Electroacupuncture alleviates diabetes-induced mechanical allodynia and downregulates bradykinin B1 receptor expression in spinal cord dorsal horn.

OBJECTIVE: Diabetic neuropathic pain (DNP) is one of the most prevalent symptoms of diabetes. The alteration of proteins in the spinal cord dorsal horn (SCDH) plays a significant role in the genesis and the development of DNP. Our previous study has shown electroacupuncture could effectively relieve DNP. However, the potential mechanism inducing DNP's genesis and development remains unclear and needs further research. METHODS: This study established DNP model rats by intraperitoneally injecting a single high-dose streptozotocin; 2 Hz electroacupuncture was used to stimulate Zusanli (ST36) and Kunlun (BL60) of DNP rats daily from day 15 to day 21 after streptozotocin injection. Behavioral assay, quantitative PCR, immunofluorescence staining, and western blotting were used to study the analgesic mechanism of electroacupuncture. RESULTS: The bradykinin B1 receptor (B1R) mRNA, nuclear factor-κB p65 (p65), substance P, and calcitonin gene-related peptide (CGRP) protein expression were significantly enhanced in SCDH of DNP rats. The paw withdrawal threshold was increased while body weight and fasting blood glucose did not change in DNP rats after the electroacupuncture treatment. The expression of B1R, p65, substance P, and CGRP in SCDH of DNP rats was also inhibited after the electroacupuncture treatment. CONCLUSION: This work suggests that the potential mechanisms inducing the allodynia of DNP rats were possibly related to the increased expression of B1R, p65, substance P, and CGRP in SCDH. Downregulating B1R, p65, substance P, and CGRP expression levels in SCDH may achieve the analgesic effect of 2 Hz electroacupuncture treatment.

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.

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.

Soluble CD13 induces inflammatory arthritis by activating the bradykinin receptor B1.

CD13, an ectoenzyme on myeloid and stromal cells, also circulates as a shed, soluble protein (sCD13) with powerful chemoattractant, angiogenic, and arthritogenic properties, which require engagement of a G protein-coupled receptor (GPCR). Here we identify the GPCR that mediates sCD13 arthritogenic actions as the bradykinin receptor B1 (B1R). Immunofluorescence and immunoblotting verified high expression of B1R in rheumatoid arthritis (RA) synovial tissue and fibroblast-like synoviocytes (FLSs), and demonstrated binding of sCD13 to B1R. Chemotaxis, and phosphorylation of Erk1/2, induced by sCD13, were inhibited by B1R antagonists. In ex vivo RA synovial tissue organ cultures, a B1R antagonist reduced secretion of inflammatory cytokines. Several mouse arthritis models, including serum transfer, antigen-induced, and local innate immune stimulation arthritis models, were attenuated in Cd13-/- and B1R-/- mice and were alleviated by B1R antagonism. These results establish a CD13/B1R axis in the pathogenesis of inflammatory arthritis and identify B1R as a compelling therapeutic target in RA and potentially other inflammatory diseases.

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.

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).

Myocardial angiogenesis induced by exercise training involves a regulatory mechanism mediated by kinin receptors.

OBJECTIVE: To demonstrate that the kallikrein-kinin system (KKS) is upstream of angiogenic signaling pathway, and to determine the role of the kinin B1 and B2 receptors in myocardial angiogenesis induced by exercise training. METHODS: Forty Wistar rats were randomly assigned to an exercise control (EC) group, a B1 receptor antagonist (B1Ant) group, a B2 receptor antagonist (B2Ant) group, and a double receptor antagonist ((B1+ B2)Ant) group. A myocardial infarction model was employed. Animals in all groups received 30 min of exercise training for 4 weeks. The expression of VEGF and eNOS, capillary supply, and apoptosis rate were evaluated. RESULTS: The mRNA and protein expression of VEGF and eNOS showed similar trends in all groups, and were lowest in the (B1+ B2) Ant group, and highest in the EC group. Levels of VEGF and eNOS mRNA were significantly lower in the B1Ant group than in the B2Ant group (p< .001 and p< .05, respectively). VEGF and eNOS protein in the B1Ant group was also significantly lower (p< .01 and p< .05, respectively) than in the B2Ant group. The capillary numbers in the (B1+ B2) Ant group were significantly lower than in the EC group (395.8 ± 105 vs. 1127.9 ± 192.98, respectively). The apoptosis rate of cardiomyocytes was highest in the (B1+ B2) Ant group. CONCLUSION: KKS may act as an upstream signal transduction pathway for angiogenic factors in myocardial angiogenesis. The B1 and B2 receptors exert additive effects, and the B1 receptor has the most prominent role in mediating KKS-induced myocardial angiogenesis.

Binding Mode Exploration of B1 Receptor Antagonists' by the Use of Molecular Dynamics and Docking Simulation-How Different Target Engagement Can Determine Different Biological Effects.

The kinin B1 receptor plays a critical role in the chronic phase of pain and inflammation. The development of B1 antagonists peaked in recent years but almost all promising molecules failed in clinical trials. Little is known about these molecules' mechanisms of action and additional information will be necessary to exploit the potential of the B1 receptor. With the aim of contributing to the available knowledge of the pharmacology of B1 receptors, we designed and characterized a novel class of allosteric non-peptidic inhibitors with peculiar binding characteristics. Here, we report the binding mode analysis and pharmacological characterization of a new allosteric B1 antagonist, DFL20656. We analyzed the binding of DFL20656 by single point mutagenesis and radioligand binding assays and we further characterized its pharmacology in terms of IC50, B1 receptor internalization and in vivo activity in comparison with different known B1 antagonists. We highlighted how different binding modes of DFL20656 and a Merck compound (compound 14) within the same molecular pocket can affect the biological and pharmacological properties of B1 inhibitors. DFL20656, by its peculiar binding mode, involving tight interactions with N114, efficiently induced B1 receptor internalization and evoked a long-lasting effect in an in vivo model of neuropathic pain. The pharmacological characterization of different B1 antagonists highlighted the effects of their binding modes on activity, receptor occupancy and internalization. Our results suggest that part of the failure of most B1 inhibitors could be ascribed to a lack of knowledge about target function and engagement.

Understanding the Pathophysiology of COVID-19: Could the Contact System Be the Key?

To date the pathophysiology of COVID-19 remains unclear: this represents a factor determining the current lack of effective treatments. In this paper, we hypothesized a complex host response to SARS-CoV-2, with the Contact System (CS) playing a pivotal role in innate immune response. CS is linked with different proteolytic defense systems operating in human vasculature: the Kallikrein-Kinin (KKS), the Coagulation/Fibrinolysis and the Renin-Angiotensin (RAS) Systems. We investigated the role of the mediators involved. CS consists of Factor XII (FXII) and plasma prekallikrein (complexed to high-molecular-weight kininogen-HK). Autoactivation of FXII by contact with SARS-CoV-2 could lead to activation of intrinsic coagulation, with fibrin formation (microthrombosis), and fibrinolysis, resulting in increased D-dimer levels. Activation of kallikrein by activated FXII leads to production of bradykinin (BK) from HK. BK binds to B2-receptors, mediating vascular permeability, vasodilation and edema. B1-receptors, binding the metabolite [des-Arg9]-BK (DABK), are up-regulated during infections and mediate lung inflammatory responses. BK could play a relevant role in COVID-19 as already described for other viral models. Angiotensin-Converting-Enzyme (ACE) 2 displays lung protective effects: it inactivates DABK and converts Angiotensin II (Ang II) into Angiotensin-(1-7) and Angiotensin I into Angiotensin-(1-9). SARS-CoV-2 binds to ACE2 for cell entry, downregulating it: an impaired DABK inactivation could lead to an enhanced activity of B1-receptors, and the accumulation of Ang II, through a negative feedback loop, may result in decreased ACE activity, with consequent increase of BK. Therapies targeting the CS, the KKS and action of BK could be effective for the treatment of COVID-19.

Kallikrein-kinin blockade in patients with COVID-19 to prevent acute respiratory distress syndrome.

COVID-19 patients can present with pulmonary edema early in disease. We propose that this is due to a local vascular problem because of activation of bradykinin 1 receptor (B1R) and B2R on endothelial cells in the lungs. SARS-CoV-2 enters the cell via ACE2 that next to its role in RAAS is needed to inactivate des-Arg9 bradykinin, the potent ligand of the B1R. Without ACE2 acting as a guardian to inactivate the ligands of B1R, the lung environment is prone for local vascular leakage leading to angioedema. Here, we hypothesize that a kinin-dependent local lung angioedema via B1R and eventually B2R is an important feature of COVID-19. We propose that blocking the B2R and inhibiting plasma kallikrein activity might have an ameliorating effect on early disease caused by COVID-19 and might prevent acute respiratory distress syndrome (ARDS). In addition, this pathway might indirectly be responsive to anti-inflammatory agents.

The COVID-19 pandemic represents an unprecedented threat to global health. Millions of cases have been confirmed around the world, and hundreds of thousands of people have lost their lives. Common symptoms include a fever and persistent cough and COVID-19 patients also often experience an excess of fluid in the lungs, which makes it difficult to breathe. In some cases, this develops into a life-threatening condition whereby the lungs cannot provide the body's vital organs with enough oxygen. The SARS-CoV-2 virus, which causes COVID-19, enters the lining of the lungs via an enzyme called the ACE2 receptor, which is present on the outer surface of the lungs' cells. The related coronavirus that was responsible for the SARS outbreak in the early 2000s also needs the ACE2 receptor to enter the cells of the lungs. In SARS, the levels of ACE2 in the lung decline during the infection. Studies with mice have previously revealed that a shortage of ACE2 leads to increased levels of a hormone called angiotensin II, which regulates blood pressure. As a result, much attention has turned to the potential link between this hormone system in relation to COVID-19. However, other mouse studies have shown that ACE2 protects against a build-up of fluid in the lungs caused by a different molecule made by the body. This molecule, which is actually a small fragment of a protein, lowers blood pressure and causes fluid to leak out of blood vessels. It belongs to a family of molecules known as kinins, and ACE2 is known to inactivate certain kinins. This led van de Veerdonk et al. to propose that the excess of fluid in the lungs seen in COVID-19 patients may be because kinins are not being neutralized due to the shortage of the ACE2 receptor. This had not been hypothesized before, even though the mechanism could be the same in SARS which has been researched for the past 17 years. If this hypothesis is correct, it would mean that directly inhibiting the receptor for the kinins (or the proteins that they come from) may be the only way to stop fluid leaking into the lungs of COVID-19 patients in the early stage of disease. This hypothesis is unproven, and more work is needed to see if it is clinically relevant. If that work provides a proof of concept, it means that existing treatments and registered drugs could potentially help patients with COVID-19, by preventing the need for mechanical ventilation and saving many lives.

Reciprocal Regulatory Interaction between TRPV1 and Kinin B1 Receptor in a Rat Neuropathic Pain Model.

Kinins are mediators of pain and inflammation and evidence suggests that the inducible kinin B1 receptor (B1R) is involved in neuropathic pain (NP). This study investigates whether B1R and TRPV1 are colocalized on nociceptors and/or astrocytes to enable regulatory interaction either directly or through the cytokine pathway (IL-1ß, TNF-α) in NP. Sprague Dawley rats were subjected to unilateral partial sciatic nerve ligation (PSNL) and treated from 14 to 21 days post-PSNL with antagonists of B1R (SSR240612, 10 mg·kg-1, i.p.) or TRPV1 (SB366791, 1 mg·kg-1, i.p.). The impact of these treatments was assessed on nociceptive behavior and mRNA expression of B1R, TRPV1, TNF-α, and IL-1ß. Localization on primary sensory fibers, astrocytes, and microglia was determined by immunofluorescence in the lumbar spinal cord and dorsal root ganglion (DRG). Both antagonists suppressed PSNL-induced thermal hyperalgesia, but only SB366791 blunted mechanical and cold allodynia. SSR240612 reversed PSNL-induced enhanced protein and mRNA expression of B1R and TRPV1 mRNA levels in spinal cord while SB366791 further increased B1R mRNA/protein expression. B1R and TRPV1 were found in non-peptide sensory fibers and astrocytes, and colocalized in the spinal dorsal horn and DRG, notably with IL-1ß on astrocytes. IL-1ß mRNA further increased under B1R or TRPV1 antagonism. Data suggest that B1R and TRPV1 contribute to thermal hyperalgesia and play a distinctive role in allodynia associated with NP. Close interaction and reciprocal regulatory mechanism are suggested between B1R and TRPV1 on astrocytes and nociceptors in NP.

Diabetes-Induced Inflammation and Vascular Alterations in the Goto-Kakizaki Rat Retina.

PURPOSE: Diabetic retinopathy is characterized by multiple microcirculatory dysfunctions and angiogenesis resulting from hyperglycemia, oxidative stress, and inflammation. In this study, the retina and retinal pigmented epithelium of non-insulin-dependent diabetic Goto-Kakizaki (GK) rats were examined to detect microvascular alterations, gliosis, macrophage infiltration, lipid deposits, and fibrosis. Emphasis was given to the distribution of kinin B1 receptor (B1R) and vascular endothelial growth factor (VEGF), two major factors in inflammation and angiogenesis. MATERIALS AND METHODS: 30-week-old male GK rats and age-matched Wistar rats were used. The retinal vascular bed was examined using ADPase staining. The level of lipid accumulation was graded using triglyceride staining with Oil red O. Macrophage and retinal microglia activation, as well as other markers, were revealed by immunohistochemistry and studied with confocal laser scanning microscopy. RESULTS: Abundant lipid deposits were observed in the Bruch's membrane of GK rats. Immunohistochemistry and quantitative analysis showed significantly higher B1R, VEGF, Iba1 (microglia), CD11 (macrophages), fibronectin, and collagen I labeling in the diabetic retina. B1R immunolabeling was detected in the vascular layers of the GK retina. A strong VEGF staining within different retinal cell processes was detected and a pattern of GFAP staining suggested strong Müller cells/astrocytes reactivity. Microgliosis was apparent in the GK retina. A greater tortuosity of the retinal microvessels (an index of endothelial dysfunction) and their increased number were also observed in GK retinas. CONCLUSIONS: Data suggest retinal vascular bed alterations in spontaneous type 2 diabetic retinas at 30 weeks. Lipid and collagen accumulation in the retina and choroid, in addition to retinal upregulation of VEGF and B1R, microgliosis, and Müller cell reactivity, may contribute to vascular alterations and inflammatory processes.

Bradykinin Receptor B1 and C-Reactive Protein as Prognostic Factors for Pharyngocutaneous Fistula Development After Laryngectomy.

Pharyngocutaneous fistulae (PCF) are one of the most common complications after laryngectomy. Predisposing risk factors have been studied, yet knowledge to determine which patients are prone to developing a fistula remains scarce. This study aims to establish prognostic parameters to identify individual patients at risk for PCF development. As PCF and inflammation seem to be interwoven, this work focuses on markers able to detect an inflammatory response. We retrospectively analyzed all patients who had undergone a laryngectomy at our clinic in the years 2007 to 2017 (n = 182). Immunohistochemical expression of bradykinin type 1 and 2 receptor and vascular endothelial growth factor receptor 2 was studied in all available tumor samples. Additionally, the clinical inflammation parameters 'body temperature', 'pain', 'c-reactive protein (CRP)', and 'leucocytes' were postoperatively tracked in all patients. The times between fistula diagnosis, therapeutic approach, and hospital discharge were recorded. We found a strong correlation between inflammation and the formation of a fistula. High bradykinin 1 receptor expression in the tumor samples correlated with postoperative PCF development. Persistently elevated CRP and leukocyte levels beyond the 6th postoperative day were also risk factors. A decreased time lapse between PCF diagnosis and surgical revision clearly correlated with a shorter hospital stay. In this study, we identified a bradykinin 1 receptor positive patient group at high risk for development of PCF. We recommend close monitoring for fistula formation in these patients to ensure timely intervention.

Kinin B1 Receptor Blockade Prevents Angiotensin II-induced Neuroinflammation and Oxidative Stress in Primary Hypothalamic Neurons.

Neuroinflammation has become an important underlying factor in many cardiovascular disorders, including hypertension. Previously we showed that elevated angiotensin II (Ang II) and angiotensin II type I receptor (AT1R) expression levels can increase neuroinflammation leading to hypertension. We also found that kinin B1 receptor (B1R) expression increased in the hypothalamic paraventricular neurons resulting in neuroinflammation and oxidative stress in neurogenic hypertension. However, whether there are any potential interactions between AT1R and B1R in neuroinflammation is not clear. In the present study, we aimed to determine whether Ang II-mediated effects on inflammation and oxidative stress are mediated by the activation of B1R in mouse neonatal primary hypothalamic neuronal cultures. Gene expression and immunostaining revealed that both B1R and AT1R are expressed on primary hypothalamic neurons. Ang II stimulation significantly increased the expression of B1R, decreased mitochondrial respiration, increased the expression of two NADPH oxidase subunits (Nox2 and Nox4), increased the oxidative potential, upregulated several proinflammatory genes (IL-1ß, IL-6, and TNFα), and increased NF-kB p65 DNA binding activity. These changes were prevented by pretreatment with the B1R-specific peptide antagonist, R715. In summary, our study demonstrates a causal relationship between B1R expression after Ang II stimulation, suggesting a possible cross talk between AT1R and B1R in neuroinflammation and oxidative stress.

Kallikrein-kinin system and oxidative stress in cisplatin-induced ovarian toxicity.

Kallikrein-kinin system (KKS) is involved in vascular reactivity and inflammatory response to cytotoxic drugs. Since cisplatin is a widely used chemotherapy and its cytotoxic mechanism can trigger inflammation and oxidative damage, in this work we evaluated the role of KKS in an animal model of cisplatin-induced ovarian toxicity. Biomarkers of ovarian stem cells, activity of KKS, inflammation and oxidative damage were measured in ovarian tissue of C57BL/6 female mice treated with vehicle or cisplatin (2.5 mg/kg). Cisplatin group presented greater number of atretic follicles, and lower numbers of antral and total viable follicles. Ki67, DDX4 and OCT-4 markers were similar between groups. Cisplatin triggered plasma and ovarian tissue kallikrein generation; and increased expression of bradykinin receptors B1 and B2. Neutrophil and macrophage infiltration markers increased. Superoxide anion generation also increased, while reduced glutathione levels decreased. These results suggest that KKS is activated and contributes to ovarian injury during cisplatin treatment.