Karim Lahjouji

Cognitive and cerebrovascular improvements following kinin B1 receptor blockade in Alzheimer’s disease mice

BackgroundRecent evidence suggests that the inducible kinin B1 receptor (B1R) contributes to pathogenic neuroinflammation induced by amyloid-beta (Aβ) peptide. The present study aims at identifying the cellular distribution and potentially detrimental role of B1R on cognitive and cerebrovascular functions in a mouse model of Alzheimer’s disease (AD).MethodsTransgenic mice overexpressing a mutated form of the human amyloid precursor protein (APPSwe,Ind, line J20) were treated with a selective and brain penetrant B1R antagonist (SSR240612, 10 mg/kg/day for 5 or 10 weeks) or vehicle. The impact of B1R blockade was measured on i) spatial learning and memory performance in the Morris water maze, ii) cerebral blood flow (CBF) responses to sensory stimulation using laser Doppler flowmetry, and iii) reactivity of isolated cerebral arteries using online videomicroscopy. Aβ burden was quantified by ELISA and immunostaining, while other AD landmarks were measured by western blot and immunohistochemistry.ResultsB1R protein levels were increased in APP mouse hippocampus and, prominently, in reactive astrocytes surrounding Aβ plaques. In APP mice, B1R antagonism with SSR240612 improved spatial learning, memory and normalized protein levels of the memory-related early gene Egr-1 in the dentate gyrus of the hippocampus. B1R antagonism restored sensory-evoked CBF responses, endothelium-dependent dilations, and normalized cerebrovascular protein levels of endothelial nitric oxide synthase and B2R. In addition, SSR240612 reduced (approximately 50%) microglial, but not astroglial, activation, brain levels of soluble Aβ1-42, diffuse and dense-core Aβ plaques, and it increased protein levels of the Aβ brain efflux transporter lipoprotein receptor-related protein-1 in cerebral microvessels.ConclusionThese findings show a selective upregulation of astroglial B1R in the APP mouse brain, and the capacity of the B1R antagonist to abrogate amyloidosis, cerebrovascular and memory deficits. Collectively, these findings provide convincing evidence for a role of B1R in AD pathogenesis.

Activation of TRPV1 by capsaicin induces functional kinin B(1) receptor in rat spinal cord microglia.

BackgroundThe kinin B1 receptor (B1R) is upregulated by pro-inflammatory cytokines and oxydative stress, which are enhanced by transient receptor potential vanilloid subtype 1 (TRPV1) activation. To examine the link between TRPV1 and B1R in inflammatory pain, this study aimed to determine the ability of TRPV1 to regulate microglial B1R expression in the spinal cord dorsal horn, and the underlying mechanism.MethodsB1R expression (mRNA, protein and binding sites) was measured in cervical, thoracic and lumbar spinal cord in response to TRPV1 activation by systemic capsaicin (1-50 mg/kg, s.c) in rats pre-treated with TRPV1 antagonists (capsazepine or SB-366791), the antioxidant N-acetyl-L-cysteine (NAC), or vehicle. B1R function was assessed using a tail-flick test after intrathecal (i.t.) injection of a selective B1R agonist (des-Arg9-BK), and its microglial localization was investigated by confocal microscopy with the selective fluorescent B1R agonist, [Nα-bodipy]-des-Arg9-BK. The effect of i.t. capsaicin (1 μg/site) was also investigated.ResultsCapsaicin (10 to 50 mg/kg, s.c.) enhanced time-dependently (0-24h) B1R mRNA levels in the lumbar spinal cord; this effect was prevented by capsazepine (10 mg/kg, i.p.; 10 μg/site, i.t.) and SB-366791 (1 mg/kg, i.p.; 30 μg/site, i.t.). Increases of B1R mRNA were correlated with IL-1β mRNA levels, and they were significantly less in cervical and thoracic spinal cord. Intrathecal capsaicin (1 μg/site) also enhanced B1R mRNA in lumbar spinal cord. NAC (1 g/kg/d × 7 days) prevented B1R up-regulation, superoxide anion production and NF-kB activation induced by capsaicin (15 mg/kg). Des-Arg9-BK (9.6 nmol/site, i.t.) decreased by 25-30% the nociceptive threshold at 1 min post-injection in capsaicin-treated rats (10-50 mg/kg) while it was without effect in control rats. Des-Arg9-BK-induced thermal hyperalgesia was blocked by capsazepine, SB-366791 and by antagonists/inhibitors of B1R (SSR240612, 10 mg/kg, p.o.), glutamate NMDA receptor (DL-AP5, 10 μg/site, i.t.), substance P NK-1 receptor (RP-67580, 10 μg/site, i.t.) and nitric oxide synthase (L-NNA, 10 μg/site, i.t.). The B1R fluorescent agonist was co-localized with an immunomarker of microglia (Iba-1) in spinal cord dorsal horn of capsaicin-treated rats.ConclusionThis study highlights a new mechanism for B1R induction via TRPV1 activation and establishes a link between these two pro-nociceptive receptors in inflammatory pain.

Mechanism of cigarette smoke-induced kinin B1 receptor expression in rat airways

Pulmonary inflammation is an important pathological feature of tobacco smoke related lung diseases such as chronic obstructive pulmonary disease (COPD). Kinin type 1 and type 2 receptors (B(1)R, B(2)R) are known to be associated with inflammatory responses of the lungs and other organs. In this study, we investigated whether cigarette smoke-induced airway inflammation could up-regulate B(1)R and B(2)R in correlation with IL-1β and TNF-α. Rat lung slices treated with 5 μg/ml total particulate matter (TPM) of cigarette smoke for 24 h showed an enhanced expression of B(1)R and IL-1β by 5-fold and 30-fold, respectively, in comparison to vehicle treatment (dimethyl sulfoxide). However, higher concentrations of TPM failed to induce B(1)R. No significant increase of B(2)R or TNF-α gene induction was observed. IL-1 receptor antagonist (IL-1Ra, 2 ng/ml) significantly blocked B(1)R gene induction by TPM, while 500 μM pentoxifylline, TNF-α inhibitor, reduced it partially. Western blot analysis showed a 2-fold enhanced expression of B(1)R in rat lung slices treated with 5 μg/ml TPM for 24 h and such protein expression was totally blocked by a co-treatment with IL-1Ra but not with pentoxifylline. In addition to the lower airways, rat trachea subchronically exposed to cigarette whole smoke exhibited 11-fold B(1)R gene induction in comparison with those exposed only to air. Our results demonstrate the involvement of B(1)R in cigarette smoke-induced airway inflammation through a mechanism which is mediated by the pro-inflammatory cytokine IL-1β.

L-Carnitine transport in mouse renal and intestinal brush-border and basolateral membrane vesicles

We characterized the uptake of carnitine in brush-border membrane (BBM) and basolateral membrane (BLM) vesicles, isolated from mouse kidney and intestine. In kidney, carnitine uptake was Na(+)-dependent, showed a definite overshoot and was saturable for both membranes, but for intestine, it was Na(+)-dependent only in BLM. The uptake was temperature-dependent in BLM of both kidney and intestine. The BBM transporter in kidney had a high affinity for carnitine: apparent K(m)=18.7 microM; V(max)=7.85 pmol/mg protein/s. In kidney BLM, similar characteristics were obtained: apparent K(m)=11.5 microM and V(max)=3.76 pmol/mg protein/s. The carnitine uptake by both membranes was not affected within the physiological pH 6.5-8.5. Tetraethylammonium, verapamil, valproate and pyrilamine significantly inhibited the carnitine uptake by BBM but not by BLM. By Western blot analysis, the OCTN2 (a Na(+)-dependent high-affinity carnitine transporter) was localized in the kidney BBM, and not in BLM. Strong OCTN2 expression was observed in kidney and skeletal muscle, with no expression in intestine in accordance with our functional study. We conclude that different polarized carnitine transporters exist in kidney BBM and BLM. L-Carnitine uptake by mouse renal BBM vesicles involves a carrier-mediated system that is Na(+)-dependent and is inhibited significantly by specific drugs. The BBM transporter is likely to be OCTN2 as indicated by a strong reactivity with the anti-OCTN2 polyclonal antibody.

Modulation of retinal blood flow by kinin B1 receptor in Streptozotocin-diabetic rats

The vasoactive kinin B₁ receptor (B₁R) is overexpressed in the retina of diabetic rats in response to hyperglycemia and oxidative stress. The aim of the present study was to determine whether B₁R could contribute to the early retinal blood flow changes occurring in diabetes. Male Wistar rats were rendered diabetic with a single i.p. injection of Streptozotocin (STZ) and studied 4 days or 6 weeks after diabetes induction. The presence of B₁R in the retina was confirmed by Western blot. The impact of oral administration of the B₁R selective antagonist SSR240612 (10mg/kg) was measured on alteration of retinal perfusion in awake diabetic rats by quantitative autoradiography. Data showed that B₁R was upregulated in the STZ-diabetic retina at 4 days and 6 weeks. Retinal blood flow was not altered in 4-day diabetic rats compared with age-matched controls but was significantly decreased following SSR240612 treatment. In 6-week diabetic rats, retinal blood flow was markedly reduced compared to control rats and SSR240612 did not further decrease the blood flow. These results suggest that B₁R is upregulated in STZ-diabetic retina and has a protective compensatory role on retinal microcirculation at 4 days but not at 6 weeks following diabetes induction.

Effects of hyperosmolarity on the Na+-myo-inositol cotransporter SMIT2 stably transfected in the Madin-Darby canine kidney cell line

Myo-inositol (MI) is a compatible osmolyte used by cells to compensate for changes in the osmolarity of their surrounding milieu. In kidney, the basolateral Na(+)-MI cotransporter (SMIT1) and apical SMIT2 proteins are homologous cotransporters responsible for cellular uptake of MI. It has been shown in the Madin-Darby canine kidney (MDCK) cell line that SMIT1 expression was under the control of the tonicity-sensitive transcription factor, tonicity-responsive enhancer binding protein (TonEBP). We used an MDCK cell line stably transfected with SMIT2 to determine whether variations in external osmolarity could also affect SMIT2 function. Hyperosmotic conditions (+200 mosM raffinose or NaCl but not urea) generated an increase in SMIT2-specific MI uptake by three- to ninefold in a process that required protein synthesis. Using quantitative RT-PCR, we have determined that hyperosmotic conditions augment both the endogenous SMIT1 and the transfected SMIT2 mRNAs. Transport activities for both SMIT1 and SMIT2 exhibited differences in their respective induction profiles for both their sensitivities to raffinose, as well as in their time course of induction. Application of MG-132, which inhibits nuclear translocation of TonEBP, showed that the effect of osmolarity on transfected SMIT2 was unrelated to TonEBP, unlike the effect observed with SMIT1. Inhibition studies involving the hyperosmolarity-related MAPK suggested that p38 and JNK play a role in the induction of SMIT2. Further studies have shown that hyperosmolarity also upregulates another transfected transporter (Na(+)-glucose), as well as several endogenously expressed transport systems. This study shows that hyperosmolarity can stimulate transport in a TonEBP-independent manner by increasing the amount of mRNA derived from an exogenous DNA segment.

A heterozygote phenotype is present in the jvs +/− mutant mouse livers

The juvenile visceral steatosis (jvs) mouse, having a mutation in the carnitine transporter gene Octn2, is a model of primary systemic carnitine deficiency in humans (SCD, OMIM 212140). Like humans with SCD, homozygous jvs -/- mice have hepatic and cardiac steatoses, reduced plasma and tissue carnitines, and increased urinary carnitine clearance. Because symptomatic heterozygotes have been reported for some fatty acid oxidation disorders, including SCD, we compared the jvs heterozygotes to normal control mice. We measured the free and esterified carnitine, total cholesterol, and triglycerides in adult liver samples, myocardium, and skeletal muscle. Our results indicate significant differences between the livers of nonfasting adult normal (n = 8) vs jvs heterozygotes (n = 8) (means +/- SEM, p < 0.01) for the following parameters: free carnitine, 2.28 +/- 0.36 nmol/mg protein vs 0.41 +/- 0.13; total carnitine, 3.48 +/- 0.36 vs 1.27 +/- 0.25; triglycerides, 0.14 +/- 0.04 vs 0.39 +/- 0.02; and total cholesterol, 0.21 +/- 0.02 vs 0.39 +/- 0.04, but not for esterified carnitine, 1.18 +/- 0.17 vs 0.90 +/- 0.17 (p > 0.05). There is also a negative correlation between hepatic free carnitine and triglycerides from jvs heterozygotes (p < 0.05). Similar results were obtained with myocardium and skeletal muscle. We conclude that free and total carnitine levels are significantly lower in the heterozygote mouse liver and heart while triglyceride and total cholesterol levels are significantly higher. We speculate that in situations of lipolytic stress, some SCD heterozygotes might develop clinical symptoms of carnitine deficiency.

Cigarette smoke-induced kinin B1 receptor promotes NADPH oxidase activity in cultured human alveolar epithelial cells.

Pulmonary inflammation is an important pathological feature of tobacco smoke-related lung diseases. Kinin B1 receptor (B1R) is up-regulated in the rat trachea chronically exposed to cigarette-smoke. This study aimed at determining (1) whether exposure to total particulate matter of the cigarette smoke (TPM) can induce B1R in human alveolar epithelial A549 cells, (2) the mechanism of B1R induction, (3) the functionality of de novo synthesized B1R, and (4) the role of B1R in TPM-induced increase of superoxide anion (O₂(●⁻)) level. Results show that A549 cells exposed to 10 μg/ml TPM increased O₂(●⁻) level along with B1R (protein and mRNA) and IL-1β mRNA. In contrast, B2R and TNF-α mRNA were not affected by TPM. The increasing effect of TPM on O₂(●⁻) level was not significantly affected by the B1R antagonist SSR240612. TPM-increased B1R mRNA was prevented by co-treatments with N-acetyl-l-cysteine (potent antioxidant), diphenyleneiodonium (NADPH oxidase inhibitor), IL-1Ra (interleukin-1R antagonist) and SN-50 (specific inhibitor of NF-kB activation) but not by pentoxifylline (TNF-α release inhibitor), indomethacin and niflumic acid (COX-1 and -2 inhibitors). Stimulation of B1R with a selective agonist (des-Arg⁹-BK, 10 μM; 30 min) increased O₂(●⁻)production which was prevented by apocynin and diphenyleneiodonium (NADPH oxidase inhibitors). Data suggest that the increased expression of B1R by TPM in A549 cells is mediated by oxidative stress, IL-1β and NF-kB but not by cyclooxygenases or TNF-α. The amplification of O₂(●⁻) levels via the activation of B1R-NADPH oxidase may exacerbate pulmonary inflammation and contribute to the chronicity of tobacco smoke-related lung diseases.

Effects of Alpha-Lipoic Acid on Oxidative Stress and Kinin Receptor Expression in Obese Zucker Diabetic Fatty Rats

Objective To investigate the impact of alpha-lipoic acid on superoxide anion production and NADPH oxidase activity as well as on the expression of kinin B1 and B2 receptors in key organs of obese Zucker Diabetic Fatty rats. Methods Superoxide anion production was measured by lucigenin chemiluminescence. Kinin B1 and B2 receptors expression was measured at protein and mRNA levels by western blot and qRT-PCR in key organs of Zucker Diabetic Fatty and Zucker lean control rats treated for a period of 6 weeks with a standard diet or a diet containing the antioxidant α-lipoic acid (1 g/kg). Results Superoxide anion production and NADPH oxidase activity were significantly enhanced in aorta and adipose tissue of Zucker Diabetic Fatty rats. Kinin B1 and B2 receptors expression levels were also significantly increased in the liver and the gastrocnemius muscle of Zucker Diabetic Fatty rats. Expression of both receptors was not altered in the pancreas of Zucker Diabetic Fatty rats and was undetectable in white retroperitoneal adipose tissue. Alpha-lipoic acid prevented the rise in NADPH oxidase activity in aorta and epididymal adipose tissue of Zucker Diabetic Fatty rats and the upregulation of kinin B1 receptor in liver and gastrocnemius muscle and that of kinin B2 receptor in the liver. Alpha-lipoic acid treatment was found to prevent the final body weight increase without affecting significantly hyperglycemia, hyperinsulinemia and insulin resistance index in Zucker Diabetic Fatty rats. Conclusion Findings support the hypothesis that oxidative stress is implicated in the induction of kinin B1 receptor in Zucker Diabetic Fatty rats. The ability of α-lipoic acid to blunt the body weight gain appears to be mediated in part by preventing NADPH oxidase activity rise in adipose tissue and reversing the hepatic upregulation of kinin B1 receptor in Zucker Diabetic Fatty rats.

Bradykinin B1 receptor blockade in the treatment of Alzheimer's disease: improvement of cognitive and cerebrovascular functions and reduction of amyloidosis

dementia and Alzheimer disease (AD) in elderly African Americans. Methods: A community-based sample of 1473 African Americans aged 70 or older residing in Indianapolis, Indiana was assessed in 2001, 2004, 2007, and 2009. Subjects were evaluated using the Community Screening Interview for Dementia (CSI-D), and an in-home assessment including a brief neurological examination, neuropsychological testing, and informant interview. Medication use, including statins, was recorded using participant and informant report, medical record review, and in-home inspection of medications. Incident dementia and AD were defined as the first time a subject was diagnosed with dementia or AD in 2004, 2007, or 2009; diagnoses were determined in consensus diagnosis conferences. Measurements of low-density lipoprotein cholesterol (LDL) and C-reactive protein (CRP) were obtained from baseline blood samples. 974 subjects with complete information on statin use, APOE status, LDL, and CRP were included in this analysis. Results: After controlling for age at diagnosis, education level, and the presence of the ApoE E4 allele, continuous use of statins since baseline was associated with a significantly decreased risk of incident AD (OR1⁄40.27; p1⁄40.0339), and a trend toward a decreased risk of incident dementia (OR1⁄40.40; p1⁄40.0599) compared to those who had never taken stains. Use of statins during only a portion of the assessment period did not confer a significant reduction in risk of either incident AD (OR1⁄40.73; p1⁄40.3123) or for incident dementia (OR1⁄40.72; p1⁄40.2548). There was no significant interaction with baseline LDL or CRP level. Conclusions: In this study, exposure to statin medications conferred a reduced risk for incident AD and a trend toward a reduced risk for incident dementia, but only with consistent use of the medication. The potential mechanism for this risk reduction has not been elucidated completely, but is likely to extend beyond statins’ lipid-lowering effects.