Neutral Endopeptidase Research Articles

Calcium‑dependent activation of PHEX, MEPE and DMP1 in osteocytes

Calcium (Ca2+) signaling is the first messenger signal exhibited by osteocytes. The present study aimed to better understand the link between Ca2+ concentration, and the levels of bone mineralization regulator proteins [phosphate‑regulating neutral endopeptidase on chromosome X (PHEX), matrix extracellular phosphoglycoprotein (MEPE) and dentin matrix protein 1 (DMP1)] and the levels of oxidative stress in osteocytes. The viability of MLO‑Y4 cells was determined using the live/dead assay following treatment with various Ca2+ concentrations (1.8, 6, 12, 18, 24 and 50mM) for different durations (15 and 60min, and 24h). Superoxide dismutase (SOD), catalase (CAT), glutathione (GSH) and NADPH oxidase (NOX) enzymes were analyzed using a colorimetric method. Apoptosis was detected by caspase‑3 analysis. Furthermore, the protein expression levels of PHEX, MEPE and DMP1 were analyzed using immunoblotting, and oxidative stress was examined using the total antioxidant and total oxidant status (TOS) assay. Notably, after 15min, there were more live cells than dead cells; however, after 60min, the number of dead cells was increased following treatment with 24 and 50mM Ca2+. After 24h, there were more dead cells than live cells following treatment with 50mM Ca2+. After 24h of Ca2+ treatment, the highest protein expression levels of PHEX, MEPE and DMP1 were measured in cells treated with 24mM Ca2+. In addition, as Ca2+ concentration increased, the TOS and the oxidative stress index values were also increased. In conclusion, these results suggested that 24mM Ca2+ may trigger bone mineralization proteins, such as PHEX, MEPE and DMP1, and could be considered an applicable dosage for the treatment of bone damage in the future.

Molecular Mechanism of Blood Pressure Regulation through the Atrial Natriuretic Peptide.

Simple SummaryAtrial natriuretic peptide (ANP) is a cardiac peptide hormone that was identified by Kangawa and Matsuo in 1984. In Japan, ANP has been used as an intravenous drug for the treatment of acute heart failure since 1995. Because ANP has a hypotensive effect, it is important to avoid excessive lowering of blood pressure when ANP is used. Recently, a compound that inhibits neutral endopeptidase, the enzyme that degrades ANP, has been developed (angiotensin receptor-neprilysin inhibitor (ARNI)). ARNI has been approved worldwide for the treatment of chronic heart failure and has been authorized in Japan as an antihypertensive drug. However, it is not understood exactly how ANP exerts its hypotensive effect. In this review, we discuss the molecular mechanism of the blood pressure-regulating effects of ANP, focusing on our recent findings.Natriuretic peptides, including atrial natriuretic peptide (ANP), brain natriuretic peptide (BNP), and C-type natriuretic peptide (CNP), have cardioprotective effects and regulate blood pressure in mammals. ANP and BNP are hormones secreted from the heart into the bloodstream in response to increased preload and afterload. Both hormones act through natriuretic peptide receptor 1 (NPR1). In contrast, CNP acts through natriuretic peptide receptor 2 (NPR2) and was found to be produced by the vascular endothelium, chondrocytes, and cardiac fibroblasts. Based on its relatively low plasma concentration compared with ANP and BNP, CNP is thought to function as both an autocrine and a paracrine factor in the vasculature, bone, and heart. The cytoplasmic domains of both NPR1 and NPR2 display a guanylate cyclase activity that catalyzes the formation of cyclic GMP. NPR3 lacks this guanylate cyclase activity and is reportedly coupled to Gi-dependent signaling. Recently, we reported that the continuous infusion of the peptide osteocrin, an endogenous ligand of NPR3 secreted by bone and muscle cells, lowered blood pressure in wild-type mice, suggesting that endogenous natriuretic peptides play major roles in the regulation of blood pressure. Neprilysin is a neutral endopeptidase that degrades several vasoactive peptides, including natriuretic peptides. The increased worldwide clinical use of the angiotensin receptor-neprilysin inhibitor for the treatment of chronic heart failure has brought renewed attention to the physiological effects of natriuretic peptides. In this review, we provide an overview of the discovery of ANP and its translational research. We also highlight our recent findings on the blood pressure regulatory effects of ANP, focusing on its molecular mechanisms.

Membranous nephropathy: Systems biology-based novel mechanism and traditional Chinese medicine therapy.

Membranous nephropathy (MN) is a renal-limited non-inflammatory autoimmune disease in the glomerulus, which is the second or third main cause of end-stage kidney diseases in patients with primary glomerulonephritis. Substantial achievements have increased our understanding of the aetiology and pathogenesis of murine and human MN. The identification of nephritogenic autoantibodies against neutral endopeptidase, phospholipase A2 receptor (PLA2R) and thrombospondin type-1 domain-containing 7A (THSD7A) antigens provide more specific concept-driven intervention strategies for treatments by specific B cell-targeting monoclonal antibodies to inhibit antibody production and antibody-antigen immune complex deposition. Furthermore, additional antibody specificities for antigens have been discovered, but their pathogenic effects are uncertain. Although anti-PLA2R and anti-THSD7A antibodies as a diagnostic marker is widely used in MN patients, many questions including autoimmune response development, antigenic epitopes, and podocyte damage signalling pathways remain unresolved. This review describes the current available evidence regarding both established and novel molecular mechanisms based on systems biology approaches (gut microbiota, long non-coding RNAs, metabolite biomarkers and DNA methylation) in MN, with an emphasis on clinical findings. This review further summarizes the applications of traditional Chinese medicines such as Tripterygium wilfordii and Astragalus membranaceus for MN treatment. Lastly, this review considers how the identification of novel antibodies/antigens and unresolved questions and future challenges reveal the pathogenesis of MN.

Central administration of human opiorphin alleviates dextran sodium sulfate-induced colitis in mice through activation of the endogenous opioid system.

The opioid system plays a crucial role in maintaining gastrointestinal homeostasis. Endogenous opioid peptide enkephalins have anti-inflammatory effect and participate in the treatment of inflammatory bowel diseases (IBDs). Here, we investigated the effect of natural enkephalinase inhibitor human opiorphin (HO) on dextran sodium sulfate (DSS)-induced colitis in mice. Our results showed that central administration of HO attenuated DSS-induced colitis, as indicated by the reduction of disease activity index (DAI) scores, macroscopic scores, histological scores, and the myeloperoxidase (MPO) activity. Moreover, HO alleviated DSS-induced inflammation by decreasing inflammatory cytokines TNF-α, IL-6, and IL-1β, and increasing anti-inflammatory cytokine IL-10 in both serum and colon tissues in DSS-treated mice. The potential anti-inflammatory effect of HO at a dose of 40 μg/kg was observed as evidenced by a decrease in nuclear factor κB (NF-κB) p65, toll-like receptor-4 (TLR-4), iNOS, and COX-2. HO also improved intestinal barrier function by enhancing the expression of tight junction proteins. Furthermore, HO treatment significantly inhibited activities of neutral endopeptidase (NEP) and aminopeptidase N (APN), elevated serum enkephalins concentrations, and increased expressions of mu and delta opioid receptors. In addition, pretreatment with opioid receptor antagonist naloxone hydrochloride (NH) compromised the protective effect of HO and aggravated colitis symptoms, as indicated by inhibited anti-inflammatory effects, disrupted intestinal barrier function, and decreased opioid receptor activity. In conclusion, these data indicate that HO protects against DSS-induced colitis by inhibiting TLR4/NF-κB pathway activation and improving intestinal barrier function through activation of the endogenous opioid system. Therefore, targeting the opioid system with peptidase inhibitors intervention would be a novel strategy in the therapy of IBD.

Abstract P252: Differential Expression Of ACE2, Neprilysin And ACE In Extracellular Vesicles Isolated From Human Urine

The Ang II convertase ACE2 is present in urine and may correlate with various renal pathologies. We reported functional ACE2 in urinary exosomes and now extend our studies to distinguish exosomes (EXOs) and microvesicles (MVs) containing ACE2, as well as neprilysin (NEP) and ACE. EXO purification was modified by an intermediate centrifugation step (20k xg, 60 mins) to pellet MVs followed by 100k xg to pellet EXOs. The MV, EXO, and 100k supernatant (soluble, SOL) fractions were assessed for ACE2 activity with MCA-APK-DNP, and NEP and ACE activities with MCA-RPPGFSAFK-Dnp with selective inhibitors. Peptidase activities were expressed as relative fluorescent units (RFU x10 6 /min/mL), and data are means ± SEM (*P<0.05; **P<0.01, N=4). Morning collections from male subjects [51 to 65 years of age, non-smokers] were collected, treated with Cibacron blue to bind albumin, 0.2 μM filtration, and 100 kDa Amicon concentration prior to high speed and ultracentrifugation steps. ACE2 activity was >3-fold higher in the EXO versus the MV fractions with minor activity in the SOL fraction. In contrast, NEP activity was highest in the MV fraction [~2-fold > EXO] while endopeptidase activity was barely detectable in the SOL fraction. ACE activity was similar in MVs and EXOs, but NEP activity was 40-fold higher than ACE in both the MV and EXO fractions. Differential centrifugation of the 100-kDa urine concentrate reveals a unique profile of ACE2, NEP and ACE expression in MVs and EXOs. The functional significance of these peptidase activities in different vesicular populations is currently unknown; however, their origin may reflect the distinct regulation and release of ACE2 and NEP/ACE in urinary vesicles.

Efficacy of vosoritide in the treatment of achondroplasia.

Achondroplasia is the commonest form of dwarfism and results from a mutation in the fibroblast growth factor receptor 3 (FGFR3) gene on chromosome 4p16.3. The mutation is at nucleotide 1138 resulting in a G-to-A transition (134934.0001). This condition is characterized by full penetration meaning that everyone with this genetic mutation will exhibit the phenotypic characteristics of achondroplasia. It is a gain-of function mutation that causes increased inhibition of cartilage formation. C-type natriuretic peptide (CNP) acts on the growth plate through the natriuretic peptide receptor-B (NPR-B) causing the transformation of guanosine 5'-triphosphate into cyclic guanosine monophosphate. However, CNP cannot be used in the treatment of achondroplasia because it is rapidly degraded by neutral endopeptidase. Vosoritide is a modified recombinant human CNP and has a half-life 10 times that of CNP. Clinical trials have demonstrated that vosoritide is effective in significantly increasing the annualized growth velocity in children with achondroplasia before the fusion of the epiphyses.

The Potential Role of Serum Tau Protein (MAPT), Neuronal Cell Adhesion Molecule (NrCAM) and Neprilysin (NEP) in Neurodegenerative Disorders Development in Psoriasis-Preliminary Results.

Psoriasis is one of the most common dermatoses, which shortens patients’ lives because of the wide comorbidity. However, little is known about its association with neurodegenerative diseases (NDs). We aimed to investigate whether psoriatics are at increased risk of NDs. Sixty patients with plaque-type psoriasis were enrolled into the study. Serum concentrations of tau protein (MAPT), neuronal cell adhesion molecule (NrCAM) and neprilysin (NEP), which are NDs biomarkers and have been hardly studied in psoriasis before, were measured before and after 12 weeks of treatment with acitretin or methotrexate. NrCAM and NEP concentrations were significantly lower in patients than controls, whereas MAPT higher (all p < 0.05). There was no association between these markers and psoriasis severity, BMI or disease duration. After the treatment the concentration of NrCAM and NEP significantly increased and MAPT decreased (p < 0.001, p < 0.05, p < 0.01, respectively). Methotrexate had significant influence on the concentrations of all markers, hence it seems to have neuroprotective properties. Psoriasis severity and duration do not seem to affect the risk of neurodegenerative process. Our results suggest that NDs could be considered as another comorbidity of psoriasis and that further research are needed in order to establish their definite association.

In Vitro Evaluation of In Silico Screening Approaches in Search for Selective ACE2 Binding Chemical Probes.

New models for ACE2 receptor binding, based on QSAR and docking algorithms were developed, using XRD structural data and ChEMBL 26 database hits as training sets. The selectivity of the potential ACE2-binding ligands towards Neprilysin (NEP) and ACE was evaluated. The Enamine screening collection (3.2 million compounds) was virtually screened according to the above models, in order to find possible ACE2-chemical probes, useful for the study of SARS-CoV2-induced neurological disorders. An enzymology inhibition assay for ACE2 was optimized, and the combined diversified set of predicted selective ACE2-binding molecules from QSAR modeling, docking, and ultrafast docking was screened in vitro. The in vitro hits included two novel chemotypes suitable for further optimization.

Cathepsin B Dipeptidyl Carboxypeptidase and Endopeptidase Activities Demonstrated across a Broad pH Range.

Cathepsin B is a lysosomal protease that participates in protein degradation. However, cathepsin B is also active under neutral pH conditions of the cytosol, nuclei, and extracellular locations. The dipeptidyl carboxypeptidase (DPCP) activity of cathepsin B, assayed with the Abz-GIVR↓AK(Dnp)-OH substrate, has been reported to display an acidic pH optimum. In contrast, the endopeptidase activity, monitored with Z-RR-↓AMC, has a neutral pH optimum. These observations raise the question of whether other substrates can demonstrate cathepsin B DPCP activity at neutral pH and endopeptidase activity at acidic pH. To address this question, global cleavage profiling of cathepsin B with a diverse peptide library was conducted under acidic and neutral pH conditions. Results revealed that cathepsin B has (1) major DPCP activity and modest endopeptidase activity under both acidic and neutral pH conditions and (2) distinct pH-dependent amino acid preferences adjacent to cleavage sites for both DPCP and endopeptidase activities. The pH-dependent cleavage preferences were utilized to design a new Abz-GnVR↓AK(Dnp)-OH DPCP substrate, with norleucine (n) at the P3 position, having improved DPCP activity of cathepsin B at neutral pH compared to the original Abz-GIVR↓AK(Dnp)-OH substrate. The new Z-VR-AMC and Z-ER-AMC substrates displayed improved endopeptidase activity at acidic pH compared to the original Z-RR-AMC. These findings illustrate the new concept that cathepsin B possesses DPCP and endopeptidase activities at both acidic and neutral pH values. These results advance understanding of the pH-dependent cleavage properties of the dual DPCP and endopeptidase activities of cathepsin B that function under different cellular pH conditions.

Effect of moxibustion with deqi on Aβ-receptor mediated transport and enzymatic degradation in hippocampus in rats with Alzheimer's disease

To observe the clinical effect of moxibustion with deqi on Alzheimer's disease (AD) rats, and evaluate its effect on β-amyloid (Aβ) transport and enzymatic degradation proteins, to explore its molecular mechanism for improving cognitive function. Sixty SPF-grade male SD rats were randomly divided into a blank group (8 rats), a sham-operation group (8 rats) and a model establishment group (44 rats). The rats in the model establishment group were injected with Aβ1-42 at bilateral ventricles to establish AD model. Among the 38 rats with successful model establishment, 8 rats were randomly selected as the model group, and the remaining rats were treated with mild moxibustion at "Dazhui" (GV 14), once a day, 40 min each time, for 28 days. According to whether deqi appeared and the occurrence time of deqi, the rats were divided into a deqi group (12 rats), a delayed deqi group (10 rats) and a non-deqi group (8 rats). After the intervention, the Morris water maze test was applied to evaluate the cognitive function; the HE staining was applied to observe the brain morphology; the Western blot method was applied to measure the protein expression of Aβ and its receptor mediated transport [low-density lipoprotein receptor-related protein (LRP) 1, receptor for advanced glycation end products (RAGE), apolipoprotein E (ApoE)] and enzymatic degradation [neprilysin (NEP), insulin degrading enzyme (IDE), endothelin converting enzyme (ECE)-1 and angiotensin converting enzyme (ACE) 2]. Compared with the sham-operation group, in the model group, the escape latency was prolonged (P<0.01), and the times of platform crossing and the ratio of platform quadrant to total time were reduced (P<0.01); the brain tissue was seriously damaged; the expression of hippocampal Aβ and RAGE was increased (P<0.01), and the expression of hippocampal LRP1, ApoE, NEP, IDE, ECE-1 and ACE2 was decreased (P<0.01). Compared with the model group, the escape latency was shortened in the deqi group (P<0.05, P<0.01), and the escape latency in the delayed deqi group and the non-deqi group was shortened from Day 2 to Day 5 (P<0.05, P<0.01), and the times of platform crossing and the ratio of platform quadrant to total time were increased in the deqi group and the delayed deqi group (P<0.01, P<0.05); the brain damage in each moxibustion group was reduced, which was smallest in the deqi group, followed by the delayed deqi group and the non-deqi group; the expression of Aβ and RAGE was decreased (P<0.01, P<0.05) and the expression of LRP1 and IDE was increased in each moxibustion group (P<0.01, P<0.05); the expression of ApoE was increased in the deqi group and the delayed deqi group (P<0.01, P<0.05); the expression of NEP was increased in deqi group (P<0.05), and the expression of ECE-1 and ACE2 was increased in the deqi group and the delayed deqi group (P<0.05). Compared with the delayed deqi group and the non-deqi group, the escape latency in the deqi group was shortened from Day 3 to Day 5 (P<0.05), and the times of platform crossing and the ratio of platform quadrant to total time were increased (P<0.05, P<0.01). Compared with the non-deqi group, the expression of Aβ was reduced (P<0.05), the expression of LRP1 and ApoE was increased in the deqi group (P<0.05). The expression of NEP in the deqi group was higher than that in the delayed deqi group and the non-deqi group (P<0.05). Compared with non-deqi, moxibustion with deqi could promote Aβ transport and degradation, thereby reducing Aβ level in the brain and improving cognitive function for AD rats.

Induction of ICAM1 in Brain Vessels is Implicated in an Early AD Pathogenesis by Modulating Neprilysin.

Intercellular adhesion molecule 1 (ICAM1) is a vessel adhesion protein induced during brain vascular inflammation, which could be closely linked with the development of Alzheimer's disease (AD). This study investigated the effect of ICAM1 on amyloid-degrading enzymes (ADEs) in endothelial cells and their potential involvement in inflammation and AD progression. TNF-α treatment increased ICAM1 in human brain microvascular endothelial cells (HBMVECs) but decreased the neprilysin (NEP) protein level. Knock-down of ICAM1 using siRNA enhanced NEP, which increased the degradation of amyloid-β. In the brains of 4-month-old AD transgenic mice (APPswe/PSEN1dE9), there were significantly higher levels of ICAM1 expression and amyloid deposits but lower levels of NEP and insulin-degrading enzymes (IDE), demonstrating an inverse correlation of ICAM1 with NEP and IDE expression. Further studies demonstrated significantly increased GFAP protein levels in the brain, specifically localized near blood vessels, of both TNF-α-injected and 4-month-old AD transgenic mice. Taken together, the induction of ICAM1 in endothelial cells suppresses NEP expression, accelerating the accumulation of amyloid-β in blood vessels. It also enhances leukocyte adhesion to blood vessels stimulating the migration of leukocytes into the brain, subsequently triggering brain inflammation.

Actions of Novel Angiotensin Receptor Blocking Drugs, Bisartans, Relevant for COVID-19 Therapy: Biased Agonism at Angiotensin Receptors and the Beneficial Effects of Neprilysin in the Renin Angiotensin System.

Angiotensin receptor blockers (ARBs) used in the treatment of hypertension and potentially in SARS-CoV-2 infection exhibit inverse agonist effects at angiotensin AR1 receptors, suggesting the receptor may have evolved to accommodate naturally occurring angiotensin ‘antipeptides’. Screening of the human genome has identified a peptide (EGVYVHPV) encoded by mRNA, complementary to that encoding ANG II itself, which is an inverse agonist. Thus, opposite strands of DNA encode peptides with opposite effects at AR1 receptors. Agonism and inverse agonism at AR1 receptors can be explained by a receptor ‘switching’ between an activated state invoking receptor dimerization/G protein coupling and an inverse agonist state mediated by an alternative/second messenger that is slow to reverse. Both receptor states appear to be driven by the formation of the ANG II charge-relay system involving TyrOH-His/imidazole-Carboxylate (analogous to serine proteases). In this system, tyrosinate species formed are essential for activating AT1 and AT2 receptors. ANGII is also known to bind to the zinc-coordinated metalloprotease angiotensin converting enzyme 2 (ACE2) used by the COVID-19 virus to enter cells. Here we report in silico results demonstrating the binding of a new class of anionic biphenyl-tetrazole sartans (‘Bisartans’) to the active site zinc atom of the endopeptidase Neprilysin (NEP) involved in regulating hypertension, by modulating humoral levels of beneficial vasoactive peptides in the RAS such as vasodilator angiotensin (1–7). In vivo and modeling evidence further suggest Bisartans can inhibit ANG II-induced pulmonary edema and may be useful in combatting SARS-CoV-2 infection by inhibiting ACE2-mediated viral entry to cells.

Sustained high body temperature exacerbates cognitive function and Alzheimer’s disease-related pathologies

Global warming is a serious public health threat to people worldwide. High body temperature is one of the important risk factors for Alzheimer’s disease (AD), and the body temperature of AD patients has been found to be significantly higher than that of elderly control subjects. However, the effects of high body temperature on cognitive function and AD pathologies have not been completely elucidated. We report here that Tg2576 mice housed at a high ambient temperature of 30 °C for 13 months showed an increase in the body temperature, which is accompanied by memory impairment and an enhancement of amyloid-β peptides (Aβ) generation through the upregulation of β-site APP cleaving enzyme 1 (BACE1) level and decrease in the level of an Aβ-degrading enzyme, neprilysin (NEP) in the brain, compared with those of Tg2576 mice at 23 °C. High body temperature also increased the levels of heat shock proteins (HSPs), stress-stimulated kinases such as JNK, and total tau, leading to the enhancement of tau phosphorylation at 30 °C. Taken together, our findings suggest that high body temperature exacerbates cognitive function and AD pathologies, which provides a mechanistic insight for its prevention.

Seminal-Plasma-Mediated Effects on Sperm Performance in Humans.

Seminal plasma (SP) plays a crucial role in reproduction and contains a large number of proteins, many of which may potentially modify sperm functionality. To evaluate the effects of SP identity and its protein composition on human sperm function, we treated the sperm of several males with either their own or multiple foreign SPs in all possible sperm–SP combinations (full-factorial design). Then we recorded sperm motility and viability in these combinations and investigated whether the sperm performance is dependent on sperm and SP identity (or their interaction). Finally, we studied whether the above-mentioned sperm traits are affected by the abundance of three SP proteins, dipeptidyl peptidase-4 (DPP4), neutral endopeptidase (NEP), and aminopeptidase N (APN). The identity of the SP donor affected sperm swimming velocity, viability, and the proportion of hyperactivated sperm, but males’ own SP was not consistently more beneficial for sperm than foreign SPs. Furthermore, we show that sperm performance is also partly affected by the interaction between sperm and SP donor. Finally, we found that DPP4 and NEP levels in SP were positively associated with sperm swimming velocity and hyperactivation. Taken together, our results highlight the importance of seminal plasma as a potential source of biomarkers for diagnostics and therapeutic interventions for male-derived infertility.

Roles of Natriuretic Peptides and the Significance of Neprilysin in Cardiovascular Diseases.

Simple SummaryThe endocrine effects of atrial natriuretic peptide (ANP) and brain natriuretic peptide (BNP) in the vasculature, and the autocrine effects of ANP and BNP in cardiomyocytes are mediated by the common guanylyl cyclase A receptor (GC-A) expressed in various tissues and cell types. C-type natriuretic peptide (CNP) has paracrine actions that regulate vascular resistance and moderate myocardial stiffness via guanylyl cyclase B receptor (GC-B). Genetically modified mice have revealed the physiological roles of ANP and BNP in blood pressure, cardiac remodeling, and acute myocardial infarction. Molecular pathways in GC-A signaling specifically in cardiomyocytes were also investigated. ANP and BNP via the GC-A signaling phosphorylate regulator of G-protein signaling subtype 4 (RGS4) result in the inhibition of Gαq signaling coupled with angiotensin II type 1A receptor, inhibit the activation of transient receptor potential C6 (TRPC6), and attenuate genomic actions of the cardiac mineralocorticoid receptor (MR). Moreover, recent studies showed the physiological roles of CNP via GC-B in blood pressure and cardiac stiffness. Since natriuretic peptides are degraded by neprilysin (NEP), inhibiting NEP activity is expected to enhance the actions of natriuretic peptides. Experimental studies and clinical trials have shown the effect of NEP inhibition on cardiac remodeling, acute myocardial infarction, and hypertension.Atrial natriuretic peptide (ANP) and brain natriuretic peptide (BNP) activate the guanylyl cyclase A receptor (GC-A), which synthesizes the second messenger cGMP in a wide variety of tissues and cells. C-type natriuretic peptide (CNP) activates the cGMP-producing guanylyl cyclase B receptor (GC-B) in chondrocytes, endothelial cells, and possibly smooth muscle cells, cardiomyocytes, and cardiac fibroblasts. The development of genetically modified mice has helped elucidate the physiological roles of natriuretic peptides via GC-A or GC-B. These include the hormonal effects of ANP/BNP in the vasculature, autocrine effects of ANP/BNP in cardiomyocytes, and paracrine effects of CNP in the vasculature and cardiomyocytes. Neprilysin (NEP) is a transmembrane neutral endopeptidase that degrades the three natriuretic peptides. Recently, mice overexpressing NEP, specifically in cardiomyocytes, revealed that local cardiac NEP plays a vital role in regulating natriuretic peptides in the heart tissue. Since NEP inhibition is a clinically accepted approach for heart failure treatment, the physiological roles of natriuretic peptides have regained attention. This article focuses on the physiological roles of natriuretic peptides elucidated in mice with GC-A or GC-B deletion, the significance of NEP in natriuretic peptide metabolism, and the long-term effects of angiotensin receptor-neprilysin inhibitor (ARNI) on cardiovascular diseases.

Effects of BNP and Sacubitrilat/Valsartan on Atrial Functional Reserve and Arrhythmogenesis in Human Myocardium.

BackgroundAlthough the angiotensin receptor-neprilysin inhibitor (ARNI) sacubitril/valsartan started a new era in heart failure (HF) treatment, less is known about the tissue-level effects of the drug on the atrial myocardial functional reserve and arrhythmogenesis.Methods and ResultsRight atrial (RA) biopsies were retrieved from patients (n = 42) undergoing open-heart surgery, and functional experiments were conducted in muscle strips (n = 101). B-type natriuretic peptide (BNP) did not modulate systolic developed force in human myocardium during β-adrenergic stimulation, but it significantly reduced diastolic tension (p < 0.01) and the probability of arrhythmias (p < 0.01). In addition, patient's plasma NTproBNP positively correlated with isoproterenol-induced contractile reserve in atrial tissue in vitro (r = 0.65; p < 0.01). Sacubitrilat+valsartan (Sac/Val) did not show positive inotropic effects on atrial trabeculae function but reduced arrhythmogeneity. Atrial and ventricular biopsies from patients with end-stage HF (n = 10) confirmed that neprilysin (NEP) is equally expressed in human atrial and ventricular myocardium. RA NEP expression correlates positively with RA ejection fraction (EF) (r = 0.806; p < 0.05) and left ventricle (LV) NEP correlates inversely with left atrial (LA) volume (r = −0.691; p < 0.05).ConclusionBNP ameliorates diastolic tension during adrenergic stress in human atrial myocardium and may have positive long-term effects on the inotropic reserve. BNP and Sac/Val reduce atrial arrhythmogeneity during adrenergic stress in vitro. Myocardial NEP expression is downregulated with declining myocardial function, suggesting a compensatory mechanism in HF.

Identification of potential hub genes for the diagnosis and therapy of dilated cardiomyopathy with heart failure through bioinformatics analysis

Dilated cardiomyopathy (DCM) is a common cause of heart failure. However, genetic-level treatments are not available for this condition. In this study, we searched for biological markers and therapeutic targets for DCM from a genetic perspective. We chose microarray datasets of idiopathic DCM with heart failure tissues and normal function (NF) heart tissues, which were downloaded from the Gene Expression Omnibus (GEO) database. The differentially expressed genes (DEGs) were analyzed by the GEO2R tool. Gene ontology (GO) and gene set enrichment analysis were used to analyze the functions of DEGs and the pathways in which they are involved. Next, protein-protein interaction networks were built to filter out the hub genes from DEGs. The expression of hub gene was validated by other GEO datasets. Receiver operating characteristic (ROC) curves were plotted to verify the accuracy of the genetic diagnosis. In the end, the mRNA-miRNA-lncRNA network was built to find potentially correlative genes. Twenty-eight common DEGs in total were screened, and GO analysis showed that DEGs were mainly associated with neutrophil degranulation and activation, regulation of Wnt signaling pathway and the development of cardiac cell and tissue. Five hub genes (asporin [ASPN], osteoglycin [OGN], secreted frizzled-related protein 4 [SFRP4], membrane metalloendopeptidase [MME], and natriuretic peptide gene [NPPA]) were shown to be highly expressed in the validation sets and accurate in distinguish between DCM and NF by ROC curves. miRNA prediction of the hub genes revealed that hsa-mir-28b-5p was associated with SFRP4, ASPN, and MME. All of them may serve as biological diagnostic indicators and provide direction for treatment at the genetic level.

New Borane-Protected Derivatives of α-Aminophosphonous Acid as Anti-Osteosarcoma Agents: ADME Analysis and Molecular Modeling, In Vitro Studies on Anti-Cancer Activities, and NEP Inhibition as a Possible Mechanism of Anti-Proliferative Activity.

Many organophosphorus compounds (OPs), especially various α-aminophosphonates, exhibit anti-cancer activities. They act, among others, as inhibitors of the proteases implicated in cancerogenesis. Thesetypes of inhibitors weredescribed, e.g., for neutral endopeptidase (NEP) expressed in different cancer cells, including osteosarcoma (OS). The aim of the present study isto evaluate new borane-protected derivatives of phosphonous acid (compounds 1–7) in terms of their drug-likeness properties, anti-osteosarcoma activities in vitro (against HOS and Saos-2 cells), and use as potential NEP inhibitors. The results revealed that all tested compounds exhibited the physicochemical and ADME properties typical for small-molecule drugs. However, compound 4 did not show capability of blood–brain barrier penetration (Lipiński and Veber rules;SwissAdme tool). Moreover, the α-aminophosphonite-boranes (compounds 4–7) exhibited stronger anti-proliferative activity against OS cells than the other phosphonous acid-borane derivatives (compounds 1–3),especially regarding HOS cells (MTT assay). The most promising compounds 4 and 6 induced apoptosis through the activation of caspase 3 and/or cell cycle arrest at the G2 phase (flow cytometry). Compound 4 inhibited the migration and invasiveness of highly aggressive HOS cells (wound/transwell and BME-coated transwell assays, respectively). Additionally, compound 4 and, to a lesser extent, compound 6 inhibited NEP activity (fluorometric assay). This activity of compound 4 was involved in its anti-proliferative potential (BrdU assay). The present study shows that compound 4 can be considered a potential anti-osteosarcoma agent and a scaffold for the development of new NEP inhibitors.

Analyzing Corin-BNP-NEP Protein Pathway Revealing Differential Mechanisms in AF-Related Ischemic Stroke and No AF-Related Ischemic Stroke.

BackgroundThe incidence of atrial fibrillation (AF)-related stroke increases with aging. Natriuretic peptides (NPs) family, including Corin-B type natriuretic peptide (BNP)-neprilysin (NEP) protein levels increased with age and are risk markers of cardiovascular and cerebrovascular diseases, such as AF and cardioembolic stroke. Aging is also linked to epigenetics, specifically DNA methylation. However, only a few studies have investigated the effect of DNA methylation on the NP system. Thus, the present study aimed to investigate whether the Corin-BNP-NEP protein pathway is involved in the pathogenesis of AF-stroke and CpG methylation in the promoter region of the Corin protein gene has an effect on AF-related ischemic stroke.MethodsA total of 82 patients hospitalized with acute ischemic strokes were enrolled in this study. The differences in clinical information were compared between the AF-stroke (n = 37) and no AF-stroke groups (n = 45). Plasma-soluble Corin and NEP were detected using an ELISA kit. CpG methylation in the promoter region of the gene was assessed by a next-generation sequencing-based bisulfite sequencing polymerase chain reaction (BSP).Results(1) Patients in AF-stroke were older, had higher initial NIHSS score, 90-day mRs, higher D2-dimer, INR, and APTT, and low TG, TC, and HbA1c (all p < 0.05). (2) Serum levels of Corin and BNP in the AF-stroke group were significantly higher than that in the no AF-stroke group (p < 0.05). No significant difference was detected in the serum levels of NEP between the two groups. (3) The levels of CpG methylation in the promoter region of the Corin protein gene in the AF-stroke group was significantly lower than that in the no AF-stroke group (p < 0.05). The CpG sites with maximal methylation differences between the two groups were CORIN:678, CORIN:682, CORIN:694, and CORIN:700.ConclusionThe current findings raise the possibility that the Corin–BNP–NEP protein pathway may be involved in the pathogenesis of AF-related ischemic stroke. Deficient CpG methylation in the promoter region of the Corin protein gene is associated with AF-related ischemic stroke.

Comparative studies for amyloid beta degradation: “Neprilysin vs insulysin”, “monomeric vs aggregate”, and “whole Aβ40 vs its peptide fragments”

Amyloid beta (Aβ) proteins are produced from amyloid precursor protein cleaved by β- and γ-secretases, and are the main components of senile plaques pathologically found in Alzheimer's disease (AD) patient brains. Therefore, the relationship between AD and Aβs has been well studied for both therapeutic and diagnostic purposes. Several enzymes have been reported to degrade Aβs in vivo, with neprilysin (NEP) and insulysin (insulin-degrading enzyme, IDE) being the most prominent. In this article, we describe the mass spectrometric characterization of peptide fragments generated using NEP and IDE, and clarify the differences in digestion specificities between these two enzymes for non-aggregated Aβ40, aggregated Aβ40, and Aβ40 peptide fragments, including Aβ16. Our results allowed identification of all the peptide fragments from non-aggregated Aβ40: NEP, 23 peptide fragments consisting of 2–11 amino-acid residues, 17 cleavage sites; IDE, 23 peptide fragments consisting of 6–33 amino-acid residues, 15 cleavage sites. Also, we confirmed that IDE can digest only whole Aβ40, whereas NEP can digest both Aβ40 and partial structures such as Aβ16 and peptide fragments generated by the digestion of Aβ40 by IDE. Furthermore, we confirmed that IDE and NEP are unable to digest aggregated Aβ40.