Role of Endothelial Kinin B1 Receptor on the Membrane Potential of Transgenic Rat Aorta.

The kinin receptors are classically involved in inflammation, pain and sepsis. The effects of the kinin B1 receptor agonist des-Arg9-bradykinin (DBK) and lipopolysaccharide (LPS) were investigated by comparing the membrane potential responses of aortic rings from transgenic rats overexpressing the kinin B1 receptor (B1R) in the endothelium (TGR(Tie2B1)) and Sprague Dawley (SD) rats. No difference in the resting membrane potential in the aorta's smooth muscle from the transgenic and SD rats was observed. The aorta rings from SD rats hyperpolarized only to LPS but not to DBK, whereas the aorta rings from TGR(Tie2B1) responded by the administration of both drugs. DBK and LPS responses were inhibited by the B1 receptor antagonist R715 and by iberiotoxin in both cases. Thapsigargin induced a hyperpolarization in the smooth muscle of SD rats that was not reversed by R715, but was reversed by iberiotoxin and this hyperpolarization was further augmented by DBK administration. These results show that the model of overexpression of vascular B1 receptors in the TGR(Tie2B1) rats represent a good model to study the role of functional B1 receptors in the absence of any pathological stimulus. The data also show that KCa channels are the final mediators of the hyperpolarizing responses to DBK and LPS. In addition, we suggest an interaction between the B1R and TLR4, since the hyperpolarization induced by LPS could be abolished in the presence of R715.

Cardiac morphofunctional characteristics of transgenic rats with overexpression of the bradykinin B1 receptor in the endothelium.

Our aim was to evaluate whether endothelial overexpressing of the bradykinin B1 receptor could be associated with altered left ventricular and myocardial performance. Echocardiography and hemodynamic were employed to assess left ventricular morphology and function in Sprague Dawley transgenic rats overexpressing the endothelial bradykinin B1 receptor (Tie2B1 rats). The myocardial inotropism was evaluated on papillary muscles contracting in vitro. In Tie2B1 animals, an enlarged left ventricular cavity and lower fractional shortening coupled with a lower rate of pressure change values indicated depressed left ventricular performance. Papillary muscle mechanics revealed that both Tie2B1 and wild-type rat groups had the same contractile capacities under basal conditions; however, in transgenic animals, there was accentuated inotropism due to post-pause potentiation. Following treatment with the Arg(9)-BK agonist, Tie2B1 papillary muscles displayed a reduction in myocardial inotropism. Endothelial B1 receptor overexpression has expanded the LV cavity and worsened its function. There was an exacerbated response of papillary muscle in vitro to a prolonged resting pause, and the use of a B1 receptor agonist impairs myocardial inotropism.

Homozygosity for a factor XII mutation in one female and one male patient with hereditary angio-oedema.

Hereditary angio-oedema (HAE) with normal C1 inhibitor is associated with heterozygous mutations in the factor XII gene (FXII-HAE). We report two Brazilian FXII-HAE families segregating the mutation c.983 C>A (p.Thr328Lys). In each family, one patient with a homozygous mutation was found. The homozygous female patient in family 1 displayed a severe phenotype. However, this falls within the clinical phenotype spectrum reported for heterozygous female mutation carriers. The homozygous male patient in family 2 also showed a severe phenotype. This finding is intriguing, as to our knowledge, it is the first such report for a male FXII-HAE mutation carrier. In the rare instances in which male mutation carriers are affected, a mild phenotype is typical. The present findings therefore suggest that homozygous FXII-HAE mutation status leads to a severe phenotype in females and males, and to an increased risk of manifest symptoms in the latter.

Kinin B2 receptor can play a neuroprotective role in Alzheimer's disease.

Alzheimer's disease (AD) is characterized by cognitive decline, presence of amyloid-beta peptide (Aß) aggregates and neurofibrillary tangles. Kinins act through B1 and B2 G-protein coupled receptors (B1R and B2R). Chronic infusion of Aß peptide leads to memory impairment and increases in densities of both kinin receptors in memory processing areas. Similar memory impairment was observed in C57BL/6 mice (WTAß) but occurred earlier in mice lacking B2R (KOB2Aß) and was absent in mice lacking B1R (KOB1Aß). Thus, the aim of this study was to evaluate the participation of B1R and B2R in Aß peptide induced cognitive deficits through the evaluation of densitiesof kinin receptors, synapses, cell bodies and number of Aß deposits in brain ofWTAß, KOB1Aß and KOB2Aß mice. An increase in B2R density was observed in both WTAß and KOB1Aß in memory processing related areas. KOB1Aß showed a decrease in neuronal density and an increase in synaptic density and, in addition, an increase in Aß deposits in KOB2Aß was observed. In conclusion, memory preservation in KOB1Aß, could be due to the increase in densities of B2R, suggesting a neuroprotective role for B2R, reinforced by the increased number of Aß plaques in KOB2Aß. Our data point to B2R as a potential therapeutic target in AD.

Activation of HPA axis and remodeling of body chemical composition in response to an intense and exhaustive exercise in C57BL/6 mice.

Several deleterious effects may occur when intense and exhaustive exercise (IE) is not well-planned. This study aimed to investigate the effects of a short duration IE on body chemical composition and hypothalamic-pituitary-adrenal (HPA) axis. C57Bl/6 mice were distributed into four groups (10 mice per group): control (C-4D and C-10D), 4 days (E-4D), and 10 days of IE (E-10D). IE program consisted of a daily running session at 85 % of maximum speed until the animal reached exhaustion. Body weight as well as total body water, fat and protein content were determined from animal carcasses. HPA activation was assessed by plasma corticosterone levels measured by radioimmunoassay and the weight of both the adrenal glands and thymus were measured. Plasma corticosterone levels increased by 64 % in both the E-4D and E-10D groups. The weight of the adrenal glands augmented by 74 % and 45 %, at 4 and 10 days of IE, respectively, whereas thymus weight diminished by 15 % only in the E-10D group. The total carcass fat content decreased by 20 % only at 4 days IE, whereas protein content decreased by 20 % in both E-4D and E-10D groups. A relationship between corticosterone plasma levels and loss of body protein content in both E-4D and E-10D groups was observed (R(2)=0.999). We concluded that IE may be related to HPA axis activation associated with remodeling of body chemical composition in C57BL/6 mice.

Exacerbation of DSS-induced colitis in mice lacking kinin B(1) receptors through compensatory up-regulation of kinin B(2) receptors: the role of tight junctions and intestinal homeostasis.

BACKGROUND AND PURPOSE: Kinins are pro-inflammatory peptides that are released during tissue injury, including that caused by inflammatory bowel disease. Herein, we assessed the role and underlying mechanisms through which the absence of kinin B(1) receptors exacerbates the development of dextran sulfate sodium (DSS)-induced colitis in mice. EXPERIMENTAL APPROACH: B(1) and B(2) receptor antagonists and B(1) receptor knockout mice (B1(-/-) ) were used to assess the involvement of B(1) and B(2) receptor signalling in a DSS-colitis. B(1) receptor, B(2) receptor, occludin and claudin-4 expression, cytokine levels and cell permeability were evaluated in colon from wild-type (WT) and B1(-/-) mice. KEY RESULTS: DSS-induced colitis was significantly exacerbated in B1(-/-) compared with WT mice. IL-1ß, IFN-γ, keratinocyte-derived chemokine and macrophage inflammatory protein-2 were markedly increased in the colon from DSS-treated B1(-/-) compared with DSS-treated WT mice. Treatment of WT mice with a selective B(1) receptor antagonist, DALBK or SSR240612, had no effect on DSS-induced colitis. Of note, B(2) receptor mRNA expression was significantly up-regulated in colonic tissue from the B1(-/-) mice after DSS administration. Moreover, treatment with a selective B(2) receptor antagonist prevented the exacerbation of colitis in B1(-/-) mice following DSS administration. The water- or DSS-treated B1(-/-) mice showed a decrease in occludin gene expression, which was partially prevented by the B(2) receptor antagonist. CONCLUSIONS AND IMPLICATIONS: A loss of B(1) receptors markedly exacerbates the severity of DSS-induced colitis in mice. The increased susceptibility of B1(-/-) may be associated with compensatory overexpression of B(2) receptors, which, in turn, modulates tight junction expression.

Angiotensin (5-8) modulates nociception at the rat periaqueductal gray via the NO-sGC pathway and an endogenous opioid.

Angiotensins (Angs) modulate blood pressure, hydro-electrolyte composition, and antinociception. Although Ang (5-8) has generally been considered to be inactive, we show here that Ang (5-8) was the smallest Ang to elicit dose-dependent responses and receptor-mediated antinociception in the rat ventrolateral periaqueductal gray matter (vlPAG). Ang (5-8) antinociception seems to be selective, because it did not alter blood pressure or act on vascular or intestinal smooth muscle cells. The non-selective Ang-receptor (Ang-R) antagonist saralasin blocked Ang (5-8) antinociception, but selective antagonists of Ang-R types I, II, IV, and Mas did not, suggesting that Ang (5-8) may act via an unknown receptor. Endopeptidase EP 24.11 and amastatin-sensitive aminopeptidase from the vlPAG catalyzed the synthesis (from Ang II or Ang III) and inactivation of Ang (5-8), respectively. Selective inhibitors of neuronal-nitric oxide (NO) synthase, soluble guanylyl cyclase (sGC) and a non-selective opioid receptor (opioid-R) inhibitor blocked Ang (5-8)-induced antinociception. In conclusion, Ang (5-8) is a new member of the Ang family that selectively and strongly modulates antinociception via NO-sGC and endogenous opioid in the vlPAG.

Higher frequency of paraoxonase gene polymorphism and cardiovascular impairment among Brazilian Fabry Disease patients.

OBJECTIVES: Paraoxonase (PON1) plays a role in preventing the oxidation of lipoproteins and protecting against atherosclerosis. Several polymorphisms have been described in the gene encoding this enzyme, which are related to different enzymatic activities. Fabry Disease (FD) is a lysosomal storage disease associated with cardiomyopathy, early-onset stroke, renal failure, among other features. The objective of the current study was to investigate the PON1 polymorphisms Gln192Arg and Leu55Met in FD patients and correlate them with clinical symptoms. DESIGN AND METHODS: A total of 106 subjects with FD and 26 healthy individuals were selected for the study. Both polymorphisms were assessed in the DNA of blood samples using PCR-RFLP. Hardy-Weinberg equilibrium was calculated for the genotypes and statistical analyses were realized using the Chi-Squared test with Yates correction. RESULTS: The allele frequencies of the polymorphism Gln192Arg for FD patients and control were 0.38 and 0.25, respectively. A comparison of the frequencies for Gln192Arg polymorphism between FD patients and controls revealed a significant difference. The clinical information was obtained from 41 patients. Patients with the Gln192Arg polymorphism showed different cardiovascular manifestations. CONCLUSIONS: The higher frequency of the Gln192Arg polymorphism among FD patients highlighted the possibility of a correlation between the PON1 genetic variation and the phenotypes because the disease has a wide range of symptoms not explained exclusively by mutations on the GLA gene.

Carbamazepine inhibits angiotensin I-converting enzyme, linking it to the pathogenesis of temporal lobe epilepsy.

We find that a common mutation that increases angiotensin I-converting enzyme activity occurs with higher frequency in male patients suffering from refractory temporal lobe epilepsy. However, in their brains, the activity of the enzyme is downregulated. As an explanation, we surprisingly find that carbamazepine, commonly used to treat epilepsy, is an inhibitor of the enzyme, thus providing a direct link between epilepsy and the renin-angiotensin and kallikrein-kinin systems.

Endostatin gene therapy stimulates upregulation of ICAM-1 and VCAM-1 in a metastatic renal cell carcinoma model.

One of the greatest challenges in urological oncology is renal cell carcinoma (RCC), which is the third leading cause of death in genitourinary cancers. RCCs are highly vascularized and respond positively to antiangiogenic therapy. Endostatin (ES) is a fragment of collagen XVIII that possesses antiangiogenic activity. In this study, we examined the potential of ES-based antiangiogenic therapy to activate tumor-associated endothelial cells in metastatic RCC (mRCC). Balb/c-bearing Renca cells were treated with NIH/3T3-LendSN or, as a control, with NIH/3T3-LXSN cells. The T-cell subsets and lymphocyte populations of tumors, mediastinal lymph nodes and the spleen were assessed by flow cytometry. The expression of intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1) was assessed by real-time PCR, flow cytometry and immunohistochemistry analysis. ES gene therapy led to an increase in the percentage of infiltrating CD4-interferon (IFN)-γ cells (P<0.05), CD8-IFN-γ cells (P<0.01) and CD49b-tumor necrosis factor-α cells (P<0.01). In addition, ES therapy caused an increase at the mRNA level of ICAM-1 (1.4-fold; P<0.01) and VCAM-1 (1.5-fold) (control vs treated group; P<0.001). Through flow cytometry, we found a significant increase in the CD34/ICAM-1 cells (8.1-fold; P<0.001) and CD34/VCAM-1 cells (1.6-fold; P<0.05). ES gene therapy induced a significant increase in both T CD4 and CD8 cells in the lymph nodes and the spleen, suggesting that ES therapy may facilitate cell survival or clonal expansion. CD49b cells were also present in increased quantities in all of these organs. In this study, we demonstrate an antitumor inflammatory effect of ES in an mRCC model, and this effect is mediated by an increase in ICAM-1 and VCAM-1 expression in tumor-associated endothelial cells.

Effect of 12 weeks of resistance exercise on post-exercise hypotension in stage 1 hypertensive individuals.

Post-exercise hypotension (PEH), the reduction of blood pressure (BP) after a single bout of exercise, is of great clinical relevance. As the magnitude of this phenomenon seems to be dependent on pre-exercise BP values and chronic exercise training in hypertensive individuals leads to BP reduction; PEH could be attenuated in this context. Therefore, the aim of the present study was to investigate whether PEH remains constant after resistance exercise training. Fifteen hypertensive individuals (46 ± 8 years; 88 ± 16 kg; 30 ± 6% body fat; 150 ± 13/93 ± 5 mm Hg systolic/diastolic BP, SBP/DBP) were withdrawn from medication and performed 12 weeks of moderate-intensity resistance training. Parameters of cardiovascular function were evaluated before and after the training period. Before the training program, hypertensive volunteers showed significant PEH. After an acute moderate-intensity resistance exercise session with three sets of 12 repetitions (60% of one repetition maximum) and a total of seven exercises, BP was reduced post-exercise (45-60 min) by an average of aproximately -22 mm Hg for SBP, -8 mm Hg for DBP and -13 mm Hg for mean arterial pressure (P<0.05). However, this acute hypotensive effect did not occur after the 12 weeks of training (P>0.05). In conclusion, our data demonstrate that PEH, following an acute exercise session, can indeed be attenuated after 12 weeks of training in hypertensive stage 1 patients not using antihypertensive medication.

Leucurogin, a new recombinant disintegrin cloned from Bothrops leucurus (white-tailed-jararaca) with potent activity upon platelet aggregation and tumor growth.

Disintegrins and disintegrins-like proteins are able to inhibit platelet aggregation and integrin-mediated cell adhesion. The aim of this study was to produce one disintegrin-like cloned from Bothrops leucurus venom gland and to characterize it regarding biological activity. The recombinant protein was purified by one step procedure involving anion-exchange chromatography (DEAE-cellulose) and presented a molecular mass of 10.4 kDa. The purified protein was able to inhibit platelet aggregation induced by collagen (IC50 = 0.65 µM) and to inhibit growth of Ehrlich tumor implanted in mice by more than 50% after 7 days administration of 10 µg/day. No effects were observed upon adenosine 5'-diphosphate (ADP)-and arachidonic acid (AA)-induced platelet aggregation. The recombinant protein was recognized by an antibody specific for jararhagin one metalloproteinase isolated from Bothrops jararaca venom, and therefore it was named leucurogin. Anti-angiogenesis effect of leucurogin was evaluated by the sponge implant model. After 7 days administration leucurogin inhibited, in a dose dependent way, the vascularization process in the sponge. Leucurogin represents a new biotechnological tool to understand biological processes where disintegrins-like are involved and may help to characterize integrins that can be involved in development and progression of malignant cells.

Effect of kinin B2 receptor ablation on skeletal muscle development and myostatin gene expression.

Bradykinin (BK) is an active peptide that binds to the kinin B(2) receptor and induces biological events during the development and adult life. In this study we aimed to investigate the effect of kinin B(2) receptor ablation in the postnatal skeletal muscle development and body composition in adult life. For studies of skeletal muscle development, control (C57Bl6 - WT) and B(2) receptor knockout mice (B(2)(-/-)) were sacrificed at 15, 30 and 90days after birth, the gastrocnemius skeletal muscle was weighed and myostatin gene expression evaluated by real time PCR. For energy balance determination, data from control and B(2)(-/-) at 90 and 120days were collected by calorimetric method. Body composition at 120days was determined by chloroform-methanol (total body fat) and Lowry-modified method (total body protein). The results show that B(2)(-/-) have significantly increased total body weight at 15, 30 and 90days of life, when compared to WT. The weight of the gastrocnemius skeletal muscle was also significantly increased at 30 and 90days of life. Body composition analyses revealed that B(2)(-/-) mice exhibit more total corporal protein and less total corporal fat. Energy balance revealed that B(2)(-/-) have increased metabolizable energy intake and energy expenditure when compared to control mice, resulting in a lower energy gain. Interestingly, myostatin mRNA expression was significantly decreased in 15 and 30days old B(2)(-/-) mice and after icatibant treatment of WT adult mice for 5days. In conclusion, together our results show that kinin B(2) receptor deletion increases lean mass, reduces fat mass and improves metabolism efficiency in mice. The mechanism involved in this phenotype could be related to the reduction of myostatin gene expression during postnatal life.

Plasma Kallikrein and Angiotensin I-converting enzyme N- and C-terminal domain activities are modulated by the insertion/deletion polymorphism.

Angiotensin I-converting enzyme (ACE) is recognized as one of the main effector molecules involved in blood pressure regulation. In the last few years some polymorphisms of ACE such as the insertion/deletion (I/D) polymorphism have been described, but their physiologic relevance is poorly understood. In addition, few studies investigated if the specific activity of ACE domain is related to the I/D polymorphism and if it can affect other systems. The aim of this study was to establish a biochemical and functional characterization of the I/D polymorphism and correlate this with the corresponding ACE activity. For this purpose, 119 male brazilian army recruits were genotyped and their ACE plasma activities evaluated from the C- and N-terminal catalytic domains using fluorescence resonance energy transfer (FRET) peptides, specific for the C-domain (Abz-LFK(Dnp)OH), N-domain (Abz-SDK(Dnp)P-OH) and both C- and N-domains (Abz-FRK(Dnp)P-OH). Plasma kallikrein activity was measured using Z-Phe-Arg-AMC as substrate and inhibited by selective plasma kallikrein inhibitor (PKSI). Some physiological parameters previously described related to the I/D polymorphism such as handgrip strength, blood pressure, heart rate and BMI were also evaluated. The genotype distribution was II n=27, ID n=64 and DD n=28. Total plasma ACE activity of both domains in II individuals was significantly lower in comparison to ID and DD. This pattern was also observed for C- and N-domain activities. Difference between ID and DD subjects was observed only with the N-domain specific substrate. Blood pressure, heart rate, handgrip strength and BMI were similar among the genotypes. This polymorphism also affected the plasma kallikrein activity and DD group presents high activity level. Thus, our data demonstrate that the I/D ACE polymorphism affects differently both ACE domains without effects on handgrip strength. Moreover, this polymorphism influences the kallikrein-kinin system of normotensive individuals.

Delayed blockade of the kinin B1 receptor reduces renal inflammation and fibrosis in obstructive nephropathy.

Renal fibrosis is the common histological feature of advanced glomerular and tubulointerstitial disease leading to end-stage renal disease (ESRD). However, specific antifibrotic therapies to slow down the evolution to ESRD are still absent. Because persistent inflammation is a key event in the development of fibrosis, we hypothesized that the proinflammatory kinin B1 receptor (B1R) could be such a new target. Here we show that, in the unilateral ureteral obstruction model of renal fibrosis, the B1R is overexpressed and that delayed treatment with an orally active nonpeptide B1R antagonist blocks macrophage infiltration, leading to a reversal of the level of renal fibrosis. In vivo bone marrow transplantation studies as well as in vitro studies on renal cells show that part of this antifibrotic mechanism of B1R blockade involves a direct effect on resident renal cells by inhibiting chemokine CCL2 and CCL7 expression. These findings suggest that blocking the B1R is a promising antifibrotic therapy.

The relevance of kinin B1 receptor upregulation in a mouse model of colitis.

BACKGROUND AND PURPOSE: Kinins are implicated in many pathophysiological conditions, and recent evidence has suggested their involvement in colitis. This study assessed the role of the kinin B1 receptors in a mouse model of colitis. EXPERIMENTAL APPROACH: Colitis was induced in mice by 2,4,6-trinitrobenzene sulphonic acid (TNBS), and tissue damage and myeloperoxidase activity were assessed. B1 receptor induction was analysed by organ bath studies, binding assay and reverse transcription PCR. KEY RESULTS: TNBS-induced colitis was associated with tissue damage, neutrophil infiltration and time-dependent increase of colon B1 receptor-mediated contraction, with the maximal response observed at 72 h. The upregulation of the B1 receptor at this time point was also confirmed by means of binding studies. B1 receptor mRNA levels were elevated as early as 6 h after colitis induction and remained high for up to 48 h. TNBS-evoked tissue damage and neutrophil influx were reduced by the selective B1 receptor antagonist SSR240612, and in B1 receptor knockout mice. In vivo treatment with inhibitors of protein synthesis, nuclear factor-kappaB activation, inducible nitric oxide synthase (iNOS) or tumour necrosis factor alpha (TNFalpha) significantly reduced B1 receptor agonist-induced contraction. Similar results were observed in iNOS and TNF receptor 1-knockout mice. CONCLUSIONS AND IMPLICATIONS: These results provide convincing evidence on the role of B1 receptors in the pathogenesis of colitis. Therefore, the blockade of kinin B1 receptors might represent a new therapeutic option for treating inflammatory bowel diseases.