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Neprilysin (NEP) is an emerging biomarker for various diseases including heart failure (HF). However, major inter-assay inconsistency in the reported concentrations of circulating NEP and uncertainty with respect to its correlations with type and severity of disease are in part attributed to poorly characterized antibodies supplied in commercial ELISA kits. Validated antibodies with well-defined binding footprints are critical for understanding the biological and clinical context of NEP immunoassay data. To achieve this, we applied in silico epitope prediction and rational peptide selection to generate monoclonal antibodies (mAbs) against spatially distant sites on NEP. One of the selected epitopes contained published N-linked glycosylation sites at N285 and N294. The best antibody pair, mAb 17E11 and 31E1 (glycosylation-sensitive), were characterized by surface plasmon resonance, isotyping, epitope mapping, and western blotting. A validated two-site sandwich NEP ELISA with a limit of detection of 2.15 pg/ml and working range of 13.1-8000 pg/ml was developed with these mAbs. Western analysis using a validated commercial polyclonal antibody (PE pAb) and our mAbs revealed that non-HF and HF plasma NEP circulates as a heterogenous mix of moieties that possibly reflect proteolytic processing, post-translational modifications and homo-dimerization. Both our mAbs detected a ~ 33 kDa NEP fragment which was not apparent with PE pAb, as well as a common ~ 57-60 kDa moiety. These antibodies exhibit different affinities for the various NEP targets. Immunoassay results are dependent on NEP epitopes variably detected by the antibody pairs used, explaining the current discordant NEP measurements derived from different ELISA kits., (© 2024. The Author(s).)

Background: Lack of validation and standardization of research-use-only (RUO) immunoassays brings with it inherent threats to authenticity and functional quality. Poor correlation between different commercial neprilysin RUO immunoassays is concerning and discordant findings need to be resolved. We seek to identify and validate reliable neprilysin immunoassays to strengthen the scientific rigor and reproducibility of neprilysin-related investigation and of biomarker research in general., Methods: Soluble neprilysin (sNEP) concentrations were determined in cohorts (n = 532) from Spain (Cohort 1), New Zealand (NZ, Cohort 2) and Singapore (Cohort 3), using commercial kits from six vendors. Apparent sNEP concentrations were correlated between different assays and with plasma neprilysin activity. Assay reliability was further validated by performance verification, MS analysis and cross-reactivity tests., Results: sNEP in Cohorts 1 and 2 measured concurrently in Spain and NZ showed significant inter-laboratory correlation only for the Aviscera Bioscience sNEP ELISA SK00724-01. Neprilysin concentrations obtained with the R&D systems and SK00724-01 ELISAs correlated with each other but not with neprilysin activity. In Cohort 3, sNEP concentrations from the Perkin Elmer AlphaLISA and Biotechne ELLA assays agreed (r = 0.89) and both correlated with neprilysin activity (r = 0.87, 0.77 respectively). MS analysis detected authentic neprilysin in the AlphaLISA kit calibrator and in antibody pull-down material from human plasma. The AlphaLISA assay performed within acceptable limits (spike and recovery, dilutional linearity, inter- and intra-assay CV) and showed no cross-reactivity against neprilysin substrates and closely-related analogues., Conclusion: AlphaLISA and ELLA assays provide reliable measures of sNEP concentrations. Reliability of other commercial neprilysin assays remains in question., (Copyright © 2022. Published by Elsevier Inc.)

Myocardial restoration using tissue-engineered grafts to regenerate the ischemic myocardium offers improved donor cell retention, yet a limited cell survival resulting from poor vascularization needs to be addressed. A cell type derived from the subamnion, namely, cord-lining mesenchymal stem cells (CL-MSC), has recently been identified. Here we present a restorative strategy that combines a fibrin graft containing human CL-MSC and omental flap providing, thereby, cell-, structural-, and angiogenic support to the injured myocardium. The graft consisted of a mixture of 2×10(6) CL-MSC-GFP-Fluc and fibrin. Myocardial infarction (MI) was induced in nude rats and following confirmation of ensued heart failure with echocardiography 2 weeks after injury, therapeutic intervention was performed as follows: untreated (MI, n=7), CL-MSC graft (CL-MSCG, n=8), CL-MSCG and omental flap (CL-MSCG+OM, n=11), and omental flap (OM, n=8). In vivo bioluminescence imaging at 1, 3, 7, and 14 days post-treatment indicated comparable early donor cell viability between the CL-MSCG and CL-MSCG+OM. Treatment with CL-MSCG+OM improved the myocardial function as assessed by the measurement of end-diastolic left ventricular (LV) pressure (3.53±0.34 vs. 5.21±0.54 mmHg, p<0.05), contractility (+dP/dt, 3383.8±250.78 mmHg vs. 2464.9±191.8 mmHg, p<0.05), and the relaxation rate (-dP/dt, -2707.2±250.7 mmHg vs. 1948.7±207.8 mmHg, p<0.05), compared to MI control 6 weeks after ischemic injury. Furthermore, evidence of a 20.32% increase in the ejection fraction was observed in CL-MSCG+OM rats from week 2 to 6 after injury. Both CL-MSCG and CL-MSCG+OM led to an enhanced cardiac output (p<0.05), and attenuated the infarct size (35.7%±4.2% and 34.7%±4.8%), as compared to MI (60.7%±3.1%; p<0.01 and p<0.001, respectively). All treated groups had a higher arteriole density than controls. Yet, a higher amount of functional blood vessels, and a 20-fold increase in arteriole numbers were found in CL-MSCG+OM. Altogether, CL-MSCGs supplemented with vascular supply have the potential to repair the failing, chronically ischemic heart by improving myocardial revascularization, attenuating remodeling, and ameliorating cardiac dysfunction.

Rationale Myocardial infarction (MI) triggers a dynamic microRNA response with the potential of yielding therapeutic targets. Objective We aimed to identify novel aberrantly expressed cardiac microRNAs post-MI with potential roles in adverse remodeling in a rat model, and to provide post-ischemic therapeutic inhibition of a candidate pathological microRNA in vivo. Methods and results Following microRNA array profiling in rat hearts 2 and 14 days post-MI, we identified a time-dependent up-regulation of miR-31 compared to sham-operated rats. A progressive increase of miR-31 (up to 91.4 ± 11.3 fold) was detected in the infarcted myocardium by quantitative real-time PCR. Following target prediction analysis, reporter gene assays confirmed that miR-31 targets the 3´UTR of cardiac troponin-T ( Tnnt2 ), E2F transcription factor 6 ( E2f6 ), mineralocorticoid receptor ( Nr3c2 ) and metalloproteinase inhibitor 4 ( Timp4 ) mRNAs. In vitro, hypoxia and oxidative stress up-regulated miR-31 and suppressed target genes in cardiac cell cultures, whereas LNA-based oligonucleotide inhibition of miR-31 (miR-31i) reversed its repressive effect on target mRNAs. Therapeutic post-ischemic administration of miR-31i in rats silenced cardiac miR-31 and enhanced expression of target genes, while preserving cardiac structure and function at 2 and 4 weeks post-MI. Left ventricular ejection fraction (EF) improved by 10% (from day 2 to 30 post-MI) in miR-31i-treated rats, whereas controls receiving scrambled LNA inhibitor or placebo incurred a 17% deterioration in EF. miR-31i decreased end-diastolic pressure and infarct size; attenuated interstitial fibrosis in the remote myocardium and enhanced cardiac output. Conclusion miR-31 induction after MI is deleterious to cardiac function while its therapeutic inhibition in vivo ameliorates cardiac dysfunction and prevents the development of post-ischemic adverse remodeling. [ABSTRACT FROM AUTHOR]

Natriuretic peptide receptor 3 (NPR3) is the clearance receptor for the cardiac natriuretic peptides (NPs). By modulating the level of NPs, NPR3 plays an important role in cardiovascular homeostasis. Although the physiological functions of NPR3 have been explored, little is known about its regulation in health or disease. MicroRNAs play an essential role in the post-transcriptional expression of many genes. Our aim was to investigate potential microRNA-based regulation of NPR3 in multiple models. Hypoxic challenge elevated levels of NPPB and ADM mRNA, as well as NT-proBNP and MR-proADM in human left ventricle derived cardiac cells (HCMa), and in the corresponding conditioned medium, as revealed by qRT-PCR and ELISA. NPR3 was decreased while NPR1 was increased by hypoxia at mRNA and protein levels in HCMa. Down-regulation of NPR3 mRNA was also observed in infarct and peri-infarct cardiac tissue from rats undergoing myocardial infarction. From microRNA microarray analyses and microRNA target predictive databases, miR-100 was selected as a candidate regulator of NPR3 expression. Further analyses confirmed up-regulation of miR-100 in hypoxic cells and associated conditioned media. Antagomir-based silencing of miR-100 enhanced NPR3 expression in HCMa. Furthermore, miR-100 levels were markedly up-regulated in rat hearts and in peripheral blood after myocardial infarction and in the blood from heart failure patients. Results from this study point to a role for miR-100 in the regulation of NPR3 expression, and suggest a possible therapeutic target for modulation of NP bioactivity in heart disease. [ABSTRACT FROM AUTHOR]