Neutral endopeptidase depletion decreases colon cancer cell proliferation and TGF-B1 synthesis in indirect co-cultures with normal colon fibroblasts

Purpose Although recent studies have suggested that neutral endopeptidase (NEP) is implicated in the regulation of colon cancer (CC) cell growth and metastasis, the influence of the tumor microenvironment on this role of NEP has not been investigated so far. Normal colon fibroblasts (NCFs) constitute a component of the stroma surrounding a tumor in an early stage of its development. NCFs can influence transformed cells via different paracrine factors, including TGF-β1. This in vitro study was undertaken to evaluate the role of NEP in CC promotion in conditions of indirect co-culture of CC cells (LS180 and SW620) with NCFs (CCD-18Co) or their conditioned medium (CM-18Co). Methods We examined cell proliferation (with the BrdU assay) and invasiveness (using BME-coated inserts, 8 µm) of NEP-expressing, NEP-silenced (siRNA), and NEP-inhibited (with thiorphan, i.e. a NEP specific inhibitor) CC cells cultured alone or co-cultured with CCD-18Co or with their conditioned medium. The Western blot and ELISA methods were used to assess the level of TGF-β1. Results The results showed that the co-culture of the NEP-depleted CC cells with NCFs or their conditioned medium resulted in a significant decrease in cell proliferation in comparison with the proliferative potential of NEP-silenced/inhibited CC cells cultivated alone. In contrast, the NEP depletion did not influence the invasiveness of CC cells in the co-cultures. The co-culture of CC cells with CCD-18Co or CM-C18Co resulted in increased synthesis of TGF-β1, while the NEP downregulation decreased the synthesis of TGF-β1 in CC cells and abolished the stimulatory effect of the co-cultures on TGF-β1 production. Conclusions The results suggest that the expression of NEP by colon cancer cells is essential for their proliferation and TGF-β1 synthesis during paracrine interactions with NCFs (AU)

Neutral endopeptidase depletion decreases colon cancer cell proliferation and TGF-ß1 synthesis in indirect co-cultures with normal colon fibroblasts.

PURPOSE: Although recent studies have suggested that neutral endopeptidase (NEP) is implicated in the regulation of colon cancer (CC) cell growth and metastasis, the influence of the tumor microenvironment on this role of NEP has not been investigated so far. Normal colon fibroblasts (NCFs) constitute a component of the stroma surrounding a tumor in an early stage of its development. NCFs can influence transformed cells via different paracrine factors, including TGF-ß1. This in vitro study was undertaken to evaluate the role of NEP in CC promotion in conditions of indirect co-culture of CC cells (LS180 and SW620) with NCFs (CCD-18Co) or their conditioned medium (CM-18Co). METHODS: We examined cell proliferation (with the BrdU assay) and invasiveness (using BME-coated inserts, 8 µm) of NEP-expressing, NEP-silenced (siRNA), and NEP-inhibited (with thiorphan, i.e. a NEP specific inhibitor) CC cells cultured alone or co-cultured with CCD-18Co or with their conditioned medium. The Western blot and ELISA methods were used to assess the level of TGF-ß1. RESULTS: The results showed that the co-culture of the NEP-depleted CC cells with NCFs or their conditioned medium resulted in a significant decrease in cell proliferation in comparison with the proliferative potential of NEP-silenced/inhibited CC cells cultivated alone. In contrast, the NEP depletion did not influence the invasiveness of CC cells in the co-cultures. The co-culture of CC cells with CCD-18Co or CM-C18Co resulted in increased synthesis of TGF-ß1, while the NEP downregulation decreased the synthesis of TGF-ß1 in CC cells and abolished the stimulatory effect of the co-cultures on TGF-ß1 production. CONCLUSIONS: The results suggest that the expression of NEP by colon cancer cells is essential for their proliferation and TGF-ß1 synthesis during paracrine interactions with NCFs.

TaleNeprilysin and Neprilysin inhibition in chronic kidney disease.

PURPOSE OF REVIEW: Chronic kidney disease (CKD) is associated with increased risk of progression to end-stage kidney disease and cardiovascular events. There is limited evidence that available treatments have beneficial effects on cardiorenal outcomes in all people with nondiabetic CKD. Neprilysin inhibition (NEPi) is a new therapeutic strategy with potential to improve outcomes for patients with CKD. RECENT FINDINGS: NEPi enhances the activity of the natriuretic peptide system producing natriuresis, diuresis and inhibition of the renin-angiotensin system and sympathetic nervous system. Sacubitril/valsartan is the first Angiotensin receptor-neprilysin inhibitor (ARNI) to be produced and has been shown to substantially improve cardiovascular outcomes in heart failure and delay progression of kidney disease in this population. Although ARNIs have not shown similar effects on kidney function in the short-to-medium term in people with CKD, they are associated with substantial reductions in cardiac biomarkers and blood pressure in CKD. SUMMARY: These data suggest that NEPi with an ARNI could benefit patients with CKD by reducing the risk of cardiovascular disease and have the possibility of retarding the progression of CKD (hence delaying the need for renal replacement therapy).

Angiotensin Receptor-Neprilysin Inhibition Based on History of Heart Failure and Use of Renin-Angiotensin System Antagonists.

BACKGROUND: The PIONEER-HF (comParIson Of sacubitril/valsartaN versus Enalapril on Effect on nt-pRo-bnp in patients stabilized from an acute Heart Failure episode) trial demonstrated the efficacy and safety of sacubitril/valsartan (S/V) in stabilized patients with acute decompensated heart failure (HF) and reduced ejection fraction. OBJECTIVES: The study sought to determine whether and how prior HF history and treatment with an angiotensin-converting enzyme (ACE) inhibitor or angiotensin receptor blocker (ARB) affected the results. METHODS: The PIONEER-HF trial was a prospective, multicenter, double-blind, randomized clinical trial enrolling 881 patients with an ejection fraction ≤40%. Patients were randomly assigned 1:1 to in-hospital initiation of S/V (n = 440) versus enalapril (n = 441). Pre-specified subgroup analyses were performed based on prior HF history (i.e., de novo HF vs. worsening chronic HF) and treatment with an ACE inhibitor or ARB (i.e., ACE inhibitor or ARB-yes vs. ACE inhibitor or ARB-no) at admission. RESULTS: At enrollment, 303 (34%) patients presented with de novo HF and 576 (66%) patients with worsening chronic HF. A total of 421 (48%) patients had been treated with an ACE inhibitor or ARB, while 458 (52%) had not been treated with an ACE inhibitor or ARB. N-terminal pro-B-type natriuretic peptide declined significantly in all 4 subgroups (p < 0.001), with greater decreases in the S/V versus the enalapril arm (p < 0.001). There was no interaction between prior HF history (p = 0.350) or ACE inhibitor or ARB treatment (p = 0.880) and the effect of S/V versus enalapril on cardiovascular death or rehospitalization for HF. The incidences of adverse events were comparable between S/V and enalapril across all 4 subgroups. CONCLUSIONS: Among patients admitted for acute decompensated HF, S/V was safe and well tolerated, led to a significantly greater reduction in N-terminal pro-B-type natriuretic peptide, and improved clinical outcomes compared with enalapril irrespective of previous HF history or ACE inhibitor or ARB treatment. (Comparison of Sacubitril/Valsartan Versus Enalapril on Effect of NT-proBNP in Patients Stabilized From an Acute Heart Failure Episode [PIONEER-HF]; NCT02554890).

The heart regulates the endocrine response to heart failure: cardiac contribution to circulating neprilysin.

Aims: Heart failure (HF) is accompanied by major neuroendocrine changes including the activation of the natriuretic peptide (NP) pathway. Using the unique model of patients undergoing implantation of the CARMAT total artificial heart and investigating regional differences in soluble neprilysin (sNEP) in patients with reduced or preserved systolic function, we studied the regulation of the NP pathway in HF. Methods and results: Venous blood samples from two patients undergoing replacement of the failing ventricles with a total artificial heart were collected before implantation and weekly thereafter until post-operative week 6. The ventricular removal was associated with an immediate drop in circulating NPs, a nearly total disappearance of circulating glycosylated proBNP and furin activity and a marked decrease in sNEP. From post-operative week 1 onwards, NP concentrations remained overall unchanged. In contrast, partial recoveries in glycosylated proBNP, furin activity, and sNEP were observed. Furthermore, while in patients with preserved systolic function (n = 6), sNEP concentrations in the coronary sinus and systemic vessels were similar (all P > 0.05), in patients with reduced left-ventricular systolic function, sNEP concentration, and activity were ∼three-fold higher in coronary sinus compared to systemic vessels (n = 21, all P < 0.0001), while the trans-pulmonary gradient was neutral (n = 5, P = 1.0). Conclusion: The heart plays a pivotal role as a regulator of the endocrine response in systolic dysfunction, not only by directly releasing NPs but also by contributing to circulating sNEP, which in turn determines the bioavailability of other numerous vasoactive peptides.

Renal Sympathetic Denervation Protects the Failing Heart Via Inhibition of Neprilysin Activity in the Kidney.

BACKGROUND: Sustained sympathetic activation contributes to the progression of myocardial cell injury, cardiac fibrosis, and left ventricular (LV) dysfunction in heart failure (HF). OBJECTIVES: This study investigated the effects of radiofrequency renal nerve denervation (RF-RDN) on the pathobiology of HF and the interaction between the renal sympathetic nerves and natriuretic peptide (NP) metabolism. METHODS: Spontaneously hypertensive rats (SHR) and normotensive Wistar-Kyoto rats (WKY) were subjected to 45 min of coronary artery ligation and reperfusion for 12 weeks. At 4 weeks post-reperfusion, SHR and WKY underwent either bilateral RF-RDN or sham-RDN. RESULTS: Following RF-RDN in both strains, LV ejection fraction remained significantly above those levels in respective sham-RDN rats, and at the end of the 12-week study, rats in both strains had significantly reduced LV fibrosis and improved vascular function. RF-RDN therapy significantly improved vascular reactivity to endothelium-dependent and -independent vasodilators as well as vascular compliance in the setting of severe HF. Improvements in LV function were accompanied by significant elevations in circulating NP as compared to those associated with sham-RDN. Further investigation into the cause of increased circulating NP levels demonstrated that RF-RDN significantly inhibited renal neprilysin activity in SHR and WKY with HF. Likewise, chronic treatment with the beta1 antagonist bisoprolol inhibited renal neprilysin activity and increased circulation NP levels in WKY with HF. CONCLUSIONS: This study identifies a novel endogenous pathway by which the renal nerves participate in the degradation of cardioprotective NP. Furthermore, removal of the influence of the renal nerves on kidney function attenuates renal neprilysin activity, augments circulating NP levels, reduces myocardial fibrosis, and improves LV function in the setting of HF.

Neprilysin Is Required for Angiotensin-(1-7)'s Ability to Enhance Insulin Secretion via Its Proteolytic Activity to Generate Angiotensin-(1-2).

Recent work has renewed interest in therapies targeting the renin-angiotensin system (RAS) to improve ß-cell function in type 2 diabetes. Studies show that generation of angiotensin-(1-7) by ACE2 and its binding to the Mas receptor (MasR) improves glucose homeostasis, partly by enhancing glucose-stimulated insulin secretion (GSIS). Thus, islet ACE2 upregulation is viewed as a desirable therapeutic goal. Here, we show that, although endogenous islet ACE2 expression is sparse, its inhibition abrogates angiotensin-(1-7)-mediated GSIS. However, a more widely expressed islet peptidase, neprilysin, degrades angiotensin-(1-7) into several peptides. In neprilysin-deficient mouse islets, angiotensin-(1-7) and neprilysin-derived degradation products angiotensin-(1-4), angiotensin-(5-7), and angiotensin-(3-4) failed to enhance GSIS. Conversely, angiotensin-(1-2) enhanced GSIS in both neprilysin-deficient and wild-type islets. Rather than mediating this effect via activation of the G-protein-coupled receptor (GPCR) MasR, angiotensin-(1-2) was found to signal via another GPCR, namely GPCR family C group 6 member A (GPRC6A). In conclusion, in islets, intact angiotensin-(1-7) is not the primary mediator of beneficial effects ascribed to the ACE2/angiotensin-(1-7)/MasR axis. Our findings warrant caution for the concurrent use of angiotensin-(1-7) compounds and neprilysin inhibitors as therapies for diabetes.

Therapeutic options of Angiotensin Receptor Neprilysin inhibitors (ARNis) in chronic heart failure with reduced ejection fraction: Beyond RAAS and sympathetic nervous system inhibition.

In heart failure, in addition to the renin-angiotensin-aldosterone system and sympathetic nervous system, the natriuretic peptide (NP) system plays a fundamental role among compensating mechanisms. The NPs undergo rapid enzymatic degradation that limits their vasorelaxant, natriuretic, and diuretic actions. Degradation of NPs is partially due to the action of neprilysin, which is a membrane-bound endopeptidase found in many tissues. This article summarizes recent findings on a new natriuretic peptide-enhancing drug and their implication for future pharmacological treatment of patients suffering from heart failure with reduced ejection fraction.

Hypothesizing That Neuropharmacological and Neuroimaging Studies of Glutaminergic-Dopaminergic Optimization Complex (KB220Z) Are Associated With "Dopamine Homeostasis" in Reward Deficiency Syndrome (RDS).

BACKGROUND: There is need for better treatments of addictive behaviors, both substance and non-substance related, termed Reward Deficiency Syndrome (RDS). While the FDA has approved pharmaceuticals under the umbrella term Medication Assisted Treatment (MAT), these drugs are not optimal. OBJECTIVES: It is our contention that these drugs work well in the short-term by blocking dopamine function leading to psychological extinction. However, use of buprenorphine/Naloxone over a long period of time results in unwanted addiction liability, reduced emotional affect, and mood changes including suicidal ideation. METHODS: We are thus proposing a paradigm shift in addiction treatment, with the long-term goal of achieving "Dopamine Homeostasis." While this may be a laudable goal, it is very difficult to achieve. Nevertheless, this commentary briefly reviews past history of developing and subsequently, utilizing a glutaminergic-dopaminergic optimization complex [Kb220Z] shown to be beneficial in at least 20 human clinical trials and in a number of published and unpublished studies. RESULTS: It is our opinion that, while additional required studies could confirm these findings to date, the cited studies are indicative of achieving enhanced resting state functional connectivity, connectivity volume, and possibly, neuroplasticity. Conclusions/Importance: We are proposing a Reward Deficiency Solution System (RDSS) that includes: Genetic Addiction Risk Score (GARS); Comprehensive Analysis of Reported Drugs (CARD); and a glutaminergic-dopaminergic optimization complex (Kb220Z). Continued investigation of this novel strategy may lead to a better-targeted approach in the long-term, causing dopamine regulation by balancing the glutaminergic-dopaminergic pathways. This may potentially change the landscape of treating all addictions leading us to the promised land.

Major amyloid-ß-degrading enzymes, endothelin-converting enzyme-2 and neprilysin, are expressed by distinct populations of GABAergic interneurons in hippocampus and neocortex.

Impaired clearance of amyloid-ß peptide (Aß) has been postulated to significantly contribute to the amyloid accumulation typical of Alzheimer's disease. Among the enzymes known to degrade Aß in vivo are endothelin-converting enzyme (ECE)-1, ECE-2, and neprilysin (NEP), and evidence suggests that they regulate independent pools of Aß that may be functionally significant. To better understand the differential regulation of Aß concentration by its physiological degrading enzymes, we characterized the cell and region-specific expression pattern of ECE-1, ECE-2, and NEP by in situ hybridization and immunohistochemistry in brain areas relevant to Alzheimer's disease. In contrast to the broader distribution of ECE-1, ECE-2 and NEP were found enriched in GABAergic neurons. ECE-2 was majorly expressed by somatostatin-expressing interneurons and was active in isolated synaptosomes. NEP messenger RNA was found mainly in parvalbumin-expressing interneurons, with NEP protein localized to perisomatic parvalbuminergic synapses. The identification of somatostatinergic and parvalbuminergic synapses as hubs for Aß degradation is consistent with the possibility that Aß may have a physiological function related to the regulation of inhibitory signaling.

Neprilysin and Natriuretic Peptide Regulation in Heart Failure.

Neprilysin is acknowledged as a key player in neurohormonal regulation, a cornerstone of modern drug therapy in chronic heart failure. In the cardiovascular system, neprilysin cleaves numerous vasoactive peptides, some with mainly vasodilating effects (natriuretic peptides, adrenomedullin, bradykinin) and other with mainly vasoconstrictor effects (angiotensin I and II, endothelin-1). For decades, neprilysin has been an important biotarget. Academia and industry have combined active efforts to search for neprilysin inhibitors (NEPIs) that might be useful in clinical practice. NEPI monotherapy was initially tested with little success due to efficacy issues. Next, combination of NEPI and ACE-inhibiting activity agents were abandoned due to safety concerns. Recently, the combination of NEPI and ARB, also known as ARNI, has shown better than expected results in heart failure with reduced ejection fraction, and multitude of ongoing studies are set to prove its value across the heart failure spectrum.

Neprilysin gene transfer: A promising therapeutic approach for Alzheimer's disease.

Alzheimer's disease (AD) is characterized by widespread neurodegeneration throughout the association cortex and limbic system, deposition of amyloid-ß (Aß) in the neuropil and around blood vessels, and formation of neurofibrillary tangles. Aß accumulation is considered the major pathological change in AD progression. In recent years, several therapeutic strategies for treating AD have focused on reducing the Aß burden in the brain. Among these approaches, the expression of Aß-degrading enzymes in the brain has been effective but, so far, impractical for treating patients. Neprilysin (NEP), the most prominent of the Aß-degrading enzymes in vivo, has been successfully delivered intracranially by viral vectors and is a promising therapeutic approach for reducing Aß accumulation and treating AD. However, some challenges are associated with the use of viral and nonviral vectors, including secondary toxicity, activation of the immune response, and low efficiency. Therefore, safe and efficient NEP delivery systems that could avoid the viral problems with minor injury and high transfection efficiency are required to deliver AD medical applications. This Mini-Review summarizes NEP gene transfer technologies that use viral and nonviral vectors and discusses the rationale and benefits of these delivery systems for AD treatment trials, providing a reference for basic and clinical studies on AD.

Role of neprilysin inhibitor combinations in hypertension: insights from hypertension and heart failure trials.

Neprilysin is a neutral endopeptidase and its inhibition increases bioavailability of natriuretic peptides, bradykinin, and substance P, resulting in natriuretic, vasodilatatory, and anti-proliferative effects. In concert, these effects are prone to produce a powerful ventricular unloading and antihypertensive response. LCZ696 (Valsartan/sacubitril) is a first-in-class angiotensin II-receptor neprilysin inhibitor. LCZ696 is a novel drug not only for the treatment of heart failure but it is also likely to be a useful antihypertensive drug and may have a preferential effect on systolic pressure. This review discusses (i) the mechanism of action, pharmacokinetics, and pharmacodynamics of this novel drug, (ii) the efficacy, safety, and tolerability of LCZ696 in treatment of hypertension from the available trials, (iii) evidence from other contemporary trials on combined Neprilysin inhibitors, (iv) future trials and areas of research to identify hypertensive patient populations that would most benefit from LCZ696.

Natriuretic peptides and cardio-renal disease.

The natriuretic peptide (NP) system is an important endocrine, autocrine and paracrine system, consisting of a family of peptides which provide cardiac, renal and vascular effects that, through their beneficial physiological actions, play a key role in maintaining overall cardiovascular health. Traditionally, the pathophysiological origins of cardio-renal disease have been viewed as the domain of the renin-angiotensin-aldosterone system (RAAS) and the sympathetic nervous system (SNS), with inappropriate activation of both systems leading to deleterious changes in cardio-renal function and structure. Therapies designed to suppress the RAAS and the SNS have been routinely employed to address the consequences of cardio-renal disease. However, it is now becoming increasingly apparent that enhancing the beneficial physiological effects of the NP system may represent an attractive alternative therapeutic approach to counter the pathophysiological effects of disease. In particular, innovative therapeutic strategies aimed at enhancing the physiological benefits afforded by NPs while simultaneously suppressing the RAAS are generating increasing interest as potential treatment options for the management of cardio-renal disease.

The Aß-clearance protein transthyretin, like neprilysin, is epigenetically regulated by the amyloid precursor protein intracellular domain.

Proteolytic cleavage of the amyloid precursor protein (APP) by the successive actions of ß- and γ-secretases generates several biologically active metabolites including the amyloid ß-peptide (Aß) and the APP intracellular domain (AICD). By analogy with the Notch signalling pathway, AICD has been proposed to play a role in transcriptional regulation. Among the cohort of genes regulated by AICD is the Aß-degrading enzyme neprilysin (NEP). AICD binds to the NEP promoter causing transcriptional activation by competitive replacement with histone deacetylases (HDACs) leading to increased levels of NEP activity and hence increased Aß clearance. We now show that the Aß-clearance protein transthyretin (TTR) is also epigenetically up-regulated by AICD. Like NEP regulation, AICD derived specifically from the neuronal APP isoform, APP695 , binds directly to the TTR promoter displacing HDAC1 and HDAC3. Cell treatment with the tyrosine kinase inhibitor Gleevec (imatinib) or with the alkalizing agent NH4 Cl causes an accumulation of 'functional' AICD capable of up-regulating both TTR and NEP, leading to a reduction in total cellular Aß levels. Pharmacological regulation of both NEP and TTR might represent a viable therapeutic target in Alzheimer's disease.

Study on Aß34 biology and detection in transgenic mice brains.

The ß-amyloid precursor protein undergoes cleavages by ß- and γ-secretasses yielding amyloid-ß peptides (Aß) that accumulate in Alzheimer's disease. Subsequently, Aß peptides are targets of additional truncations or endoproteolytic cleavages explaining the diversity of Aß-related fragments recovered in cell media or pathologic human fluids. Here, we focused on Aß1-34 (Aß34) that has been detected both in vitro and in vivo and that derives from the hydrolysis of Aß by ß-secretase. We have obtained and fully characterized by immunologic and biochemical approaches, a polyclonal antibody that specifically recognizes the C-terminus of Aßx-34. We present immunohistochemical evidence for the presence of Aßx-34 in the brain of 3xTg mice and Alzheimer's disease-affected human brains. Finally, we demonstrate a neprilysin-mediated degradation process of Aß34 and the ability of synthetic Aß34 to protect HEK cells overexpressing either wild type or Swedish-mutated ß-amyloid precursor protein from apoptosis.

Neprilysins: an evolutionarily conserved family of metalloproteases that play important roles in reproduction in Drosophila.

Members of the M13 class of metalloproteases have been implicated in diseases and in reproductive fitness. Nevertheless, their physiological role remains poorly understood. To obtain a tractable model with which to analyze this protein family's function, we characterized the gene family in Drosophila melanogaster and focused on reproductive phenotypes. The D. melanogaster genome contains 24 M13 class protease homologs, some of which are orthologs of human proteases, including neprilysin. Many are expressed in the reproductive tracts of either sex. Using RNAi we individually targeted the five Nep genes most closely related to vertebrate neprilysin, Nep1-5, to investigate their roles in reproduction. A reduction in Nep1, Nep2, or Nep4 expression in females reduced egg laying. Nep1 and Nep2 are required in the CNS and the spermathecae for wild-type fecundity. Females that are null for Nep2 also show defects as hosts of sperm competition as well as an increased rate of depletion for stored sperm. Furthermore, eggs laid by Nep2 mutant females are fertilized normally, but arrest early in embryonic development. In the male, only Nep1 was required to induce normal patterns of female egg laying. Reduction in the expression of Nep2-5 in the male did not cause any dramatic effects on reproductive fitness, which suggests that these genes are either nonessential for male fertility or perform redundant functions. Our results suggest that, consistent with the functions of neprilysins in mammals, these proteins are also required for reproduction in Drosophila, opening up this model system for further functional analysis of this protein class and their substrates.