Neprilysin Levels Research Articles

Investigate the impact of ICAM-2 and Neprilysin biomarkers in prostate cancer patients infected with JC virus in AL-Najaf AL-Ashraf Province

A total of 74 clinical samples Formalin-Fixed Paraffin-Embedded (FFPE) were collected from patients diagnosed with prostate cancer (PCa) aged between 41 and 90 years and these samples were obtained from patients treated at notable medical institutions like Al-Sadr Medical City and leading clinical laboratories in Al-Najaf City, Iraq, during the period of January to December 2023. The current study indicated the potential role of the JCV virus in provoking prostatitis, which may lead to the emergence and development of prostate cancer in males compared to males who do not suffer from viral infection. The present study showed the presence of JCV virus DNA, as the percentage of positive samples reached (11, 14.864%) compared to negative samples (63, 85.135%). The current study showed a significant increase in the level of ICAM-2 biomarker in patients with JCV-positive prostate cancer, reaching (818.500±42.748 pg/ml) compared with patients with JCV-negative, reaching (502.925±58.037 pg/ml). Neprilysin (NEP) levels in the current study were significantly high in for JCV-positive patients and those with prostate cancer, reaching (4.275±0.376 pg/ml) compared with JCV-negative patients, reaching (3.131±0.232 pg/ml).

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.

Increased Expression of Neprilysin Is Associated with Inflammation in Preeclampsia

Preeclampsia (PE) is associated with a finely tuned equilibrium between trophoblast cell invasion and fetal-maternal immunological tolerance. An imbalance between proinflammatory (IL-6) and anti-inflammatory (IL-10) cytokines is a hallmark of PE. Neprilysin (NEP), a membrane-bound metalloprotease, is vulnerable to the inflammatory environment and plays a significant role in modulating vascular tone. The aim of this study was to determine the correlation between NEP (mRNA and protein) levels and the inflammatory status in PE patients compared to healthy pregnant women and to identify the role of NEP in evaluating the severity of preeclampsia. The study group comprised 52 pregnant women with PE while the control group comprised 47 normotensive pregnant women. After a caesarean section, placental tissue samples from patients and controls were collected to measure the expression levels of IL-6, TGF-β, IL-10, and NEP mRNA. In addition, an enzyme-linked immunosorbent assay was used to assess the quantity of NEP protein in blood samples. Our results revealed a significant positive correlation between NEP (mRNA and protein) and proinflammatory markers IL-6 and TGF-β levels in patients compared to controls and a significant inverse correlation between NEP and anti-inflammatory cytokine IL-10. Moreover, this is the first study to find a strong positive correlation between NEP level and PE severity. In conclusion, in PE patients, there is a substantial relationship between NEP, the degree of inflammation, and PE severity. NEP could act as a potential biomarker for diagnosis and prognosis of PE.

Hippocampal subregion‐specific proteomic differences between Alzheimer disease (AD) and primary age‐related tauopathy (PART)

AbstractBackgroundAlthough Alzheimer disease (AD) and primary age‐related tauopathy (PART) both harbor 3R/4R‐tau immunopositive Alzheimer‐type neurofibrillary degeneration, they differ in the spatial development of neurofibrillary tangles (NFTs) in the hippocampus. PART displays an early predilection for neurofibrillary degeneration in the CA2 subregion of the hippocampus, whereas AD typically develops NFTs in the entorhinal cortex and CA1 subregion initially (Figures 1‐2).MethodUsing Nanostring’s GeoMx™ Digital Spatial Profiling (DSP), we compared hippocampal subregion protein expression differences between AD and PART. The DSP panel allows for spatial analysis of the level of expression of 73 different proteins in multiple regions of interest (ROIs) on formalin‐fixed paraffin embedded sections. Our ROIs were NFT‐bearing neurons and non‐NFT‐bearing neurons in the entorhinal cortex, CA1 and CA2 hippocampal subregions, as well as the neuronal microenvironments in these regions.ResultAnalyses identified several proteins displaying differential expression in tangle‐bearing neurons when comparing AD to PART. These included higher levels of synaptic and neuronal markers in PART, such as Synaptophysin, MAP2, Calbindin and NRGN, as well as higher levels of proteins involved in degradation pathways, such as CTSD, GPNMB, Park7 and FUS. PART also displayed higher levels of Neprilysin in CA2, and higher IDE in CA1, which are proteins that help degrade β‐amyloid (Figure 3). Many of these proteins were also differentially expressed in the non‐tangle bearing neurons. In addition, AD demonstrated higher levels of p‐tau T231, S214, S199, and S396 in non‐tangle bearing neurons of CA1, as well as higher expression of Aβ1‐40. In the microenvironment, NEFL, NRGN, CLEC7a, GPNMB, and CTSD were higher in PART, while p‐tau S396 and BACE1 were higher in AD.ConclusionIn conclusion, PART cases displayed higher levels of neuronal and synaptic markers, suggesting better maintenance of neuronal and synaptic integrity than AD, as well as higher levels of proteins important for degradation of detrimental proteins. AD cases expressed higher levels of certain p‐tau epitopes, especially in and around CA1 non‐tangle bearing neurons. In addition, AD displayed higher levels of BACE1, whereas PART displayed higher levels of CTSD, Neprilysin and IDE, which could explain the differences in β‐amyloid deposition in these cases.

Integrated in-silico and in-vitro assessments of HDAC6 inhibitor efficacy in mitigating amyloid beta pathology in Alzheimer’s disease

Alzheimer’s disease, marked by memory loss and cognitive decline, is associated with amyloid-beta (Aβ) peptide accumulation in the brain. The enzyme neprilysin (NEP), crucial for Aβ degradation, decreases with age and in sporadic Alzheimer’s disease, leading to increased Aβ build-up. This study hypothesized the targeting of enzyme HDAC6, believed to influence NEP activity. An in-silico study was conducted using an FDA-approved drug database, with the focus on their interaction with the HDAC6 structure. Among tested ligands, Panobinostat showed the most favourable interaction with HDAC6. In-vitro experiments on the SH-SY5Y neuronal cell line confirmed these findings, with Panobinostat inhibiting HDAC6, enhancing NEP levels, and reducing Aβ load. The study suggests Panobinostat as a potential Alzheimer’s therapeutic agent, mitigating Aβ accumulation via NEP upregulation. Further research is required for comprehensive understanding and validation. Communicated by Ramaswamy H. Sarma

SAT032 Circulating Neprilysin Activity Is Increased In Type 2 Diabetes And Fatty Liver Disease, And Reduces Following Bariatric Surgery And Liraglutide Therapy

Abstract Disclosure: S.A. Kjeldsen: None. L. Gluud: None. M. Werge: None. J. Pedersen: None. F. Bendtsen: None. K. Alexiadou: None. T. Tan: None. S.S. Torekov: None. E.W. Iepsen: None. N.J. Jensen: None. M. Richter: None. B. Hartmann: None. J.J. Holst: None. B. Holst: None. J. Holt: None. S. Madsbad: None. M.S. Svane: None. K.N. Bojsen-Møller: None. N.J. Wewer Albrechtsen: None. The neutral endopeptidase neprilysin (NEP) cleaves peptides important for metabolism, including GLP-1. Plasma levels of GLP-1 are reduced in patients with T2D, but the reason is unknown. Long-term treatment with the NEP inhibitor sacubitril (in combination with valsartan) improves glycemic control in individuals with T2D and heart failure. Here, we investigate plasma activity and protein levels of NEP in patients with obesity, T2D, and/or NAFLD diagnosed using biopsy or ultrasound, following bariatric surgery (RYGB and sleeve gastrectomy), and after treatment with GLP-1. We hypothesized that NEP activity (NEPa) is increased in patients with T2D, and that GLP-1 agonism reduces NEPa in a weight loss-independent manner. Here we show that plasma levels of NEPa are increased by 51% in patients with T2D (n=33) compared to individuals with normal glucose tolerance (NGT, n=160) (mean ± SD; 2734 ± 3646 vs. 1809 ± 2039 pmol/ml/min, P=0.02). Plasma NEPa was not increased in obesity (n=188) compared to lean controls (n=15) (1944 ± 2392 vs. 1373 ± 1918, P=0.4), but was increased by 76% in NAFLD (n=51) compared to non-NAFLD controls (n=43) (2629 ± 3219 vs. 1497 ± 1404, P=0.02). There were no differences in plasma NEP protein between any of the comparator groups, suggesting that changes in NEPa in metabolic disease are post-translational modifications of the enzyme or other mechanisms influencing NEPa. Bariatric surgery (RYGB, n=66 and SG, n=20) reduced plasma NEPa by 60% (P<0.001), while plasma NEP protein levels were unaltered or increased in individuals with and without preoperative T2D. Six weeks GLP-1 (liraglutide) treatment (3 mg/day) in individuals with overweight and obesity (n=14) reduced NEPa by 28% (P=0.04; apparent after 4 weeks), and at follow-up (3 weeks after discontinuation) NEPa levels returned to baseline, whereas NEP protein levels were unchanged throughout the study. In a study of individuals with obesity randomized to complete a 52-week weight maintenance intervention by diet or liraglutide (1.2 mg/day) following an 8-week diet-induced weight loss period, plasma NEPa was not affected by the initial 12% weight loss (n=48, P=0.3). However, plasma NEPa reduced in response to 52-week liraglutide therapy (n=15, P=0.05). A 90-minute low dose infusion of native GLP-1 (7-36 amide,1 pmol/kg/min) in individuals with overweight and obesity (n=19) did not alter plasma NEPa, which suggests that the attenuating effects on NEPa with GLP-1R agonism is not acute. In conclusion, our data support that plasma NEPa is increased in T2D and NAFLD independent of obesity but may be normalized by bariatric surgery, and that chronic treatment with GLP-1 reduces NEPa. Altered NEPa may therefore contribute to altered metabolism of GLP-1 in diabetes and following bariatric surgery. Whether liraglutide-induced reduction in NEPa contributes to its metabolic effects demands further investigation. Presentation: Saturday, June 17, 2023

Changes in Aβ42, Neprilysin, and γ-Secretase in the Hippocampus of Male Rats Alzheimer’s model: The Effects of Aerobic Training and Omega-3 Intake

Background: Alzheimer›s disease (AD) is characterized by excessive deposition of the amyloid-β peptide (Aβ) in the central nervous system and reducing its level is the goal of many medications. This study aimed to investigate the effect of aerobic training and omega-3 intake on Aβ42, neprilysin, and γ-secretase levels in the hippocampus of male rats Alzheimer›s model. Methods: Fifty male Wistar rats (age: 12 weeks-old and weight: 222.31±11.91 g), were divided into the five groups including control Alzheimer’s (AC), Alzheimer’s with omega-3 intake (AO), Alzheimer’s training (AT), Alzheimer’s with omega-3 intake and training (AOT) and Healthy Control (HC). AD was induced by the injection of homocysteine (60mM) into the rat brain ventricle. Training on the treadmill with a speed of 20 m/min (60 minutes and 5 days/week) was applied. The supplement group received omega-3 supplement 800 mg/kg of body weight, daily for eight weeks. Levels of Aβ42, γ-secretase, and neprilysin protein were measured using ELISA method. In data analysis, one-way ANOVA and Tukey test as post hoc were used (P<0.05). Results: The obtained results showed that the level of Aβ42 in the hippocampus of AC group was significantly higher than that of the HC group (P=0.001). Also, the level of Aβ42 in the hippocampus of AC group was significantly higher as compared to AO, AT, and AOT groups (P values 0.001, 0.007, and 0.003 respectively). The γ-Secretase level in the hippocampus of AC group was significantly higher than that in the HC group (P=0.001). Moreover, the γ-secretase levels in the hippocampus of the AC group were significantly higher compared to AO, AT, and AOT groups (P values: 0.002, 0.001, and 0.001 respectively). There was no significant difference in neprilysin levels of the hippocampus among the research groups (P=0.534). Conclusion: It appears that exercise training and omega-3 consumption, can affect amyloidogenic pathways through reducing the level of γ-secretase, and lead to reduced level of hippocampus Aβ in AD subjects. Therefore, aerobic exercise training and omega-3 intake can be studied as a complementary therapy in Alzheimer’s patients.

Adipose tissue-derived mesenchymal stem cells ameliorate cognitive impairment in Alzheimer's disease rat model: Emerging role of SIRT1.

Alzheimer's disease (AD) is a complex form of neurodegenerative dementia. Growing body of evidence supports the cardinal role of sirtuin1 (SIRT1) in neurodegeneration and AD development. Recently, adipose tissue-derived mesenchymal stem cells (Ad-MSCs) have made their mark for a wide array of regenerative medicine applications, including neurodegenerative disorders. Therefore, the present study aimed to investigate the therapeutic potential of Ad-MSCs in AD rat model, and to explore the possible implication of SIRT1. Ad-MSCs were isolated from rat epididymal fat pads and properly characterized. Aluminum chloride was used to induce AD in rats, and afterward, a group of AD-induced rats received a single dose of Ad-MSCs (2 × 106 cell, I.V per rat). One month after Ad-MSCs transplantation, behavioral tests were done, brain tissues were collected, then histopathological and biochemical assessments were performed. Amyloid beta and SIRT1 levels were determined by enzyme-linked immunosorbent assay. Whereas expression levels of neprilysin, BCL2 associated X protein, B-cell lymphoma-2, interleukin-1β, interleukin-6, and nerve growth factor in hippocampus and frontal cortex brain tissues were assessed using reverse transcriptase quantitative polymerase chain reaction. Our data demonstrated that transplantation of Ad-MSCs alleviated cognitive impairment in AD rats. Additionally, they exhibited anti-amyloidogenic, antiapoptotic, anti-inflammatory, as well as neurogenic effects. Furthermore, Ad-MSCs were found to possibly mediate their therapeutic effects, at least partially, via modulating both central and systemic SIRT1 levels. Hence, the current study portrays Ad-MSCs as an effective therapeutic approach for AD management and opens the door for future investigations to further elucidate the role of SIRT1 and its interrelated molecular mediators in AD.

СВЯЗЬ ПОЛИМОРФНОГО ВАРИАНТА RS989692 ГЕНА НЕПРИЛИЗИНА (MME) С УРОВНЕМ НАТРИЙУРЕТИЧЕСКИХ ПЕПТИДОВ И ЕГО КЛИНИЧЕСКОЕ ЗНАЧЕНИЕ У ПАЦИЕНТОВ С СЕРДЕЧНОЙ НЕДОСТАТОЧНОСТЬЮ И ФИБРИЛЛЯЦИЕЙ ПРЕДСЕРДИЙ

Background. The variability in the activity of natriuretic peptides (NUPs) is determined genetically, as evidenced by the association of polymorphic variants encoding brain natriuretic peptide (BNP) and atrial natriuretic peptide (ANP) with an increased risk of cardiovascular (CV) disease. The aim of the study. To determine the frequency of alleles and genotypes of the SNP rs989692 of the neprilysin gene (MME). To determine the association of SNP rs989692 in MME with the concentration of NUP and soluble neprilysin and to evaluate its prognostic value in relation to the development of adverse cardiovascular events in patients with heart failure (HF) associated with a left ventricular ejection fraction (LVEF) <50% and persistent or long-term persistent atrial fibrillation (AF). Material and methods. The main group included 152 patients with HF, the control group included 35 individuals without CV disease. The levels of ANP, BNP, NT-proBNP and soluble neprilysin were determined for all patients. The genetic study of the SNP rs989692 in the MME gene was carried out by polymerase chain reaction. The endpoint: hospitalization due to HF progression. The composite endpoint: hospitalization due to HF progression, new onset or progressive exertional angina pectoris, myocardial infarction. Results. The frequency distribution of genotypes and alleles of the SNP rs989692 in MME did not differ significantly between the control and experimental groups. The levels of ANP, BNP and neprilysin in patients with HF in combination with AF did not differ depending on the SNP rs989692 in MME genotype. Patients with HF associated with LVEF <50%, AF and TT genotype rs989692 in MME had higher levels of NT-proBNP (those with CC genotype – 964 [655.1; 1724] pg/ml, those with TC genotype – 1074.1 [857; 1944] pg/ml, those with the TT genotype – 2992 [886; 4885] pg/ml, p<0.05). The presence of the homozygous TT genotype in patients with HF combined with LVEF <50% and AF was associated with an increased risk of developing adverse CV events, OR=1.9 [95% CI from 1.2 to 3.09]. Conclusion. Patients with HF associated with LVEF <50% in combination with permanent or long-term persistent AF with homozygous TT genotype rs989692 of the MME gene have higher levels of NT-proBNP and a higher risk of developing adverse cardiovascular events.

Evaluation of Serum Level of Neprilysin in Patients with Acne Vulgaris

Evaluation of Serum Level of Neprilysin in Patients with Acne Vulgaris

Association between soluble neprilysin and diabetes: Findings from a prospective longitudinal study.

The potential role of neprilysin (NEP) in glucose metabolism has been found by basic studies but lacks population evidence. The objective of this study was to examine the association between serum NEP and diabetes in Chinese adults. In a prospective longitudinal cohort study - the Gusu cohort (n=2,286, mean age: 52 years, 61.5% females), the cross-sectional, longitudinal, and prospective associations between serum NEP and diabetes were systemically examined by logistic regression adjusting for conventional risk factors. Serum NEP was measured at baseline using commercial ELISA assays. Fasting glucose was repeatedly measured 4 years apart. The cross-sectional analysis found a positive association between serum NEP and fasting glucose at baseline (β=0.08, P=0.004 for log-transformed NEP). This association persisted after controlling for the dynamic risk profiles during follow-up (β=0.10, P=0.023 for log-transformed NEP). The prospective analysis found that a higher level of serum NEP at baseline was associated with a higher risk of diabetes during follow-up (OR=1.79, P=0.039 for log-transformed NEP). Serum NEP was not only associated with prevalent diabetes but also predicted the future risk of diabetes development in Chinese adults, independent of many behavioral and metabolic factors. Serum NEP may be a predictor and even a new therapeutic target for diabetes. However, the casualty and mechanisms of NEP in the development of diabetes require further investigation.

Inhibition of CDK4/6 regulates AD pathology, neuroinflammation and cognitive function through DYRK1A/STAT3 signaling

Repurposing approved drugs is an emerging therapeutic development strategy for Alzheimer's disease (AD). The CDK4/6 inhibitor abemaciclib mesylate is an FDA-approved drug for breast cancer treatment. However, whether abemaciclib mesylate affects Aβ/tau pathology, neuroinflammation, and Aβ/LPS-mediated cognitive impairment is unknown. In this study, we investigated the effects of abemaciclib mesylate on cognitive function and Aβ/tau pathology and found that abemaciclib mesylate improved spatial and recognition memory by regulating the dendritic spine number and neuroinflammatory responses in 5xFAD mice, an Aβ-overexpressing model of AD. Abemaciclib mesylate also inhibited Aβ accumulation by enhancing the activity and protein levels of the Aβ-degrading enzyme neprilysin and the α-secretase ADAM17 and decreasing the protein level of the γ-secretase PS-1 in young and aged 5xFAD mice. Importantly, abemaciclib mesylate suppressed tau phosphorylation in 5xFAD mice and tau-overexpressing PS19 mice by reducing DYRK1A and/or p-GSK3β levels. In wild-type (WT) mice injected with lipopolysaccharide (LPS), abemaciclib mesylate rescued spatial and recognition memory and restored dendritic spine number. In addition, abemaciclib mesylate downregulated LPS-induced microglial/astrocytic activation and proinflammatory cytokine levels in WT mice. In BV2 microglial cells and primary astrocytes, abemaciclib mesylate suppressed LPS-mediated proinflammatory cytokine levels by downregulating AKT/STAT3 signaling. Taken together, our results support repurposing the anticancer drug, CDK4/6 inhibitor abemaciclib mesylate as a multitarget therapeutic for AD pathologies.

Long-term use of metformin and Alzheimer's disease: beneficial or detrimental effects.

Alzheimer's disease (AD) is the most common neurodegenerative disease characterized by extracellular deposition of amyloid beta (Aβ) leading to cognitive decline. Evidence from epidemiological studies has shown the association between type 2 diabetes mellitus (T2DM) and the development of AD. T2DM and peripheral insulin resistance (IR) augment the risk of AD with the development of brain IR with inhibition of neuronal insulin receptors. These changes impair clearance of Aβ, increase secretion of Aβ1-42, reduce brain glucose metabolism, and abnormal deposition of Aβ plaques. Insulin-sensitizing drug metformin inhibits aggregation of Aβ by increasing the activity of the insulin-degrading enzyme (IDE) and neprilysin (NEP) levels. Additionally, different studies raised conflicting evidence concerning long-term metformin therapy in T2DM patients, as it may increase the risk of AD or it may prevent the progression of AD. Therefore, the objective of this review was to clarify the beneficial and detrimental effects of long-term metformin therapy in T2DM patientsandrisk of AD. Evidence from clinical trial studies revealed the little effect of metformin on AD. Various animal studies showed that metformin increases Aβ formation by activation of amyloid precursor protein (APP)-cleaving enzymes with the generation of insoluble tau species. Of note, the metformin effect on cognitive function relative to AD pathogenesis is mostly assessed in animal model studies. The duration of metformin therapy was short in most animal studies, this finding cannot apply to the long-term duration of metformin in humans. Therefore, large-scale prospective and comparative studies involving long-term metformin therapy in both diabetic and non-diabetic patients are required to exclude the effect of T2DM-induced AD.

Systemic Administration of Lipopolysaccharide from Porphyromonas gingivalis Decreases Neprilysin Expression in the Mouse Hippocampus.

Alzheimer's disease is the most common type of neurodegenerative disorder in elderly individuals worldwide. Increasing evidence suggests that periodontal diseases are involved in the pathogenesis of Alzheimer's disease, and an association between periodontitis and amyloid-β deposition in elderly individuals has been demonstrated. The aim of the present study was to examine the effects of systemic administration of Porphyromonas gingivalis-derived lipopolysaccharide (PG-LPS) on neprilysin expression in the hippocampus of adult and senescence-accelerated mice. PG-LPS diluted in saline was intraperitoneally administered to male C57BL/6J and senescence-accelerated mouse prone 8 (SAMP8) mice at a dose of 5 mg/kg every 3 days for 3 months. Both C57BL/6J and SAMP8 mice administered saline without PG-LPS comprised the control group. The mRNA expression levels of neprilysin and interleukin (IL)-10 were evaluated using the quantitative reverse transcriptase-polymerase chain reaction. The protein levels of neprilysin were assessed using western blotting. Sections of the brain tissues were immunohistochemically stained. The serum IL-10 concentration significantly increased in both mouse strains after stimulation with PG-LPS. Neprilysin expression at both mRNA and protein levels was significantly lower in the SAMP8 PG-LPS group than those in the SAMP8 control group; however, they did not differ in PG-LPS-treated or non-treated C57BL/6J mice. Additionally, the immunofluorescence intensity of neprilysin in the CA3 region of the hippocampus in PG-LPS-treated SAMP8 mice was significantly lower than that in control SAMP8 mice. Porphyromonas gingivalis may reduce the expression of neprilysin in elderly individuals and thus increase amyloid-β deposition.

Urinary neprilysin - Another marker to predict postoperative acute kidney injury.

Dear Editor, Perioperative acute kidney injury (AKI) is a serious complication which is multifactorial, which leads to increased hospital and intensive care unit stay, possibility of chronic kidney disease and hemodialysis, increased cost of treatment, significant morbidity, and mortality. Neprilysin or neutral endopeptidase (NEP) is a single-pass membrane glycoprotein with zinc-dependent endopeptidase activity and a short cytosolic tail. NEP is found predominantly within the kidneys, gastrointestinal tract, liver, male genital organs, lungs, adipose tissue, brain, and heart. The renal brush border of proximal tubule on the luminal side is rich in membrane-bound NEP. NEP is involved in the degradation of both atrial and B-type natriuretic peptides, adrenomedullin, endothelin-1 and angiotensin II, and also nonvasoactive peptides, such as glucagon, glucagon-like peptide, enkephalins, and somatostatin. Circulating NEP estimation in serum has been used as a biomarker in acute and chronic heart failure patients for predicting cardiovascular death and time-to-first admission for an acute event.[1] The prospective comparison of angiotensin receptor neprilysin inhibitor with angiotensin-converting enzyme (ACE) inhibitor (ACEi) to determine impact on global mortality and morbidity in heart failure (PARADIGM-HF) study reported significant clinical benefits with the combination of an angiotensin receptor blocker valsartan with a neprilysin inhibitor sacubitril over ACEi in heart failure with reduced ejection fraction.[2] However, trends of serum NEP could never correlate NT-pro-BNP and even clinically. In AKI, NEP is flushed out from the kidneys due to shedding of the tubular brush border and thus is available for estimation in the urine as a marker. Bernardi et al.[3] conducted a prospective, observational cohort study involving 96 patients undergoing elective cardiac surgeries with cardiopulmonary bypass. They estimated urinary neprilysin levels in the immediate postoperative period and on day 1. The authors concluded that estimation of urinary neprilysin levels after cardiac surgeries has comparable discriminatory power to presently studied AKI biomarkers and has potential as a biomarker for the early detection of AKI after cardiac surgery. Pajenda et al.[4] analyzed urinary neprilysin levels of 90 critically ill patients admitted to intensive or intermediate care unit over 2–5 days. On analysis, they found that NEP levels who suffered AKI according to the KDIGO (Kidney Disease Improving Global Outcomes) criteria were significantly elevated in comparison to healthy controls. In a comparative study performed by Gutta et al.[5] between 20 nondiabetics and 40 diabetic patients, authors compared urinary ACE2, NEP, disintegrin and metalloproteinase 17 (ADAM17) between two groups. The authors concluded that urinary ACE2 and NEP can be used as early biomarkers to predict the incidence or progression of chronic kidney disease at early stages among individuals with type 2 diabetes. The normal values of urinary neprilysin derived from recent studies are in the range of 0.5–1 ng/mL. To conclude, urinary NEP needs to be validated with adequately powered randomized controlled studies in various postoperative scenarios like thoraco-abdominal aneurysm repair, major laparotomies including cytoreductive surgeries and heated intraperitoneal chemotherapy. Financial support and sponsorship Nil. Conflicts of interest There are no conflicts of interest.

Sirt3 deficiency induced down regulation of insulin degrading enzyme in comorbid Alzheimer’s disease with metabolic syndrome

SIRT3 deacetylates mitochondrial proteins, thereby enhancing their function. We have previously demonstrated that Sirt3 gene deletion leads to brain mitochondrial dysfunction and neuroinflammation. We also reported that silencing of Sirt3 gene in APP/PS1 mice results in exacerbation of insulin resistance, neuroinflammation and β amyloid plaque deposition. To further understand how metabolic syndrome and amyloid pathology interact, we performed RNA-seq analysis of the brain samples of APP/PS1/Sirt3-/- mice. Gene expression patterns were modulated in metabolic and inflammatory pathways by Sirt3 gene deletion, amyloid pathology, and the combination. Following Sirt3 gene deletion, a key finding was the decreased expression of insulin-degrading enzyme (IDE), an enzyme that regulates the levels of insulin and Aβ peptides. Western diet feeding of Sirt3-/- and APP/PS1 mice resulted in decrease of IDE protein, parallel to Sirt3 downregulation. Conversely, activation of SIRT3 by nicotinamide riboside in vivo and in vitro resulted in IDE upregulation. SIRT3 activation in vivo also increased the levels of neprilysin, another Aβ degrading enzyme and decreased the levels of BACE1 which generates Aβ peptide suggesting SIRT3’s role in amyloid plaque reduction. Our findings provide a plausible mechanism linking metabolic syndrome and amyloid pathology. SIRT3 may be a potential therapeutic target to treat AD.

Poly(I:C) promotes neurotoxic amyloid β accumulation through reduced degradation by decreasing neprilysin protein levels in astrocytes.

Inflammation associated with viral infection of the nervous system has been involved in the pathogenesis of neurodegenerative diseases, such as Alzheimer's disease (AD) and multiple sclerosis. Polyinosinic:polycytidylic acid (poly[I:C]) is a Toll-like receptor 3 (TLR3) agonist that mimics the inflammatory response to systemic viral infections. Despite growing recognition of the role of glial cells in AD pathology, their involvement in the accumulation and clearance of amyloid β (Aβ) in the brain of patients with AD is poorly understood. Neprilysin (NEP) and insulin-degrading enzyme (IDE) are the main Aβ-degrading enzymes in the brain. This study investigated whether poly(I:C) regulated Aβ degradation and neurotoxicity by modulating NEP and IDE protein levels through TLR3 in astrocytes. To this aim, primary rat primary astrocyte cultures were treated with poly(I:C) and inhibitors of the TLR3 signaling. Protein levels were assessed by Western blot. Aβ toxicity to primary neurons was measured by lactate dehydrogenase release. Poly(I:C) induced a significant decrease in NEP levels on the membrane of astrocytes as well as in the culture medium. The degradation of exogenous Aβ was markedly delayed in poly(I:C)-treated astrocytes. This delay significantly increased the neurotoxicity of exogenous Aβ1-42. Altogether, these results suggest that viral infections induce Aβ neurotoxicity by decreasing NEP levels in astrocytes and consequently preventing Aβ degradation.

Abstract 9449: Increased Neprilysin Expression Links Atrial Fibrotic Remodeling in Cardiovascular Surgery Patients

Introduction: Deposition of extracellular matrix (ECM) such as collagen leads to fibrosis, which plays essential roles in structural remodeling in atrial fibrillation (AF). Neprilysin (NEP) is an integral membrane-bound metallopeptidase with a wide spectrum of substrates and physiological functions. Hypothesis: We investigated the association of NEP and the atrial fibrosis-related genes in cardiovascular surgery patients including heart valve disease. Methods: Atrial mRNA expression including collagen and NEP were analyzed by real-time reverse transcription polymerase chain reaction (RT-PCR) in 59 cases (sinus rhythm (SR) 24 patients, AF 35 patients) in which LA tissues were removed from the appendage during surgery. NEP mRNA expression was also analyzed in adipose tissues (AT) obtained from both epicardial and subcutaneous adipose tissues. The NEP protein expression was measured by western blotting analysis and immunohistochemistry staining, and serum level of NEP was measured by ELISA. Results: The immunohistochemical staining showed the expression of NEP in both AT and LA. Higher expression of NEP mRNA and protein in adipose tissues was observed, compared with LA. Serum NEP concentration did not significantly correlate with NEP mRNA and protein expression of AT and LA. The significant correlations were observed among ECM (COL1A1, COL3A1, fibronectin) and the five selected fibrosis-related genes (TGFβ1, CTGF, miR21, NOX2 and BAX) attributed to the fibrosis of myocardium in LA. NEP mRNA expression of LA also positively correlated with the ECM and the fibrosis-related gene expression level. The multiple regression analysis revealed TGFβ1, NOX2, and NEP as independent predictors of atrial COL1A1 mRNA expression level. In addition, it revealed NEP as an independent predictor of COL1A1 mRNA expression level, even adjusted by age, sex, and BMI. Serum NEP concentration did not correlate with the ECM and the fibrosis-gene expression level. Patients with AF had higher atrial expression of NEP protein, compared to those with SR. Conclusions: Increased atrial expression of NEP links atrial fibrotic remodeling and developing AF in cardiovascular surgery patients. However, serum NEP did not reflect atrial NEP expression in LA.

Vitamin D3 supplementation ameliorates cognitive impairment and alters neurodegenerative and inflammatory markers in scopolamine induced rat model.

A multifaceted approach can be effective for the treatment of dementia including the most common form, Alzheimer's disease (AD). However, currently, it involves only symptomatic treatment with cholinergic drugs. Beneficial effects of high Vitamin D3 levels or its intake in the prevention and treatment of cognitive disorders have been reported. Thus, the present study examined the preventive effect of Vitamin D3 (Calcitriol) supplementation on cognitive impairment and evaluated its impact on the accumulation or degradation of Aβ plaques. A single intraperitoneal injection of scopolamine was used to induce cognitive impairment in rats. Treatment of Vitamin D3 was provided for 21days after the injection. Various behavioral parameters like learning, spatial memory and exploratory behavior, biochemical alterations in the brain homogenate and histology of the hippocampus were investigated. Our results indicated that scopolamine-induced rats depicted cognitive deficits with high Aβ levels and hyperphosphorylated tau proteins in the brain tissue, while Vitamin D supplementation could significantly improve the cognitive status and lower these protein levels. These results were supported by the histopathological and immunohistochemical staining of the hippocampal brain region. Furthermore, mechanistic analysis depicted that Vitamin D supplementation improved the Aβ protein clearance by increasing the neprilysin levels. It also reduced the accumulation of Aβ plaques by lowering neuroinflammation as well as oxidative stress. The present findings indicate that Vitamin D3 supplementation can ameliorate cognitive deficits and thereby delay AD progression by increasing Aβ plaque degradation, reducing inflammation and oxidative stress.

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.