Differential Gene Expression of Bradykinin Receptors 1 and 2 in Peripheral Monocytes from Patients with Essential Hypertension

ORIGINAL ARTICLE Differential gene expression of bradykinin receptors 1 and 2 in peripheral monocytes from patients with essential hypertension

ME Marketou1, J Kontaraki1, E Zacharis1, F Parthenakis1, S Maragkoudakis1, I Gavras2, H Gavras2 and PE Vardas1

Bradykinin participates in various hypertensive processes, exerted via its type 1 and type 2 receptors (BKR1 and BKR2). The aim of the study was to investigate BKR1 and BK2R gene expression in peripheral monocytes in patients with essential hypertension compared with healthy individuals. Seventeen hypertensive patients (9 males, age 56±7 years) and 12 healthy individuals (7 males, age 55±6) participated. Mononuclear cells isolated using anti-CD14 þ antibodies and mRNAs of BKR1 and BKR2 were estimated by real-time quantitative reverse transcription-PCR. Both BKR1 and BKR2 showed signiﬁcantly upregulated gene expression in the group of hypertensive patients. Speciﬁcally, BKR1 gene expression was 142.1±42.2 in hypertensives versus 20.2±8 in controls (P ¼ 0.024) and BKR2 was 1222.2±361.6 in hypertensives versus 259.5±99.1 in controls (P ¼ 0.038). Antihypertensive treatment resulted in a decrease in BKR1 (from 142.1±42.2 to 55.2±17.1, P ¼ 0.065) and in BKR2 (from 1222.2±361.6 to 256.8±81.8, P ¼ 0.014) gene expression. BKR1 and BKR2 gene expression on peripheral monocytes is upregulated in essential hypertension. This may lead to functional changes in monocytes and contribute to the development of target organ damage in hypertensive patients.

Journal of Human Hypertension (2014) 28, 450–455; doi:10.1038/jhh.2013.133; published online 9 January 2014 Keywords: bradykinin receptors; monocytes; essential hypertension

INTRODUCTION of BKR1 promotes monocyte chemotaxis and arteriogenesis, 9 The various vasoactive and metabolic effects of bradykinin are whereas BKR2 signaling governs monocyte recruitment in vitro. mediated by the cell surface B1 and B2 receptors. Biological In view of the key role of monocytes in hypertension and its effects of bradykinin receptor signaling have an important role in complications, we examined BKR1 and BKR2 gene expression in vascular homeostasis, inflammation, and vessel wall remodeling, peripheral monocytes isolated from patients with essential arteriogenesis, leukocyte recruitment and production of cytokines, hypertension, before and after BP control with antihypertensive and have been shown to mediate cell proliferation and capillary medication. In order to examine the factors that might alter the sprouting.1,2 Most of the physiologically significant actions of gene expression of BKR1 and BKR2 in monocytes, we also isolated bradykinin are exerted via activation of its constitutively present and cultured peripheral monocytes from hypertensive patients bradykinin type 2 receptor (BKR2), whereas its bradykinin type 1 and healthy volunteers, and their mRNA levels were assessed after receptor (BKR1) is believed to be minimally expressed under activation with angiotensin II and a highly potent, selective and 10 normal conditions, but may be inducible by lipopolysaccharides, long-acting BKR2 agonist. bacterial toxins and inflammatory mediators resulting from tissue injury.3,4 The BKR1 and BKR2 are particularly important in blood pressure (BP) regulation and in the pathophysiolology of essential MATERIALS AND METHODS hypertension.5 Notably, the bradykinin system is involved in the Study population mediation and modulation of the vasoconstrictor renin– We prospectively enrolled subjects with untreated grade 1 or grade 2 angiotensin system and has a major role in the modulation of essential hypertension and with no indications of other organic heart hypertension by regulating sodium–water balance, renal and disease. The study population was recruited from the cardiology 6 outpatient department from January 2011 until December 2012 and the cardiac hemodynamics, and BP. diagnosis of hypertension was based on three outpatient measurements of On the other hand, arterial hypertension is accompanied by BP 4140/90 mm Hg at intervals of no longer than 2 weeks. Participants functional changes in circulating peripheral monocytes, whose with BP 4140/90 mm Hg on the final visit underwent 24-h ambulatory BP activation has an important role in the early stages of monitoring. To be eligible for inclusion in the study, a mean 24-h BP atherosclerotic lesion formation and which have a crucial role in 4130/80 mm Hg was required. The patients had not previously taken any the cardiovascular complications of hypertensives.7,8 antihypertensive medication and did not take any other drugs for 3 weeks BKR1 and BKR2 have been found to be expressed in numerous before the studies. All of the participants underwent routine clinical cell types. However, our knowledge of the existence and assessments, including chest X-ray and echocardiography. The following regulation of these receptors in monocytes is still limited. were criteria for exclusion: smokers, diabetics, pregnant or lactating women or women of childbearing potential, previous history or medica- Circulating monocytes have been shown to be the pivotal cellular tion for hypertension, patients with grade 3 hypertension or secondary factor during adaptive arteriogenesis, accelerating the remodeling 4 hypertension, tachy- or bradyarrhythmias, coronary artery disease, process of collateral arterial tissue. Although the presence of cerebrovascular, liver or renal disease, albumin excretion rate 4200 mg BKR1 and BKR2 in human monocytes has not been studied in min–1, history of drug or alcohol abuse, any chronic inflammatory or other depth, previous studies have noted their important role. Activation infectious disease during the last 6 months, thyroid gland disease and

1Cardiology Department, Heraklion University Hospital, Crete, Greece and 2Hypertension and Atherosclerosis Section, Department of Medicine, Boston University School of Medicine, Boston, MA, USA. Correspondence: Dr ME Marketou, Cardiology Department, Heraklion University Hospital, PO Box 1352, Stavrakia, Heraklion, Greece. E-mail: [email protected] Received 17 July 2013; revised 7 November 2013; accepted 19 November 2013; published online 9 January 2014 Bradykinin receptors in hypertension ME Marketou et al 451 body mass index 430 kg m–2. In addition, vascular, metabolic or neoplastic The adjusted t-test (Tukey’s HSD) was used to estimate differences conditions were ruled out by a careful examination of the history and between pairs of groups. Data are presented as means ±s.e.m. All tests routine laboratory tests. The exclusion criteria also included symptoms of were two-sided and the significance level of Po0.05 was considered to heart failure or an ejection fraction o55%. Antihypertensive drug indicate statistical significance. Statistical analysis was performed with a treatment was left to the physicians’ discretion and included combinations commercially available statistical package (IBM SPSS Statistics 20.0; of angiotensin-converting enzyme inhibitors, angiotensin receptor block- Chicago, IL, USA). ers, calcium antagonists and diuretics. In addition, we included a control group consisting of 12 normotensive individuals whose clinical and laboratory examinations were normal. Blood samples were drawn from all participants at baseline and at 12 weeks after RESULTS initiation of treatment in the group of hypertensives. The clinical characteristics of the participants are presented in All patients gave written informed consent. The Institutional Ethics Table 1. Hypertensive patients had significantly higher systolic and Committee approved the study. diastolic BP, which decreased by 12 weeks after the initiation of treatment. No changes in the patients’ physical characteristics— Blood collection and monocyte separation body weight or body mass index—were observed at the end of Blood samples were collected into EDTA (ethylenediaminetetraacetic acid) the 3-month follow-up period (data not shown). No significant collection tubes. Peripheral blood mononuclear cells were isolated by adverse events were experienced in the treatment group. Ficoll–Paque plus (Stem Cell Technologies Inc., Vancouver, BC, Canada) BKR1 and BKR2 mRNA levels are shown in Figure 1. Hyperten- centrifugation and CD14 þ monocytes were purified from peripheral blood sive patients revealed significantly higher BKR1 and BKR2 mRNA mononuclear cells by positive selection using MACS high-gradient levels in peripheral monocytes compared with normotensive magnetic separation columns type MS and positive magnetic bead individuals, as expressed in arbitrary units (for BKR1: 142.1±42.2 selection (Miltenyi Biotec, Bergisch Gladbach, Germany). Purity assessed in hypertensives versus 20.2±8 in controls, P ¼ 0.024; and for by fluorescence-activated cell sorting (FACSCalibur, Becton Dickinson, BKR2: 1222.2±361.6 in hypertensives versus 259.5±99.1 in Franklin Lakes, NJ, USA) analysis was 495%. controls, P ¼ 0.038). In addition, the ratio of BKR1/BKR2 mRNA levels in peripheral monocytes was significantly higher in Monocyte culture and treatment hypertensives compared with the control group (0.24±0.04 Peripheral monocytes from three hypertensive patients and three controls versus 0.09±0.02, P ¼ 0.002), indicating a more prominent were isolated by density gradient centrifugation, plated on six-well plates upregulation of BKR1 gene transcripts in hypertension. in RPMI, GlutaMAX, HEPES containing 10% fetal bovine serum (Life Antihypertensive treatment resulted in a decrease in BKR1 that 1 Technologies, Carlsbad, CA, USA), and incubated for 2 h (37 C, 5% CO2). trends to be statistically significant (from 142.1±42.2 to Non-adherent cells were removed and the remaining cells were further 55.2±17.1, P ¼ 0.065) and to a significant decrease in BKR2 (from cultured in the above medium supplemented with 10% human serum for 1222.2±361.6 to 256.8±81.8, P 0.014) gene expression. Nota- 24 h before treatment. The cells were divided into four groups according to ¼ whether or not they were activated by angiotensin II, a BKR2-agonist or bly, their mRNA levels reached values very close to those of ± ± both. More specifically, monocytes were treated with 100 nM AngII (Sigma- normotensives (BKR1: 55.2 17.1 versus 20.2 8, P ¼ 0.12) and 10 Aldrich, St Louis, MO, USA) and/or 10 nM B2-agonist, and after 48-h BKR2: 256.8±81.8 versus 259.5±99.1, P ¼ 0.98). culture were collected for analysis. The BKR2-agonist chosen was (Hyp3, Thi5, NChg7, Thi8)-bradykinin, kindly provided by Dr Fernand Gobeil from Sherbrooke University, Canada, as it is the most potent and long-acting Peripheral monocyte isolation and culture agent described in his studies. Three independent experimental studies BKR1 mRNA levels isolated from cultured peripheral monocytes of were performed each time. normotensives did not show any significant difference when stimulated by angiotensin II (fold induction 1.32±0.19, P ¼ 0.971), RNA isolation and quantitative reverse transcriptase-PCR BKR2-agonist (fold induction 2.58±0.74, P ¼ 0.129), or both fold Total RNA was isolated from monocytes using the TRI-Reagent (Ambion, induction 2.83±0.84, P ¼ 0.111; Figure 2a). On the other hand, Life Technologies, Carlsbad, CA, USA) and reverse-transcribed using the BKR1 in monocytes isolated from hypertensive subjects showed a PrimeScript RT reagent Kit (Takara Bio Inc., Otsu, Shiga, Japan). Measure- different response (Figure 2b). Angiotensin II stimulation resulted ments of mRNA levels were performed by quantitative PCR (qPCR) using in an increase in gene expression by fold induction to 1.49±0.11, the Corbett Research 6000 detection system. qPCR assays were performed P ¼ 0.028, whereas exposure to the BKR2-agonist resulted in a using the KAPA SYBR FAST qPCR Kit (Kapa Biosystems, Woburn, MA, USA). slight but not statistically significant decrease (fold induction: The housekeeping gene GAPDH (glyceraldehyde-3-phosphate dehydrogen- 0.77±0.16, P ¼ 0.514). The presence of BKR2-agonist in angioten- ase) was used as an endogenous reference gene and relative quantification sin II exposure prevented the increase in BKR1 (fold induction was done by normalizing the BKR1 and BKR2 absolute quantification signals with the GAPDH signal. Primers used were 50-GGGCCGCAAGGATAG 0 0 0 CAAGAC-3 (sense) and 5 -GCAGGCCCAGGTCAATGAAGT-3 (antisense) for Table 1. Participants’ demographic and clinical data BKR1,50-ACACAGGGCAGTCATTCAGCAC-30 (sense) and 50-CATGCATAGGGC CGCTCTTC-30 (antisense) for BKR2,50-CCATCTTCCAGGAGCGAG-30 (sense) 0 0 Hypertensive Healthy P-value and 5 -GCAGGAGGCATTGCTGAT-3 (antisense) for GAPDH. All samples patients volunteers were run in duplicate. The standard curve method was used for absolute (n ¼ 17) (n ¼ 12) quantification of the amplification products and specificity was determined by performing a melting curve analysis. mRNA levels of BKR1 and BKR2 Age (years) 54±755±6NS were expressed as arbitrary units, whereas their levels before and after Sex (male/female) 9/8 7/5 NS treatment were expressed as fold induction (ratio of mRNA after treatment Systolic blood pressure 155±5 126±5 o0.001 to mRNA before treatment). (mm Hg) Diastolic blood pressure 94±784±6 o0.001 Statistical analysis (mm Hg) Heart rate (b.p.m.) 72±869±8NS For participants’ demographic and clinical data, continuous variables are –1 Fasting glucose (mg dl ) 109±22 105±29 NS summarized as mean values (s.d.), and discrete variables as counts and –1 Creatinine (mg dl ) 1.03±0.13 1.13±0.11 NS proportions. Variables were compared between groups using independent –1 2 Uric acid (mg dl ) 7.4±3.4 7.1±5.2 NS samples t-tests, Fisher’s exact or w tests as appropriate. For group-wise –1 Total cholesterol (mg dl ) 243±46 207±54 o0.001 comparisons, the significance of mRNA level differences was evaluated by independent sample Student’s t-test. One-way analysis of variance was Abbreviation: NS, not significant. used to explore differences when comparing more than two groups.

& 2014 Macmillan Publishers Limited Journal of Human Hypertension (2014) 450 – 455 Bradykinin receptors in hypertension ME Marketou et al 452 with essential hypertension, which under the inﬂuence of 200 p=0.024 antihypertensive therapy returns to levels similar to those of normotensive individuals. On the other hand, short-term exposure of isolated cultured monocytes from hypertensive patients to 150 angiotensin II resulted in upregulation of BKR1 and downregulation BKR2, a phenomenon that was blunted in the presence of a selective BKR2-agonist. Dysregulation of the kinin–kallikrein system and its receptors is 100 involved in many pathological conditions, such as inﬂammation

BKR1 and cardiovascular diseases. The expression of BKR1 and BKR2 is regulated by a wide range of conditions, but their role in cardiovascular diseases is complex and still needs to be 50 elucidated. The kinin system contributes to the pathogenesis of hypertension. The bradykinin system is involved in the mediation of the vasoconstrictor renin–angiotensin system and the vasodi- 0 lators prostaglandin, prostacyclin and nitric oxide in regulating salt–water balance and BP. Kinins are involved in vasodilatation, Controls Hypertensives vascular permeability, inflammation, and tissue repair, which are mediated via the constitutively expressed BKR2. The BKR1 is weakly expressed under physiological conditions, but is strongly 2000 induced in response to pathological stimuli such as inflammation or tissue injury.3,11–13 It has been postulated that the kinin p=0.038 receptor signaling pathway may act as a molecular link between 1500 changes in hemodynamic forces and the activation of inflammatory pathways. Monocytes and macrophages can release various inflammatory substances following BKRs stimulation.14 As monocyte activation 1000 has a critical role in the pathophysiology of cardiovascular disease,

BKR2 we aimed to clarify the role of kinin receptors in peripheral monocytes in patients with essential hypertension. It has been suggested that BKR2 have a role in the recruitment and 500 maturation of immature monocytes. In mice, activation of BKR2 on monocytes regulates chemokine expression and leukocyte recruitment in the cerebral microvasculature during autoimmune encephalomyelitis.15 In humans, BKR2 activation, via the 0 production of chemokines, could be involved in the recruitment of human circulating progenitor cells with neovascularization Controls Hypertensives potential.16 Figure 1. BKR1 and BKR2 mRNA levels in peripheral monocytes in BKR2 is constitutively expressed on most cell types and is hypertensive patients compared with normotensives. Data are rapidly internalized upon agonist binding, followed by its presented as means±s.e.m. P-values from independent Student’s recycling to the cell membrane. BKR1, on the other hand, is t-test are presented. (a) BKR1mRNA levels in controls and expressed at very low levels under physiological conditions, but is hypertensive patients, (b) BKR2 mRNA levels in controls and induced upon pathologic stimulus.17 Upon expression on the cell hypertensive patients. BKR1: bradykinin type 1 receptor, BKR2: surface—for instance, following stimulation with proinflammatory bradykinin type 2 receptor substances, such as interleukin 1 or endotoxins—BKR1 exhibits high ligand-independent, constitutive activity that is further 0.72±0.14 versus 1.49±0.11, Po0.001), whose levels of gene enhanced by agonist binding.18 Upregulation of the BKR1 is also expression were decreased in a similar way (fold induction: demonstrated when the BK2R is absent, which, as the two BKRs 0.72±0.14, P ¼ 0.379). seem to have some overlapping functions, may in part Regarding the levels of BKR2 gene expression, we observed that compensate for the loss of the BKR2.5 the short-term effect of angiotensin II on the cell cultures was to Monocytes and macrophages have been mostly used for the reduce them but not significant in controls (fold induction: analysis of bradykinin-evoked responses. However, until now little 0.65±0.10, P ¼ 0.431) and even more in hypertensives (fold has been known about bradykinin receptors in human monocytes induction: 0.38±0.04, Po0.001). The presence of a BKR2-agonist in cardiovascular disease. Alterations in BKR1 and BKR2 mRNA prevented this decrease in hypertensives (fold induction expression in peripheral blood mononuclear cells have been 0.55±0.03 versus 0.38±0.04, P ¼ 0.001) although in hypertensives reported in patients with cardiac X syndrome19 and acute there was a significant reduction, to slightly higher levels (fold coronary syndromes.20 A previous study by Bourdet et al.21, induction: 0.55±0.03, Po0.001; Figure 2d). This prevention was which investigated only BKR2 in human monocytes, revealed also observed in controls, however, it did not reach statistical increased expression in treated hypertensive women, but not in significance (Figure 2c). Exposure to a b2-agonist reduced the men. However, the sample of men was very small and all the levels of BKR2 gene expression only in cultured monocytes subjects were under treatment, which may have prevented the isolated from hypertensive subjects (fold induction: 0.41±0.02, demonstration of any differences. Po0.001; Figures 2c and d). BKR1 are induced by inflammatory mediators and tissue damage.5 Studies using BKR2 antagonists indicate that endogenous bradykinin contributes to the beneficial effects of DISCUSSION angiotensin converting enzyme inhibition on vasodilation and This study shows for the first time that BKR1 and BKR2 gene BP,22,23 as well as on endothelial tissue-type plasminogen activator expression in peripheral monocytes is upregulated in patients release.24 On the other hand, kinins are implicated in endothelial

Journal of Human Hypertension (2014) 450 – 455 & 2014 Macmillan Publishers Limited Bradykinin receptors in hypertension ME Marketou et al 453 3.5 3.5 3.0 3.0 2.5 2.5 2.0 2.0 1.5 1.5 1.0 1.0 BKR1 (fold induction) 0.5 BKR1 (fold induction) 0.5 0.0 0.0 Ang II b2-agonist Ang II + Ang II b2-agonist Ang II + b2-agonist b2-agonist Normal Hypertensive

1.2 1.2

1.0 1.0

0.8 0.8

0.6 0.6

0.4 0.4

0.2 0.2 BKR2 (fold induction) BKR2 (fold induction)

0.0 0.0 Ang II b2-agonist Ang II + Ang II b2-agonist Ang II + b2-agonist b2-agonist

Normal Hypertensive Figure 2. BKR1 and BKR2 mRNA level changes in cultured monocytes isolated from hypertensives and normotensives after exposure to angiotensin II, B2R agonist or both. Data are presented as mean fold induction±s.e.m. (ratio of mRNA levels after treatment to mRNA levels without treatment). (a) BKR1 fold induction in normotensives, (b) BKR1 fold induction in hypertensives, (c) BKR2 fold induction in normotensives, (d) BKR2 fold induction in hypertensives. BKR1: bradykinin type 1 receptor, BKR2: bradykinin type 2 receptor. function and vascular integrity and via the recruitment of angio- Angiotensin II and kinin receptor interrelationships were supportive cells, mainly mediated by the BKR2.1 In vivo and in vitro also indicated in animal experiments where both BKR1 and studies have suggested that BKR1 activation on neutrophils in BKR2 were evaluated.28 Freshly isolated monocytes from inflammatory situations directly promotes neutrophil adhesion hypertensives were more sensitive to angiotensin II stimulation, and migration into the perivascular tissue.25 Inflammation which resulted in rapid activation and upregulation of BRK1.29 On mediators can regulate the mRNA of both receptors and the other hand, angiotensin II exposure resulted in a significant enhance their expression on a promonocytic U937 cell line.26 downregulation of BKR2. Endogenous angiotensin II upregulates Hypertension and its atherosclerotic complications are closely BKR1 via the AT1 receptor in hypertensive rats,30 it has been linked to inflammation.27 The fact that we found higher levels of shown to regulate BKR2 expression in previous experimental gene expression of BKR1 and BKR2 in peripheral monocytes in studies31 and our results show that the same applies to human hypertensive patients is consistent with previous reports peripheral monocytes. investigated the effect of inflammatory mediators in BKRs.26 The Apart from angiotensin, we attempted to activate the BKR2 increase in the gene expression of BKR1 may be considered receptors in order to see whether some compensatory effect responsible for the pathological activation of monocytes, which could be achieved. In vivo, bradykinin is rapidly degraded, which ultimately are implicated in the vicious cycle of atheromatosis limits its clinical use. For this reason, we used a highly potent, pathology. It is not entirely clear how or why the increase in BKR2 selective and long-acting BKR2 agonist.10 The presence of b2- occurs. It could possibly be a compensatory protective mechanism agonist appeared to limit the effect of angiotensin II on in response to chronic activation of the renin–angiotensin– monocytes as to both the upregulation of BKR1 and the aldosterone system, which finally does not prevail, as the ratio downregulation of BKR2. of BKR1/BKR2 mRNA levels in peripheral monocytes is significantly The high heterogeneity of peripheral monocytes may limit the higher in hypertensives. This increased ratio suggests a shift to interpretation of our findings. The study of the gene expression of sustained BKR1-mediated chronic inflammation in essential these receptors after treatment did not include a placebo control. hypertension. Nevertheless, our findings are indicative and we considered it In order to study the short-term results of the exposure of unethical to include a hypertensive group that would not receive monocytes to angiotensin II and its effect on bradykinin receptors, therapy. The hypertensive population included in the study was we obtained tissue cultures of monocytes isolated from hyper- treated with different antihypertensive drugs. We did not tensive patients and normal controls. Notably, the behavior of investigate the effect of either angiotensin-converting enzyme these receptors in monocytes after exposure to various agents inhibitor or angiotensin receptor blockers on BKR1 and BKR2 differed between hypertensives and normotensives, probably mRNA expression as this was not our aim. However, this is of great because their long-term exposure to conditions with increased importance and deserves further investigation. In addition, we activation of the renin–angiotensin–aldosterone system had cannot rule out that different antihypertensive drugs might have a already altered their gene expression and perhaps also their different effect in our findings. Another limitation of our study is function. the small number of participants. However, even this small

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& 2014 Macmillan Publishers Limited Journal of Human Hypertension (2014) 450 – 455