NEP Activity Research Articles

ADAM10 and Neprilysin level decreases in immune cells of mice bearing metastatic breast carcinoma: Possible role in cancer inflammatory response

Objective and designADAM10 and Neprilysin, proteases, play critical role in inflammatory disease, however their role in cancer immune response is not clear. We here evaluated changes in immune response using an experimental model for breast cancer. Material and methodHighly metastatic breast cancer cells (4T1-derived) were injected orthotopically (mammary-pad of Balb-c mice) to induce tumors. Changes in enzyme level and activity as well as alterations in inflammatory cytokine release in the presence or absence of ADAM10 and NEP activity was determined using specific inhibitors and recombinant proteins. Cytokine response was evaluated using mix leucocyte cultures obtained from control and tumor-bearing mice. ANOVA with Dunnett's posttest was used for statistical analysis. ResultsADAM10 and NEP expression was decreased markedly in lymph nodes and spleens of tumor-bearing mice. ADAM10 activity was reduced together with apparent alterations of ADAM10 processing. ADAM10 and NEP activity decreased TNF-α, IL-6 and IFN-ɣ secretion. Suppression of these inflammatory cytokines were more prominent in cultures obtained from control mice demonstrating counteracting factors that are exist in tumor-bearing mice. ConclusionLoss of ADAM10 and NEP activity in immune cells during breast cancer metastasis might be one of the main factors involved in induction of chronic inflammation by tumors.

PS-C30-10: EFFECT OF OBESITY ON SERUM PROFILES OF THE RENIN ANGIOTENSIN ALDOSTERONE SYSTEM (RAAS) IN PREGNANCY

Objective: Hypertensive disorders of pregnancy (HDP) are a leading cause of maternal and infant morbidities and mortalities. Activity of the renin angiotensin aldosterone system (RAAS) is critical for the physiology of pregnancy, and suppressed RAAS during pregnancy is associated with adverse outcomes, such as preeclampsia. Obesity is a major contributor to adverse outcomes of pregnancy, and obesity modulates the RAAS in non-pregnant conditions. It is not known how the RAAS is regulated with obesity during pregnancy. We quantified serum biomarkers of the classical and alternative arms of the RAAS in a cohort of pregnant women to determine associations between obesity and activity of the RAAS during pregnancy. Design and Methods: We recruited a prospective cohort of n = 36 pregnant women in the first trimester with no clinical risk factors. Patients were separated into two groups, greater (n = 23) or less than (n = 13) body mass index (BMI) of 30 kg/m2 at enrollment. We quantified 6 angiotensin peptides, aldosterone, and enzyme activities of ACE2 and NEP in serum using LC-MS/MS in the first and third trimester. PRA, S was calculated as: Ang I + Ang II (pmol/L). Data are median [IQR]. Results: PRA, S and aldosterone increased with pregnancy in both groups, but the change from first to third trimester was reduced in the BMI > 30 group compared to those with BMI < 30 (PRA-S: 24.1 [-45.4 to 194.7] versus 70.9 [9.2 to 113.1]) pmol/L, respectively; Aldo: 234.3 [45.5 to 532.7] versus 648.2 [182.1 to 1207.3] pmol/L, respectively; P < 0.05). ACE2 activity was increased during pregnancy, with no differences between groups, however, NEP activity was elevated in the BMI > 30 group at both time points during pregnancy compared to the BMI < 30 group (1st trim: 13.4 [8.1 to 20.38] versus 8.4 [3.7 to 12.03]; 3rd trim: 21.3 [11.7 to 26.5] versus 13.47 [10.5 to 22.8] ng/mL; P < 0.01). In the BMI < 30 group, n = 2 developed gestational hypertension and n = 1 developed postpartum preeclampsia. In contrast, in the BMI > 30 group, n = 7 developed gestational hypertension, n = 2 developed preeclampsia/postpartum preeclampsia. Conclusion: Activity of the classical RAAS is reduced in women with 1st trimester BMI > 30 compared to those with BMI < 30. In contrast, the alternative RAS is not suppressed by obesity during pregnancy. Further, elevated 1st trimester BMI was associated with more adverse outcomes. Reduced activity of the classical RAAS with obesity may contribute to adverse outcomes in pregnancy.

Alpha Ketoglutarate Downregulates the Neutral Endopeptidase and Enhances the Growth Inhibitory Activity of Thiorphan in Highly Aggressive Osteosarcoma Cells.

Since natural substances are widely explored as epigenetic modulators of gene expression and epigenetic abnormalities are important causes of cancerogenesis, factors with pro-tumor activities subjected to epigenetic control, e.g., neutral endopeptidase (NEP, neprilysin), are promising anticancer targets for potential therapies acting via epigenetic regulation of gene expression. Alpha-ketoglutarate (AKG) is a naturally occurring co-substrate for enzymes involved in histone and DNA demethylation with suggested anti-cancer activity. Hence, we investigated a potential effect of AKG on the NEP expression in cells derived from various cancers (cervical, colon, osteosarcoma) and normal epithelial cells and osteoblasts. Moreover, the overall methylation status of histone H3 was explored to establish the molecular target of AKG activity. Additionally, it was investigated whether AKG in combination with thiorphan (NEP specific inhibitor) exhibited enhanced anticancer activity. The results revealed that AKG downregulated the expression of NEP at the protein level only in highly aggressive osteosarcoma HOS cells (flow cytometry and fluorometric assays), and this protease was found to be involved in AKG-induced growth inhibition in osteosarcoma cells (siRNA NEP silencing, BrdU assay, flow cytometry). Unexpectedly, AKG-induced hypermethylation of H3K27 in HOS cells, which was partially dependent on EZH2 activity. However, this effect was not implicated in the AKG-induced NEP downregulation (flow cytometry). Finally, the combined treatment with AKG and thiorphan was shown to significantly enhance the growth inhibitory potential of each one towards HOS cells (BrdU assay). These preliminary studies have shown for the first time that the downregulation of NEP expression is a promising target in therapies of NEP-implicating HOS cells. Moreover, this therapeutic goal can be achieved via AKG-induced downregulation of NEP and synergistic activity of AKG with thiorphan, i.e., a NEP specific inhibitor. Furthermore, this study has reported for the first time that exogenous AKG can influence the activity of histone methyltransferase, EZH2. However, this issue needs further investigation to elucidate the mechanisms of this phenomenon.

Tripchlorolide attenuates β-amyloid generation by inducing NEP activity in N2a/APP695 cells

Background and purposeAlzheimer’s disease (AD) is a neurodegeneration disease. The previous work from our research group demonstrated the neuroprotective effects of tripchlorolide (T4) in AD animal models.Materials and methodsNeprilysin (NEP) is known as an important physiological amyloid-β protein (Aβ) peptide-degrading enzyme in the brain due to its apparent rate-limiting function. In this study, we explored the effect of NEP on AD model N2a/APP695 cells. Western blots and enzyme-linked immunosorbent assays were performed to assess the expression of proteins, while quantitative real-time polymerase chain reaction assays were used to evaluate RNA levels. Cell vitality was detected by the MTT assay, and reactive oxygen species (ROS) levels were assessed using a ROS activity assay kit.ResultsWe discovered that T4 was able to enhance the enzyme activity of NEP. T4 administration decreased the protein levels of the soluble amyloid precursor protein. In further experiments, we found that by using thiorphan the secretion of Aβ, oxidative stress, nitrosative stress, and inflammatory factors, which were suppressed by T4, were reversed. Due to its ability to attenuate Aβ generation and to protect neurons against the neurotoxicity of Aβ, T4 may be a potential therapy in the regulation of Aβ-related pathology in AD by affecting NEP activity.ConclusionTripchlorolide attenuates Aβ generation by inducing NEP activity in N2a/APP695 cells.

Role of neprilysin in synaptic plasticity and memory

Neprilysin (NEP, neutral endopeptidase, endopeptidase-24.11) is an important enzyme being most abundant in the kidney and liver and participating in the degradation of a variety of biologically active peptides. It is essential for the regulation of blood pressure and the normal function of practically all organs in the body, and its dysregulation contributes to the development of such diseases like heart failure, cancer, diabetes, and chronic inflammation. It is also a critical neuropeptidase that terminates the action of various neuropeptides such as enkephalins, substance P, somatostatin, which participate in signaling and regulatory events in the brain. NEP is a major amyloid-degrading enzyme, which makes it an important therapeutic target in Alzheimer's disease (AD). Our studies have revealed that NEP expression declines with age in the brain structures participating in memory and which are mainly affected by AD pathogenesis. Moreover, NEP expression decreases under hypoxic conditions, and prenatal hypoxia leads to its deficit in some brain structures both during development and adulthood, which correlates with a reduced number of labile dendritic spines and cognitive deficit in rats. Using the model of prenatal hypoxia in rats, we have tested the effectiveness of several compounds (inhibitors of caspases, histone deacetylases, tyrosine kinases or nuclear retinoid X receptor agonists as well as an antioxidant, epigallocatechin gallate) which, via different mechanisms, were able to restore NEP expression and activity reduced after prenatal hypoxia and improve cognitive functions in rats. In this paper, we provide an overview of the literature and our data on the role of NEP in synaptic plasticity, neuronal activity, and cognitive functions and outline current approaches to regulate its expression and activity in the brain.

P5449Neurohumoral regulation of the low-, medium- and high-renin HFrEF phenotypes

Abstract Background Previous investigations of plasma Renin-Angiotensin-System (RAS) fingerprints of patients with heart failure with reduced ejection fraction (HFrEF) revealed the existence of low, medium and high renin phenotypes independently of disease severity. Plasma renin serves as an excellent surrogate for angiotensin levels. The different renin phenotypes could not only hypothetically respond differently to RAS blockade but associated alterations of other vasoactive peptide systems could elucidate disease mechanisms and novel targets for heart failure therapy. The study aimed to investigate the relation between RAS regulation and pathophysiologically relevant vasoactive peptide systems based on different renin phenotypes. Methods We prospectively enrolled 369 patients with stable HFrEF. Laboratory markers including NT-proBNP and active renin concentration (ARC) were assessed. Plasma NEP levels (sNEP), bioactive adrenomedullin (bio-ADM) and big-endothelin1 (bigET-1) were measured by ELISA (R&D systems, UK; Sphingotec GmbH, Germany and Eagle Biosciences, Austria). NEP activity was determined by a fluorimetric peptide cleavage assay. The correlation between biomarkers and association with all-cause mortality was assessed. sNEP, bio-ADM and bigET-1 levels as well as NEP activity between the different renin phenotypes (i.e. <15. percentile, 15.-85. percentile and >85. percentile of ARC) was compared. Results Median age was 65 (IQR 53–73) years, 75% of patients were male. Median NT-proBNP levels were 1936 (IQR 855–4126) pg/mL. Median ARC was 155 (29–569) μIE/mL, the low, medium and high renin HFrEF phenotypes showed median ARC levels of 4.2μIE/mL (IQR 2.0–7.8), 155.1μIE/mL (IQR 43.3–353.5) and 2360μIE/mL (IQR 1483–3250) μIE/mL. Median bigET-1 was 0.62pmol/L (IQR 0.42–1.10), bio-ADM 26.0pg/mL (IQR 16.1–46.7), sNEP 413pg/mL (IQR 0–4111) and NEP activity 2.36nmol/mL/min (IQR 1.16–4.59). There was no correlation between sNEP and NEP activity [r=0.09, p=0.088]. ARC did not show a meaningful correlation with any of the four biomarkers [p=ns for sNEP, NEP activity and bigET-1; r=0.13, p=0.018 for bio-ADM]. In the univariate analysis ARC, bigET-1, bio-ADM but not sNEP and NEP activity, were associated with outcome. This association remained significant after adjustment for age, gender and kidney function for all three markers and for ARC after adding NT-proBNP [adj. HR per 1-IQR increase of ARC 1.27 (95% CI 1.04–1.22), p=0.003]. There were no differences in bigET-1, bio-ADM and sNEP or NEP activity stratified by the different renin phenotypes (Figure1). Figure 1 Conclusions ARC is a risk factor for mortality in HFrEF patients, independently of NT-proBNP. Plasma NEP levels and activity neither correlated with each other nor were associated with outcome. Bio-ADM and bigET-1 were strong risk factors for all-cause mortality. Interestingly, neither NEP nor bio-ADM or bigET-1 were related to RAS-activation, suggesting that there is no direct relationship with RAS regulation.

P1620Association of granulocyte neprilysin (CD10) expression with prognosis in heart failure with reduced ejection fraction

Abstract Background The exact mechanism of action of neprilysin inhibition (NEPi) is still a subject of debate. The soluble form of the enzyme (sNEP), detectable in plasma, is discussed controversially as a potential biomarker in heart failure with reduced ejection fraction (HFrEF). NEP is present on solid tissues but identically to CD10, expressed on the surface of leukocytes under physiological conditions. The possible impact of NEP expression on peripheral leukocytes on prognosis and its association with sNEP levels have not been investigated yet. Methods 99 stable HFrEF patients were prospectively enrolled and clinically followed-up. Laboratory markers including NT-proBNP were assessed. NEP (CD10) expression on peripheral blood cells were measured by flow cytometry using a combination of six antibodies with fluorescence minus one samples as control [CD3 (#555339), CD19 (#555413), CD56 (#335826), CD16 (#561306), CD14 (#562692), +/−CD10 (#332777); BD Biosciences,USA]. sNEP levels were determined by a specific ELISA [SEB785Hu, USCN, China].Associations between NEP expression and heart failure severity, sNEP levels and all-cause mortality were determined. Results Median age was 65 years (IQR: 55–73), 75% were male. Median NT-proBNP level was 1700pg/ml (IQR: 794–4009).NEP was expressed on granulocytes with 94.8% (IQR: 90.5–97.4) of CD10+ cells and measurable on B-cells and monocytes with 8.5% (IQR: 5.3–13.5) and 0.8% (IQR: 0.4–1.5) of CD10+ cells of the respective leukocyte subtype. NEP expression on T-cells was not detectable. The mean fluorescence intensity (MFI) of CD10+ cells was 5461 (IQR: 4028–6904) for granulocytes, 640 (IQR: 535–740) for B-cells and 1589 (IQR: 1395–1975) for monocytes. Granulocyte NEP expression, but not NEP expression on B-cells or monocytes, correlated inversely with heart failure severity reflected by NT-proBNP level (r=−0.46, p<0.001) and NYHA class (p=0.013) (Figure A). sNEP concentrations correlated weakly with NEP expression on granulocytes (r=0.22, p=0.030) as well as the MFI of CD10+ granulocytes (r=0.31, p=0.003). 15% of the patients died during a median FUP of 24 (IQR: 23–28) months. Increased NEP expression on granulocytes was indicative for better overall survival even after adjustment for age and kidney function [adj. HR per 1-IQR increase of MFI 0.41 (95% CI: 0.18–0.94), p=0.035]. Kaplan-Meier analysis illustrates the impact of granulocyte NEP expression on outcome graphically (Figure B). Figure 1 Conclusions Albeit beneficial effects of NEPi by ARNI therapy, NEP expression on granulocytes is inversely correlated with heart failure severity and mortality. The results support the inverse relationship between BNP and plasma NEP activity reported for a mixed population of heart failure patients. The positive correlation of granulocyte NEP expression and sNEP indicates a possible contribution of shed membrane NEP molecules to plasma NEP levels as a surrogate marker. The utility of granulocyte NEP expression or sNEP as biomarkers in HFrEF have to be further evaluated.

Neprilysin degrades murine Amyloid-β (Aβ) more efficiently than human Aβ: Further implication for species-specific amyloid accumulation

For over a century, aggregated forms of amyloid-β protein (Aβ) have been viewed as a key hallmark of brains affected by Alzheimer’s disease (AD). Today, it remains unknown whether Aβ aggregates (oligomers, fibrils or plaques) originate from increased production or decreased catabolism of Aβ. Neprilysin (NEP, neutral endopeptidase) is a ubiquitously distributed peptidase, known to degrade Aβ, amongst other peptides. In this study, we identified differences in NEP-mediated catabolism of murine and human forms of Aβ, using recombinant human NEP, membrane-bound NEP from cells overexpressing the murine peptidase or from human organ preparations with high NEP activity, and purified soluble bovine NEP. NEP degraded murine Aβ (mAβ) faster than human Aβ (hAβ). These findings were observed with full-length Aβ containing 40 or 42 amino acids (Aβ1−40 and Aβ1−42) and a truncated form (Aβ4–15), which (i) contains one of the main NEP cleavage sites for Aβ (between positions 9 and 10), (ii) harbours all three amino acid differences between murine and human Aβ sequences, and (iii) is less prone to aggregation and thus might be a simpler model to investigate Aβ biochemistry. While it has previously been shown that mAβ has a far lower propensity to aggregate than hAβ, evidence from this study suggests that a faster NEP-mediated turnover of mAβ may provide additional protection against Aβ aggregation in murine species.

Polyhydroxycurcuminoids but not curcumin upregulate neprilysin and can be applied to the prevention of Alzheimer's disease.

Neprilysin (NEP) is the most important Aβ-degrading enzyme. Its expression level decreases with age and inversely correlated with amyloid accumulation, suggesting its correlation with the late-onset of Alzheimer’s disease. Recently, many reports showed that upregulating NEP level is a promising strategy in the prevention and therapy of Alzheimer’s disease. Here, we used a sensitive fluorescence-based Aβ digestion assay to screen 25 curcumin analogs for their ability to upregulate NEP activity. To our surprise, four compounds, dihydroxylated curcumin, monohydroxylated demethoxycurcumin, and mono- and di-hydroxylated bisdemethoxycurcumin, increased NEP activity, while curcumin did not. The ability of these polyhydroxycurcuminoids to upregulate NEP was further confirmed by mRNA and protein expression levels in the cell and mouse models. Finally, feeding monohydroxylated demethoxycurcumin (also named demethylcurcumin) or dihydroxylated bisdemethoxycurcumin (also named bisdemethylcurcumin) to APPswe/PS1dE9 double transgenic mice upregulated NEP levels in the brain and reduced Aβ accumulation in the hippocampus and cortex. These polyhydroxycurcuminoids offer hope in the prevention of Alzheimer’s disease.

Des-Arg1]-Proctolin: A novel NEP-like enzyme inhibitor identified in Tityus serrulatus venom

The scorpion Tityus serrulatus venom comprises a complex mixture of molecules that paralyzes and kills preys, especially insects. However, venom components also interact with molecules in humans, causing clinic envenomation. This cross-interaction may result from homologous molecular targets in mammalians and insects, such as (NEP)-like enzymes. In face of these similarities, we searched for peptides in Tityus serrulatus venom using human NEP as a screening tool. We found a NEP-inhibiting peptide with the primary sequence YLPT, which is very similar to that of the insect neuropeptide proctolin (RYLPT). Thus, we named the new peptide [des-Arg1]-proctolin. Comparative NEP activity assays using natural substrates demonstrated that [des-Arg1]-proctolin has high specificity for NEP and better inhibitory activity than proctolin. To test the initial hypothesis that molecular homologies allow Tityus serrulatus venom to act on both mammal and insect targets, we investigated the presence of a NEP-like in cockroaches, the main scorpion prey, that could be likewise inhibited by [des-Arg1]-proctolin. Indeed, we detected a possible NEP-like in a homogenate of cockroach heads whose activity was blocked by thiorphan and also by [des-Arg1]-proctolin. Western blot analysis using a human NEP monoclonal antibody suggested a NEP-like enzyme in the homogenate of cockroach heads. Our study describes for the first time a proctolin-like peptide, named [des-Arg1]-proctolin, isolated from Tityus serrulatus venom. The tetrapeptide inhibits human NEP activity and a NEP-like activity in a cockroach head homogenate, thus it may play a role in human envenomation as well as in the paralysis and death of scorpion preys.

Mo1236 Early Rebleeding and Mortality in Post HCV Cirrhotic Patients With Esophageal Variceal Bleeding After Band Ligation: Predictive Factors and Value of Vascular Endothelial Growth Factor

and human clinical trials, and is approved for use in ASD in some countries. However, it suffers from shortcomings that might be improved upon with next generation NEPi that have progressed to the clinic for other indications, but which have not been evaluated for anti-diarrheal efficacy. To identify a potentially superior NEPi for future clinical investigation, we tested their activities in a standard rat ASD model and compared the pharmacokinetic and pharmacodynamic (PK/PD) relationships of the most promising compounds. Methods: Racecadotril and 6 other clinical-stage inhibitors of NEP or ACE/NEP were acquired or synthesized for testing. Specificity of NEP inhibition was assessed by determination of IC50 concentrations in an assay panel using commercially available purified peptidase enzymes. Compounds were administered to fasted male Wistar rats by oral gavage at indicated times prior to oral administration of castor oil to induce diarrhea. Stool weight was recorded every 15 minutes up to 4 hours. To assess PK/PD properties, select compounds were administered to healthy or castor oil-treated rats by oral gavage, blood and tissue samples collected at multiple time points, and concentrations of active compound were determined by mass spectroscopy. NEP enzyme activity was measured in tissue homogenates. Results: Three previously untested clinical NEPi significantly delayed diarrhea onset and reduced total stool output. All were shown to be very potent highly specific inhibitors of the NEP enzyme. The three efficacious next generation NEPi exhibited greater suppression of NEP activity in intestinal and non-intestinal tissues than racecadotril, and sustained this inhibition for longer duration. Conclusion: The results suggest newer clinical-stage NEPi inhibitors originally developed as therapies for other indications may offer advantages over racecadotril such as less frequent dosing and potentially improved efficacy for ASD therapy.

Genetic Deficiency in Neprilysin or Its Pharmacological Inhibition Initiate Excessive Stress-Induced Alcohol Consumption in Mice

Both acquired and inherited genetic factors contribute to excessive alcohol consumption and the corresponding development of addiction. Here we show that the genetic deficiency in neprilysin [NEP] did not change the kinetics of alcohol degradation but led to an increase in alcohol intake in mice in a 2-bottle-free-choice paradigm after one single stress stimulus (intruder). A repetition of such stress led to an irreversible elevated alcohol consumption. This phenomenon could be also observed in wild-type mice receiving an orally active NEP inhibitor. We therefore elucidated the stress behavior in NEP-deficient mice. In an Elevated Plus Maze, NEP knockouts crossed more often the area between the arms, implicating a significant stronger stress response. Furthermore, such animals showed a decreased locomotor activity under intense light in a locomotor activity test, identifying such mice to be more responsive in aversive situations than their wild-type controls. Since the reduction in NEP activity itself does not lead to significant signs of an altered alcohol preference in mice but requires an environmental stimulus, our findings build a bridge between stress components and genetic factors in the development of alcoholism. Therefore, targeting NEP activity might be a very attractive approach for the treatment of alcohol abuse in a society with increasing social and financial stress.

A Humanin Derivative Reduces Amyloid Beta Accumulation and Ameliorates Memory Deficit in Triple Transgenic Mice

Humanin (HN), a 24-residue peptide, was identified as a novel neuroprotective factor and shows anti-cell death activity against a wide spectrum of Alzheimer's disease (AD)-related cytotoxicities, including exposure to amyloid beta (Abeta), in vitro. We previously demonstrated that the injection of S14G-HN, a highly potent HN derivative, into brain ameliorated memory loss in an Abeta-injection mouse model. To fully understand HN's functions under AD-associated pathological conditions, we examined the effect of S14G-HN on triple transgenic mice harboring APPswe, tauP310L, and PS-1M146V that show the age-dependent development of multiple pathologies relating to AD. After 3 months of intranasal treatment, behavioral analyses showed that S14G-HN ameliorated cognitive impairment in male mice. Moreover, ELISA and immunohistochemical analyses showed that Abeta levels in brains were markedly lower in S14G-HN-treated male and female mice than in vehicle control mice. We also found the expression level of neprilysin, an Abeta degrading enzyme, in the outer molecular layer of hippocampal formation was increased in S14G-HN-treated mouse brains. NEP activity was also elevated by S14G-HN treatment in vitro. These findings suggest that decreased Abeta level in these mice is at least partly attributed to S14G-HN-induced increase of neprilysin level. Although HN was identified as an anti-neuronal death factor, these results indicate that HN may also have a therapeutic effect on amyloid accumulation in AD.

Genetic Targeting Aromatase in Male Amyloid Precursor Protein Transgenic Mice Down-Regulates β-Secretase (BACE1) and Prevents Alzheimer-Like Pathology and Cognitive Impairment

As brain testosterone plays both androgenic and estrogenic actions due to its conversion into estrogen via aromatase naturally, it is unclear that the age-related reduction of testosterone increased risk of Alzheimer's disease (AD) in men is mediated through androgen alone or both androgen and estrogen mechanisms. Our previous studies using a gene-based approach in mouse model to block the conversion of testosterone into estrogen (aromatase gene knock-out, ArKO), found a depletion of estrogen and increase in testosterone endogenously in males. Here, we use crossing the ArKO mice with APP23 transgenic mice, a mouse model of AD, to produce APP23/Ar(+/-) mice to study the estrogen-independent effect of testosterone on AD. We found a significant reduction in brain plaque formation, improved cognitive function and increase NEP activity in male APP23/Ar(+/-) mice compared with age-matched male APP23 controls. In addition, we found, for the first time, a reduction of beta-secretase (BACE1) enzyme activity, mRNA level and protein expression in the male APP23/Ar(+/-) mice, suggesting that endogenous testosterone, independent from estrogen, may protect against AD in males via two major mechanisms, downregulation of BACE1 activities at transcriptional level to reduce beta amyloid production and upregulation of NEP activities to enhance bate amyloid degradation.

Retracted: Dynorphin inhibits NEP activity in R1.1 mouse thymoma cells

NEP/CALLA or CD10 is an endopeptidase (E.C. 3.4.24.11) that inactivates numerous neuropeptides, including dynorphin. Dynorphin is an endogenous opioid polypeptide that binds to kappa-opioid receptors with greatest affinity. R1.1 mouse thymoma cells highly express kappa-opioid receptors. In this study, on R1.1 cells, NEP activity was inhibited by kappa-opioid polypeptide dynorphin (10(-8)-10(-6) M) and by thiorphan (2 x 10(-4) M), a known inhibitor of NEP (30 min treatment). NEP inhibition by dynorphin was stronger than by thiorphan. A non-opioid opioid mechanism of action was mostly involved in this inhibition.

Gene cloning and heterologous synthesis of a haloalkaliphilic extracellular protease of Natrialba magadii (Nep)

The gene encoding the protease Nep secreted by the haloalkaliphilic archaeon Natrialba magadii was cloned and sequenced. Upstream of the nep gene, a region related to haloarchaeal TATA-box and BRE-like consensus sequences was identified. The nep-encoded polypeptide had a molecular mass of 56.4 kDa, a pI of 3.77 and included a 121-amino acid propeptide not present in the mature Nep. A Tat motif (GRRSVL) was also identified at residues 10-15 suggesting it is a substrate of the Tat pathway. The primary sequence of Nep was closely related to serine proteases of the subtilisin family from archaea and bacteria (50-85% similarity). The nep gene was expressed in Escherichia coli and Haloferax volcanii resulting in production of active Nep protease. In contrast to the recombinant E. coli strains in which Nep activity was only detected in cell lysate, high levels of Nep protein and activity were detected in the culture medium of stationary phase recombinant Hfx. volcanii strains. The Hfx. volcanii synthesized protease was active in high salt, high pH and high DMSO. This study provides the first molecular characterization of a halolysin-like protease from alkaliphilic haloarchaea and is the first description of a recombinant system that facilitates high-level secretion of a haloarchaeal protease.

Biosynthesis, processing, trafficking, and enzymatic activity of mouse neprilysin 2

Neprilysin 2 (NEP2) has been recently identified as a new member of the M13 subfamily of zinc-dependent metalloproteases and shares a highly homologous amino acid sequence with neprilysin (EC 3.4.24.11, NEP). NEP2 has been reported to exist as membrane-bound and soluble secreted variants. To investigate mechanisms of regulating NEP2 activity, we developed a simple and sensitive method for measuring NEP2 activity using synthetic substrates with a fluorescent probe. NEP2 only cleaved Suc-Ala-Ala-Phe-AMC, while NEP cleaved both Dansyl-D-Ala-Gly-p-nitro-Phe-Gly and Suc-Ala-Ala-Phe-AMC. Using HEK293 cells stably expressing mouse NEP2, we evaluated the effects of various reagents affecting post-translational modification and protein trafficking on extracellular NEP2 activity secreted into the culture medium. Inhibition of N-glycosylation by tunicamycin reduced both the enzymatic activity of extracellular NEP2 and the molecular size of intracellular NEP2. Disruption of the Golgi apparatus with brefeldin A markedly reduced extracellular NEP2 activity in parallel with intracellular NEP2 protein level in HEK293 cells. In contrast, the cytoskeleton disrupting reagents, nocodazole and cytochalasin B barely affected NEP2 activity. Two distinct calcium-perturbing reagents, a calcium ionophore A23187 and thapsigargin, reduced extracellular NEP2 activity. However, A23187-mediated down-regulation was not rescued by co-treatment with inhibitors of MAPK, calmodulin, or the proteasome/calpains. In conclusion, we established a simple and sensitive protocol which was able to discriminate NEP2 and NEP activity, and showed that intracellular transport and secretion of NEP2 is regulated by processes such as glycosylation, ER-Golgi transport, and intracellular calcium levels.

Anti-Progressive Effect of Neutral Endopeptidase 24.11 (NEP/CD10) on Cervical Carcinoma in vitro and in vivo

Objectives: Neutral endopeptidase 24.11 (NEP) is known to play important roles in the maintenance of homeostasis or in neoplastic transformation and tumor progression in certain human malignancies through the enzymatic inactivation of bioactive peptides such as endothelin-1 (ET-1), angiotensin-II, and bombesin. Methods: In this study, we first investigated NEP expression in cervical carcinoma by immunohistochemical staining and Western blot analysis. Next, we examined NEP functions in vitro and in vivo by generating NEP-overexpressing cervical carcinoma cells. Results: We found a significant decrease in cellular proliferative and invasive abilities with a reduced ET-1 concentration in the conditioned medium by NEP overexpression in cervical carcinoma CaSki cells, which have an ET-1 autocrine loop. In addition, these potentials were cancelled by blockade of NEP activity with a specific inhibitor. Although vector-transfected CaSki cells could grow even in serum-free media, NEP-overexpressing cells failed to proliferate in these media. Furthermore, we demonstrated that NEP suppressed tumor formation of subcutaneous xenografts using nude mice. Conclusions: Our results indicated that NEP functions as a tumor-suppressor gene in cervical carcinoma cells, and its expression may have prognostic significance. Further elucidation of the mechanism underlying the observed effect of NEP will contribute to a better understanding of its role in the pathophysiology of cervical carcinoma.

Substance P and neutral endopeptidase in development of acute respiratory distress syndrome following fire smoke inhalation.

To characterize the tachykininergic effects in fire smoke (FS)-induced acute respiratory distress syndrome (ARDS), we designed a series of studies in rats. Initially, 20 min of FS inhalation induced a significant increase of substance P (SP) in bronchoalveolar lavage fluid (BALF) at 1 h and persisted for 24 h after insult. Conversely, FS disrupted 51.4, 55.6, 46.3, and 43.0% enzymatic activity of neutral endopeptidase (NEP, a primary hydrolyzing enzyme for SP) 1, 6, 12, and 24 h after insult, respectively. Immunolabeling density of NEP in the airway epithelium largely disappeared 1 h after insult due to acute cell damage and shedding. These changes were also accompanied by extensive influx of albumin and granulocytes/lymphocytes in BALF. Furthermore, levels of BALF SP and tissue NEP activity dose dependently increased and decreased, respectively, following 0, low (10 min), and high (20 min) levels of FS inhalation. However, neither the time-course nor the dose-response study observed a significant change in the highest affinity neurokinin-1 receptor (NK-1R) for SP. Finally, treatment (10 mg/kg im) with SR-140333B, an NK-1R antagonist, significantly prevented 20-min FS-induced hypoxemia and pulmonary edema 24 h after insult. Further examination indicated that SR-140333B (1.0 or 10.0 mg/kg im) fully abolished early (1 h) plasma extravasation following FS. Collectively, these findings suggest that a combination of sustained SP and NEP inactivity induces an exaggerated neurogenic inflammation mediated by NK-1R, which may lead to an uncontrolled influx of protein-rich edema fluid and cells into the alveoli as a consequence of increased vascular permeability.

Neprilysin levels in plasma and synovial fluid of juvenile idiopathic arthritis patients.

Neprilysin (neutral endopeptidase, 3:4:24:11, CD10) (NEP) is a Zn metallopeptidase linked to controlling inflammation through the degradation of neuropeptides involved in neurogenic inflammation of chronic rheumatic diseases. The aim of our study was to evaluate circulating activity and cellular expression of NEP in the plasma of 58 children with juvenile idiopathic arthritis (JIA) and 52 controls. In 20 subjects requiring local steroid injection, NEP was measured in synovial fluid. Plasma and synovial NEP were evaluated using a fluorimetric technique. Neprilysin, expressed as the antigen CD10, was determined on circulating and synovial fluid cells as mean fluorescence intensity (MFI) and as percentage of positive cells by two-color immunofluorescence. Circulating NEP levels were lower in JIA patients than in controls (42.0+/-16.6 vs 76.5+/-24 pmol/ml per min, P<0.001), while synovial fluid NEP values were higher than circulating levels (241.4+/-86.2 vs 40+/-15.3 pmol/ml per min, P<0.001). In monocytes, the percentage of CD10-positive circulating cells and the MFI in JIA were lower than in controls (11.6+/-5.2% vs 41.4+/-13%, P<0.001 and 18.1+/-7.5 vs 31.2+/-5.4, P<0.05, respectively). On synovial monocytes, the percentage of CD10-positive cells and the MFI were higher than on circulating monocytes (35.2+/-14.6% vs 9.1+/-2.4%, P<0.001 and 66.4+/-5.4 vs 22.8+/-14.7, P<0.001, respectively). The downregulation of CD10 expression in monocytes and the reduction in NEP activity may be linked to the enzyme's role in the control of peptides involved in the inflammation. The increased levels of NEP, MFI, and CD10-positive monocytes in synovial fluid, even though in plasma, might reflect a reactive effort to control synovial proliferation.