Increased Reactive Oxygen Species Generation Contributes to the Atherogenic Activity of the B2 Bradykinin Receptor

Atherosclerosis and ensuing cardiovascular disease are major causes of death with insufficient treatment options. In search for pathomechanisms of atherosclerosis, we investigated the impact of the B2 bradykinin receptor, Bdkrb2, on atherosclerotic lesion formation, because to date it is not clear whether the B2 bradykinin receptor is atheroprotective or atherogenic. As a model of atherosclerosis, we used hypercholesterolemic ApoE-deficient (apolipoprotein E-deficient) mice, which develop atherosclerotic lesions in the aorta with increasing age. The role of Bdkrb2 in atherosclerosis was studied in ApoE-deficient mice, which were either Bdkrb2-deficient, or had moderately increased aortic B2 bradykinin receptor protein levels induced by transgenic BDKRB2 expression under control of the ubiquitous CMV promoter. We found that Bdkrb2 deficiency led to a significantly decreased atherosclerotic plaque area whereas transgenic BDKRB2 expression enhanced atherosclerotic lesion formation in the aorta of ApoE-deficient mice at an age of 8 months. Concomitantly, the aortic content of reactive oxygen species (ROS) was higher in BDKRB2-expressing mice whereas Bdkrb2 deficiency decreased aortic ROS levels of ApoE-deficient mice. In addition, aortic nitrate as a marker of nitric oxide activity and the endothelial nitric oxide synthase (eNOS) co-factor, tetrahydrobiopterin (BH4) were reduced in BDKRB2-expressing ApoE-deficient mice. The decreased aortic BH4 content could be a consequence of increased ROS generation and down-regulated aortic expression of the BH4-synthesizing enzyme, Gch1 (GTP cyclohydrolase 1). In agreement with a causal involvement of decreased BH4 levels in the atherogenic function of BDKRB2, we found that treatment with the BH4 analog, sapropterin, significantly retarded atherosclerotic plaque formation in BDKRB2-expressing ApoE-deficient mice. Together our data show that the B2 bradykinin receptor is atherogenic, and the atherosclerosis-promoting function of BDKRB2 is partially caused by decreased aortic BH4 levels, which could account for eNOS uncoupling and further enhancement of ROS generation.

Atherosclerosis and ensuing cardiovascular disease are major causes of death with insufficient treatment options. In search for pathomechanisms of atherosclerosis, we investigated the impact of the B2 bradykinin receptor, Bdkrb2, on atherosclerotic lesion formation, because to date it is not clear whether the B2 bradykinin receptor is atheroprotective or atherogenic. As a model of atherosclerosis, we used hypercholesterolemic ApoE-deficient (apolipoprotein E-deficient) mice, which develop atherosclerotic lesions in the aorta with increasing age. The role of Bdkrb2 in atherosclerosis was studied in ApoE-deficient mice, which were either Bdkrb2-deficient, or had moderately increased aortic B2 bradykinin receptor protein levels induced by transgenic BDKRB2 expression under control of the ubiquitous CMV promoter. We found that Bdkrb2 deficiency led to a significantly decreased atherosclerotic plaque area whereas transgenic BDKRB2 expression enhanced atherosclerotic lesion formation in the aorta of ApoE-deficient mice at an age of 8 months. Concomitantly, the aortic content of reactive oxygen species (ROS) was higher in BDKRB2-expressing mice whereas Bdkrb2 deficiency decreased aortic ROS levels of ApoE-deficient mice. In addition, aortic nitrate as a marker of nitric oxide activity and the endothelial nitric oxide synthase (eNOS) co-factor, tetrahydrobiopterin (BH4) were reduced in BDKRB2-expressing ApoE-deficient mice. The decreased aortic BH4 content could be a consequence of increased ROS generation and down-regulated aortic expression of the BH4-synthesizing enzyme, Gch1 (GTP cyclohydrolase 1). In agreement with a causal involvement of decreased BH4 levels in the atherogenic function of BDKRB2, we found that treatment with the BH4 analog, sapropterin, significantly retarded atherosclerotic plaque formation in BDKRB2-expressing ApoE-deficient mice. Together our data show that the B2 bradykinin receptor is atherogenic, and the atherosclerosis-promoting function of BDKRB2 is partially caused by decreased aortic BH4 levels, which could account for eNOS uncoupling and further enhancement of ROS generation.
Keywords: BDKRB2, reactive oxygen species, atherosclerosis, hypercholesterolemia, GCH1, Apoe, AGTR1, ACE INTRODUCTION Atherosclerosis and cardiovascular disease are leading causes of death worldwide (1,2). The high morbidity and mortality of atherosclerotic vascular disease is in part attributed to limited treatment options (1,2). Elucidation of pathomechanisms with the identification of potential new targets to improve the pharmacotherapy of atherosclerosis therefore is of great interest (1)(2)(3). To study pathomechanisms of atherosclerosis, hypercholesterolemic, apolipoprotein E (ApoE)-deficient mice are often used as a model because these mice reproduce major features of atherosclerotic vascular disease such as hypercholesterolemia-induced atherosclerotic lesion formation in the vascular system with increasing age (4)(5)(6)(7)(8).
In this study, we investigated the role of the B2 bradykinin receptor (BDKRB2) in atherosclerotic lesion formation. To date it is not known whether BDKRB2 is atheroprotective or atherogenic. Due to the blood pressure-lowering and nitric oxide-(NO)-generating activity, the B2 bradykinin receptor is considered to exert cardioprotection (9,10). This cardioprotective potential of B2 bradykinin receptor stimulation by the agonist bradykinin and related kinins of the kinin-kallikrein system is exploited therapeutically with ACE (angiotensin-converting enzyme) inhibitors. ACE inhibition not only blunts the generation of the vasopressor angiotensin II but also prevents the proteolytic degradation of bradykinin (9,10). The contribution of bradykinin to the antihypertensive activity of ACE inhibitors is well-documented in experimental models and patients (11,12). On the other hand, the atherosclerosis-decreasing potential of ACE-inhibition is reportedly independent from bradykinin and B2 bradykinin receptor stimulation (13). Moreover, bradykinin and related kinins are pro-inflammatory peptides, and inflammation is an established risk factor of atherosclerosis (14,15). In addition, beneficial B2 bradykinin receptor-stimulated nitric oxide (NO) generation and vasodilation are impaired in atherosclerosis (16). Hypercholesterolemia and atherosclerosis are known to cause endothelial dysfunction with concomitant uncoupling of endothelial nitric oxide synthase (eNOS), which then generates atherosclerosis-promoting reactive oxygen species (ROS) instead of atheroprotective NO (17)(18)(19).
In view of this scenario, we investigated the impact of the B2 bradykinin receptor on atherosclerotic lesion development.
To study the role of the B2 bradykinin receptor (Bdkrb2) in atherosclerosis, we used (i) ApoE-/-mice with endogenously expressed Bdkrb2, (ii) ApoE-/-mice with Bdkrb2 deficiency, and (iii) ApoE-/-mice with moderately increased transgenic BDKRB2 expression level. We found that transgenic B2 receptor expression enhanced atherosclerotic plaque formation in the aorta of ApoE-/-mice whereas Bdkrb2 deficiency retarded the development of atherosclerosis.

Experimental Model of Atherosclerosis, and Generation of Transgenic Mice
The study was performed with three groups of male mice, i.e., (i) apolipoprotein E-deficient (ApoE-/-) mice in B6 (C57Bl/6J) background (4)(5)(6)(7)(8), (ii) double-deficient Bdkrb2-/-ApoE-/-mice (B2-/-ApoE-/-), and (iii) ApoE-/-mice with transgenic expression of BDKRB2 under control of the ubiquitous CMV immediate-early promoter/enhancer (derived from plasmid pcDNA3, Invitrogen AG -Thermo Fisher Scientific). Bdkrb2deficient mice in B6 background were obtained by backcross breeding of Bdkrb2-/-mice (20) for ten generations into B6 background. Double-deficient, Bdkrb2-/-and ApoE-/-mice, were subsequently generated by cross-breeding, and identified by genotyping PCR. For transgenic expression of BDKRB2, the BDKRB2 transgene (2 ng/µL) was injected into the pronucleus of fertilized oocytes isolated from super-ovulated ApoE-/-mice with B6 background followed by transfer of 2-cell embryos into pseudo-pregnant CD-1 foster mice similarly as described (21). After weaning at an age of 3-4 weeks, PCR genotyping was performed with ear-punch biopsies. Founder mice of the FO generation were identified with stable integration of the transgenic DNA into the genomic mouse DNA and used for further breeding. Due to the pathologic phenotype in ApoE-/-background, Tg-CMVBDKRB2 (Tg-B2++) mice with ApoE-deficiency only were used for phenotyping but otherwise, the colony of Tg-CMVBDKRB2 mice was maintained on the B6 background without ApoE-deficiency (C57BL/6-Tg-(CMVBDKRB2)Sjaa; Janvier No. 181.281 ETH Zurich). Phenotyping was determined with 8-month-old mice. Mice were kept on a 12 h light/12 h dark cycle, had free access to food and water, and were fed a standard rodent chow diet (Ain-93-based diet without addition of tocopherol acetate) containing 7% fat and 0.15% cholesterol. As indicated, 3-month-old mice received sapropterin (10 mg/kg/d, in drinking water, freshly prepared, every day) for 5 months. Treatment of Tg-B2++ApoE-/-mice and ApoE-/-mice with the ACE inhibitor, captopril (20 mg/kg/d, in drinking water, freshly prepared, every day) was started at an age of 3 months and continued for 5 months until the end of the observation period at 8 months. At the end of the study, anesthetized mice (ketamine/xylazine 100 mg/10 mg per kg body-weight) were perfused intracardially with ice-cold, sterile PBS, the aorta was rapidly dissected on ice and processed for further analysis. All animal experiments were performed according to NIH guidelines, and reviewed and approved by the local committee on animal care and use (Cantonal Veterinary office, Zurich).

Biochemical Assays and Aortic Atherosclerotic Lesion Determination
The atherosclerotic lesion area was determined in the aorta by quantitative image analysis of oil red O-stained aortas opened longitudinally (7,8). The area of the aortic intima with pathologic intimal atherosclerotic lesions was determined with hematoxylin-eosin-stained, aortic paraffin sections of the aortic arch. The aortic content of BH4 was determined as described (22). The content of reactive oxygen species (ROS) was determined by quantitative fluorescence evaluation of dihydroethidium-(DHE)-stained aortic cryosections (8,23). The aortic expression of Gch1 (GTP cyclohydrolase 1) was assessed after reverse transcription of mRNA into cDNA followed by quantitative real-time qRT-PCR using a LightCycler 480 Instrument (Roche). For quantitative real-time qRT-PCR, total aortic RNA was isolated by the RNeasy Mini kit according to the protocol of the manufacturer (Qiagen). RNA purity was confirmed by an absorbance ratio A260/280 of ∼2.0. The absence of RNA degradation and RNA quality were further controlled by the presence of bright bands of 18S and 28S ribosomal RNA in denaturing RNA electrophoresis. RNA was reverse transcribed into cDNA by the Transcriptor High Fidelity cDNA Synthesis Kit and subjected to qRT-PCR using the LightCycler R 480 System with the LightCycler R 480 SYBR Green I Master reaction mix according to the protocol of the manufacturer (Roche Molecular Systems). Primer sequences used for determination of Gch1 expression by qRT-PCR were as follows: Gch1 forward 5 ′ -GCC GCTTACTCGTCCATTCT-3 ′ , and Gch1 reverse 5-CCACCG CAATCTGTTTGGTG-3 ′ . Specific amplification of the Gch1 fragment of 358 bp was controlled by agarose gel electrophoresis. Total number of B2 bradykinin receptor binding sites was determined with aortic smooth muscle cells in HEPES-buffered DMEM (supplemented with 1% BSA, protease inhibitors and enalaprilat) by saturation radioligand binding (for 2 h at 4 • C) with increasing concentrations (0.1-10 nM) of [2,3-prolyl-3,4-3 H(N)]bradykinin (79-96 Ci/mmol; Perkin Elmer) in the absence and presence of 10 µM HOE140 to determine non-specific binding. Likewise, the number AT1 receptor binding sites was determined with Sar 1 ,[ 125 I]Tyr 4 ,Ile 8 -angiotensin II (2200 Ci/mmol; Perkin Elmer) in the absence and presence of 10 µM losartan. Aortic vascular smooth muscle cells were isolated from aortas of ApoE-/-, Bdkrb2-/-ApoE-/-, Tg-B2++ApoE-/-and non-transgenic B6 control mice at an age of 6-10 days. For vascular smooth muscle cell (VSMC) isolation, aortas from 10 to 15 mice were dissected. Aortas were endothelium-denuded and minced with a scalpel followed by a washing step with HEPESbuffered DMEM (supplemented with 1 mM L-glutamine, 100 I.U./ml penicillin and 100 µg/ml streptomycin) and digestion at 37 • C with collagenase (2 mg/ml of collagenase type 2) under constant agitation with a magnetic stirrer. The first digestion step was discarded and in subsequent steps, the enzyme solution was exchanged every 5 min. Cells were filtered through a nylon mesh (pore size 40 µm) and collected in DMEM supplemented with 10% FCS. After the aortic tissue was digested, cell fractions were pooled. Thereafter, cells were collected by centrifugation (300 x g), plated on cell culture plates and cultured in DMEM supplemented with 10% FCS as described (24). Total inositol phosphates of aortic smooth muscle cells were determined as described (25). The specific B2 bradykinin receptor-stimulated signal is given and was determined by bradykinin stimulation (100 nM) in the absence and presence of 10 µM of the B2specific antagonist, HOE140. Aortic Gch1 protein contents were determined by immunoblot detection with GCH1/Gch1specific antibody (GCH1 monoclonal antibody M01, clone 4A12, Cat. No. H00002643-M01, Abnova) similarly as detailed previously (25). Aortic tissue nitrate content was determined with a fluorometric assay kit according to the protocol of the manufacturer (Cayman Chemical).

Statistical Analysis
All data are presented as mean ± s.d. Analysis of variance followed by a Post-test as indicated was performed to determine statistical significance between more than two groups. Statistical significance was set at a p-value of < 0.05. Statistical evaluation was performed with GraphPad PRISM 7.0.

Increased Number of Aortic B2 Bradykinin Receptors in Tg-B2++ApoE-/-Mice
To investigate the role of the B2 bradykinin receptor in atherosclerosis, we used hypercholesterolemic ApoE-/-mice as an experimental model of atherosclerosis. These mice develop atherosclerotic lesions in the vascular system and the aorta with increasing age (4)(5)(6)(7)(8). In frame of our study, we compared three groups of ApoE-/-mice with different expression levels of the B2 bradykinin receptor, i.e., (i) ApoE-/-mice in B6 (C57Bl/6J) background with endogenous Bdkrb2 expression level, (ii) ApoE-/-mice in B6 background, which are deficient in the B2 bradykinin receptor gene, Bdkrb2-/-ApoE-/ (B2-/-ApoE-/-), and (iii) ApoE-/-mice in B6 background with transgenic expression of BDKRB2 under control of the ubiquitous CMV promoter, Tg-B2++ApoE-/mice ( Figure 1A). Because the genetic background has a strong influence on the atherosclerotic phenotype of ApoE-/-mice (26), all groups of transgenic mice used in our study had identical B6 background. To obtain Bdkrb2-/-mice with pure B6 (C57Bl/6J) background, we performed 2 years of backcrossing of Bdkrb2-/-mice into the B6 background for more than 10 generations. These Bdkrb2-/-mice with B6 background were then used for cross-breeding with ApoE-/-mice, to finally obtain homozygous mice, which were double-deficient in Bdkrb2 and ApoE (Bdkrb2-/-and ApoE-/-). To generate mice with moderately increased BDKRB2 level, we generated BDKRB2transgenic mice in ApoE-/-background with expression of BDKRB2 under control of the ubiquitous CMV promoter. We used the CMV promoter, because the endogenous B2 bradykinin receptor is also ubiquitously expressed. Male offspring of these three study groups of mice with different expression levels of the B2 bradykinin receptor were used to analyze the impact of this receptor on the pathogenesis of atherosclerosis ( Figure 1A).
B2 bradykinin receptors of aortic smooth muscle cells were functional and mediated an increase in total inositol phosphate levels upon bradykinin stimulation ( Figure 1C).
Together these data show that transgenic BDKRB2 expression under control of the ubiquitous CMV promoter led to a moderately increased number of B2 bradykinin receptor binding sites on aortic smooth muscle cells of Tg-B2++ApoE-/-mice whereas the B2 receptor was absent in B2-/-ApoE-/-mice with deficiency of the B2 bradykinin receptor gene, Bdkrb2.
Transgenic BDKRB2 Expression Enhances Atherosclerotic Lesion Formation Whereas Bdkrb2 Deficiency Leads to a Decreased Atherosclerotic Plaque Area in the Aorta of ApoE-/-Mice To investigate the impact of the B2 bradykinin receptor on atheroma formation, we determined the atherosclerotic lesion area in the aorta of 8-month-old ApoE-/-mice. As outlined before, the study compared three different groups of mice, i.e., (i) ApoE-/-mice with endogenous Bdkrb2 expression level, (ii) B2-/-ApoE-/-mice with Bdrkb2 deficiency, and (iii) Tg-B2++ApoE-/-mice with moderately increased B2 receptor level due to transgenic BDKRB2 expression. All mice had B6 background. Atherosclerotic lesion area in the aorta was quantified by oil red O staining (Figure 2A). We found that transgenic BDKRB2 expression led to a significantly enhanced atherosclerotic plaque formation in the aorta of Tg-B2++ApoE-/-mice, i.e., the atherosclerotic lesion area was increased 2.2 ± 0.4-fold in Tg-B2++ApoE-/-mice compared to ApoE-/mice with endogenous Bdkrb2 level ( Figure 2B). In contrast, B2 bradykinin receptor deficiency led to a significantly decreased atherosclerotic plaque area in double-deficient, B2-/-ApoE-/mice compared to ApoE-/-mice with endogenously expressed Bdkrb2 ( Figure 2B). These data provide strong evidence that the B2 bradykinin receptor enhances the progression of atherosclerotic plaque formation in the aorta of 8-month-old ApoE-/-mice as an experimental model of atherosclerosis.
Transgenic BDKRB2 Expression Leads to an Increased Aortic ROS Content in Tg-B2++ApoE-/-Mice Enhanced generation of reactive oxygen species (ROS) is a major contributor to atherosclerotic plaque formation in experimental models of atherosclerosis and involved in the pathogenesis of atherosclerosis in patients (8,18,27). We asked whether the B2 bradykinin receptor led to an increased aortic ROS content of Tg-B2++ApoE-/-mice. Aortic ROS was detected in situ by dihydroethidium (DHE) staining (8,23). Quantitative image analysis shows that the ROS production in the aorta of BDKRB2-expressing Tg-B2++ApoE-/-mice was significantly increased compared to ApoE-/-mice with endogenous B2 receptor expression level, i.e., aortic ROS levels of Tg-B2++ApoE-/-mice were 1.6 ± 0.3-fold higher than those of ApoE-/-mice (Figures 3A,B). Vice versa, there was a significant decrease in the aortic ROS content of doubledeficient B2-/-ApoE-/-mice compared to ApoE-/-mice with intact Bdkrb2 gene (Figures 3A,B). Taken together, the B2 bradykinin receptor mediates an enhanced production of ROS in the aorta of atherosclerosis-prone Tg-B2++ApoE-/-mice with hypercholesterolemia.
The Aortic Nitrate Content as a Marker of NO Activity and the Nitric Oxide Synthase (NOS) Cofactor Tetrahydrobiopterin (BH4) Are Decreased in Tg-B2++ApoE-/-Mice On vascular endothelial cells, the B2 bradykinin receptor is known for its nitric oxide (NO)-stimulating function (10,28,29). Nitric oxide is considered as a major atheroprotective factor released from intact endothelium (30,31). On the other hand, cardiovascular risk factors such as hypercholesterolemia and atherosclerosis trigger endothelial dysfunction (29,30), which leads to endothelial nitric oxide synthase (eNOS) uncoupling (19). Uncoupled eNOS produces atherogenic superoxide instead of atheroprotective NO (19). In agreement with previous data on defective NO generation in hypercholesterolemic ApoE-/-mice (32), the aortic tissue nitrate content as a marker of NO activity in vivo (33), was significantly decreased in ApoE-/-mice compared to non-transgenic B6 mice, i.e the aortic nitrate of ApoE-/mice was 539 ± 89 nmol/g compared to 1031 ± 158 nmol/g in B6 mice ( Figure 4A). Transgenic expression of BDKRB2 further decreased the aortic nitrate content in Tg-B2++ApoE-/-mice compared to ApoE-/-mice with endogenous Bdkrb2 expression ( Figure 4A). Vice versa, deficiency of Bdkrb2 led to a significantly increased aortic nitrate content of Bdkrb2-/-ApoE-/-mice compared to ApoE-/-mice ( Figure 4A). This finding is complementary to previous data, which show that deficiency of Bdkrb2 increases the serum nitrate level (34). Nevertheless, the aortic nitrate content of Bdkrb2-/-ApoE-/mice was not normalized to B6 control level by B2 bradykinin receptor deficiency (Figure 4A), most likely because defective NO generation in atherosclerotic Bdkrb2-/-ApoE-/-mice has additional causes, which are independent of Bdkrb2, e.g., ApoE deficiency-induced hypercholesterolemia. Together these data show that the B2 bradykinin receptor mediates a decrease in aortic nitrate content as a marker of dysfunctional aortic (e)NOS activity in Tg-B2++ApoE-/-mice whereas deficiency of Bdkrb2 increases the aortic nitrate level in Bdkrb2-/-ApoE-/-mice. The eNOS uncoupling during the pathogenesis of atherosclerosis was partially attributed to down-regulation of the (e)NOS cofactor, tetrahydrobiopterin, BH4 (35,36). We asked whether the decreased aortic nitrate content of BDKRB2transgenic Tg-B2++ApoE-/-mice was accompanied by changes in aortic BH4 levels. We measured the aortic BH4 content and found that transgenic BDKRB2 expression led to a decrease in the aortic BH4 content of Tg-B2++ApoE-/-mice, which was 2.48 ± 0.56 pmol/mg in Tg-B2++ApoE-/-mice compared to 5.23 ± 1.09 pmol/mg in ApoE-/-mice with endogenous Bdkrb2 expression level (Figure 4B). For comparison, aortic BH4 levels were not significantly different between ApoE-/-mice with Bdkrb2-deficiency and non-transgenic B6 mice (Figure 4B). This observation could be due to the fact that there are two opposing mechanisms acting on the aortic BH4 level in Bdkrb2-/-ApoE-/-mice, i.e., (i) ApoE deficiency, which decreases the aortic BH4 content, and (ii) Bdkrb2 deficiency, which counteracts the ApoE deficiency-induced BH4 decrease. The net result is a normalization of aortic BH4 content in Bdkrb2-/-ApoE-/-mice toward the level of non-transgenic B6 mice (Figure 4B).
Histologic evaluation of hematoxylin-eosin-stained aortic specimens from the different groups of ApoE-/-mice confirmed the enhanced atherosclerosis progression of Tg-B2++/ApoE-/mice by an increased intimal atherosclerotic lesion area in the aortic arch of Tg-B2++ApoE-/-mice with transgenic BDKRB2 expression compared to ApoE-/-mice with endogenous Bdkrb2 expression, and double-deficient B2-/-ApoE-/-mice (Figures 4C,D). Together these data show a decrease in (e)NOS activity as documented by a reduced aortic content of nitrate and depletion of the (e)NOS co-factor, BH4, by the B2 bradykinin receptor in hypercholesterolemic Tg-B2++ApoE-/-mice with concomitantly enhanced atherosclerotic lesion development.
The Major BH4-Synthesizing Enzyme, Gch1 (GTP Cyclohydrolase 1) Is Down-Regulated by BDKRB2 in Tg-B2++ApoE-/-Mice We searched for the mechanism underlying the aortic decrease in the (e)NOS cofactor, BH4, which was triggered by the B2 bradykinin receptor. BH4 deficiency in atherosclerosis could be caused by at least two different mechanisms, i.e., by (i) BH4 oxidation as a consequence of increased ROS levels (cf. Figure 3), and (ii) impaired BH4 synthesis due to decreased levels/activity of the GTP cyclohydrolase 1 (Gch1), which is the rate-limiting enzyme in tetrahydrobiopterin (BH4) biosynthesis (36,37). Previous data have shown that endothelial Gch1 activity and expression are decreased by various cardiovascular risk factors (37). Therefore, we determined the aortic expression level of Gch1 in Tg-B2++ApoE-/-mice. Our data show that aortic Gch1 expression levels were significantly decreased in BDKRB2expressing Tg-B2++ApoE-/-mice compared to ApoE-/-mice with endogenous Bdkrb2 levels, i.e., aortic Gch1 levels were 2.1fold lower in Tg-B2++ApoE-/-mice compared to ApoE-/-mice ( Figure 5A). For comparison, the aortic Gch1 expression level of B2-/-ApoE-/-mice with Bdkrb2 deficiency was not significantly different from non-transgenic B6 mice (Figure 5A). As a control, immunoblot detection of the aortic Gch1 protein confirmed the significant decrease of the aortic Gch1 content in BDKRB2expressing Tg-B2++ApoE-/-mice compared to ApoE-/-mice with and without endogenously expressed Bdkrb2 (Figure 5B).

ACE Inhibition With Captopril Inhibits Atherosclerotic Plaque Accumulation in Tg-B2++ApoE-/-Mice
The angiotensin II AT1 receptor is a major contributor to atherosclerosis-promoting ROS generation in ApoE-/-mice (8,(38)(39)(40). Atherogenic functions of the AT1 receptor are enhanced by BDKRB2 (25,41), and atherosclerosis-related endothelial dysfunction can be prevented by inhibition of angiotensin II AT1 receptor stimulation with an inhibitor of the angiotensin II-generating ACE (42,43). Because BDKRB2 enhanced the endothelial dysfunction of Tg-B2++ApoE-/-mice, we asked whether inhibition of ACE-dependent angiotensin II generation in Tg-B2++ApoE-/-mice could retard the BDKRB2-enhanced atherosclerosis progression. To address this question, we treated Tg-B2++ApoE-/-mice with the ACE inhibitor, captopril (20 mg/kg/d) for 5 months. Atherosclerotic lesion area was evaluated of oil red O-stained aortas and revealed that captopril largely prevented the accumulation of atherosclerotic plaques in 8 month-old Tg-B2++ApoE-/-mice (Figures 7A,B). In agreement with previous studies (7,8), captopril also inhibited the formation of atherosclerotic lesions in ApoE-/-control mice with endogenous Bdkrb2 expression (Figures 7A,B). Taken together, inhibition of ACE-dependent angiotensin II AT1 receptor activation is capable to prevent the BDKRB2enhanced atherosclerotic lesion development in Tg-B2++ApoE-/-mice. Consequently, the atherogenic function of the B2 bradykinin receptor in ApoE-/-mice involves a synergistic interplay between the B2 bradykinin receptor and angiotensin II-stimulated AT1 receptor activation.

DISCUSSION
In this study, we investigated the impact of the B2 bradykinin receptor on atherosclerotic lesion formation in hypercholesterolemic ApoE-/-mice as a model of atherosclerosis. We found that a moderately increased BDKRB2 level in Tg-B2++ApoE-/-mice led to a significantly enhanced progression of atherosclerotic lesion development whereas deficiency of Bdkrb2 retarded the accumulation of atherosclerotic plaques in Bdkrb2-/-ApoE-/-mice.
In search for pathomechanisms underlying the enhanced atherogenesis triggered by BDKRB2, we detected an increased aortic ROS content in Tg-B2++ApoE-/-mice compared to ApoE-/-mice with endogenous Bdkrb2 expression level. In addition, the endogenous Bdkrb2 contributes to enhanced ROS generation in ApoE-/-mice because the aortic ROS content was significantly lower in double-deficient Bdkrb2-/-ApoE-/-mice compared to ApoE-/-mice with intact endogenous Bdkrb2 gene. The increased vascular ROS level could directly contribute to enhanced atherosclerotic lesion formation because ROS is known to accelerate atherogenesis in animal models of atherosclerosis and patients with atherosclerotic vascular disease (8,18,27). Aortic ROS generation in ApoE-/-mice was previously attributed to the atherosclerosis-enhancing function of the angiotensin II AT1 receptor, which generates ROS by activation of NADPH oxidases (8,38). Activation of the AT1 receptor in experimental models of atherosclerosis and patients with cardiovascular disease is increased due to hypercholesterolemia-induced up-regulation of the systemic renin angiotensin system (39,40). Our study identifies the B2 bradykinin receptor as another player involved in enhanced ROS generation in ApoE-/-mice. B2 bradykinin receptor-enhanced ROS generation could as well be mediated by the AT1 receptor, which becomes hyperactive by protein complex formation with the B2 bradykinin receptor (25,41). Notably, endogenous Bdkrb2 levels are sufficient to sensitize the AT1 receptor-stimulated response, and the AT1-sensitizing function of the B2 bradykinin receptor does not require bradykinin (25,41). In agreement with a bradykinin-independent function of the B2 bradykinin receptor in atherosclerosis, treatment of ApoE-/-mice with the B2 bradykinin receptor-specific antagonist, HOE140, did not alter atherosclerosis progression (13). However, the (brady)kiningenerating kallikrein system was not active in this study, because treatment with the B2-specific antagonist, HOE140, had no effect on blood pressure, neither under basal conditions nor upon blood pressure lowering with the ACE inhibitor, ramipril (13). Thus, bradykinin-dependent and bradykinin-independent effects need to be considered in the atherosclerosis-promoting activity of the B2 bradykinin receptor depending on the activation state of the kinin-kallikrein system.
As a consequence of the AT1-receptor sensitizing activity, the B2 bradykinin receptor could mediate a decrease in the nitric oxide synthase (NOS) cofactor, BH4, which is inactivated by ROS (37). Decreased BH4 leads to uncoupled eNOS, which in turn generates more ROS. The decrease in aortic BH4 content could synergistically be aggravated by downregulation of Gch1, which is the rate-limiting enzyme in BH4 synthesis. In concert with hypercholesterolemia, BDKRB2 and Bdkrb2 could decrease vascular Gch1 expression and protein level by activation of Gi-coupled signaling because hypercholesterolemia and Gicoupled signaling are both known to down-regulate Gch1 (44)(45)(46). The ensuing depletion of vascular BH4 could contribute to eNOS uncoupling in the pathogenesis of atherosclerosis (35). The uncoupled eNOS generates detrimental ROS instead of atheroprotective NO (Figure 8). Transgenic animal models support that Gch1 deficiency and reduced vascular BH4 accelerate atherosclerosis whereas supplementation of BH4 or Gch1 expression reverse these deficits and retard atherosclerosis (19,35,47). In agreement with these findings, we found that treatment with BH4 decreased aortic ROS levels and dampened the atherosclerosis-enhancing effect of BDKRB2.
It is well-established that the bradykinin-potentiating effect of ACE inhibitors contributes to cardio-protection [ Figure 8A, (9,10)]. However, the atherosclerosis-lowering potential of ACE inhibition does not necessarily rely on bradykinin-stimulated Bdkrb2 activation, e.g., when there is no activation of the (brady)kinin-generating kallikrein system (13). A B2-specific antagonist, HOE140, also had no effect on atherosclerosis progression in ApoE-/-mice without ACE inhibition (13), most likely because the beneficial NO-generating capacity of endothelial B2 bradykinin receptor stimulation is impaired in atherosclerosis and cardiovascular disease [ Figure 8B, (42,43,48)]. Because atherosclerosis progression in ApoE-/-mice is not affected by the B2-specific antagonist, HOE140 (13), atherosclerosis promotion by Bdkrb2 in ApoE-/-mice could largely be mediated by the angiotensin II AT1 receptorsensitizing function of Bdkrb2, which is bradykinin-independent (25,41). In agreement with this conclusion, our study shows that inhibition of angiotensin II AT1 receptor stimulation by an ACE inhibitor completely prevents atherosclerotic lesion formation in Tg-B2++ApoE-/-mice. Thus, under hypercholesterolemia, the B2 bradykinin receptor gene is atherogenic, leads to increased vascular ROS, mediates a decrease in (e)NOS activity as evidenced by a reduced aortic nitrate content, and enhances aortic BH4 deficiency ( Figure 8B). BH4 depletion contributes to the atherogenic activity of the B2 bradykinin receptor because supplementation with BH4 was sufficient to inhibit aortic ROS and retard atherosclerosis progression triggered by transgenic BDKRB2 expression (Figure 8B).
Enhanced ROS generation in hypercholesterolemia and atherosclerosis is largely attributed to angiotensin II AT1 receptor stimulation (8,38). Consequently, inhibition of detrimental AT1 receptor-stimulated ROS generation by an ACE inhibitor or AT1 antagonist in animal models and patients could exert a dual protective function regarding the atherogenic activity of BDKRB2, i.e., (i) restoration of protective B2 bradykinin receptor signaling, and (ii) neutralization of the AT1-sensitizing function of BDKRB2. Symptoms of the herein deduced atherogenic activity of the B2 bradykinin receptor can be prevented by ACE inhibition, which inhibits sensitized AT1 receptor signaling, blunts exaggerated ROS generation, and heals uncoupled eNOS in atherosclerosis and cardiovascular disease (42,43,47). Consequently, the agonist-stimulated B2 bradykinin receptor could become anti-atherogenic upon treatment with an ACE inhibitor, which prevents endothelial dysfunction triggered by oxidized low-density lipoproteins in a B2 bradykinin receptor stimulation-dependent manner (49).
Taken together, our study shows that the atherosclerosisenhancing function of the ubiquitously expressed B2 bradykinin receptor involves a synergistic interplay between enhanced ROS generation, dysfunctional NO activity and AT1 receptor activation. Pathomechanisms identified in this study could act in concert with already established B2 and/or AT1 receptor-dependent atherogenic activities. Notably, the following mechanisms mediated by the B2 and/or AT1 receptor could play a direct or indirect role in B2 bradykinin receptorenhanced atherosclerosis: (I) The B2 bradykinin receptor could promote atherosclerosis by the reduced activation of atheroprotective AT1-inhibitory receptors, AGTR2 and MAS1 (34,(50)(51)(52), which are directly down-regulated by the B2 bradykinin receptor and angiotensin II AT1 receptor (34,53) and indirectly dampened via angiotensin II AT1mediated ACE2 down-regulation (54). (II) In addition, the B2 bradykinin receptor could enhance atherogenesis by decreasing the expression of the vasculoprotective endothelial Kruppel-like factor 4, KLF4 (55), which is directly downregulated by angiotensin II AT1 stimulation [(56); NCBI GEO dataset GSE19286 in Abd Alla et al. (7)], and indirectly down-regulated by reduced Mas receptor activation (57). (III) And finally, the B2 bradykinin receptor-stimulated atherogenesis could involve the enhanced aortic infiltration with macrophages and inflammatory immune cells, which is promoted by angiotensin II AT1 receptor signaling directly (7,58), and indirectly by B2 bradykinin and angiotensin II AT1 receptor-mediated downregulation of Mas, which accounts for a decreased expression of atheroprotective sirtuin 1, SIRT1 (34,57,59). In view of this panoply of different atherogenic functions, specific targeting of the detrimental B2 -AT1 receptor axis could be envisaged as a potential approach to treat not only atherosclerosis but also related pro-thrombotic activities triggered by the B2 bradykinin and angiotensin II AT1 receptors (34,60,61).