B1 Receptor Expression Research Articles

Experimental periodontitis in rats potentiates inflammation at a distant site: Role of B1 kinin receptor

It has been demonstrated that periodontitis, a chronic oral disease, can induce systemic inflammation which is associated with systemic diseases, including rheumatoid arthritis. Here, we evaluated whether periodontitis can modulate the inflammatory response at a site distant to the oral cavity. Wistar rats were subjected to ligature-induced experimental periodontitis. Fourteen days after the procedure, paw edema was induced by carrageenan or by different receptor-specific inflammatory mediators. Blood and the tissue of the paws were obtained for TNF-α and IL-1β measurement. It was observed that carrageenan-induced paw edema and leukocyte migration was potentiated in periodontitis animals. The edema induced by carrageenan, bradykinin and des-Arg9-BK (B1 agonist) was also potentiated in periodontitis animals and blocked by a B1 antagonist. Ligature-induced periodontitis increased plasma levels of TNF-α and tissue IL-1β. Periodontitis also up-regulated kinin B1 receptor expression in paw tissue. Additionally, the treatment of ligature animals with anti-TNF-α, etanercept, completely abolished the potentiation of edema induced by des-Arg9-BK. Taken together, these results show that experimental periodontitis in rats can induce systemic inflammation through the up-regulation of kinin B1 receptors at a site distant from the oral cavity, modifying the inflammatory response.

Kininase 1 As a Preclinical Therapeutic Target for Kinin B1 Receptor in Insulin Resistance.

Kinin B1 receptor (B1R) contributes to insulin resistance, an early event in type 2 diabetes, through the upregulation and activation of the inducible form of nitric oxide synthase (iNOS), pro-inflammatory cytokines and the oxidative stress. This study addresses the hypothesis that inhibition of kininase 1 (carboxypeptidase M, CPM), the key enzyme involved in the biosynthesis of B1R agonists, could exert the same beneficial effects to B1R antagonism in insulin resistance. Male Sprague-Dawley rats were made insulin resistant with a drinking solution containing 10% D-glucose for a period of 9 weeks. Control rats received tap water. During the last week, kininase 1 was blocked with Mergetpa (1 mg kg−1 twice daily, s.c.) and the impact was determined on insulin resistance (HOMA index), metabolic hormone levels, oxidative stress and the expression of several markers of inflammation by western blot and qRT-PCR. Glucose-fed rats displayed hyperglycemia, hyperinsulinemia, hyperleptinemia, insulin resistance, hypertension, positive body weight gain, and enhanced expression of B1R, CPM, iNOS, and IL-1β in renal cortex, aorta and liver. Markers of oxidative stress (superoxide anion and nitrotyrosine expression) were also enhanced in aorta and renal cortex. Mergetpa reversed and normalized most of those alterations, but failed to affect leptin levels and hypertension. Pharmacological blockade of kininase 1 (CPM) exerted similar beneficial effects to a 1-week treatment with a B1R antagonist (SSR240612) or an iNOS inhibitor (1,400 W). These data reinforce the detrimental role of B1R in insulin resistance and recommend CPM as a new therapeutic target.

Bradykinin does not acutely sensitize the reflex pressor response during hindlimb skeletal muscle stretch in decerebrate rats.

Hindlimb skeletal muscle stretch (i.e., selective activation of the muscle mechanoreflex) in decerebrate rats evokes reflex increases in blood pressure and sympathetic nerve activity. Bradykinin has been found to sensitize mechanogated channels through a bradykinin B2 receptor-dependent mechanism. Moreover, bradykinin B2 receptor expression on sensory neurons is increased following chronic femoral artery ligation in the rat (a model of simulated peripheral artery disease). We tested the hypothesis that injection of bradykinin into the arterial supply of a hindlimb in decerebrate, unanesthetized rats would acutely augment (i.e., sensitize) the increase in blood pressure and renal sympathetic nerve activity during hindlimb muscle stretch to a greater extent in rats with a ligated femoral artery than in rats with a freely perfused femoral artery. The pressor response during static hindlimb muscle stretch was compared before and after hindlimb arterial injection of 0.5 µg of bradykinin. Injection of bradykinin increased blood pressure to a greater extent in "ligated" (n = 10) than "freely perfused" (n = 10) rats. The increase in blood pressure during hindlimb muscle stretch, however, was not different before vs. after bradykinin injection in freely perfused (14 ± 2 and 15 ± 2 mmHg for pre- and post-bradykinin, respectively, P = 0.62) or ligated (15 ± 3 and 14 ± 2 mmHg for pre- and post-bradykinin, respectively, P = 0.80) rats. Likewise, the increase in renal sympathetic nerve activity during stretch was not different before vs. after bradykinin injection in either group of rats. We conclude that bradykinin did not acutely sensitize the pressor response during hindlimb skeletal muscle stretch in freely perfused or ligated decerebrate rats.

Blockade of the kinin B1 receptor affects the cytokine/chemokine profile in rat brain subjected to autoimmune encephalomyelitis.

Kinins are bioactive peptides which provide multiple functions, including critical regulation of the inflammatory response. Released during tissue injury, kinins potentiate the inflammation which represents a hallmark of numerous neurological disorders, including those of autoimmune origin such as multiple sclerosis (MS). In the present work, we assess the expression of B1 receptor (B1R) in rat brain during the course of experimental autoimmune encephalomyelitis (EAE) which is an animal model of MS. We apply pharmacological inhibition to investigate the role of this receptor in the development of neurological deficits and in shaping the cytokine/chemokine profile during the course of the disease. Overexpression of B1R is observed in brain tissue of rats subjected to EAE, beginning at the very early asymptomatic phase of the disease. This overexpression is suppressed by a specific antagonist known as DALBK. The involvement of B1R in the progression of neurological symptoms in immunized rats is confirmed. Analysis of an array of cytokines/chemokines identified a sub-group as being B1R-dependent. Increase of the protein levels for the proinflammatory cytokines (Il-6, TNF-α but not IL-1β), chemokines attracting immune cells into nervous tissue (MCP-1, MIP-3α, LIX), and protein levels of fractalkine and vascular endothelial growth factor observed in EAE rats, were significantly diminished after DALBK administration. This may indicate the protective potential of pharmacological inhibition of B1R. However, simultaneously reduced protein levels of anti-inflammatory and neuroprotective factors (IL-10, IL-4, and CNTF) was noticed. The results show that B1R-mediated signaling regulates the cellular response profile following neuroinflammation in EAE.

Expression and Distribution of Kinin B2 Receptor in Medulla and Spinal Cord Tissues of Rats Treated with Capsaicin

Expression and Distribution of Kinin B2 Receptor in Medulla and Spinal Cord Tissues of Rats Treated with Capsaicin

Analgesic Effect of Photobiomodulation on Bothrops Moojeni Venom-Induced Hyperalgesia: A Mechanism Dependent on Neuronal Inhibition, Cytokines and Kinin Receptors Modulation.

BackgroundEnvenoming induced by Bothrops snakebites is characterized by drastic local tissue damage that involves an intense inflammatory reaction and local hyperalgesia which are not neutralized by conventional antivenom treatment. Herein, the effectiveness of photobiomodulation to reduce inflammatory hyperalgesia induced by Bothrops moojeni venom (Bmv), as well as the mechanisms involved was investigated.Methodology/Principal FindingsBmv (1 μg) was injected through the intraplantar route in the right hind paw of mice. Mechanical hyperalgesia and allodynia were evaluated by von Frey filaments at different time points after venom injection. Low level laser therapy (LLLT) was applied at the site of Bmv injection at wavelength of red 685 nm with energy density of 2.2 J/cm2 at 30 min and 3 h after venom inoculation. Neuronal activation in the dorsal horn spinal cord was determined by immunohistochemistry of Fos protein and the mRNA expression of IL-6, TNF-α, IL-10, B1 and B2 kinin receptors were evaluated by Real time-PCR 6 h after venom injection. Photobiomodulation reversed Bmv-induced mechanical hyperalgesia and allodynia and decreased Fos expression, induced by Bmv as well as the mRNA levels of IL-6, TNF-α and B1 and B2 kinin receptors. Finally, an increase on IL-10, was observed following LLLT.Conclusion/SignificanceThese data demonstrate that LLLT interferes with mechanisms involved in nociception and hyperalgesia and modulates Bmv-induced nociceptive signal. The use of photobiomodulation in reducing local pain induced by Bothropic venoms should be considered as a novel therapeutic tool for the treatment of local symptoms induced after bothropic snakebites.

Abstract 076: Brain-targeted Bradykinin B1 Receptor Blockade Prevents DOCA-salt Hypertension by Oxidative Stress and ERK1/2 Phosphorylation Mediated Signaling Mechanisms

DOCA-salt hypertension is associated with increased expression of the pro-inflammatory bradykinin B1 receptor (B1R) in the brain. We previously reported that DOCA-salt hypertension is attenuated in global B1R knockout mice. In this study, we further examined the role of B1R within the central nervous system, on hypertension and cardiac hypertrophy, and explored the contribution of downstream signaling mechanisms. DOCA-salt treatment (1 mg/g body weight DOCA, 1% saline in drinking water, 3 weeks) of C57Bl6/J male mice produced a significant rise in mean arterial pressure (MAP; telemetry) in these animals (145 ±3 mmHg, n=8, p<0.01). Intracerebroventricular (ICV) infusion of R715 (70 μg/kg/day, 3 weeks), a specific B1R antagonist, attenuated the DOCA-salt-induced hypertension (125 ±3 mmHg, n=8, p<0.01). Moreover, DOCA-salt treatment resulted in cardiac hypertrophy in C57 compared to sham-treated mice (heart weight/tibia length; 9.2 ±0.3 vs. 7.2 ±0.2 mg/mm), which was prevented by B1R blockade with R715 (7.4 ±0.3 mg/mm; P <0.01 vs. DOCA). DOCA-salt-treated mice had increased inflammation (qRT-PCR), as shown by elevated gene expression of TNF, IL-1β and IL-6 (3-, 8- and 5-fold, respectively, n=9, p<0.01 vs. sham) in the hypothalamic paraventricular nucleus (PVN), which was attenuated in R715-treated mice. C57 mice treated with DOCA-salt showed increased oxidative stress, as indicated by increased gene expression of Nox-2 and iNOS (5- and 15-fold increase, respectively, n=4, p<0.01 vs. sham) in the PVN which was prevented by central B1R blockade with R715 (1.9- and 4.4-fold increase, respectively, n=4, p<0.01 vs. DOCA). DOCA-salt-induced increase in PVN Nox-2 protein expression was also prevented by central B1R blockade (2.2 vs. 1.1-fold change, p<0.01). Furthermore, phosphorylation of ERK1/2 was increased in the PVN of DOCA-salt-treated mice (66 ±3 % increase, n=4, p<0.05 vs. sham), which was blunted in R715 treated mice. Taken together, these data provide evidence that blockade of brain B1R attenuates DOCA-salt-induced hypertensive and hypertrophic effects via oxidative stress and ERK1/2 phosphorylation-mediated signaling mechanisms. (Funding: AHA 15SDG25720021 (SS), NIH/NHLBI (HL093178) (EL), and P30GM106392).

Exogenous kallikrein protects against diabetic nephropathy

The kallikrein-kinin system has been shown to be involved in the development of diabetic nephropathy, but specific mechanisms are not fully understood. Here, we determined the renal-protective role of exogenous pancreatic kallikrein in diabetic mice and studied potential mechanisms in db/db type 2 diabetic and streptozotocin-induced type 1 diabetic mice. After the onset of diabetes, mice were treated with either pancreatic kallikrein (db/db+kallikrein, streptozotocin+kallikrein) or saline (db/db+saline, streptozotocin+saline) for 16 weeks, while another groupof streptozotocin-induced diabetic mice received thesame treatment after onset of albuminuria (streptozotocin'+kallikrein, streptozotocin'+saline). Db/m littermates or wild type mice were used as non-diabetic controls. Pancreatic kallikrein had no effects on body weight, blood glucose and blood pressure, but significantly reduced albuminuria among all three groups. Pathological analysis showed that exogenous kallikrein decreased thethickness of the glomerular basement membrane, protected against the effacement of foot process, the lossof endothelial fenestrae, and prevented the loss of podocytes in diabetic mice. Renal fibrosis, inflammation and oxidative stress were reduced in kallikrein-treated micecompared to diabetic controls. The expression of kininogen1, tissue kallikrein, kinin B1 and B2 receptors were all increased in the kallikrein-treated compared to saline-treated mice. Thus, exogenous pancreatic kallikrein both prevented and ameliorated diabetic nephropathy, which may be mediated by activating the kallikrein-kinin system.

The effects of magnesium valproate on expression of the kinin B1 and B2 receptors in the hippocampus of the juvenile rats submitted to pilocarpine model of epilepsy

Objective To investigate the mechanisms and the effects of magnesium Valproate on the expressions of the kinin B1 and B2 receptors in the hippocampus of the juvenile rats submitted to pilocarpine model of epilepsy. Methods 35 healthy Wistar juvenile rats were randomly divided into six groups, that is the model groups: Ⅰgroup, Ⅱgroup, Ⅲgroup, and intraperitoneal injection of saline water control groups : Ⅰa group, Ⅱa group, Ⅲa group , after succession of 15 rats to kindle to establish the model of epilepsy by pilocarpine. To collect hippocampus tissue after the rats were to put to death, and to compared the expression levels of kinin B1 and B2 receptor mRNA by RT-PCR and western blot in the hippocampus of rats. Results By treated with magnesium valproate, kinin B1 receptor mRNA (0.38±0.051)and protein expressions(0.58±0.057)decreased and kinin B2 receptor mRNA (0.48±0.056)and protein expressions(0.48±0.044)increased in Ⅰ group, compared with that(0.76±0.068, 0.89±0.034; 0.28±0.034, 0.32±0.039) of Ⅰa group(P<0.05). Compared with control group, there were more significant upregulation of kinin B1 receptor mRNA and protein expressions (P<0.05) in the Ⅰ and the Ⅱ groups and there were no alteration in Ⅲ group.The expressional levels of B2 receptor mRNA and protein were upregulated in the Ⅰ, Ⅱ and Ⅲ groups. Conclusion The kinin B1 and B2 receptor may play a role in the onset and maintenance of epilepsy. The magnesium valproate increased the expressional levels of kinin B2 receptor, and decreased the expressional levels of kinin B1 receptor. Key words: Kinin B receptor; Magnesium valproate; Epilepsy model; Pilocarpine

Knockdown of Bradykinin B1 Receptor Reduces Neuro‐inflammation and Prevents the Development of Salt‐Sensitive Hypertension

We previously reported that DOCA‐salt hypertension is associated with increased bradykinin B1 receptor (B1R) expression in the brain. In the present study, we tested the hypothesis that B1R knockdown prevents the development of hypertension. To test this hypothesis, wild‐type (WT) and B1R knockout (KO) mice were implanted with telemetry probes for conscious blood pressure (BP) monitoring. DOCA (1 mg/g, sc) was implanted following baseline BP recording and drinking water was replaced by 1% NaCl solution. Autonomic function was analyzed using a pharmacological method involving ip injection of propranolol (4 mg/kg) and atropine (1 mg/kg). Changes in heart rate (ΔHR) were used as index of sympathetic and parasympathetic tones. Baseline mean arterial pressure (MAP) was not different between WT and B1R KO mice (102±2 vs. 104±5 mmHg), nor were ΔHR following assessment of sympathetic (−73±6 vs. −75±15 beats/minute) or parasympathetic tone (+166±7 vs. +153±12 beats/minute). Three weeks after DOCA‐salt treatment, MAP was significantly increased in WT mice (138±3 mmHg, P&lt;0.01 vs. baseline). However, this increase was blunted in B1R KO mice (121±2 mmHg, P&lt;0.05 vs. WT+DOCA). In addition, DOCA‐salt mediated enhancement of cardiac sympathetic drive (increased bradycardia to propranolol, −145±21 beats/minutes, P&lt;0.01 vs. baseline) and reduction of parasympathetic tone (reduced tachycardia to atropine, +111±8 beats/minute, P&lt;0.01 vs. baseline) were normalized in B1R KO mice (−72±13 and +165±6 beats/minute, respectively, P&lt;0.05 vs. WT+DOCA). DOCA‐salt treatment resulted in sympathoexcitation in WT mice, as indicated by increased urinary norepinephrine levels (299±23 ng/ml, P&lt;0.05 vs. control), but this increase was blunted in B1R KO mice with DOCA (172±30 ng/ml, P&lt;0.05 vs. WT+DOCA). Moreover, pro‐inflammatory high‐mobility group box 1 (HMGB1) and cyclooxygenase 2 protein levels were significantly upregulated in the hypothalamus following DOCA treatment in WT mice (2.5 and 2 fold, respectively, P&lt;0.05 vs. control) but not in B1R KO mice. WT mice treated with DOCA‐salt showed increased inflammation as indicated by increased gene expression of TNF (3 fold, n=9, p&lt;0.001 vs. WT), IL‐1beta (8 fold, n=9, p&lt;0.001 vs. WT) and IL‐6 (5 fold, n=9, p&lt;0.01 vs. WT) in the hypothalamic paraventricular nucleus (PVN) which was attenuated in B1R KO mice. Taken together, these data suggest that B1R knockdown prevents the development of hypertension, possibly through decreased sympathetic activity, reducing HMGB1 expression, and diminishing its downstream neuroinflammatory cascade.Support or Funding Information15SDG25720021 (SS), NIH/NHLBI (HL093178) (EL), and P30GM106392

Severe asthma, shrimp allergy and impact of maternal fatty acid status on their children

Clinical & Experimental AllergyVolume 46, Issue 3 p. 386-386 Editor-in-Chief Editorial - March 2016Free Access Severe asthma, shrimp allergy and impact of maternal fatty acid status on their children First published: 24 February 2016 https://doi.org/10.1111/cea.12716Citations: 1AboutSectionsPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinkedInRedditWechat Specific IgE to Staphylococcus aureus enterotoxin is a marker of a specific severe asthma phenotype In a well-characterized cohort of older people with asthma, Song et al.1 found that high levels of specific IgE to Staphylococcus aureus enterotoxin were associated with severe asthma. This was independent of other factors. These results represent further evidence linking Staphylococcus aureus enterotoxin-specific IgE to a specific phenotype of severe asthma characterized by late onset, coexisting chronic rhinosinusitis, eosinophilia and elevated total IgE levels. The constellation of features is illustrated in their paper (reproduced in Fig. 1). This cohort contained many non-atopic individuals, defined on the basis of negative skin prick testing to common aeroallergens. Interestingly, many of these non-atopic subjects still had positive specific IgE to Staphylococcus aureus enterotoxin; so perhaps, this particular enterotoxin might be able to generate Th2 inflammation, as a superantigen, bypassing the usual allergic pathways? Fan Chung, in his accompanying editorial, discusses this interesting hypothesis and potential therapeutic implications 2. Woo-Yung Song Multiple correspondence analyses plot for the interrelationships clinical features. Severe asthma is situated close to high Staphylococcus aureus enterotoxin-specific IgE (SE-IgE), chronic rhinosinusitis without nasal polyps (CRSwNP) and sputum eosinophilia. Reproduced from 1. Peptide-based immunotherapy successfully suppresses allergic responses in a mouse model of shrimp allergy Peptide-based immunotherapy, utilizing T-cell epitopes, has the theoretical potential to deliver desensitization without causing allergic adverse effects as this approach does not cross link specific IgE. In this issue, Wai et al. 3 describe how they identified the immunodominant T-cell epitopes of shrimp tropomyosin and evaluated their therapeutic impact in a mouse model of shrimp allergy. In their experimental model, they were able to reduce allergic symptoms (Fig. 2), plus antibody and cytokine responses. Can this strategy be translated into human shrimp and other shellfish allergy? Christine Yee Yan Wai Amelioration of systemic allergic symptoms following oral peptide-based immunotherapy. Reproduced from 3. Heterogeneous association of maternal fatty acid status with childhood atopic disease Rucci et al. 4 examined the relationship between maternal fatty acid status during pregnancy and the risk of childhood atopic diseases, while taking into account childhood fatty acid status in the large Generation R birth cohort (Fig. 3). They found heterogeneous associations, for example total polyunsaturated fatty acid (PUFA) and total n-6 PUFA were associated with a reduced risk of asthma but a higher risk of eczema. Associations were independent of child's status. What is the mechanism? Emanuela Rucci Association of maternal plasma PUFA levels with current asthma in children aged 6 years. Reproduced from 4. References 1Song W-J, Sintobin I, Sohn K-H et al. Staphylococcal enterotoxin IgE sensitization in late-onset severe eosinophilic asthma in the elderly. Clin Exp Allergy 2016; 46: 411– 21. 2Chung KF. Staphylococcal enterotoxin specific IgE: a biomarker for a distinct phenotype of severe asthma? Clin Exp Allergy 2016; 46: 387– 9. 3Wai CYY, Leung NYH, Leung PSC, Chu KH. T cell epitope immunotherapy ameliorates allergic responses in a murine model of shrimp allergy. Clin Exp Allergy 2016; 46: 491– 503. 4Rucci E, den Dekker HT, de Jongste JC. Maternal fatty acid levels during pregnancy, childhood lung function and atopic diseases. The Generation R Study. Clin Exp Allergy 2016; 46: 461– 71. Caption to cover illustration: Confocal microscopy analysis of bradykinin B2 receptor (B2R) and α-smooth muscle actin (α-SMA) expression in the bronchial lamina propria of an individual with asthma after diluent or allergen challenge and from one control individual. [see figure 2b in F. L. M. Ricciardolo et al. (pp. 428–438)]. This logo highlights the Editor-in-Chief Editorial articles on the cover and the first page of each of the articles. Citing Literature Volume46, Issue3March 2016Pages 386-386 ReferencesRelatedInformation

Bradykinin B2 receptor expression in the bronchial mucosa of allergic asthmatics: the role of NF-kB.

Bradykinin (BK) mediates acute allergic asthma and airway remodelling. Nuclear factor-kappa B (NF-kB) is potentially involved in BK B2 receptor (B2R) regulation. In this observational cross-sectional study, B2R and NF-kB expression was evaluated in bronchial biopsies from mild asthmatics (after diluent/allergen challenge) and healthy controls, examining the role of NF-kB in B2R expression in primary human fibroblasts from normal and asthmatic subjects (HNBFb and HABFb). B2R and NF-kB (total and nuclear) expression was analysed by immunohistochemistry in biopsies from 10 mild intermittent asthmatics (48 h after diluent/allergen challenge) and 10 controls undergoing bronchoscopy. B2R co-localization in 5B5(+) and αSMA(+) mesenchymal cells was studied by immunofluorescence/confocal microscopy, and B2R expression in HABFb/HNBFb incubated with interleukin (IL)-4/IL-13 with/without BK, and after NF-kB inhibitor, by Western blotting. Bronchial mucosa B2R and nuclear NF-kB expression was higher in asthmatics after diluent (B2R only) and allergen challenge than in controls (P < 0.05), while B2R and NF-kB (total and nuclear) increased after allergen compared with after diluent (P < 0.05). Allergen exposure increased B2R expression in 5B5(+) and αSMA(+) cells. Constitutive B2R protein expression was higher in HABFb than in HNBFb (P < 0.05) and increased in both cell types after IL-13 or IL-4/IL-13 and BK treatment. This increase was suppressed by a NF-kB inhibitor (P < 0.05). Bronchial B2R expression is constitutively elevated in allergic asthma and is further increased after allergen exposure together with NF-kB expression. NF-kB inhibitor blocked IL-4/IL-13-induced increase in B2R expression in cultured fibroblasts, suggesting a role as potential anti-asthma drug.

Cellular localisation of the kinin B1R in the pancreas of streptozotocin-treated rat and the anti-diabetic effect of the antagonist SSR240612

The mechanism by which kinin B1 receptor (B1R) contributes to type 1 diabetes is addressed by determining the impact of its inhibition on diabetes and on its pancreatic expression and cellular localisation on immunocompetent cells and primary sensory C-fibres. Rats were made diabetic with streptozotocin (STZ). On day 4, they were treated daily for 7 days with a B1R antagonist (SSR240612, 10 mg/kg) or its vehicle. The surviving β-cells were measured by immunostaining. The expression of B1R, iNOS, TNF-α, macrophages, TCD4+, CGRP and TRPV1 was measured by Western blotting, qRT-PCR and immunofluorescence. Macrophages and TCD4+ lymphocytes were absent in control, but distributed abundantly in the pancreas of STZ-diabetic rats. B1R was upregulated on these immune cells infiltrating the diabetic rat pancreas while it was not expressed on primary sensory C-fibres even if the expression of TRPV1 and CGRP was enhanced. SSR240612 prevented the infiltration of macrophages and TCD4+ lymphocytes and the upregulation of B1R, iNOS, TNF-α and TRPV1. SSR240612 corrected hyperglycaemia and hypoinsulinaemia by improving the Langerhans islets survival or regeneration. It is concluded that kinin B1R antagonism exerts anti-diabetic action by preventing the infiltration of immune cells in the pancreas and by preserving the integrity of Langerhans islets β-cells.

Utero-placental cellular and nuclear expression of bradykinin B2 receptors in normal and preeclamptic pregnancies.

The bradykinin type 2 receptor (B2R), main effector of the pleiotropic kallikrein-kinin system (KKS), has been localized in the key sites related to placentation in human, rat and guinea pig utero-placental units. The present study was directed to characterize the content, the cellular and subcellular localization of B2R in the villi and basal plate of placentas from normal and preeclamptic pregnancies by means of western blotting, immunohistochemistry and immunoelectron microscopy. The protein content of B2R was demonstrated in both placental zones. The villous placenta of normal and preeclamptic pregnancies expressed B2R in syncytiotrophoblast and fetal endothelium; the basal plate displayed B2R in extravillous trophoblasts and decidual cells. Lastly, immunogold electron microscopy revealed B2R in fetal endothelium, syncytiotrophoblast, extravillous cytotrophoblasts and decidual cells; in all cell types the receptor was mainly located in the cytosol and nucleus. The protein content of placental homogenates and the immunoreactivity in the different cells types did not differ between both study groups; however the abundance of nuclear immunogold B2R positive beads in extravillous trophoblasts was greater in the normal than in the preeclamptic placentas. The purpose of describing nuclear B2R in the utero-placental unit, and its increment in normal extravillous trophoblasts, is to stimulate the study of the functional pathways that may be relevant to understand the local role of the B2R in normal and preeclamptic gestation.

Kinin B1 receptor antagonist BI113823 reduces allergen-induced airway inflammation and mucus secretion in mice

Kinin B1 receptors are implicated in asthmatic airway inflammation. Here we tested this hypothesis by examining the anti-inflammatory effects of BI113823, a novel non-peptide orally active kinin B1 receptor antagonist in mice sensitized to ovalbumin (OVA). Male Balb-c mice were randomly assigned to four study groups: (1) control, (2) OVA+vehicle, (3) OVA+BI113823, (4) OVA+dexamethasone. Mice were sensitized intraperitoneally with 75μg ovalbumin on days 1 and 8. On days 15–17, mice were challenged intranasally with 50μg of ovalbumin. Mice received vehicle, BI113823, or dexamethasone (positive control) on days 16–18. On day 19, bronchoalveolar lavage (BAL) and lung tissue were collected for biochemical and immuno-histological analysis. Compared to controls treatment with BI113823 significantly reduced the numbers of BAL eosinophils, macrophages, neutrophils and lymphocytes by 58.3%, 61.1%, 66.4% and 56.0%, respectively. Mice treated with dexamethasone showed similar reductions in BAL cells. Treatment with BI113823 and dexamethasone also significantly reduced total protein content, IgE, TNF-α and IL-1β in lavage fluid, reduced myeloperoxidase activity, mucus secretion in lung tissues, and reduced the expression of B1 receptors, matrix metalloproteinase (MMP)-2 and cyclooxygenase (COX)-2 compared to vehicle-treated mice. Only BI113823 reduced MMP-9 and inducible nitric oxide synthase (iNOS). BI113823 effectively reduced OVA-induced inflammatory cell, mediator and signaling pathways equal to or greater than that seen with steroids in a mouse asthma model. BI113823 might be useful in modulating inflammation in asthma.

Glioblastoma-mesenchymal stem cell communication modulates expression patterns of kinin receptors: Possible involvement of bradykinin in information flow.

The most aggressive subtype of brain tumors is glioma WHO grade IV, the glioblastoma (GBM). The present work aims to elucidate the role of kinin receptors in interactions between GBM cells and mesenchymal stem cells (MSC). The GBM cell line U87-MG was stably transfected to express dsRed protein, single cell cloned, expanded, and cultured with MSC, both in the direct co-cultures (DC) and indirect co-cultures (IC) at equal cell number ratio for 72 h. Up- and down-regulation of matrix metalloproteases (MMP)-9 expression in U87-MG and MSC cells, respectively, in direct co-culture points to possible MSC participation in tumor invasion. MMP9 expression is in line with significantly increased expression of kinin B1 (B1R) and B2 receptor (B2R) in U87-MG cells and their decreased levels in MSC, as confirmed by quantitative assessment using flow cytometric analysis. Similarly, in indirect cultures (IC), lacking the contact between GBM and MSC cells, an increase of B1 and B2 receptor expression was again noted in U87-MG cells, and no significant changes in kinin receptors in MSC was observed. Functionality of kinin-B1 and B2 receptors was evidenced by stimulation of intracellular calcium fluxes by their respective agonists, des-Arg9-bradykinin (DBK) and bradykinin (BK). Moreover, BK showed a feedback control on kinin receptor expression in mono-cultures, direct and indirect co-cultures. The treatment with BK resulted in down-regulation of B1 and B2 receptors in MSC, with simultaneous up-regulation of these receptors in U87-MG cells, suggesting that functions of BK in information flow between these cells is important for tumor progression and invasion. © 2015 International Society for Advancement of Cytometry.

Impairing effects of angiotensin-converting enzyme inhibitor Captopril on bone of normal mice

There are contradicting results about the effects of angiotensin-converting enzyme inhibitors (ACEIs) on bones. This study was aimed to investigate the effect of ACEI, Captopril, on bone metabolism and histology as well as the action of Captopril on skeletal renin–angiotensin system (RAS) and bradykinin receptor pathway in normal male mice. The urine, serum, tibias and femurs from normal control mice and Captopril-treated (10mg/kg) mice were collected for biochemical, histological and molecular analyses after drug administration for eight weeks. The mice after the treatment with Captopril had a significant decrease of serum testosterone level. The histological measurements showed the loss of trabecular bone mass and trabecular bone number, and the breakage of trabecular bone network as well as the changes of chondrocyte zone at epiphyseal plate in Captopril-treated mice. The defect of Captopril on trabecular bone was reflected by the quantitative bio-parameters from micro-CT. The expression of renin receptor and bradykinin B2 receptor (B2R) was significantly up-regulated in tibia of mice upon to the Captopril treatment, which decreased the ratio of OPG/RANKL and the expression of osteoblastic factor RUNX2. Furthermore, Captopril treatment resulted in the increase of pAkt/Akt and pNFκB expression in tibia. The present study revealed the impairing effects of Captopril on bone via interfering with the circulating sex hormone level and B2R pathway, which suggests that the bone metabolism of patients need to be carefully monitored when being prescribed for ACEIs.

Abstract P113: Angiotensin Converting Enzyme 2 Modulates Bradykinin B1 Receptor Function During DOCA-Salt Hypertension

DOCA-salt hypertension is associated with reduced angiotensin converting enzyme 2 (ACE2) and increased bradykinin B1 receptor (B1R) expression in the brain. ACE2 hydrolyzes des-Arg(9)-bradykinin, the endogenous B1R agonist, into inactive metabolites. Therefore, we hypothesized that ACE2 overexpression or deletion modulates B1R function in the brain during neurogenic hypertension. To test this hypothesis, we used mice overexpressing ACE2 in neurons (SA), ACE2 knockout and B1R knockout mice. Blood pressure (BP) was monitored using telemetry probes in conscious animals. While baseline BP was not different between strains, DOCA-salt treatment (1 mg/g body weight DOCA, 1% NaCl for 3 weeks) resulted in a significantly lower BP in B1R knockout mice (121 ±2 mmHg, n=5) compared to wildtype (WT) mice (138 ±3 mmHg, n=8). DOCA-salt hypertension resulted in 27% decrease (74 ±6 vs. 54 ±2 Fluorescence Units (FU)/min/μg of protein, p&lt;0.05) in ACE2 activity in the hypothalamus, but not in B1R knockout mice with DOCA (69 ±6 vs. 65 ±3 FU/min/μg of protein). In DOCA-treated WT mice, B1R mRNA (Real time PCR) and protein expression (Western blot) in the hypothalamus were increased by 3 and 2 fold (n=6, p&lt;0.01), respectively. This increased B1R expression was blunted in SA mice with DOCA (3.2 ±0.4 vs. 0.9 ±0.1 fold, p&lt;0.001) but not in ACE2 knockout mice with DOCA (3.2 ±0.4 vs. 3.1 ±0.9 fold). Moreover, DOCA-salt treatment resulted in an increased expression of pro-inflammatory ADAM17 in the brain by 2.2 fold (n=6, p&lt;0.01 vs. WT). ACE2 overexpression or B1R knockdown blunted this ADAM17 expression (1.4 ±0.1 and 1.1 ±0.6 fold, respectively, n=3, p&lt;0.01 vs. WT+DOCA), while it was remain increased in ACE2 knockout mice (2.6 ±0.7 fold, n=3, p&lt;0.01) compared to control mice. Together, our data provide novel evidence to support a role for ACE2 in the modulation of central B1R function in the development of DOCA-salt hypertension.

Bradykinin inhibits oxidative stress-induced senescence of endothelial progenitor cells through the B2R/AKT/RB and B2R/EGFR/RB signal pathways.

Circulating endothelial progenitor cells (EPCs) have multiple protective effects that facilitate repair of damage to tissues and organs. However, while various stressors are known to impair EPC function, the mechanisms of oxidative stress-induced EPC senescence remains unknown. We demonstrated that B2 receptor (B2R) expression on circulating CD34(+) cells was significantly reduced in patients with diabetes mellitus (DM) as compared to healthy controls. Furthermore, CD34(+) cell B2R expression in patients with DM was inversely correlated with plasma myeloperoxidase concentrations. Bradykinin (BK) treatment decreased human EPC (hEPC) senescence and intracellular oxygen radical production, resulting in reduced retinoblastoma 1 (RB) RNA expression in H2O2-induced senescent hEPCs and a reversal of the B2R downregulation that is normally observed in senescent cells. Furthermore, BK treatment of H2O2-exposed cells leads to elevated phosphorylation of RB, AKT, and cyclin D1 compared with H2O2-treatment alone. Antagonists of B2R, PI3K, and EGFR signaling pathways and B2R siRNA blocked BK protective effects. In summary, this study demonstrates that BK significantly inhibits oxidative stress-induced hEPC senescence though B2R-mediated activation of PI3K and EGFR signaling pathways.

Inhibition of kinin B1 receptors attenuates pulmonary hypertension and vascular remodeling.

This study examined whether the kinin B1 receptor is involved in the pathogenesis of pulmonary hypertension, and whether its inhibition could reduce inflammation, pulmonary hypertension, vascular remodeling, and right heart dysfunction. Male Wistar rats underwent left pneumonectomy. Seven days later, the rats were injected subcutaneously with monocrotaline (60 mg/kg). The rats were then randomly assigned to receive treatment with vehicle or with BI113823 (a selective B1 receptor antagonist, 30 mg/kg, twice per day) via oral gavage from the day of monocrotaline injection to day 28. By day 28, BI113823-treated rats had significantly lower mean pulmonary artery pressure, less right ventricular hypertrophy, and pulmonary arterial neointimal formation than that of the vehicle-treated rats. Real-time polymerase chain reaction revealed that there was a significant increase in mRNA expression of B1 receptors in the lungs of monocrotaline-challenged pneumonectomized rats. Treatment with BI113823 significantly reduced macrophage recruitment, as measured via bronchoalveolar lavage. It also markedly reduced CD-68 positive macrophages and proliferating cell nuclear antigen positive cells in the perivascular areas, reduced expression of inducible nitric oxide synthase, matrix metalloproteinase 2 and 9, and B1 receptors compared with measurements in vehicle-treated rats. These findings demonstrate that kinin B1 receptors represent a novel therapeutic target for pulmonary arterial hypertension.