Evaluation of Novel B1R/B2R Agonists Containing TRIOZAN™ Nanoparticles for Targeted Brain Delivery of Antibodies in a Mouse Model of Alzheimer Disease.

The blood-brain barrier (BBB) is a major obstacle to the development of effective therapeutics for central nervous system (CNS) disorders, including Alzheimer's disease (AD). This has been particularly true in the case of monoclonal antibody (mAbs) therapeutic candidates, due to their large size. To tackle this issue, we developed new nanoformulations, comprising bio-based Triozan polymers along with kinin B1 and B2 receptor (B1R and B2R) peptide agonist analogues, as potent BBB-permeabilizers to enhance brain delivery of a new anti-C1q mAb for AD (ANX005). The prepared B1R/B2R-TRIOZAN™ nanoparticles (NPs) displayed aqueous solubility, B1R/B2R binding capacity and uniform sizes (~130-165 nm). The relative biodistribution profiles of the mAb loaded into these NPs versus the naked mAb were assessed in vivo through two routes of administrations (intravenous (IV), intranasal (IN)) in the Tg-SwDI mouse model of AD. At 24 h post-administration, brain levels of the encapsulated mAb were significantly increased (up to 12-fold (IV) and 5-fold (IN), respectively) compared with free mAb in AD brain affected regions, entorhinal cortex and hippocampus of aged mice. Liver uptakes remained relatively low with similar values for the nanoformulations and free mAb. Our findings demonstrate the potential of B1R/B2R-TRIOZAN™ NPs for the targeted delivery of new CNS drugs, which could maximize their therapeutic effectiveness.

Development of a multigram synthesis of the bradykinin receptor 2 agonist FR-190997 and analogs thereof.

Using Fujisawa's B2R agonist FR-190997, we recently demonstrated for the first time that agonism at the bradykinin receptor type 2 (B2R) produces substantial antiproliferative effects. FR-190997 elicited an EC50 of 80 nM in the triple-negative breast cancer cell line MDA-MB-231, a much superior performance to that exhibited by most approved breast cancer drugs. Consequently, we initiated a program aiming primarily at synthesizing adequate quantities of FR-190997 to support further in vitro and in vivo studies toward its repurposing for various cancers and, in parallel, enable the generation of novel FR-190997 analogs for an SAR study. Prerequisite for this endeavor was to address the synthetic challenges associated with the FR-190997 scaffold, which the Fujisawa chemists had constructed in 20 steps, 13 of which required chromatographic purification. We succeeded in developing a 17-step synthesis amenable to late-stage diversification that eliminated all chromatography and enabled access to multigram quantities of FR-190997 and novel derivatives thereof, supporting further anticancer research based on B2R agonists.

Function and structure of bradykinin receptor 2 for drug discovery.

Type 2 bradykinin receptor (B2R) is an essential G protein-coupled receptor (GPCR) that regulates the cardiovascular system as a vasodepressor. Dysfunction of B2R is also closely related to cancers and hereditary angioedema (HAE). Although several B2R agonists and antagonists have been developed, icatibant is the only B2R antagonist clinically used for treating HAE. The recently determined structures of B2R have provided molecular insights into the functions and regulation of B2R, which shed light on structure-based drug design for the treatment of B2R-related diseases. In this review, we summarize the structure and function of B2R in relation to drug discovery and discuss future research directions to elucidate the remaining unknown functions of B2R dimerization.

Sex-specific Regulation of Prolactin Secretion by Pituitary Bradykinin Receptors.

Sex differences in the control of prolactin secretion are well documented. Sex-related differences in intrapituitary factors regulating lactotroph function have recently attracted attention. Sex differences in prolactinoma development are well documented in clinic, prolactinomas being more frequent in women but more aggressive in men, for poorly understood reasons. Kallikrein, the enzyme releasing kinins has been found in the pituitary, but there is no information on pituitary kinin receptors and their function. In the present work, we characterized pituitary bradykinin receptors (BRs) at the messenger RNA and protein levels in 2 mouse models of prolactinoma, Drd2 receptor gene inactivation and hCGß gene overexpression, in both males and females, wild type or genomically altered. BR B2 (B2R) accounted for 97% or more of total pituitary BRs in both models, regardless of genotype, and was present in lactotrophs, somatotrophs, and gonadotrophs. Male pituitaries displayed higher level of B2R than females, regardless of genotype. Pituitary B2R gene expression was downregulated by estrogen in both males and females but only in females by dopamine. Activation of B1R or B2R by selective pharmacological agonists induced prolactin release in male pituitaries but inhibited prolactin secretion in female pituitaries. Increased B2R content was observed in pituitaries of mutated animals developing prolactinomas, compared to their respective wild-type controls. The present study documents a novel sex-related difference in the control of prolactin secretion and suggests that kinins are involved, through B2R activation, in lactotroph function and prolactinoma development.

Potent antiproliferative activity of bradykinin B2 receptor selective agonist FR-190997 and analogue structures thereof: A paradox resolved?

Βradykinin stimulation of B2 receptor is known to activate the oncogenic ERK pathway and overexpression of bradykinin receptors B1 and B2 has been reported to occur in glioma, colorectal and cervical cancers. B1R and B2R antagonists have been shown to reverse tumor proliferation and invasion. Paradoxically, B1R and B2R agonism has also been reported to elicit antiproliferative benefits. In order to complement the data accumulated to date with the natural substrate bradykinin and peptidic B2R antagonists, we decided to examine for the first time the response elicited by B2R stimulation in breast cancer lines with a non-peptidic small molecule B2R agonist. We synthesized and assessed the highly selective and potent B2R partial agonist FR-190997 in MCF-7 and MDA-MBA-231 breast cancer lines and found it possessed significant antiproliferative activity (IC50 2.14 and 0.08 µΜ, respectively). The modular nature of FR-190997 allowed us to conduct a focused SAR study and discover compound 10 which exhibits subnanomolar antiproliferative activity (IC 50 0.06 nΜ) in the TNBC MDA-MBA-231 cell line. This performance surpasses, in most cases by several orders of magnitude, those of established anticancer agents and FDA-approved breast cancer drugs. In line with the established literature we suggest that this remarkable activity precipitates from a dual mode of action involving agonist-induced receptor internalization/degradation combined with sequestration of functional intracellular B2 receptors and inhibition of the associated endosomal signaling. The latter mode may be realized by appropriate ligands regardless of B2R agonist/antagonist designation which only relates to membrane residing GCPRs. Under this prism the controversy over the antiproliferative effects of B2 agonists and antagonists is potentially neutralized.

Identification of a new myotropic decapeptide from the skin secretion of the red-eyed leaf frog, Agalychnis callidryas.

Bradykinin-related peptides (BRPs) family is one of the most significant myotropic peptide families derived from frog skin secretions. Here, a novel BRP callitide was isolated and identified from the red-eyed leaf frog, Agalychnis callidryas, with atypical primary structure FRPAILVRPK-NH2. The mature peptide was cleaved N-terminally at a classic propeptide convertase cleavage site (-KR-) and at the C-terminus an unusual -GKGKGK sequence was removed using the first G residue as an amide donor for the C-terminally-located K residue. Thereafter, the synthetic replicates of callitide were assessed the myotropic activity and showed a significant contraction of balder, with the 0.63 nM EC50 value, more potent than most discovered myotropic peptides. The binding mode was further speculated by molecular docking and stimulation. The result indicated that the C-terminal of callitide might selectively bind to bradykinin receptor B2 (BKRB2). Further investigation of the callitide needs to be done in the future to be exploited as potential future drug leads.

Discovery of a Bradykinin B2 Partial Agonist Profile of Raloxifene in a Drug Repurposing Campaign.

Covid-19 urges a deeper understanding of the underlying molecular mechanisms involved in illness progression to provide a prompt therapeutical response with an adequate use of available drugs, including drug repurposing. Recently, it was suggested that a dysregulated bradykinin signaling can trigger the cytokine storm observed in patients with severe Covid-19. In the scope of a drug repurposing campaign undertaken to identify bradykinin antagonists, raloxifene was identified as prospective compound in a virtual screening process. The pharmacodynamics profile of raloxifene towards bradykinin receptors is reported in the present work, showing a weak selective partial agonist profile at the B2 receptor. In view of this new profile, its possible use as a therapeutical agent for the treatment of severe Covid-19 is discussed.

Bradykinin sensitizes the cough reflex via a B2 receptor dependent activation of TRPV1 and TRPA1 channels through metabolites of cyclooxygenase and 12-lipoxygenase.

BACKGROUND: Inhaled bradykinin (BK) has been reported to both sensitize and induce cough but whether BK can centrally sensitize the cough reflex is not fully established. In this study, using a conscious guinea-pig model of cough, we investigated the role of BK in the central sensitization of the cough reflex and in airway obstruction. METHODS: Drugs were administered, to guinea pigs, by the intracerebroventricular (i.c.v.) route. Aerosolized citric acid (0.2 M) was used to induce cough in a whole-body plethysmograph box, following i.c.v. infusion of drugs. An automated analyser recorded both cough and airway obstruction simultaneously. RESULTS: BK, administered by the i.c.v. route, dose-dependently enhanced the citric acid-induced cough and airway obstruction. This effect was inhibited following i.c.v. pretreatment with a B2 receptor antagonist, TRPV1 and TRPA1 channels antagonists and cyclooxygenase (COX) and 12-lipoxygenase (12-LOX) inhibitors. Furthermore, co-administration of submaximal doses of the TRPV1 and TRPA1 antagonists or the COX and 12-LOX inhibitors resulted in a greater inhibition of both cough reflex and airway obstruction. CONCLUSIONS: Our findings show that central BK administration sensitizes cough and enhances airway obstruction via a B2 receptor/TRPV1 and/or TRPA1 channels which are coupled via metabolites of COX and/or 12-LOX enzymes. In addition, combined blockade of TRPV1 and TRPA1 or COX and 12-LOX resulted in a greater inhibitory effect of both cough and airway obstruction. These results indicate that central B2 receptors, TRPV1/TRPA1 channels and COX/12-LOX enzymes may represent potential therapeutic targets for the treatment of cough hypersensitivity.

Influence of bradykinin B2 receptor and dopamine D2 receptor on the oxidative stress, inflammatory response, and apoptotic process in human endothelial cells.

Endothelial dysfunction is a hallmark of a wide range of cardiovascular diseases and is often linked to oxidative stress and inflammation. Our earlier study reported the formation of a functional heterodimer between bradykinin receptor 2 (B2R) and dopamine receptor 2 (D2R) that may modulate cell responses, dependent on intracellular signaling. Here, for the first time, we showed a cooperative effect of these receptors on the modulation of processes involved in oxidative stress, inflammation, and apoptosis in endothelial cells. Sumanirole, a specific D2R agonist, was shown to diminish the excessive production of reactive oxygen species induced by bradykinin, a proinflammatory B2R-activating peptide. This effect was accompanied by modified activities of antioxidant enzymes and increased phosphorylation of endothelial nitric oxide synthase, leading to enhance NO production. In turn, endothelial cell co-stimulation with B2R and D2R agonists inhibited the release of interleukin-6 and endothelin-1 and modulated the expression of apoptosis markers, such as Bcl-2, Bcl-xL, Bax, and caspase 3/7 activity. All these observations argue that the D2R agonist counteracts the pro-oxidative, pro-inflammatory, and pro-apoptotic effects induced through B2R, finally markedly improving endothelial functions.

Bradykinin B2 receptor and dopamine D2 receptor cooperatively contribute to the regulation of neutrophil adhesion to endothelial cells.

Leukocyte adhesion to the vascular endothelium contributes to many immunological and inflammatory disorders. These processes have been shown to be mediated by bradykinin receptor type 2 (B2R) and dopamine receptor type 2 (D2R). In a previous study, we reported the formation of a B2R-D2R heterodimer, possibly altering cellular functions. Hence, in the present study, we examined the effect of co-activation of endothelial cells with B2R and D2R agonists on the interaction of these cells with neutrophils. Bradykinin, the main B2R agonist, significantly increased cell adhesion, and this effect was reversed when the endothelial cells were additionally co-treated with a selective D2R agonist, sumanirole. These results were dependent on the incubation time, showing an opposite tendency after prolonged stimulation. Significant changes in the expression of adhesion proteins, such as E-selectin and intercellular adhesion molecule 1 in endothelial cells were observed. Additionally, the cells preincubated with tumor necrosis factor-α showed decreased cell adhesion and IL-8 release after long incubation with both agonists. The modulation of cell adhesion by D2R and B2R seem to be mediated via STAT3 phosphorylation. In summary, this study demonstrated a protective role of D2R in neutrophil-endothelial cell adhesion induced by bradykinin, especially in cytokine-stimulated endothelial cells.

Kinin Receptors Sensitize TRPV4 Channel and Induce Mechanical Hyperalgesia: Relevance to Paclitaxel-Induced Peripheral Neuropathy in Mice.

Kinin B1 (B1R) and B2 receptors (B2R) and the transient receptor potential vanilloid 4 (TRPV4) channel are known to play a critical role in the peripheral neuropathy induced by paclitaxel (PTX) in rodents. However, the downstream pathways activated by kinin receptors as well as the sensitizers of the TRPV4 channel involved in this process remain unknown. Herein, we investigated whether kinins sensitize TRPV4 channels in order to maintain PTX-induced peripheral neuropathy in mice. The mechanical hyperalgesia induced by bradykinin (BK, a B2R agonist) or des-Arg9-BK (DABK, a B1R agonist) was inhibited by the selective TRPV4 antagonist HC-067047. Additionally, BK was able to sensitize TRPV4, thus contributing to mechanical hyperalgesia. This response was dependent on phospholipase C/protein kinase C (PKC) activation. The selective kinin B1R (des-Arg9-[Leu8]-bradykinin) and B2R (HOE 140) antagonists reduced the mechanical hyperalgesia induced by PTX, with efficacies and time response profiles similar to those observed for the TRPV4 antagonist (HC-067047). Additionally, both kinin receptor antagonists inhibited the overt nociception induced by hypotonic solution in PTX-injected animals. The same animals presented lower PKCε levels in skin and dorsal root ganglion samples. The selective PKCε inhibitor (εV1-2) reduced the hypotonicity-induced overt nociception in PTX-treated mice with the same magnitude observed for the kinin receptor antagonists. These findings suggest that B1R or B2R agonists sensitize TRPV4 channels to induce mechanical hyperalgesia in mice. This mechanism of interaction may contribute to PTX-induced peripheral neuropathy through the activation of PKCε. We suggest these targets represent new opportunities for the development of effective analgesics to treat chronic pain.

Neuroprotective effect of kinin B1 receptor activation in acute cerebral ischemia in diabetic mice.

Activation of the kallikrein-kinin system enhances cardiac and renal tolerance to ischemia. Here we investigated the effects of selective agonists of kinin B1 or B2 receptor (R) in brain ischemia-reperfusion in diabetic and non-diabetic mice. The role of endogenous kinins was assessed in tissue kallikrein deficient mice (TK-/-). Mice underwent 60min-middle cerebral artery occlusion (MCAO), eight weeks after type 1-diabetes induction. Treatment with B1R-, B2R-agonist or saline was started at reperfusion. Neurological deficit (ND), infarct size (IS), brain water content (BWC) were measured at day 0, 1 and 2 after injury. MCAO induced exaggerated ND, mortality and IS in diabetic mice. B2R-agonist increased ND and mortality to 60% and 80% in non-diabetic and diabetic mice respectively, by mechanisms involving hemodynamic failure and renal insufficiency. TK-/- mice displayed reduced ND and IS compared to wild-type littermate, consistent with suppression of B2R activity. B1R mRNA level increased in ischemic brain but B1R-agonist had no effect on ND, mortality or IS in non-diabetic mice. In contrast, in diabetic mice, B1R-agonist tested at two doses significantly reduced ND by 42-52% and IS by 66-71%, without effect on BWC or renal function. This suggests potential therapeutic interest of B1R agonism for cerebral protection in diabetes.

Bradykinin B2 and dopamine D2 receptors form a functional dimer.

In recent years a wide range of studies have shown that G protein-coupled receptors modulate a variety of cell functions through the formation of dimers. For instance, there is growing evidence for the dimerization of bradykinin or dopamine receptors, both as homodimers and heterodimers. A discovery of direct interactions of angiotensin II receptors with bradykinin 2 receptor (B2R) or dopamine D2 (D2R) receptor has led to a hypothesis on a potential dimerization between two latter receptors. In this study, we have demonstrated a constitutive colocalization of receptors on the membranes of HEK293 cells transiently transfected with plasmid vectors encoding B2R and D2R, fused with fluorescent proteins. The receptor colocalization was significantly enhanced by specific agonists of B2R or D2R after 5min following the addition, whereas simultaneous stimulation with these agonists did not influence the B2R/D2R colocalization level. In addition, B2R-D2R heterodimerization was confirmed with FLIM-FRET technique. The most characteristic signaling pathways for B2R and D2R, dependent on intracellular Ca2+ and cAMP concentration, respectively, were analyzed in cells presenting similar endogenous expression of B2R and D2R. Significant changes in receptors' signaling were observed after simultaneous stimulation with agonists, suggesting transformations in proteins' conformation after dimerization. The evidence of B2R-D2R dimerization may open new perspectives in the modulation of diverse cellular functions which depend on their activation.

Pharmacology of Bradykinin-Evoked Coughing in Guinea Pigs.

Bradykinin has been implicated as a mediator of the acute pathophysiological and inflammatory consequences of respiratory tract infections and in exacerbations of chronic diseases such as asthma. Bradykinin may also be a trigger for the coughing associated with these and other conditions. We have thus set out to evaluate the pharmacology of bradykinin-evoked coughing in guinea pigs. When inhaled, bradykinin induced paroxysmal coughing that was abolished by the bradykinin B2 receptor antagonist HOE 140. These cough responses rapidly desensitized, consistent with reports of B2 receptor desensitization. Bradykinin-evoked cough was potentiated by inhibition of both neutral endopeptidase and angiotensin-converting enzyme (with thiorphan and captopril, respectively), but was largely unaffected by muscarinic or thromboxane receptor blockade (atropine and ICI 192605), cyclooxygenase, or nitric oxide synthase inhibition (meclofenamic acid and N(G)-nitro-L-arginine). Calcium influx studies in bronchopulmonary vagal afferent neurons dissociated from vagal sensory ganglia indicated that the tachykinin-containing C-fibers arising from the jugular ganglia mediate bradykinin-evoked coughing. Also implicating the jugular C-fibers was the observation that simultaneous blockade of neurokinin2 (NK2; SR48968) and NK3 (SR142801 or SB223412) receptors nearly abolished the bradykinin-evoked cough responses. The data suggest that bradykinin induces coughing in guinea pigs by activating B2 receptors on bronchopulmonary C-fibers. We speculate that therapeutics targeting the actions of bradykinin may prove useful in the treatment of cough.

Modulation of Kinin B2 Receptor Signaling Controls Aortic Dilatation and Rupture in the Angiotensin II-Infused Apolipoprotein E-Deficient Mouse.

OBJECTIVE: Abdominal aortic aneurysm (AAA) is an important cause of mortality in older adults. Activity of the local kallikrein-kinin system may be important in cardiovascular disease. The effect of kinin B2 receptor (B2R) agonist and antagonist peptides on experimental AAA was investigated. APPROACH AND RESULTS: AAA was induced in apolipoprotein E-deficient mice via infusion of angiotensin II (1.0 µg/kg per minute SC). B2R agonists or antagonists were given via injection (2 mg/kg IP) every other day. The B2R agonist (B9772) promoted aortic rupture in response to angiotensin II associated with an increase in neutrophil infiltration of the aorta in comparison to controls. Mice receiving a B2R/kinin B1 receptor antagonist (B9430) were relatively protected from aortic rupture. Neutrophil depletion abrogated the ability of the B2R agonist to promote aortic rupture. Progression of angiotensin II-induced aortic dilatation was inhibited in mice receiving a B2R antagonist (B9330). Secretion of metalloproteinase-2 and -9, osteoprotegerin, and osteopontin by human AAA explant was reduced in the presence of the B2R antagonist (B9330). B2R agonist and antagonist peptides enhanced and inhibited, respectively, angiotensin II-induced neutrophil activation and aortic smooth muscle cell inflammatory phenotype. The B2R antagonist (B9330; 5 µg) delivered directly to the aortic wall 1 week post-AAA induction with calcium phosphate in a rat model reduced aneurysm growth associated with downregulation of aortic metalloproteinase-9. CONCLUSIONS: B2R signaling promotes aortic rupture within a mouse model associated with the ability to stimulate inflammatory phenotypes of neutrophils and vascular smooth muscle cells. B2R antagonism could be a potential therapy for AAA.

Mechanisms of bradykinin-induced expression of connective tissue growth factor and nephrin in podocytes.

Diabetic nephropathy (DN) is the main cause of morbidity and mortality in diabetes and is characterized by mesangial matrix deposition and podocytopathy, including podocyte loss. The risk factors and mechanisms involved in the pathogenesis of DN are still not completely defined. In the present study, we aimed to understand the cellular mechanisms through which activation of B2 kinin receptors contribute to the initiation and progression of DN. Stimulation of cultured rat podocytes with bradykinin (BK) resulted in a significant increase in ROS generation, and this was associated with a significant increase in NADPH oxidase (NOX)1 and NOX4 protein and mRNA levels. BK stimulation also resulted in a signicant increase in the phosphorylation of ERK1/2 and Akt, and this effect was inhibited in the presence of NOX1 and Nox4 small interfering (si)RNA. Furthermore, podocytes stimulated with BK resulted in a significant increase in protein and mRNA levels of connective tissue growth factor (CTGF) and, at the same time, a significant decrease in protein and mRNA levels of nephrin. siRNA targeted against NOX1 and NOX4 significantly inhibited the BK-induced increase in CTGF. Nephrin expression was increased in response to BK in the presence of NOX1 and NOX4 siRNA, thus implicating a role for NOXs in modulating the BK response in podocytes. Moreover, nephrin expression in response to BK was also significantly increased in the presence of siRNA targeted against CTGF. These findings provide novel aspects of BK signal transduction pathways in pathogenesis of DN and identify novel targets for interventional strategies.

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.

Enhanced Ca(2+) response and stimulation of prostaglandin release by the bradykinin B2 receptor in human retinal pigment epithelial cells primed with proinflammatory cytokines.

Kallikrein, kininogen and kinin receptors are present in human ocular tissues including the retinal pigment epithelium (RPE), suggesting a possible role of bradykinin (BK) in physiological and/or pathological conditions. To test this hypothesis, kinin receptors expression and function was investigated for the first time in human fetal RPE cells, a model close to native RPE, in both control conditions and after treatment with proinflammatory cytokines. Results showed that BK evoked intracellular Ca(2+) transients in human RPE cells by activating the kinin B2 receptor. Pretreatment of the cells with TNF-α and/or IL-1ß enhanced Ca(2+) response in a time- and concentration-dependent additive manner, whereas the potency of BK and that of the selective B2 receptor antagonist, fasitibant chloride, both in the nanomolar range, remained unaffected. Cytokines have no significant effect on cell number and viability and on the activity of other GPCRs such as the kinin B1, acetylcholine, ATP and thrombin receptors. Immunoblot analysis and immunofluorescence studies revealed that cytokines treatment was associated with an increase in both kinin B2 receptor and COX-2 expression and with the secretion of prostaglandin E1 and E2 into the extracellular medium. BK, through activation of the kinin B2 receptor, potentiated the COX-2 mediated prostaglandin release in cytokines-primed RPE cells while new protein synthesis and prostaglandin production contribute to the potentiating effect of cytokines on BK-induced Ca(2+) response. In conclusion, overall data revealed a cross-talk between the kinin B2 receptor and cytokines in human RPE in promoting inflammation, a key feature in retinal pathologies including diabetic retinopathy and macular edema.

The cough reflex is upregulated by lisinopril microinjected into the caudal nucleus tractus solitarii of the rabbit.

We have previously shown that cough potentiation induced by intravenous administration of the AT1 receptor antagonist losartan is lower than that induced by the ACE inhibitor lisinopril in anesthetized and awake rabbits. Since losartan and lisinopril cross the blood-brain barrier, their central action on the cough reflex can be hypothesized. Mechanical stimulation of the tracheobronchial tree and citric acid inhalation were used to induce cough reflex responses in pentobarbital sodium-anesthetized, spontaneously breathing rabbits. Bilateral microinjections (30-50 nl) of losartan (5mM), lisinopril (1mM), bradykinin (0.05 mM), HOE-140 (0.2mM, a bradykinin B2 receptor antagonist) and CP-99,994 (1mM, an NK1 receptor antagonist) were performed into the caudal nucleus tractus solitarii, the predominant site of termination of cough-related afferents. Lisinopril, but not losartan increased the cough number. This effect was reverted by HOE-140 or CP-99,994. Cough potentiation was also induced by bradykinin. The results support for the first time a central protussive action of lisinopril mediated by an accumulation of bradykinin and substance P.

Human Urinary Kallidinogenase Promotes Angiogenesis and Cerebral Perfusion in Experimental Stroke.

Angiogenesisis a key restorative mechanism in response to ischemia, and pro-angiogenic therapy could be beneficial in stroke. Accumulating experimental and clinical evidence suggest that human urinary kallidinogenase (HUK) improves stroke outcome, but the underlying mechanisms are not clear. The aim of current study was to verify roles of HUK in post-ischemic angiogenesis and identify relevant mediators. In rat middle cerebral artery occlusion (MCAO) model, we confirmed that HUK treatment could improve stroke outcome, indicated by reduced infarct size and improved neurological function. Notably, the 18F-FDG micro-PET scan indicated that HUK enhanced cerebral perfusion in rats after MCAO treatment. In addition, HUK promotespost-ischemic angiogenesis, with increased vessel density as well as up-regulated VEGF andapelin/APJ expression in HUK-treated MCAO mice. In endothelial cell cultures, induction of VEGF and apelin/APJ expression, and ERK1/2 phosphorylation by HUK was further confirmed. These changes were abrogated by U0126, a selective ERK1/2 inhibitor. Moreover, F13A, a competitive antagonist of APJ receptor, significantly suppressed HUK-induced VEGF expression. Furthermore, angiogenic functions of HUK were inhibited in the presence of selective bradykinin B1 or B2 receptor antagonist both in vitro and in vivo. Our findings indicate that HUK treatment promotes post-ischemic angiogenesis and cerebral perfusion via activation of bradykinin B1 and B2 receptors, which is potentially due to enhancement expression of VEGF and apelin/APJ in ERK1/2 dependent way.