essential kinin-generating pathway in the vessel wall and that the contribution of other kinin-forming enzymes such as adsorbed circulating prekallikrein is minimal.19

In wild-type mice, FDD is mediated by NO and possibly by the EDHF, as the response is inhibited by L-NAME and by K+ but is unaffected by a cyclooxygenase inhibitor. In preparations from TK−/− mice, the response to flow is also mediated by NO and EDHF. Interestingly, a flow-dependent vasoconstrictor response is revealed in TK−/− mice at low flow rates, which can be abolished by diclofenac, indicating the involvement of vasoconstrictive cyclooxygenase products.

The present study demonstrates that the vascular kallikrein-kinin system participates in FDD, a fundamental mechanism in large arteries. This conclusion is based on several experimental observations. First of all, bradykinin B2 receptors mediate part of the FDD, as can be seen from the significant impairment of this response by the B2 receptor antagonist in wild-type mice. This is consistent with results obtained for canine and human arteries.24 25 In addition, FDD is significantly reduced in TK+/−, and even more so in TK−/−, as compared with TK+/+ carotid arteries. Finally, the amplitude of this response in TK−/− is comparable with that observed after blockade of bradykinin B2 receptors in wild-type arteries. Our results therefore show that TK is involved in FDD in murine arteries and probably in other species, including human. Although HOE-140 also exhibits inverse agonist properties affecting the basal activity of B2 receptors,38 this is unlikely to be the case under the present experimental conditions. Indeed, HOE-140 does not affect FDD in TK−/− arteries, although these preparations do express functional bradykinin B2 receptors. Furthermore, the present study demonstrates that FDD is impaired in arteries from mice lacking B2 receptors, similar to our observations on preparations from TK−/− mice.

Several mechanisms may explain the active contribution of the vascular kinin-kallikrein system to FDD. For instance, increasing flow may favor the interaction of bradykinin with its receptor by augmenting its boundary layer mass transport, therefore offsetting the possible degradation of the peptide at the plasma membrane level.4 This interpretation is consistent with the lack of effect of kinin-degradation pathway inhibitors on FDD in mice carotid arteries (S.B., unpublished observations, 2000). Alternatively, we cannot exclude the possibility that the shear stress–induced rearrangement of the endothelial cytoskeleton might modulate the interaction of either TK with kininogen or bradykinin with its receptor.4 30 Other peptides might be involved as well. Indeed, activation of AT2, which stimulates kinin release,39 40 is also implicated in FDD of rodent resistance artery.41 Interestingly, overexpression of AT2 in vascular smooth muscle cells stimulates kininogenase activity and aortic kinin formation, leading to bradykinin B2 receptor activation.42

Although the exact mechanism involving kinins and TK in FDD deserves further investigation, this study reveals the contribution of TK to vascular kinin formation and to flow-dependent regulation of arterial diameter through activation of B2 receptors coupled to endothelial NO release. The contribution of endogenously formed kinins to FDD may appear inconsistent with previous studies showing different intracellular mechanisms for shear stress and bradykinin in endothelial cells.43 However, recent evidence suggests that bradykinin, like shear stress, may activate Akt protein kinase to alter endothelial NO synthase phosphorylation and cause NO release, although this remains to be confirmed.44 45

Acknowledgments

This study was supported by the Institut National de la Santé et de la Recherche Scientifique, the Association Claude Bernard, and the Bristol Myers Squibb Institute for Medical Research.

Footnotes

  • Original received September 13, 2000; resubmission received December 8, 2000; revised resubmission received February 1, 2001; accepted February 2, 2001.

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  • Decreased Flow-Dependent Dilation in Carotid Arteries of Tissue Kallikrein–Knockout Mice
    Sonia Bergaya, Pierre Meneton, May Bloch-Faure, Eric Mathieu, François Alhenc-Gelas, Bernard I. Lévy and Chantal M. Boulanger
    Circulation Research. 2001;88:593-599, originally published March 30, 2001
    https://doi.org/10.1161/01.RES.88.6.593
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