Increased plasma bradykinin level is associated with cognitive impairment in Alzheimer's patients.

Alzheimer's disease (AD) is characterized by the presence of proteinaceous brain deposits, brain atrophy, vascular dysfunction, and chronic inflammation. Along with cerebral inflammation, peripheral inflammation is also evident in many AD patients. Bradykinin, a proinflammatory plasma peptide, is also linked to AD pathology. For example, bradykinin infusion into the hippocampus causes learning and memory deficits in rats, and blockade of the bradykinin receptor lessens cognitive impairment in AD mouse models. Even though it has been hypothesized that plasma bradykinin could contribute to inflammation in AD, the level of plasma bradykinin and its association with beta-amyloid (Aß) pathology in AD patients had not been explored. Here, we assessed plasma bradykinin levels in AD patients and age-matched non-demented (ND) control individuals. We found significantly elevated plasma bradykinin levels in AD patients compared to ND subjects. Additionally, changes in plasma bradykinin levels were more profound in many AD patients with severe cognitive impairment, suggesting that peripheral bradykinin could play a role in dementia most likely via inflammation. Bradykinin levels in the cerebrospinal fluid (CSF) were reduced in AD patients and exhibited an inverse correlation with the CSF Aß40/Aß42 ratio. We also report that bradykinin interacts with the fibrillar form of Aß and co-localizes with Aß plaques in the post-mortem human AD brain. These findings connect the peripheral inflammatory pathway to cerebral abnormalities and identify a novel mechanism of inflammatory pathology in AD.

Activation of bradykinin B2 receptor induced the inflammatory responses of cytosolic phospholipase A2 after the early traumatic brain injury.

Phospholipase A2 is a known aggravator of inflammation and deteriorates neurological outcomes after traumatic brain injury (TBI), however the exact inflammatory mechanisms remain unknown. This study investigated the role of bradykinin and its receptor, which are known initial mediators within inflammation activation, as well as the mechanisms of the cytosolic phospholipase A2 (cPLA2)-related inflammatory responses after TBI. We found that cPLA2 and bradykinin B2 receptor were upregulated after a TBI. Rats treated with the bradykinin B2 receptor inhibitor LF 16-0687 exhibited significantly less cPLA2 expression and related inflammatory responses in the brain cortex after sustaining a controlled cortical impact (CCI) injury. Both the cPLA2 inhibitor and the LF16-0687 improved CCI rat outcomes by decreasing neuron death and reducing brain edema. The following TBI model utilized both primary astrocytes and primary neurons in order to gain further understanding of the inflammation mechanisms of the B2 bradykinin receptor and the cPLA2 in the central nervous system. There was a stronger reaction from the astrocytes as well as a protective effect of LF16-0687 after the stretch injury and bradykinin treatment. The protein kinase C pathway was thought to be involved in the B2 bradykinin receptor as well as the cPLA2-related inflammatory responses. Rottlerin, a Protein Kinase C (PKC) δ inhibitor, decreased the activity of the cPLA2 activity post-injury, and LF16-0687 suppressed both the PKC pathway and the cPLA2 activity within the astrocytes. These results indicated that the bradykinin B2 receptor-mediated pathway is involved in the cPLA2-related inflammatory response from the PKC pathway.

Bradykinin in blood and cerebrospinal fluid after acute cerebral lesions: correlations with cerebral edema and intracranial pressure.

Bradykinin (BK) was shown to stimulate the production of physiologically active metabolites, blood-brain barrier disruption, and brain edema. The aim of this prospective study was to measure BK concentrations in blood and cerebrospinal fluid (CSF) of patients with traumatic brain injury (TBI), subarachnoid hemorrhage (SAH), intracerebral hemorrhage (ICH), and ischemic stroke and to correlate BK levels with the extent of cerebral edema and intracranial pressure (ICP). Blood and CSF samples of 29 patients suffering from acute cerebral lesions (TBI, 7; SAH,: 10; ICH, 8; ischemic stroke, 4) were collected for up to 8 days after insult. Seven patients with lumbar drainage were used as controls. Edema (5-point scale), ICP, and the GCS (Glasgow Coma Score) at the time of sample withdrawal were correlated with BK concentrations. Though all plasma-BK samples were not significantly elevated, CSF-BK levels of all patients were significantly elevated in overall (n=73) and early (≤72 h) measurements (n=55; 4.3±6.9 and 5.6±8.9 fmol/mL), compared to 1.2±0.7 fmol/mL of controls (p=0.05 and 0.006). Within 72 h after ictus, patients suffering from TBI (p=0.01), ICH (p=0.001), and ischemic stroke (p=0.02) showed significant increases. CSF-BK concentrations correlated with extent of edema formation (r=0.53; p<0.001) and with ICP (r=0.49; p<0.001). Our results demonstrate that acute cerebral lesions are associated with increased CSF-BK levels. Especially after TBI, subarachnoid and intracerebral hemorrhage CSF-BK levels correlate with extent of edema evolution and ICP. BK-blocking agents may turn out to be effective remedies in brain injuries.

Lipopolysaccharide activates the kallikrein-kinin system in mouse choroid plexus cell line ECPC4.

Regulation of the kallikrein-kinin system in cerebral inflammation is still unclear. Here, we used reverse-transcription polymerase chain reaction (RT-PCR) techniques to show that lipopolysaccharide (LPS) activates the kallikrein-kinin system by enhancing liberation of bradykinin (BK), and alters mRNA levels of kallikrein-kinin system components, including high molecular weight (H-) and low molecular weight (L-) kininogens, in ECPC4 cells, a cell line of mouse choroid plexus epithelium. LPS treatment increased liberation of immunoreactive bradykinin in the supernatant of ECPC4 cells, and addition of LPS (500 ng/ml) to cultures resulted in elevation of H- and L-kininogen mRNA levels in ECPC4 cells within 24-48 h. Furthermore, LPS treatment elevated bradykinin type 2 and type 1 receptor mRNA levels within 4h, but did not change tissue kallikrein or plasma kallikrein mRNA levels. On the other hand, expression of pro-inflammatory mediators interleukin-1beta (IL-1beta), tumor necrosis factor-alpha (TNF-alpha), and cyclooxygenase-2 mRNA increased within 4-8h after addition of LPS to ECPC4 cells. The addition of IL-1beta and TNF-alpha to investigate the major mediator for kininogen expression in ECPC4 cells remarkably induced expression of H- and L-kininogen mRNAs in ECPC4 cells. These results suggest that LPS activates the kallikrein-kinin system in the choroid plexus via autocrine induction of IL-1beta and TNF-alpha.

Assay of bradykinin-related peptides in human body fluids using capillary electrophoresis with laser-induced fluorescence detection.

A CE with LIF detection was developed for separation and determination of bradykinin (BK)-related peptides, such as BK, kallidin (Kal), and neurokinin A (NKA). BK-related peptides were derivatized with FITC prior to CE-LIF analysis. Sodium borate 10 mmol/L at pH 9.5 was selected as derivatization media in order to get the high efficiency. Three peptides were baseline-separated within 10 min by using 110 mmol/L sodium borate-sodium hydroxide solution at pH 10.0 as the running buffer. Concentration detection limits (S/N = 3) for BK, Kal, and NKA were 0.08, 0.5, and 0.2 nmol/L, respectively. Meanwhile we have also developed a simple, quick, and sensitive large-volume sample stacking (LVSS) technique for CE-LIF detection of BK, Kal, and NKA. By using this stacking technique, the detection limits (S/N = 3) for BK, Kal, and NKA were 0.02, 0.05, and 0.04 nmol/L, respectively. This method has been applied to the assay of human saliva and cerebrospinal fluid with satisfactory results.

Mediators involved in the febrile response induced by Tityus serrulatus scorpion venom in rats.

Tityus serrulatus venom (Tsv) was intraperitoneally (ip) injected at doses of 75, 150 and 300mug/kg and IL-1beta (2.0 microg/kg) was given intravenously (iv) to male Wistar rats. Rectal temperature was measured by radiotelemetry. Vagotomy was performed according to Bluthe et al. [1994. Lipopolysaccharide induces sickness behaviour in rats by a vagal mediated mechanism. C R Acad. Sci. 317(6), 499-503]. Cerebrospinal fluid (CSF) and peritoneal fluid (PF) levels of bradykinin (BK) were measured by ELISA. B(1) (des-Arg(9)-[Leu(8)]-BK; DALBK) and B(2) kinin receptor (icatibant) antagonists (1.0 mg/kg each), the induced nitric oxide synthase inhibitor aminoguanidine (50.0 mg/kg), the neuronal nitric oxide synthase inhibitor 7-nitroindazole (30.0 mg/kg), the dual cyclooxygenase inhibitor ibuprofen (10.0 mg/kg), the selective interleukin-1 receptor antagonist IL-ra (2.0 mg/kg) and dipyrone (120 mg/kg) were given ip. Celecoxib (5 mg/kg) was given per os (po). Tsv at doses of 75 microg/kg evoked no change in rectal temperature while at doses of 150 and 300 microg/kg it promoted long-lasting fever (2 degrees C+/-0.1). Tsv (150 microg/kg) increased by nearly 3 and 5 times, respectively BK concentration in the CSF and in the PF. Subdiaphragmatic vagotomy or 7-nitroindazole reduced, icatibant, DALBK, IL-1ra, aminoguanidine and dipyrone abolished, while ibuprofen and celecoxib failed to affect Tsv-induced fever. These results suggest that PGs do not play a relevant role, whereas, kinins via their B(1) and B(2) receptors, IL-1, nitric oxide and vagal neurotransmission are involved in Tsv-induced fever.

A single dose, three-arm, placebo-controlled, phase I study of the bradykinin B2 receptor antagonist Anatibant (LF16-0687Ms) in patients with severe traumatic brain injury.

Traumatic brain injury (TBI) mortality and morbidity remains a public health challenge. Because experimental studies support an important role of bradykinin (BK) in the neurological deterioration that follows TBI, a double-blind, randomized, placebo-controlled study of Anatibant (LF16- 0687Ms), a selective and potent antagonist of the BK B(2) receptor, was conducted in severe (Glasgow Coma Scale [GCS] < 8) TBI patients (n = 25) at six sites in the United States. At 8-12 h after injury (9.9 +/- 2.8 h), patients received a single subcutaneous injection of Anatibant (3.75 mg or 22.5 mg, n = 10 each) or placebo (n = 5). The primary objective was to investigate the pharmacokinetics of Anatibant; general safety, local tolerability, levels of the bradykinin metabolite BK1-5 in plasma and cerebrospinal fluid (CSF), intracranial pressure (ICP), and cerebral perfusion pressure were also assessed. We observed a dose-proportionality of the pharmacokinetics, Cmax, and AUC of Anatibant. V(d)/F, Cl/F, and t(1/2) were independent on the dose and protein binding was >97.7%. Anatibant, administered as single subcutaneous injections of 3.75 g and 22.5 mg, was well tolerated in severe TBI patients with no unexpected clinical adverse events or biological abnormalities observed. Interestingly, plasma and CSF levels of BK1-5 were significantly and markedly increased after trauma (e.g., 34,700 +/- 35,300 fmol/mL in plasma vs. 34.9 +/- 5.6 fmol/mL previously reported for normal volunteers), supporting the use of Anatibant as a treatment of secondary brain damage. To address this issue, a dose-response trial that would investigate the effects of Anatibant on the incidence of raised ICP and on functional outcome in severe TBI patients is needed.

Central bradykininergic system in normotensive and hypertensive rats.

1. The kinin antagonist des-Arg9-[Leu8]bradykinin, injected into the lateral ventricle, caused a long-lasting, dose-dependent reduction in arterial blood pressure and heart rate in spontaneously hypertensive rats but not in normotensive Wistar-Kyoto rats; the antagonist also blocked the pressor response to ventricularly infused bradykinin in both strains. 2. Bradykinin content was increased in the hypothalamus and septum and decreased in the dorsal medulla of spontaneously hypertensive rats when compared with those of normotensive Wistar-Kyoto rats, whereas similar bradykinin contents were observed in the pineal gland, hypophysis and rostroventrolateral medulla of both rat strains. 3. Increased concentrations of bradykinin and its precursor kininogen were found in the cerebrospinal fluid of spontaneously hypertensive rats. 4. Bradykinin receptor numbers, measured as the binding of [125I-Tyr1]bradykinin to nervous tissue, were found to be increased in the dorsal medulla and hypophysis, and to be decreased in the pineal gland, of spontaneously hypertensive rats. 5. Therefore, the central kinin system may participate, by both pre- and post-synaptic mechanisms, in the maintenance of hypertension in spontaneously hypertensive rats.

Role of endogenous brain kinins in the cardiovascular response to intracerebroventricular melittin.

Intracerebroventricular infusion of the peptide melittin increases immunoreactive kinins in the cerebrospinal fluid of anesthetized dogs, probably secondary to activation of brain or cerebrospinal fluid kininogenases. Intracerebroventricular melittin also increases blood pressure and heart rate, possibly mediated by brain kinins, since intracerebroventricular bradykinin also increases blood pressure and heart rate. We tested whether the effects of centrally administered melittin on blood pressure and heart rate could be blocked by simultaneous infusion of a kinin receptor antagonist, [DArg0]Hyp3-Thi5,8[DPhe7]bradykinin, in normotensive awake rats. In the controls, intracerebroventricular infusion of kinin receptor antagonist given for 1 hour at a rate of 10 micrograms/hr blocked bradykinin-induced increases in blood pressure and heart rate by 80%. Basal blood pressure and heart rate were not affected by the kinin receptor antagonist alone. After a 30-minute infusion of melittin (8 micrograms/30 min), cerebrospinal fluid kininogenase activity (n = 17) rose from 0.13 +/- 0.05 to 0.43 +/- 0.1 ng/ml/min (p less than 0.02). Although cerebrospinal fluid kinins increased from below sensitivity (0.02 ng/ml, n = 12) to 0.19 +/- 0.1 ng/ml (n = 17), this change was due to drastic increases in three rats, whereas in 12 of them kinins were below sensitivity. Incubation of bradykinin (10 ng) with 0.1 ml rat cerebrospinal fluid for 5 minutes destroyed 70% of kinins, suggesting that rapid destruction may have made detection of increased CSF kinins difficult.(ABSTRACT TRUNCATED AT 250 WORDS)

Activation of the coagulation system in the subarachnoid space after subarachnoid haemorrhage: serial measurement of fibrinopeptide A and bradykinin of cerebrospinal fluid and plasma in patients with subarachnoid haemorrhage.

Fibrinopeptide A (FPA) levels as an indicator of thrombin activity in the cerebrospinal fluid (CSF) and plasma of 25 patients with subarachnoid haemorrhage (SAH) were measured serially by radioimmunoassay (RIA). FPA levels in CSF were extremely high on days 0-1 (1253 +/- 269 ng/ml, mean +/- standard error) but decreased rapidly (11.3 +/- 3.9 ng/ml on days 2-4, 10.7 +/- 5.9 ng/ml on days 5-7, and 6.3 +/- 1.5 ng/ml on days 8-14). In the controls the FPA concentration in CSF was 1.2 +/- 0.9 ng/ml (mean +/- standard deviation). Plasma FPA levels in patients with SAH showed no statistically significant changes with time. The bradykinin (BK) concentration in CSF and plasma in 27 patients with SAH was measured serially by RIA. The concentrations in CSF were 122.7 +/- 22.7 pg/ml (mean +/- standard error) on day 0, 38.6 +/- 6.1 pg/ml on day 1, 22.7 +/- 6.3 pg/ml on day 2, and 17.1 +/- 3.0 pg/ml or less thereafter. Plasma BK levels in patients with SAH were higher than those in the control group, but there was no statistically significant change over time. From the measurement of FPA it was apparent that the coagulation system in the subarachnoid space is strongly activated in the early stage of SAH. The formation of BK in CSF after SAH is thought to be due to the contact activation of Hageman factor (intrinsic factor) in the subarachnoid space. Trabeculae as collagen bundles in the subarachnoid space were considered to have a possible role in activating the Hageman factor of the coagulation system in SAH.

[Serial measurement of bradykinin of the cerebrospinal fluid in patients with subarachnoid hemorrhage].

The bradykinin (BK) concentration of the cerebrospinal fluid (CSF) in 21 patients with subarachnoid hemorrhage (SAH) was measured serially by radioimmunoassay (RIA). The values were 134.9 +/- 93.9 pg/ml (mean +/- standard deviation) on the day of onset, 38.3 +/- 31.8 pg/ml on the first day after onset, 23.4 +/- 22.8 pg/ml on the second day, 16.6 +/- 9.9 pg/ml on the third day, 15.5 +/- 6.0 pg/ml on the fourth day, and 14.8 +/- 5.9 pg/ml on the fifth day. In the controls the BK concentration in the CSF was 8.0 +/- 3.3 pg/ml (n = 10). On the other hand, none of 5 patients with intraventricular hematoma due to intracerebral hemorrhage had a high level of BK in the bloody CSF at the initial stage (15.2-26.0 pg/ml). This shows that BK is not produced only by the mixture of CSF and blood. BK is produced by the activation of Hageman factor that is considered to be activated by trabecula of collagen bundles in the subarachnoid space in the case of SAH.