Bradykinin protects nucleus pulposus cells from tert-butyl hydroperoxide-induced damage and delays intervertebral disc degeneration.

Intervertebral disc degeneration (IVDD) is a leading cause of degenerative spinal disorders, involving complex biological processes. This study investigates the role of the kallikrein-kinin system (KKS) in IVDD, focusing on the protective effects of bradykinin (BK) on nucleus pulposus cells (NPCs) under oxidative stress. Clinical specimens were collected, and experiments were conducted using human and rat primary NPCs to elucidate BK's impact on tert-butyl hydroperoxide (TBHP)-induced oxidative stress and damage. The results demonstrate that BK significantly inhibits TBHP-induced NPC apoptosis and restores mitochondrial function. Further analysis reveals that this protective effect is mediated through the BK receptor 2 (B2R) and its downstream PI3K/AKT pathway. Additionally, BK/PLGA sustained-release microspheres were developed and validated in a rat model, highlighting their potential therapeutic efficacy for IVDD. Overall, this study sheds light on the crucial role of the KKS in IVDD pathogenesis and suggests targeting the B2R as a promising therapeutic strategy to delay IVDD progression and promote disc regeneration.

Role of kinin receptors in skin pigmentation.

Previous studies have shown that all kinin system is constitutively expressed in the normal and inflamed skin, with a potential role in both physiological and pathological processes. However, the understanding regarding the involvement of the kinin system in skin pigmentation and pigmentation disorders remains incomplete. In this context, the present study was designed to determine the role of kinins in the Monobenzone (MBZ)-induced vitiligo-like model. Our findings showed that MBZ induces higher local skin depigmentation in kinin receptors knockout mice (KOB1R, KOB2R and KOB1B2R) than in wild type (WT). Remarkably, lower levels of melanin content and reduced ROS generation were detected in KOB1R and KOB2R mice treated with MBZ. In addition, both KOB1R and KOB2R show increased dermal cell infiltrate in vitiligo-like skin, when compared to WT-MBZ. Additionally, lack of B1R was associated with greater skin accumulation of IL-4, IL-6, and IL-17 by MBZ, while KOB1B2R presented lower levels of TNF and IL-1. Of note, the absence of both kinin B1 and B2 receptors demonstrates a protective effect by preventing the increase in polymorphonuclear and mononuclear cell infiltrations, as well as inflammatory cytokine levels induced by MBZ. In addition, in vitro assays confirm that B1R and B2R agonists increase intracellular melanin synthesis, while bradykinin significantly enhanced extracellular melanin levels and proliferation of B16F10 cells. Our findings highlight that the lack of kinin receptors caused more severe depigmentation in the skin, as well as genetic deletion of both B1/B2 receptors seems to be linked with changes in levels of constitutive melanin levels, suggesting the involvement of kinin system in crucial skin pigmentation pathways.

Effects of Bradykinin B2 Receptor Ablation from Tyrosine Hydroxylase Cells on Behavioral and Motor Aspects in Male and Female Mice.

The kallikrein-kinin system is a versatile regulatory network implicated in various biological processes encompassing inflammation, nociception, blood pressure control, and central nervous system functions. Its physiological impact is mediated through G-protein-coupled transmembrane receptors, specifically the B1 and B2 receptors. Dopamine, a key catecholamine neurotransmitter widely distributed in the CNS, plays a crucial role in diverse physiological functions including motricity, reward, anxiety, fear, feeding, sleep, and arousal. Notably, the potential physical interaction between bradykinin and dopaminergic receptors has been previously documented. In this study, we aimed to explore whether B2R modulation in catecholaminergic neurons influences the dopaminergic pathway, impacting behavioral, metabolic, and motor aspects in both male and female mice. B2R ablation in tyrosine hydroxylase cells reduced the body weight and lean mass without affecting body adiposity, substrate oxidation, locomotor activity, glucose tolerance, or insulin sensitivity in mice. Moreover, a B2R deficiency in TH cells did not alter anxiety levels, exercise performance, or motor coordination in female and male mice. The concentrations of monoamines and their metabolites in the substantia nigra and cortex region were not affected in knockout mice. In essence, B2R deletion in TH cells selectively influenced the body weight and composition, leaving the behavioral and motor aspects largely unaffected.

TRPV4 Activation and its Intracellular Modulation Mediated by Kinin Receptors Contribute to Painful Symptoms Induced by Anastrozole.

Anastrozole, an aromatase inhibitor, induces painful musculoskeletal symptoms, which affect patients' quality of life and lead to therapy discontinuation. Efforts have been made to understand the mechanisms involved in these painful symptoms to manage them better. In this context, we explored the role of the Transient Receptor Potential Vanilloid 4 (TRPV4), a potential transducer of several nociceptive mechanisms, in anastrozole-induced musculoskeletal pain in mice. Besides, we evaluated the possible sensibilization of TRPV4 by signalling pathways downstream, PLC, PKC and PKCε from kinin B2 (B2R) and B1 (B1R) receptors activation in anastrozole-induced pain. Anastrozole caused mechanical allodynia and muscle strength loss in mice. HC067047, TRPV4 antagonist, reduced the anastrozole-induced mechanical allodynia and muscle strength loss. In animals previously treated with anastrozole, the local administration of sub-nociceptive doses of the TRPV4 (4α-PDD or hypotonic solution), B2R (Bradykinin) or B1R (DABk) agonists enhanced the anastrozole-induced pain behaviours. The sensitizing effects induced by local injection of the TRPV4, B2R and B1R agonists in animals previously treated with anastrozole were reduced by pre-treatment with TRPV4 antagonist. Furthermore, inhibition of PLC, PKC or PKCε attenuated the mechanical allodynia and muscle strength loss induced by TRPV4, B2R and B1R agonists. The generation of painful conditions caused by anastrozole depends on direct TRPV4 activation or indirect, e.g., PLC, PKC and PKCε pathways downstream from B2R and B1R activation. Thus, the TRPV4 channels act as sensors of extracellular and intracellular changes, making them potential therapeutic targets for alleviating pain related to aromatase inhibitors use, such as anastrozole.

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.

Kinin B1 and B2 receptors mediate cancer pain associated with both the tumor and oncology therapy using aromatase inhibitors.

Pain caused by the tumor or aromatase inhibitors (AIs) is a disabling symptom in breast cancer survivors. Their mechanisms are unclear, but pro-algesic and inflammatory mediators seem to be involved. Kinins are endogenous algogenic mediators associated with various painful conditions via B1 and B2 receptor activation, including chemotherapy-induced pain and breast cancer proliferation. We investigate the involvement of the kinin B1 and B2 receptors in metastatic breast tumor (4T1 breast cancer cells)-caused pain and in aromatase inhibitors (anastrozole or letrozole) therapy-associated pain. A protocol associating the tumor and antineoplastic therapy was also performed. Kinin receptors' role was investigated via pharmacological antagonism, receptors protein expression, and kinin levels. Mechanical and cold allodynia and muscle strength were evaluated. AIs and breast tumor increased kinin receptors expression, and tumor also increased kinin levels. AIs caused mechanical allodynia and reduced the muscle strength of mice. Kinin B1 (DALBk) and B2 (Icatibant) receptor antagonists attenuated these effects and reduced breast tumor-induced mechanical and cold allodynia. AIs or paclitaxel enhanced breast tumor-induced mechanical hypersensitivity, while DALBk and Icatibant prevented this increase. Antagonists did not interfere with paclitaxel's cytotoxic action in vitro. Thus, kinin B1 or B2 receptors can be a potential target for treating the pain caused by metastatic breast tumor and their antineoplastic therapy.

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.

An overview of kinin mediated events in cancer progression and therapeutic applications.

Kinins are bioactive peptides generated in the inflammatory milieu of the tissue microenvironment, which is involved in cancer progression and inflammatory response. Kinins signals through activation of two G-protein coupled receptors; inducible Bradykinin Receptor B1 (B1R) and constitutive receptor B2 (B2R). Activation of kinin receptors and its cross-talk with receptor tyrosine kinases activates multiple signaling pathways, including ERK/MAPK, PI3K, PKC, and p38 pathways regulating cancer hallmarks. Perturbations of the kinin-mediated events are implicated in various aspects of cancer invasion, matrix remodeling, and metastasis. In the tumor microenvironment, kinins initiate fibroblast activation, mesenchymal stem cell interactions, and recruitment of immune cells. Albeit the precise nature of kinin function in the metastasis and tumor microenvironment are not completely clear yet, several kinin receptor antagonists show anti-metastatic potential. Here, we showcase an overview of the complex biology of kinins and their role in cancer pathogenesis and therapeutic aspects.

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.

Early reduction of SARS-CoV-2-replication in bronchial epithelium by kinin B2 receptor antagonism.

SARS-CoV-2 has evolved to enter the host via the ACE2 receptor which is part of the kinin-kallikrein pathway. This complex pathway is only poorly understood in context of immune regulation but critical to control infection. This study examines SARS-CoV-2-infection and epithelial mechanisms of the kinin-kallikrein-system at the kinin B2 receptor level in SARS-CoV-2-infection that is of direct translational relevance. From acute SARS-CoV-2-positive study participants and -negative controls, transcriptomes of nasal curettages were analyzed. Primary airway epithelial cells (NHBEs) were infected with SARS-CoV-2 and treated with the approved B2R-antagonist icatibant. SARS-CoV-2 RNA RT-qPCR, cytotoxicity assays, plaque assays, and transcriptome analyses were performed. The treatment effect was further studied in a murine airway inflammation model in vivo. Here, we report a broad and strong upregulation of kallikreins and the kinin B2 receptor (B2R) in the nasal mucosa of acutely symptomatic SARS-CoV-2-positive study participants. A B2R-antagonist impeded SARS-CoV-2 replication and spread in NHBEs, as determined in plaque assays on Vero-E6 cells. B2R-antagonism reduced the expression of SARS-CoV-2 entry receptor ACE2, G protein-coupled receptor signaling, and ion transport in vitro and in a murine airway inflammation in vivo model. In summary, this study provides evidence that treatment with B2R-antagonists protects airway epithelial cells from SARS-CoV-2 by inhibiting its replication and spread, through the reduction of ACE2 levels and the interference with several cellular signaling processes. Future clinical studies need to shed light on the airway protection potential of approved B2R-antagonists, like icatibant, in the treatment of early-stage COVID-19. KEY MESSAGES: Induction of kinin B2 receptor in the nose of SARS-CoV-2-positive patients. Treatment with B2R-antagonist protects airway epithelial cells from SARS-CoV-2. B2R-antagonist reduces ACE2 levels in vivo and ex vivo. Protection by B2R-antagonist is mediated by inhibiting viral replication and spread.

Cryo-EM structures of human bradykinin receptor-Gq proteins complexes.

The type 2 bradykinin receptor (B2R) is a G protein-coupled receptor (GPCR) in the cardiovascular system, and the dysfunction of B2R leads to inflammation, hereditary angioedema, and pain. Bradykinin and kallidin are both endogenous peptide agonists of B2R, acting as vasodilators to protect the cardiovascular system. Here we determine two cryo-electron microscopy (cryo-EM) structures of human B2R-Gq in complex with bradykinin and kallidin at 3.0 Å and 2.9 Å resolution, respectively. The ligand-binding pocket accommodates S-shaped peptides, with aspartic acids and glutamates as an anion trap. The phenylalanines at the tail of the peptides induce significant conformational changes in the toggle switch W2836.48, the conserved PIF, DRY, and NPxxY motifs, for the B2R activation. This further induces the extensive interactions of the intracellular loops ICL2/3 and helix 8 with Gq proteins. Our structures elucidate the molecular mechanisms for the ligand binding, receptor activation, and Gq proteins coupling of B2R.

Tissue Kallikrein Protects Rat Prostate against the Inflammatory Damage in a Chronic Autoimmune Prostatitis Model via Restoring Endothelial Function in a Bradykinin Receptor B2-Dependent Way.

OBJECTIVE: The aim of this study was to investigate whether tissue kallikrein (KLK1) can protect the prostate from inflammatory damage and the mechanism involved in it. METHODS: A total of 50 male Wistar rats were used in this study. Initially, 20 rats were sacrificed to obtain the prostate antigen to induce experimental autoimmune prostatitis (EAP), and the remaining 30 rats were randomly divided into 5 experimental groups (normal control group (NC group), NC+KLK1 group (NCK group), EAP group, EAP+KLK1 group (EAPK group), and EAP+KLK1+HOE140 group (EAPKH group); n = 6). It should be explained that KLK1 mainly exerts its biological effects through bradykinin, and HOE140 is a potent and selective bradykinin receptor B2 (BDKRB2) antagonist. EAP was induced by intradermal injection of 15 mg/ml prostate antigen and complete Freund's adjuvant on days 0, 14, and 28. KLK1 was injected via tail vein at a dose of 1.5 × 10-3 PAN U/kg once a day, and HOE140 was administered by intraperitoneal injection at 20 µg/kg once every two days. Rats were sacrificed on day 42. The RNA and protein of the rat prostate were extracted to analyze the expression differences of KLK1, as well as the inflammation-, fibrosis-, and oxidative stress-related genes. The inflammatory cell infiltration and microvessel density of the prostate were also analyzed by pathological examination. In addition, pathological analysis was performed on prostate samples from patients undergoing benign prostate hyperplasia (BPH) surgery. RESULTS: The expression of KLK1 in the prostate decreased in the EAP group as well as BPH patients with obvious inflammation. KLK1 administration significantly inhibited inflammatory cell infiltration and reduced the production of inflammatory cytokines in the EAPK group. Prostate samples from the EAP group showed increased infiltration of T cells and macrophages, as well as gland atrophy, hypoxia, fibrosis, and angiogenesis. KLK1 administration upregulated endothelial nitric oxide synthase (eNOS) expression and suppressed oxidative stress, as well as transforming growth factor ß1 (TGF-ß) signaling pathways and the proangiogenic vascular endothelial growth factor (VEGF) in the EAPK group. However, in the EAPKH group in which HOE140 blocked BDKRB2, the beneficial effects of KLK1 were all cancelled. In addition, KLK1 intervention in normal rats had no obvious side effects. CONCLUSION: The KLK1 expression is inhibited in the inflamed prostates of humans and rats. Exogenous KLK1 restored endothelial function via a BDKRB2-dependent way and then played a role in improving microcirculation and exerted anti-inflammatory, antifibrotic, and antioxidative stress effects in the rat chronic-inflamed prostate.

In vitro pharmacological profile of PHA-022121, a small molecule bradykinin B2 receptor antagonist in clinical development.

PHA-022121 is a novel small molecule bradykinin B2 receptor antagonist, in clinical development for the treatment and prevention of hereditary angioedema attacks. The present study describes the in vitro pharmacological characteristics of PHA-022121 and its active metabolite, PHA-022484 (M2-D). In mammalian cell lines, PHA-022121 and PHA-022484 show high affinity for the recombinant human bradykinin B2 receptor with Ki values of 0.47 and 0.70 nM, respectively, and potent antagonism of the human bradykinin B2 receptor with Kb values of 0.15 and 0.26 nM, respectively (calcium mobilization assay). Antagonist potency at the recombinant cynomolgus monkey bradykinin B2 receptor is similarly high (Kb values of 1.42 and 1.12 nM for PHA-022121 and PHA-022484, respectively), however, potency at rat, mouse, rabbit and dog bradykinin B2 receptors is at least 100-fold lower than the potency at the human receptor for both compounds. In the human umbilical vein contractility assay, both PHA-022121 and PHA-022484 show a potent, surmountable and reversible B2 antagonist activity with pA2 values of 0.35 and 0.47 nM, respectively. The in vitro off-target profile of PHA-022121 and PHA-022484 demonstrates a high degree of selectivity over a wide range of molecular targets, including the bradykinin B1 receptor. It is concluded that PHA-022121 is a novel, low-molecular weight, competitive antagonist of the human bradykinin B2 receptor with high affinity, high antagonist potency, and high selectivity. It is about 20-fold more potent than icatibant at the human bradykinin B2 receptor as assessed using recombinant or endogenously expressed receptors.

Pathological AT1R-B2R Protein Aggregation and Preeclampsia.

Preeclampsia is one of the most frequent and severe complications of pregnancy. Symptoms of preeclampsia usually occur after 20 weeks of pregnancy and include hypertension and kidney dysfunction with proteinuria. Up to now, delivery of the infant has been the most effective and life-saving treatment to alleviate symptoms of preeclampsia because a causative treatment does not exist, which could prolong a pregnancy complicated with preeclampsia. Preeclampsia is a complex medical condition, which is attributed to a variety of different risk factors and causes. Risk factors account for insufficient placentation and impaired vasculogenesis and finally culminate in this life-threatening condition of pregnancy. Despite progress, many pathomechanisms and causes of preeclampsia are still incompletely understood. In recent years, it was found that excessive protein complex formation between G-protein-coupled receptors is a common sign of preeclampsia. Specifically, the aberrant heteromerization of two vasoactive G-protein-coupled receptors (GPCRs), the angiotensin II AT1 receptor and the bradykinin B2 receptor, is a causative factor of preeclampsia symptoms. Based on this knowledge, inhibition of abnormal GPCR protein complex formation is an experimental treatment approach of preeclampsia. This review summarizes the impact of pathological GPCR protein aggregation on symptoms of preeclampsia and delineates potential new therapeutic targets.

Role of nitric oxide, bradykinin B2 receptor, and TRPV1 in the airway alterations caused by simvastatin in rats.

Dry cough has been reported in patients receiving statin therapy. However, the underlying mechanism or other possible alterations in the airways induced by statins remain unknown. Thus, the aim of this study was to evaluate whether simvastatin promotes alterations in airways, such as bronchoconstriction and plasma extravasation, as well as the mechanism involved in these events. Using methods to detect alterations in airway resistance and plasma extravasation, we demonstrated that simvastatin [20 mg/kg, intravenous (i.v.)] caused plasma extravasation in the trachea (79.8 + 14.8 µg/g/tissue) and bronchi (73.3 + 8.8 µg/g/tissue) of rats, compared to the vehicle (34.2 + 3.6 µg/g/tissue and 29.3 + 5.3 µg/g/tissue, respectively). NG-nitro-L-arginine methyl ester (L-NAME, 30 mg/kg, intraperitoneal), a nitric oxide (NO) synthase inhibitor, Icatibant [HOE 140, 10 nmol/50 µl, intratracheal (i.t.)], a bradykinin B2 antagonist, and capsazepine (100 nmol/50 µl, i.t.), a TRPV1 antagonist, attenuated simvastatin-induced plasma extravasation. Simvastatin (5, 10 and 20 mg/kg) did not cause bronchoconstriction per se, but exacerbated the bronchoconstrictive response to bradykinin (30 nmol/kg, i.v.), a B2 agonist (0.7 + 0.1 ml/H2O), or capsaicin (30 nmol/kg, i.v.), a TRPV1 agonist (0.8 + 0.1 ml/H2O), compared to the vehicle (0.1 + 0.04 ml/H2O and 0.04 + 0.01 ml/H2O, respectively). The bronchoconstriction elicited by bradykinin (100 nmol/kg, i.v.) in simvastatin non-treated rats was inhibited by L-NAME. The exacerbation of bronchoconstriction induced by bradykinin or capsaicin in simvastatin-treated rats was inhibited by L-NAME, HOE 140 or capsazepine. These results suggest that treatment with simvastatin promotes the release of bradykinin, which, via B2 receptors, releases NO that can then activate the TRPV1 to promote plasma extravasation and bronchoconstriction.

Bradykinin Protects Human Endothelial Progenitor Cells from High-Glucose-Induced Senescence through B2 Receptor-Mediated Activation of the Akt/eNOS Signalling Pathway.

BACKGROUND: Circulating endothelial progenitor cells (EPCs) play important roles in vascular repair. However, the mechanisms of high-glucose- (HG-) induced cord blood EPC senescence and the role of B2 receptor (B2R) remain unknown. METHODS: Cord blood samples from 26 patients with gestational diabetes mellitus (GDM) and samples from 26 healthy controls were collected. B2R expression on circulating CD34+ cells of cord blood mononuclear cells (CBMCs) was detected using flow cytometry. The plasma concentrations of 8-isoprostaglandin F2α (8-iso-PGF2α) and nitric oxide (NO) were measured. EPCs were treated with HG (40 mM) alone or with bradykinin (BK) (1 nM). The B2R and eNOS small interfering RNAs (siRNAs) and the PI3K antagonist LY294002 were added to block B2R, eNOS, and PI3K separately. To determine the number of senescent cells, senescence-associated ß-galactosidase (SA-ß-gal) staining was performed. The level of mitochondrial reactive oxygen species (ROS) in EPCs was assessed by Mito-Sox staining. Cell viability was evaluated by Cell Counting Kit-8 (CCK-8) assays. Mitochondrial DNA (mtDNA) copy number and the relative length of telomeres were detected by real time-PCR. The distribution of human telomerase reverse transcriptase (hTERT) in the nucleus, cytosol, and mitochondria of EPCs was detected by immunofluorescence. The expression of B2R, p16, p21, p53, P-Ser473AKT, T-AKT, eNOS, and hTERT was demonstrated by Western blot. RESULTS: B2R expression on circulating CD34+ cells of CBMCs was significantly reduced in patients with GDM compared to healthy controls. Furthermore, B2R expression on circulating CD34+ cells of CBMCs was inversely correlated with plasma 8-iso-PGF2α concentrations and positively correlated with plasma NO levels. BK treatment decreased EPC senescence and ROS generation. Furthermore, BK treatment of HG-exposed cells led to elevated P-Ser473AKT and eNOS protein expression compared with HG treatment alone. BK reduced hTERT translocation in HG-induced senescent EPCs. B2R siRNA, eNOS siRNA, and antagonist of the PI3K signalling pathway blocked the protective effects of BK. CONCLUSION: BK, acting through PI3K-AKT-eNOS signalling pathways, reduced hTERT translocation, increased the relative length of telomeres while reducing mtDNA copy number, and finally protected against EPC senescence induced by HG.

Human urinary kininogenase reduces the endothelial injury by inhibiting Pyk2/MCU pathway.

The injury of endothelial cells is one of the initiating factors in restenosis after endovascular treatment. Human urinary kallidinogenase (HUK) is a tissue kallikrein which is used for ischemia-reperfusion injury treatment. Studies have shown that HUK may be a potential therapeutic agent to prevent stenosis after vascular injury, however, the precise mechanisms have not been fully established. This study is to investigate whether HUK can protect endothelial cells after balloon injury or H2O2-induced endothelial cell damage through the proline-rich tyrosine kinase 2 (Pyk2)/mitochondrial calcium uniporter (MCU) pathway. Intimal hyperplasia, a decrease of pinocytotic vesicles and cell apoptosis were found in the common carotid artery balloon injury and H2O2-induced endothelial cell damage, Pyk2/MCU was also up-regulated in such pathological process. HUK could prevent these injuries partially via the bradykinin B2 receptor by inhibiting Pyk2/MCU pathway, which prevented the mitochondrial damage, maintained calcium balance, and eventually inhibited cell apoptosis. Furthermore, MCU expression was not markedly increased if Pyk2 was suppressed by shRNA technique in the H2O2 treatment group, and cell viability was significantly better than H2O2-treated only. In short, our results indicate that the Pyk2/MCU pathway is involved in endothelial injury induced by balloon injury or H2O2-induced endothelial cell damage. HUK plays an protective role by inhibiting the Pyk2/MCU pathway in the endothelial injury.

Surface megalin expression is a target to the inhibitory effect of bradykinin on the renal albumin endocytosis.

Megalin-mediated albumin endocytosis plays a critical role in albumin reabsorption in proximal tubule (PT) epithelial cells (PTECs). Some studies have pointed out the modulatory effect of bradykinin (BK) on urinary protein excretion, but its role in PT protein endocytosis has not yet been determined. Here, we studied the possible correlation between BK and albumin endocytosis in PT. Using LLC-PK1 cells, a model of PTECs, we showed that BK specifically inhibited megalin-mediated albumin endocytosis. This inhibitory effect of BK was mediated by B2 receptor (B2R) because it was abolished by HOE140, an antagonist of B2R, but it was not affected by Lys-des-Arg9-BK, an antagonist of B1. BK induced the stall of megalin in EEA1+ endosomes, but not in LAMP1+ lysosomes, leading to a decrease in surface megalin expression. In addition, we showed that BK, through B2R, activated calphostin C-sensitive protein kinase C, which mediated its effect on the surface megalin expression and albumin endocytosis. These results reveal an important modulatory mechanism of PT albumin endocytosis by BK, which opens new possibilities to understanding the effect of BK on urinary albumin excretion.

Kinins and Their Receptors as Potential Therapeutic Targets in Retinal Pathologies.

The kallikrein-kinin system (KKS) contributes to retinal inflammation and neovascularization, notably in diabetic retinopathy (DR) and neovascular age-related macular degeneration (AMD). Bradykinin type 1 (B1R) and type 2 (B2R) receptors are G-protein-coupled receptors that sense and mediate the effects of kinins. While B2R is constitutively expressed and regulates a plethora of physiological processes, B1R is almost undetectable under physiological conditions and contributes to pathological inflammation. Several KKS components (kininogens, tissue and plasma kallikreins, and kinin receptors) are overexpressed in human and animal models of retinal diseases, and their inhibition, particularly B1R, reduces inflammation and pathological neovascularization. In this review, we provide an overview of the KKS with emphasis on kinin receptors in the healthy retina and their detrimental roles in DR and AMD. We highlight the crosstalk between the KKS and the renin-angiotensin system (RAS), which is known to be detrimental in ocular pathologies. Targeting the KKS, particularly the B1R, is a promising therapy in retinal diseases, and B1R may represent an effector of the detrimental effects of RAS (Ang II-AT1R).