Neprilysin Activity Research Articles

Process-Specific Blood Biomarkers and Outcomes in COVID-19 Versus Non-COVID-19 ARDS (APEL-COVID Study): A Prospective, Observational Cohort Study.

Background: Severe acute respiratory syndrome (SARS) and acute respiratory distress syndrome (ARDS) are often considered separate clinico-radiological entities. Whether these conditions also present a single process-specific systemic biomolecular phenotype and how this relates to patient outcomes remains unknown. A prospective cohort study was conducted, including adult patients admitted to the ICU and general floors for COVID-19-related (COVID+) or non-COVID-19-related (COVID-) acute respiratory failure during the main phase of the pandemic. The primary objective was to study blood biomarkers and outcomes among different groups and severity subsets. Results: A total of 132 patients were included, as follows: 67 COVID+, 54 COVID- (with 11 matched control subjects for biomarker reference), and 58 of these patients allowed for further pre- and post-analysis. The baseline apelin (APL) levels were higher in COVID+ patients (p < 0.0001 vs. COVID- patients) and in SARS COVID+ patients (p ≤ 0.02 vs. ARDS), while the IL-6 levels were higher in ARDS COVID- patients (p ≤ 0.0001 vs. SARS). Multivariable logistic regression analyses with cohort biomarkers and outcome parameters revealed the following: (i) log-transformed neprilysin (NEP) activity was significantly higher in COVID+ patients (1.11 [95% CI: 0.4-1.9] vs. 0.37 [95% CI: 0.1-0.8], fold change (FC): 1.43 [95% CI: 1.04-1.97], p = 0.029) and in SARS patients (FC: 1.65 [95% CI: 1.05-2.6], p = 0.032 vs. non-SARS COVID+ patients, and 1.73 [95% CI: 1.19-2.5], p = 0.005 vs. ARDS COVID- patients) and (ii) higher lysyl oxidase (LOX) activity and APL levels were respectively associated with death and a shorter length of hospital stay in SARS COVID+ patients (Odds Ratios (OR): 1.01 [1.00-1.02], p = 0.05, and OR: -0.007 [-0.013-0.0001], p = 0.048). Conclusion: Process-specific blood biomarkers exhibited distinct profiles between COVID+ and COVID- patients, and across stages of severity. NEP and LOX activities, as well as APL levels, are particularly linked to COVID+ patients and their outcomes (ClinicalTrials.gov Identifier: NCT04632732).

Metabolic resistance of Aβ3pE-42, a target epitope of the anti-Alzheimer therapeutic antibody, donanemab.

The amyloid β peptide (Aβ), starting with pyroglutamate (pE) at position 3 and ending at position 42 (Aβ3pE-42), predominantly accumulates in the brains of Alzheimer's disease. Consistently, donanemab, a therapeutic antibody raised against Aβ3pE-42, has been shown to be effective in recent clinical trials. Although the primary Aβ produced physiologically is Aβ1-40/42, an explanation for how and why this physiological Aβ is converted to the pathological form remains elusive. Here, we present experimental evidence that accounts for the aging-associated Aβ3pE-42 deposition: Aβ3pE-42 was metabolically more stable than other Aβx-42 variants; deficiency of neprilysin, the major Aβ-degrading enzyme, induced a relatively selective deposition of Aβ3pE-42 in both APP transgenic and App knock-in mouse brains; Aβ3pE-42 deposition always colocalized with Pittsburgh compound B-positive cored plaques in APP transgenic mouse brains; and under aberrant conditions, such as a significant reduction in neprilysin activity, aminopeptidases, dipeptidyl peptidases, and glutaminyl-peptide cyclotransferase-like were up-regulated in the progression of aging, and a proportion of Aβ1-42 may be processed to Aβ3pE-42. Our findings suggest that anti-Aβ therapies are more effective if given before Aβ3pE-42 deposition.

A Peptide Motif Covering Splice Site B in Neuroligin-1 Binds to Aβ and Acts as a Neprilysin Inhibitor.

The most common cause of dementia among elderly people is Alzheimer's disease (AD). The typical symptom of AD is the decline of cognitive abilities, which is caused by loss of synaptic function. Amyloid-β (Aβ) oligomers play a significant role in the development of this synaptic dysfunction. Neuroligin-(NL)1 is a postsynaptic cell-adhesion molecule located in excitatory synapses and involved in the maintenance and modulation of synaptic contacts. A recent study has found that Aβ interacts with the soluble N-terminal fragment of NL1. The present study aimed to elucidate the role of NL1 in Aβ-induced neuropathology. Employing surface plasmon resonance and competitive ELISA, we confirmed the high-affinity binding of NL1 to the Aβ peptide. We also identified a sequence motif representing the NL1-binding site for the Aβ peptide and showed that a synthetic peptide modeled after this motif, termed neurolide, binds to the Aβ peptide with high affinity, comparable to the NL1-Aβ interaction. To assess the effect of neurolide in vivo, wild-type and 5XFAD mice were subcutaneously treated with this peptide for 10weeks. We observed an increase in Aβ plaque formation in the cortex of neurolide-treated 5XFAD mice. Furthermore, we showed that neurolide reduces the activity of neprilysin, the predominant Aβ-degrading enzyme in the brain. Accordingly, we suggest that neurolide is the NL1-binding site for Aβ peptide, and acts as an inhibitor of neprilysin activity. Based on these data, we confirm the involvement of NL1 in the development of AD and suggest a mechanism for NL1-induced Aβ plaque formation.

The dopaminergic system promotes neprilysin-mediated degradation of amyloid-β in the brain.

Deposition of amyloid-β (Aβ) in the brain can impair neuronal function and contribute to cognitive decline in Alzheimer's disease (AD). Here, we found that dopamine and the dopamine precursor levodopa (also called l-DOPA) induced Aβ degradation in the brain. Chemogenetic approaches in mice revealed that the activation of dopamine release from ventral tegmental area (VTA) neurons increased the abundance and activity of the Aβ-degrading enzyme neprilysin and reduced the amount of Aβ deposits in the prefrontal cortex in a neprilysin-dependent manner. Aged mice had less dopamine and neprilysin in the anterior cortex, a decrease that was accentuated in AD model mice. Treating AD model mice with levodopa reduced Aβ deposition and improved cognitive function. These observations demonstrate that dopamine promotes brain region-specific, neprilysin-dependent degradation of Aβ, suggesting that dopamine-associated strategies have the potential to treat this aspect of AD pathology.

Aerobic training increases renal antioxidant defence and reduces angiotensin II levels, mitigating the high mortality in SHR-STZ model

Objectve The purpose of the research was to investigate the effects of aerobic training on renal function, oxidative stress, intrarenal renin–angiotensin system, and mortality of hypertensive and diabetic (SHR-STZ) rats. Materials and Methods Blood pressure, creatinine, urea levels, urinary glucose, urine volume, and protein excretion were reduced in trained SHR-STZ rats. Results Aerobic training not only attenuated oxidative stress but also elevated the activity of antioxidant enzymes in the kid′ney of SHR-STZ rats. Training increased intrarenal levels of angiotensin-converting enzymes (ACE and ACE2) as well as the neprilysin (NEP) activity, along with decreased intrarenal angiotensin II (Ang II) levels. Aerobic training significantly improved the survival of STZ-SHR rats. Conclusion The protective role of aerobic training was associated with improvements in the renal antioxidative capacity, reduced urinary protein excretion along with reduced intrarenal Ang II and increased NEP activity. These findings might reflect a better survival under the combined pathological conditions, hypertension, and diabetes.

Gut-specific Neprilysin Deletion Protects Against Fat-induced Insulin Secretory Dysfunction in Male Mice.

Neprilysin is a ubiquitous peptidase that can modulate glucose homeostasis by cleaving insulinotropic peptides. While global deletion of neprilysin protects mice against high-fat diet (HFD)-induced insulin secretory dysfunction, strategies to ablate neprilysin in a tissue-specific manner are favored to limit off-target effects. Since insulinotropic peptides are produced in the gut, we sought to determine whether gut-specific neprilysin deletion confers beneficial effects on insulin secretion similar to that of global neprilysin deletion in mice fed a HFD. Mice with conditional deletion of neprilysin in enterocytes (NEPGut-/-) were generated by crossing Vil-Cre and floxed neprilysin mice. Neprilysin activity was almost abolished throughout the gut in NEPGut-/- mice, and was similar in plasma, pancreas, and kidney in NEPGut-/- vs control mice. An oral glucose tolerance test was performed at baseline and following 14 weeks of HFD feeding, during which glucose tolerance and glucose-stimulated insulin secretion (GSIS) were assessed. Despite similar body weight gain at 14 weeks, NEPGut-/- displayed lower fasting plasma glucose levels, improved glucose tolerance, and increased GSIS compared to control mice. In conclusion, gut-specific neprilysin deletion recapitulates the enhanced GSIS seen with global neprilysin deletion in HFD-fed mice. Thus, strategies to inhibit neprilysin specifically in the gut may protect against fat-induced glucose intolerance and beta-cell dysfunction.

The synthetic peptide, PnPP-15, derived from the PnTx2-6 toxin of the spider Phoneutria nigriventer, induces peripheral antinociception involving neprilysin, opioid, and cannabinoid systems

BackgroundThe PnTx2–6 toxin is isolated from the Phoneutria nigriventer spider venom. The synthetic peptide PnPP-19 is derived from this toxin and induces antinociception. When topically applied, it is cleaved by proteases generating a 15 amino acid residues peptide, PnPP-15. PnPP-15 showed an antinociceptive effect in neuropathic pain developed by streptozotocin-induced diabetic mice. The present work aimed to investigate if PnPP-15 is also effective against the inflammatory pain induced by PGE2. We also evaluated its cytotoxicity in vitro and studied its possible action mechanism in the pain pathway. MethodsThe nociceptive threshold was assessed using the mechanical paw pressure test. Paw tissue was collected for protein extraction and western blot analyses. The FRET enzymatic assay measured neprilysin activity. The two-electrode voltage clamp technique measured evoked electrical currents in Xenopus laevis oocytes. Cytotoxicity was measured using multiple cell lines. ResultsPnPP-15 has peripheral antinociceptive activity via µ-opioid and CB1 cannabinoid receptors and inhibits neprilysin. In addition, it induced direct activation of mu-opioid receptors expressed in Xenopus laevis oocytes. PnPP-15 was not cytotoxic against mammalian kidney, liver, heart, nerve, or lung cells. ConclusionThis work demonstrated the antinociceptive effect of PnPP-15 in an inflammatory pain model, suggesting its use as a possible tool for developing new analgesic drugs.

Renal denervation improves cardiac function independently of afterload and restores myocardial norepinephrine levels in a rodent heart failure model

Renal nerves play a critical role in cardiorenal interactions. Renal denervation (RDN) improved survival in some experimental heart failure (HF) models. It is not known whether these favorable effects are indirect, explainable by a decrease in vascular afterload, or diminished neurohumoral response in the kidneys, or whether RDN procedure per se has direct myocardial effects in the failing heart. To elucidate mechanisms how RDN affects failing heart, we studied load-independent indexes of ventricular function, gene markers of myocardial remodeling, and cardiac sympathetic signaling in HF, induced by chronic volume overload (aorto-caval fistula, ACF) of Ren2 transgenic rats. Volume overload by ACF led to left ventricular (LV) hypertrophy and dysfunction, myocardial remodeling (upregulated Nppa, MYH 7/6 genes), increased renal and circulating norepinephrine (NE), reduced myocardial NE content, increased monoaminoxidase A (MAO-A), ROS production and decreased tyrosine hydroxylase (+) nerve staining. RDN in HF animals decreased congestion in the lungs and the liver, improved load-independent cardiac function (Ees, PRSW, Ees/Ea ratio), without affecting arterial elastance or LV pressure, reduced adverse myocardial remodeling (Myh 7/6, collagen I/III ratio), decreased myocardial MAO-A and inhibited renal neprilysin activity. RDN increased myocardial expression of acetylcholinesterase (Ache) and muscarinic receptors (Chrm2), decreased circulating and renal NE, but increased myocardial NE content, restoring so autonomic control of the heart. These changes likely explain improvements in survival after RDN in this model. The results suggest that RDN has remote, load-independent and favorable intrinsic myocardial effects in the failing heart. RDN therefore could be a useful therapeutic strategy in HF.

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

Neprilysin activity is increased in metabolic dysfunction-associated steatotic liver disease and normalizes after bariatric surgery or GLP-1 therapy

Inhibitors of neprilysin improve glycemia in patients with heart failure and type 2 diabetes (T2D). The effect of weight loss by diet, surgery, or pharmacotherapy on neprilysin activity (NEPa) is unknown. We investigated circulating NEPa and neprilysin protein concentrations in obesity, T2D, metabolic dysfunction-associated steatotic liver disease (MASLD), and following bariatric surgery, or GLP-1-receptor-agonist therapy. NEPa, but not neprilysin protein, was enhanced in obesity, T2D, and MASLD. Notably, MASLD associated with NEPa independently of BMI and HbA1c. NEPa decreased after bariatric surgery with a concurrent increase in OGTT-stimulated GLP-1. Diet-induced weight loss did not affect NEPa, but individuals randomized to 52-week weight maintenance with liraglutide (1.2mg/day) decreased NEPa, consistent with another study following 6-week liraglutide (3mg/day). A 90-min GLP-1 infusion did not alter NEPa. Thus, MASLD may drive exaggerated NEPa, and lowered NEPa following bariatric surgery or liraglutide therapy may contribute to the reported improved cardiometabolic effects.

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&amp;lt;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

Neprilysin deficiency reduces hepatic gluconeogenesis in high fat-fed mice

Neprilysin is a peptidase that cleaves glucoregulatory peptides, including glucagon-like peptide-1 (GLP-1) and cholecystokinin (CCK). Some studies suggest that its inhibition in diabetes and/or obesity improves glycemia, and that this is associated with enhanced insulin secretion, glucose tolerance and insulin sensitivity. Whether reduced neprilysin activity also improves hepatic glucose metabolism has not been explored. We sought to determine whether genetic deletion of neprilysin suppresses hepatic glucose production (HGP) in high fat-fed mice. Nep+/+ and Nep−/− mice were fed high fat diet for 16 weeks, and then underwent a pyruvate tolerance test (PTT) to assess hepatic gluconeogenesis. Since glycogen breakdown in liver can also yield glucose, we assessed liver glycogen content in fasted and fed mice. In Nep−/− mice, glucose excursion during the PTT was reduced when compared to Nep+/+ mice. Further, liver glycogen levels were significantly greater in fasted but not fed Nep−/− versus Nep+/+ mice. Since gut-derived factors modulate HGP, we tested whether gut-selective inhibition of neprilysin could recapitulate the suppression of hepatic gluconeogenesis observed with whole-body inhibition, and this was indeed the case. Finally, the gut-derived neprilysin substrates, GLP-1 and CCK, are well-known to suppress HGP. Having previously demonstrated elevated plasma GLP-1 levels in Nep−/− mice, we now measured plasma CCK bioactivity and reveal an increase in Nep−/− versus Nep+/+ mice, suggesting GLP-1 and/or CCK may play a role in reducing HGP under conditions of neprilysin deficiency. In sum, neprilysin modulates hepatic gluconeogenesis and strategies to inhibit its activity may reduce HGP in type 2 diabetes and obesity.

1546-P: Gut-Specific Neprilysin Deletion Increases Insulin Secretion in High-Fat–Fed Mice

Global deletion of the ubiquitous peptidase neprilysin (NEP) protects against high fat diet (HFD)-induced insulin secretory dysfunction. Since NEP is expressed in the gut, it can cleave locally produced insulinotropic peptides. We previously found that under physiological conditions, acute inhibition of gut NEP enhances glucose-stimulated insulin secretion (GSIS) in mice. Thus, we determined whether this beneficial effect on GSIS also occurs in mice with gut-specific NEP deletion that are fed HFD. Mice with conditional deletion of NEP in enterocytes (NEPGut-/-) were generated by crossing Vil1-cre and floxed NEP mice. NEP activity was almost abolished throughout the gut in NEPGut-/- vs control mice (5±2 vs 164±20 vs 120±20 pmol MNA/h/μg, NEP Gut-/- vs Vil1-cre vs NEPfl/fl mice; n=5-6/group; p&amp;lt;0.0001), and was similar in plasma, pancreas and kidney among all mice. At 11 weeks of age, mice were fed HFD for 14 weeks, after which an OGTT was performed and GSIS assessed as ∆insulin/∆glucose from 0 to 10 min. Despite similar body weights in NEP Gut-/- and control mice, fasting plasma glucose was lower and GSIS higher in NEP Gut-/- vs controls (Table). In sum, gut-specific NEP deletion decreases fasting plasma glucose and increases GSIS in HFD-fed mice, suggesting gut NEP impairs ß-cell function under conditions of HFD. Thus, strategies to inhibit NEP specifically in the gut may protect against fat-induced ß-cell dysfunction. Disclosure N.Esser: None. S.M.Mongovin: None. B.Barrow: None. S.Zraika: None. Funding National Institutes of Health (DK098506, (P30DK017047)

Acute Inhibition of Intestinal Neprilysin Enhances Insulin Secretion via GLP-1 Receptor Signaling in Male Mice.

The peptidase neprilysin modulates glucose homeostasis by cleaving and inactivating insulinotropic peptides, including some produced in the intestine such as glucagon-like peptide-1 (GLP-1). Under diabetic conditions, systemic or islet-selective inhibition of neprilysin enhances beta-cell function through GLP-1 receptor (GLP-1R) signaling. While neprilysin is expressed in intestine, its local contribution to modulation of beta-cell function remains unknown. We sought to determine whether acute selective pharmacological inhibition of intestinal neprilysin enhanced glucose-stimulated insulin secretion under physiological conditions, and whether this effect was mediated through GLP-1R. Lean chow-fed Glp1r+/+ and Glp1r-/- mice received a single oral low dose of the neprilysin inhibitor thiorphan or vehicle. To confirm selective intestinal neprilysin inhibition, neprilysin activity in plasma and intestine (ileum and colon) was assessed 40 minutes after thiorphan or vehicle administration. In a separate cohort of mice, an oral glucose tolerance test was performed 30 minutes after thiorphan or vehicle administration to assess glucose-stimulated insulin secretion. Systemic active GLP-1 levels were measured in plasma collected 10 minutes after glucose administration. In both Glp1r+/+ and Glp1r-/- mice, thiorphan inhibited neprilysin activity in ileum and colon without altering plasma neprilysin activity or active GLP-1 levels. Further, thiorphan significantly increased insulin secretion in Glp1r+/+ mice, whereas it did not change insulin secretion in Glp1r-/- mice. In conclusion, under physiological conditions, acute pharmacological inhibition of intestinal neprilysin increases glucose-stimulated insulin secretion in a GLP-1R-dependent manner. Since intestinal neprilysin modulates beta-cell function, strategies to inhibit its activity specifically in the intestine may improve beta-cell dysfunction in type 2 diabetes.

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.

Enhancement of Neprilysin Activity by Natural Polyphenolic Compounds and Their Derivatives in Cultured Neuroglioma Cells.

The onset of Alzheimer's disease (AD) is characterized by accumulation of amyloid β peptide (Aβ) in the brain. Neprilysin (NEP) is one of the major Aβ-degrading enzymes. Given findings that NEP expression in the brain declines from the early stage of AD before apparent neuronal losses are observed, enhancement of NEP activity and expression may be a preventive and therapeutic strategy relevant to disease onset. We screened for compounds that could enhance the activity and expression of NEP using a polyphenol library previously constructed by our research group and investigated the structure-activity relationships of the identified polyphenols. We found that amentoflavone, apigenin, kaempferol, and chrysin enhanced the activity and expression of NEP, suggesting that chemical structures involving a double bond between positions 2 and 3 in the C ring of flavones are important for NEP enhancement, while catechol or pyrogallol structures, except for the galloyl group of catechins, abolished these effects. Moreover, natural compounds, such as quercetin, were not effective per se, but were changed to effective compounds by adding a lipophilic moiety. Using our study findings, we propose improvements for dietary habits with experimental evidence, and provide a basis for the development of novel small molecules as disease-modifying drugs for AD.

Somatostatin slows Aβ plaque deposition in aged APPNL-F/NL-F mice by blocking Aβ aggregation

The neuroendocrine peptide somatostatin (SST) has long been thought of as influencing the deposition of the amyloid β peptide (Aβ) in Alzheimer’s disease (AD). Missing have been in vivo data in a relevant Aβ amyloidosis model. Here we crossed AppNL-F/NL-F mice with Sst-deficient mice to assess if and how the presence of Sst influences pathological hallmarks of Aβ amyloidosis. We found that Sst had no influence on whole brain neprilysin transcript, protein or activity levels, an observation that cannot be accounted for by a compensatory upregulation of the Sst paralog, cortistatin (Cort), that we observed in 15-month-old Sst-deficient mice. Sst-deficiency led to a subtle but significant increase in the density of cortical Aβ amyloid plaques. Follow-on western blot analyses of whole brain extracts indicated that Sst interferes with early steps of Aβ assembly that manifest in the appearance of SDS-stable smears of 55–150 kDa in Sst null brain samples. As expected, no effect of Sst on tau steady-state levels or its phosphorylation were observed. Results from this study are easier reconciled with an emerging body of data that point toward Sst affecting Aβ amyloid plaque formation through direct interference with Aβ aggregation rather than through its effects on neprilysin expression.

The systemic and hepatic alternative renin–angiotensin system is activated in liver cirrhosis, linked to endothelial dysfunction and inflammation

We aimed to assess the systemic and hepatic renin-angiotensin-system (RAS) fingerprint in advanced chronic liver disease (ACLD). This prospective study included 13 compensated (cACLD) and 12 decompensated ACLD (dACLD) patients undergoing hepatic venous pressure gradient (HVPG) measurement. Plasma components (all patients) and liver-local enzymes (n = 5) of the RAS were analyzed using liquid chromatography–tandem mass spectrometry. Patients with dACLD had significantly higher angiotensin (Ang) I, Ang II and aldosterone plasma levels. Ang 1–7, a major mediator of the alternative RAS, was almost exclusively detectable in dACLD (n = 12/13; vs. n = 1/13 in cACLD). Also, dACLD patients had higher Ang 1–5 (33.5 pmol/L versus cACLD: 6.6 pmol/L, p < 0.001) and numerically higher Ang III and Ang IV levels. Ang 1–7 correlated with HVPG (ρ = 0.655; p < 0.001), von Willebrand Factor (ρ = 0.681; p < 0.001), MELD (ρ = 0.593; p = 0.002) and interleukin-6 (ρ = 0.418; p = 0.047). Considerable activity of ACE, chymase, ACE2, and neprilysin was detectable in all liver biopsies, with highest chymase and ACE2 activity in cACLD patients. While liver-local classical and alternative RAS activity was already observed in cACLD, systemic activation of alternative RAS components occurred only in dACLD. Increased Ang 1–7 was linked to severe liver disease, portal hypertension, endothelial dysfunction and inflammation.

AW-NI-3: EFFECTS OF RENAL DENERVATION ON LEFT AND RIGHT VENTRICULAR FUNCTION IN HYPERTENSIVE RATS WITH HEART FAILURE INDUCED BY VOLUME OVERLOAD

Objective: Clinical studies showed that renal denervation (RDN) is effective in blood pressure lowering in hypertension. Previously, we found that RDN prolongs survival in a hypertensive rodent model of heart failure due to volume overload from aorto-caval fistula (ACF). Whether it is due to afterload removal, or due to intrinsic effects of RDN on the right (RV) or left (LV) ventricular function, is currently unknown and was the focus of our investigation. Design and method: 8-week-old Ren-2 transgenic male rats (model of angiotensin II-dependent hypertension) underwent ACF surgery to induce volume overload. After one week, animals underwent bilateral RDN (mechanical and chemical by topical phenol application) from laparotomy. Two weeks after RDN, the functions of both ventricles were measured by simultaneous biventricular pressure-volume analysis, and the animals were examined using echocardiography each week. Results: RDN in ACF rats only slightly reduced maximum pressure in RV but did not affect maximum pressure in LV. Yet, RDN in ACF animals significantly reduced hypertrophy and dilatation of both ventricles and decreased lung and liver congestion. RDN in ACF rats improved load-independent measures of contractility of both ventricles (end-systolic elastance and preload recruitable stroke work). RDN in ACF rats reduced renal levels of norepinephrine (NE) but restored depleted NE both in RV and LV. At the gene expression level, RDN in ACF led to favorable remodeling which was reflected as increased transcription of beta-1 adrenergic receptors in LV and beta-2 adrenergic receptors in RV and LV, decreased transcription of natriuretic peptide A gene (NPPA), collagen 1, and monoaminoxidase A (MAOA). These changes were relatively more pronounced in RV than LV. Interestingly, RDN in ACF rats also significantly inhibited the activity of neprilysin in the kidney. Conclusions: Besides the effects on pressure, RDN favorably affected load-independent measures of chamber function, markers of myocardial remodeling, and restored myocardial norepinephrine levels. These data indicate that RDN effects extend beyond pressure lowering and could be a promising method in the treatment of both right and left ventricular heart failure.

Abstract 11451: Myocardial Neprilysin Expression in Patients With Obstructive Hypertrophic Cardiomyopathy

Introduction: Hypertrophic cardiomyopathy (HCM) is characterized by left ventricular hypertrophy and fibrosis, the underlying mechanisms of which remain poorly understood. Neprilysin (NEP) inhibition has been shown to attenuate hypertrophy and fibrosis in animal models of heart failure, and NEP inhibition is an established therapeutic target in dilated and ischemic cardiomyopathy. However, the myocardial expression of NEP in HCM has not been previously reported. Hypothesis: The expression of NEP is increased in patients with obstructive HCM (oHCM). Methods/Results: We analyzed publicly available gene expression data from two independent cohorts that used myectomy samples from patients with oHCM, and healthy unused donor hearts served as controls. Cohort #1 (NCBI GEO accession number GSE36961) included 107 patients with oHCM and 39 controls and used microarray to assess gene expression. Cohort #2 (NCBI GEO accession number GSE160997) included 18 patients with oHCM and 5 controls and used RNA-Seq to assess gene expression. Both cohorts showed NEP gene expression was significantly increased in oHCM compared to controls (P&lt;0.0001 and P&lt;0.001, respectively). We then examined NEP expression in our surgical cohort (#3), which included 40 patients with oHCM and 8 controls. Using qPCR we showed NEP gene expression was significantly elevated in oHCM compared to controls (P&lt;0.001, see Figure 1). Conclusions: Myocardial NEP gene expression is significantly increased in patients with oHCM undergoing septal myectomy. Future studies are needed to determine if increased myocardial NEP gene expression is accompanied by elevated NEP protein expression and/or activity. Furthermore, future studies are required to determine the mechanism of increased NEP, and whether these findings extend to HCM patients who have not progressed to symptomatic obstruction.