Serum proteomic identification and validation of two novel atherosclerotic aortic aneurysm biomarkers, profilin 1 and complement factor D.

BACKGROUND: Effective diagnostic biomarkers for aortic aneurysm (AA) that are detectable in blood tests are required because early detection and rupture risk assessment of AA can provide insights into medical therapy and preventive treatments. However, known biomarkers for AA lack specificity and reliability for clinical diagnosis. METHODS: We performed proteome analysis of serum samples from patients with atherosclerotic thoracic AA (TAA) and healthy control (HC) subjects to identify diagnostic biomarkers for AA. Serum samples were separated into low-density lipoprotein, high-density lipoprotein, and protein fractions, and the major proteins were depleted. From the proteins identified in the three fractions, we narrowed down biomarker candidates to proteins uniformly altered in all fractions between patients with TAA and HC subjects and evaluated their capability to discriminate patients with TAA and those with abdominal AA (AAA) from HC subjects using receiver operating characteristic (ROC) analysis. For the clinical validation, serum concentrations of biomarker candidates were measured in patients with TAA and AAA registered in the biobank of the same institute, and their capability for the diagnosis was evaluated. RESULTS: Profilin 1 (PFN1) and complement factor D (CFD) showed the most contrasting profiles in all three fractions between patients with TAA and HC subjects and were selected as biomarker candidates. The PFN1 concentration decreased, whereas the CFD concentration increased in the sera of patients with TAA and AAA when compared with those of HC subjects. The ROC analysis showed that these proteins could discriminate patients with TAA and AAA from HC subjects. In the validation study, these candidates showed significant concentration differences between patients with TAA or AAA and controls. PFN1 and CFD showed sufficient area under the curve (AUC) in the ROC analysis, and their combination further increased the AUC. The serum concentrations of PFN1 and CFD also showed significant differences between patients with aortic dissection and controls in the validation study. CONCLUSION: PFN1 and CFD are potential diagnostic biomarkers for TAA and AAA and measurable in blood samples; their diagnostic performance can be augmented by their combination. These biomarkers may facilitate the development of diagnostic systems to identify patients with AA.

Sacubitril/valsartan ameliorates renal tubulointerstitial injury through increasing renal plasma flow in a mouse model of type 2 diabetes with aldosterone excess.

BACKGROUND: Aldosterone has been assumed to be one of aggravating factors in diabetic kidney disease (DKD). Natriuretic peptides/guanylyl cyclase-A/cGMP signalling has been shown to ameliorate aldosterone-induced renal injury in mice. Sacubitril/valsartan (SAC/VAL) is used clinically for chronic heart failure and hypertension, in part by augmenting natriuretic peptide bioavailability. The effects of SAC/VAL on renal pathophysiology including in DKD, however, have remained unclarified. METHODS: Eight-week-old male db/db mice fed on a high-salt diet (HSD) were treated with vehicle or aldosterone (0.2 µg/kg/min), and divided into four groups: HSD control, ALDO (aldosterone), ALDO + VAL (valsartan), and ALDO + SAC/VAL group. After 4 weeks, they were analysed for plasma atrial natriuretic peptide (ANP) levels, renal histology, and haemodynamic parameters including glomerular filtration rate (GFR) by FITC-inulin and renal plasma flow (RPF) by para-amino hippuric acid. RESULTS: The ALDO + SAC/VAL group showed significantly increased plasma ANP concentration and creatinine clearance, and decreased tubulointerstitial fibrosis and neutrophil gelatinase-associated lipocalin expression compared to ALDO and ALDO + VAL groups. SAC/VAL treatment increased GFR and RPF, and suppressed expression of Tgfb1, Il1b, Ccl2, and Lcn2 genes compared to the ALDO group. The percentage of tubulointerstitial fibrotic areas negatively correlated with the RPF and GFR. CONCLUSION: In a mouse model of type 2 diabetes with aldosterone excess, SAC/VAL increased RPF and GFR, and ameliorated tubulointerstitial fibrosis. Furthermore, RPF negatively correlated well with tubulointerstitial injury, suggesting that the beneficial effects of SAC/VAL could be through increased renal plasma flow with enhanced natriuretic peptide bioavailability.

Utility of perinatal natriuretic peptide for predicting neonatal heart failure.

BACKGROUND: We evaluated the significance of perinatal plasma natriuretic peptide (NP) levels in neonates with congenital heart defects (CHDs) or arrhythmias and determined whether measurement of perinatal plasma NP levels and echocardiographic assessment in utero could predict heart failure after birth. METHODS: The study was conducted between 2012 and 2016 to evaluate the correlation of perinatal atrial NP (ANP) and brain NP (BNP) levels at birth with the modified Ross score after birth and the cardiovascular profile (CVP) score before birth. RESULTS: A total of 122 singletons with CHDs or arrhythmias and 27 controls were analyzed. Neonatal blood sampling was performed at a median of 0.7 h (range, 0.1-1.5) after birth. The neonatal plasma ANP and BNP levels shortly after birth were significantly higher than those in the umbilical artery (UA) plasma. The ANP and BNP levels in UA and neonatal blood were correlated with the modified Ross score. The neonatal plasma ANP and BNP levels and the modified Ross scores were inversely correlated with the CVP score in neonates with CHDs or arrhythmias. The area under the receiver operating characteristic curve of UA ANP levels for predicting neonatal heart failure was highest among those for the CVP score, perinatal plasma ANP and BNP levels, and their combinations. CONCLUSIONS: The plasma ANP and BNP levels increased markedly shortly after birth. Assessment of the UA plasma ANP level at birth and the CVP score in utero may be utilized to predict neonatal heart failure.

Deficiency of Cardiac Natriuretic Peptide Signaling Promotes Peripartum Cardiomyopathy-Like Remodeling in the Mouse Heart.

BACKGROUND: The maternal circulatory system and hormone balance both change dynamically during pregnancy, delivery, and the postpartum period. Although atrial natriuretic peptides and brain natriuretic peptides produced in the heart control circulatory homeostasis through their common receptor, NPR1, the physiologic and pathophysiologic roles of endogenous atrial natriuretic peptide/brain natriuretic peptide in the perinatal period are not fully understood. METHODS: To clarify the physiologic and pathophysiologic roles of the endogenous atrial natriuretic peptide/brain natriuretic peptide-NPR1 system during the perinatal period, the phenotype of female wild-type and conventional or tissue-specific Npr1-knockout mice during the perinatal period was examined, especially focusing on maternal heart weight, blood pressure, and cardiac function. RESULTS: In wild-type mice, lactation but not pregnancy induced reversible cardiac hypertrophy accompanied by increases in fetal cardiac gene mRNAs and ERK1/2 (extracellular signaling-regulated kinase) phosphorylation. Npr1-knockout mice exhibited significantly higher plasma aldosterone level than did wild-type mice, severe cardiac hypertrophy accompanied by fibrosis, and left ventricular dysfunction in the lactation period. Npr1-knockout mice showed a high mortality rate over consecutive pregnancy-lactation cycles. In the hearts of Npr1-knockout mice during or after the lactation period, an increase in interleukin-6 mRNA expression, phosphorylation of signal transducer and activator of transcription 3, and activation of the calcineurin-nuclear factor of the activated T cells pathway were observed. Pharmacologic inhibition of the mineralocorticoid receptor or neuron-specific deletion of the mineralocorticoid receptor gene significantly ameliorated cardiac hypertrophy in lactating Npr1-knockout mice. Anti-interleukin-6 receptor antibody administration tended to reduce cardiac hypertrophy in lactating Npr1-knockout mice. CONCLUSIONS: These results suggest that the characteristics of lactation-induced cardiac hypertrophy in wild-type mice are different from exercise-induced cardiac hypertrophy, and that the endogenous atrial natriuretic peptide/brain natriuretic peptide-NPR1 system plays an important role in protecting the maternal heart from interleukin-6-induced inflammation and remodeling in the lactation period, a condition mimicking peripartum cardiomyopathy.

Manipulation of beta-adrenergic receptor in pressure-overloaded murine hearts mimics adverse and reverse cardiac remodeling.

Transverse aortic constriction (TAC) has been widely used to create pressure overload induced heart failure in mice. However, this conventional model has some limitations such as low reproducibility and long creation period of cardiac failure. In order to establish a highly reproducible cardiac failure model that mimics adverse cardiac remodeling (ACR) we combined pressure overload and beta-adrenergic receptor stimuli using isoproterenol (ISO) and explored the optimal TAC model by changing the durations of TAC and the doses of ISO. Thus we constructed a suitable model for ACR with an effective combination of 3-week TAC and subsequent one-week ISO (3 mg/kg/day) infusion. Using RNA-Seq analyses, we identified that the up-regulated genes were mainly related to fibrosis including Fbn1, C1qtnf6 and Loxl2; and that the down-regulated genes were associated with mitochondrial function including Uqcrc1, Ndufs3, and Idh2 in failing hearts of our ACR model. Next, we followed the changes in cardiac function after ceasing ISO infusion. Left ventricular function gradually recovered after cessation of ISO, suggesting cardiac reverse remodeling (CRR). Gene expression signatures of hearts, which exhibited CRR, were almost identical to that of TAC hearts without ISO. In conclusion, our new model exhibits a transition to ACR and subsequent CRR with high reproducibility. This murine model might add new insights into the experiments of heart failure technically as well as scientifically.

A New Secretory Peptide of Natriuretic Peptide Family, Osteocrin, Suppresses the Progression of Congestive Heart Failure After Myocardial Infarction.

RATIONALE: An increase of severe ischemic heart diseases results in an increase of the patients with congestive heart failure (CHF). Therefore, new therapies are expected in addition to recanalization of coronary arteries. Previous clinical trials using natriuretic peptides (NPs) prove the improvement of CHF by NPs. OBJECTIVE: We aimed at investigating whether OSTN (osteocrin) peptide potentially functioning as an NPR (NP clearance receptor) 3-blocking peptide can be used as a new therapeutic peptide for treating CHF after myocardial infarction (MI) using animal models. METHODS AND RESULTS: We examined the effect of OSTN on circulation using 2 mouse models; the continuous intravenous infusion of OSTN after MI and the OSTN-transgenic (Tg) mice with MI. In vitro studies revealed that OSTN competitively bound to NPR3 with atrial NP. In both OSTN-continuous intravenous infusion model and OSTN-Tg model, acute inflammation within the first week after MI was reduced. Moreover, both models showed the improvement of prognosis at 28 days after MI by OSTN. Consistent with the in vitro study binding of OSTN to NPR3, the OSTN-Tg exhibited an increased plasma atrial NP and C-type NP, which might result in the improvement of CHF after MI as indicated by the reduced weight of hearts and lungs and by the reduced fibrosis. CONCLUSIONS: OSTN might suppress the worsening of CHF after MI by inhibiting clearance of NP family peptides.

Design and synthesis of peptidic partial agonists of human neuromedin U receptor 1 with enhanced serum stability.

Neuromedin U (NMU) activates two receptors (NMUR1 and NMUR2) and is a promising candidate for development of drugs to combat obesity. Previously, we obtained hexapeptides as selective full NMUR agonists. Development of a partial agonist which mildly activates receptors is an effective strategy which lead to an understanding of the functions of NMU receptors. In 2014, we reported hexapeptide 3 (CPN-124) as an NMUR1-selective partial agonist but its selectivity and serum stability were unsatisfactory. Herein, we report the development of a hexapeptide-type partial agonist (8, CPN-223) based on a peptide (3) but with higher NMUR1-selectivity and enhanced serum stability. A structure-activity relationship study of synthetic pentapeptide derivatives suggested that a hexapeptide is a minimum structure consistent with both good NMUR1-selective agonistic activity and serum stability.

A chemically stable peptide agonist to neuromedin U receptor type 2.

Neuromedin U (NMU) is a peptide with appetite suppressive activity and other physiological activities via activation of the NMU receptors NMUR1 and NMUR2. In 2014, we reported the first NMUR2 selective agonist, 3-cyclohexylpropionyl-Leu-Leu-Dap-Pro-Arg-Asn-NH2 (CPN-116). However, we found that CPN-116 in phosphate buffer is unstable because of Nα-to-Nß acyl migration at the Dap residue. In this study, the chemical stability of CPN-116 was evaluated under various conditions, and it was found to be relatively stable in buffers such as HEPES and MES. We also performed a structure-activity relationship study to obtain an NMUR2-selective agonist with improved chemical stability. Consequently, CPN-219 bearing a Dab residue in place of Dap emerged as a next-generation hexapeptidic NMUR2 agonist.

Identification and mapping of brain natriuretic peptide in the normal ventricular myocardium of a desert-dwelling mammalian model, the camel (Camelus dromedarius): Immunohistochemical and ultrastructural study.

Brain natriuretic peptide (BNP) is mainly produced in the ventricular myocardium, where it is released into the circulation, producing rapid volume decrease by diuresis, natriuresis, and water shift into the extracellular space, and vasodilation. The dromedary camel, a mammalian model of the desert nomads, lives under unfavorable physiological stresses during thirst, starvation, desiccation, and hot climate, thus has a special demand for water homeostasis. The present studies characterized BNP in the ventricular myocardium of healthy camels, immunohistochemically with a specific antibody, and ultrastructurally identified the endocrine property of the cardiomyocytes and Purkinje fibers. The paranuclear, granular, immunoreactive material was not restricted to the cardiomyocytes, as it was also visible in the Purkinje fibers and their associated nerve varicosities. The intensity of immunoreactive BNP showed a transmural gradient from the subepicardium to the myocardium. Intense immunoreactivity was also noted among the perivascular cardiomyocytes. At the electron microscopic level, specific granules were demonstrated in the paranuclear cytosol of cardiomyocytes and Purkinje fibers. The current study provides the first immunohistochemical localization pattern of BNP in the camel myocardium and suggests a relationship between the intense subepicardial BNP-immunoexpression and a possible translocation of the active hormone to the pericardial fluid for further paracrine actions on the heart and its coronaries.

Wisteria floribunda agglutinin staining for the quantitative assessment of cardiac fibrogenic activity in a mouse model of dilated cardiomyopathy.

Cardiac fibrosis is a typical phenomenon in failing hearts for most cardiac diseases, including dilated cardiomyopathy (DCM), and its specific detection and quantification are crucial for the analysis of cardiac remodeling. Since cardiac fibrosis is characterized by extensive remodeling of the myocardial extracellular matrix (ECM), in which glycoproteins are the major components, we assumed that fibrosis-related alterations in the cardiac glycome and glycoproteome would be suitable targets for the detection of cardiac fibrosis. Here, we compared protein glycosylation between heart tissues of normal and DCM model mice by laser microdissection-assisted lectin microarray. Among 45 lectins, Wisteria floribunda agglutinin (WFA) was selected as the most suitable lectin for staining cardiac fibrotic tissues. Although the extent of WFA staining was highly correlated (r > 0.98) with that of picrosirius red staining, a common collagen staining method, WFA did not bind to collagen fibers. Further histochemical analysis with N-glycosidase revealed that WFA staining of fibrotic tissues was attributable to the binding of WFA to N-glycoproteins. Using a mass spectrometry-based approach, we identified WFA-binding N-glycoproteins expressed in DCM hearts, many of which were fibrogenesis-related ECM proteins, as expected. In addition, the identified glycoproteins carrying WFA-binding N-glycans were detected only in DCM hearts, suggesting their cooperative glycosylation alterations with disease progression. Among these WFA-binding ECM N-glycoproteins, co-localization of the collagen α6(VI) chain protein and WFA staining in cardiac tissue sections was confirmed with a double-staining analysis. Collectively, these results indicate that WFA staining is more suitable for the quantitative assessment of cardiac fibrogenic activity than current collagen staining methods. Furthermore, given that plasma WFA-binding glycoprotein levels were significantly correlated with the echocardiographic parameters for left ventricular remodeling, cardiac WFA-binding glycoproteins are candidate circulating glyco-biomarkers for the quantification and monitoring of cardiac fibrogenesis.

Maternal biomarkers for fetal heart failure in fetuses with congenital heart defects or arrhythmias.

BACKGROUND: Diagnosis of fetal heart failure depends primarily on fetal ultrasonography assessment. Our recent study demonstrated that plasma natriuretic peptide levels in umbilical cord blood were correlated with the severity of heart failure in fetuses with congenital heart defects or arrhythmias. However, percutaneous umbilical blood sampling is an invasive procedure, and therefore, less or noninvasive biomarkers reflecting fetal heart failure are required. OBJECTIVE: The aim of this study was to investigate the possibility of whether maternal serum biomarkers can diagnose fetal heart failure in fetuses with congenital heart defects or arrhythmias. STUDY DESIGN: This exploratory cross-sectional study was conducted at a tertiary pediatric cardiac center. A total of 50 singletons with fetal congenital heart defects or arrhythmias and 50 controls who were registered in the National Cerebral and Cardiovascular Center Biobank from 2013 to 2016 were included. Maternal serum samples obtained during the third trimester were analyzed for 2 hormones and 36 cytokines using the Bio-Plex Pro Human Cancer Biomarker panels 1 and 2. We comprehensively analyzed the association between maternal serum biomarkers and ultrasonography findings or fetal arrhythmia status. Fetal heart failure was defined as a cardiovascular profile score ≤7. RESULTS: Of 37 fetuses with congenital heart defects, heart failure was found in 1 case of tricuspid valve dysplasia with moderate tricuspid regurgitation. Of 13 fetuses with arrhythmias, 5 had heart failure at 28-33 weeks of gestation. Maternal serum cytokine and hormone concentrations were compared between patients with and without fetal heart failure at 28-33 weeks of gestation (n = 6 and n = 61, respectively). Sixty-one fetuses without heart failure consisted of 10 with congenital heart defect, 6 with arrhythmia, and 45 controls. Maternal serum concentrations of tumor necrosis factor-α, interleukin-6, soluble Fas ligand, transforming growth factor-α, and vascular endothelial growth factor-D were significantly higher when fetuses had heart failure than when they did not (P < .05), whereas maternal serum concentrations of heparin-binding epidermal growth factor-like growth factor were significantly lower when fetuses had heart failure than when they did not (P < .05). Multivariate analysis showed that maternal serum concentrations of tumor necrosis factor-α, vascular endothelial growth factor-D, and heparin-binding epidermal growth factor-like growth factor were independently associated with fetal heart failure. The cutoff values were as follows: tumor necrosis factor-α, 68 pg/mL (sensitivity of 50.0%, specificity of 93.4%, positive likelihood ratio of 7.6, negative likelihood ratio of 0.5); vascular endothelial growth factor-D, 1156 pg/mL (sensitivity of 50.0%, specificity of 93.4%, positive likelihood ratio of 7.6, negative likelihood ratio of 0.5); and heparin-binding epidermal growth factor-like growth factor, 90 pg/mL (sensitivity of 83.3%, specificity of 83.6%, positive likelihood ratio of 5.1, negative likelihood ratio of 0.2). The combination of these 3 cytokines showed sensitivity of 100%, specificity of 80.3%, positive likelihood ratio of 5.1, and negative likelihood ratio of 0. In the absence of fetal heart failure, concentrations of all maternal serum cytokines and hormones were similar in cases of fetal congenital heart defects and controls, while maternal serum soluble CD40 ligand concentrations were increased only in fetal arrhythmias. CONCLUSION: Maternal serum concentrations of tumor necrosis factor-α, vascular endothelial growth factor-D, and heparin-binding epidermal growth factor-like growth factor were associated with fetal heart failure.

Amniotic Fluid Natriuretic Peptide Levels in Fetuses With Congenital Heart Defects or Arrhythmias.

BACKGROUND: We have previously demonstrated that umbilical cord plasma natriuretic peptide (NP) levels reflect the severity of heart failure (HF) in fetuses with congenital heart defects (CHD). The aim of this study was to evaluate the significance of amniotic fluid (AF) NP levels in the assessment of HF in fetuses with CHD or arrhythmia. Methods and Results: This was a prospective observational study at a tertiary pediatric cardiac center. A total of 95 singletons with CHD or arrhythmia, and 96 controls from 2012 to 2015 were analyzed. AF concentrations of atrial NP (ANP), B-type NP (BNP) and N-terminal pro-B-type NP (NT-proBNP) at birth were compared with ultrasonographic assessment of fetal HF using the cardiovascular profile (CVP) score. Multivariate analysis showed that a CVP score ≤5 and preterm birth are independently associated with high AF NT-proBNP levels. AF NT-proBNP levels of fetuses with CHD or arrhythmia inversely correlated with CVP score (P for trend <0.01). In contrast, AF concentrations of ANP and BNP were extremely low, and it was difficult to assess the degree of fetal HF based on them. CONCLUSIONS: AF NT-proBNP concentrations increase in stepwise fashion with the severity of HF in fetuses with CHD or arrhythmia; it was the optimal NP for assessing the fetal HF.

Characterization of Gonadotrope Secretoproteome Identifies Neurosecretory Protein VGF-derived Peptide Suppression of Follicle-stimulating Hormone Gene Expression.

Reproductive function is controlled by the pulsatile release of hypothalamic gonadotropin-releasing hormone (GnRH), which regulates the expression of the gonadotropins luteinizing hormone and FSH in pituitary gonadotropes. Paradoxically, Fshb gene expression is maximally induced at lower frequency GnRH pulses, which provide a very low average concentration of GnRH stimulation. We studied the role of secreted factors in modulating gonadotropin gene expression. Inhibition of secretion specifically disrupted gonadotropin subunit gene regulation but left early gene induction intact. We characterized the gonadotrope secretoproteome and global mRNA expression at baseline and after Gαs knockdown, which has been found to increase Fshb gene expression (1). We identified 1077 secreted proteins or peptides, 19 of which showed mRNA regulation by GnRH or/and Gαs knockdown. Among several novel secreted factors implicated in Fshb gene regulation, we focused on the neurosecretory protein VGF. Vgf mRNA, whose gene has been implicated in fertility (2), exhibited high induction by GnRH and depended on Gαs In contrast with Fshb induction, Vgf induction occurred preferentially at high GnRH pulse frequency. We hypothesized that a VGF-derived peptide might regulate Fshb gene induction. siRNA knockdown or extracellular immunoneutralization of VGF augmented Fshb mRNA induction by GnRH. GnRH stimulated the secretion of the VGF-derived peptide NERP1. NERP1 caused a concentration-dependent decrease in Fshb gene induction. These findings implicate a VGF-derived peptide in selective regulation of the Fshb gene. Our results support the concept that signaling specificity from the cell membrane GnRH receptor to the nuclear Fshb gene involves integration of intracellular signaling and exosignaling regulatory motifs.

Endogenous peptide profile for elucidating biosynthetic processing of the ghrelin precursor.

Ghrelin is an orexigenic peptide primarily produced by gastric endocrine cells. The biosynthetic cleavage site of ghrelin has been well documented, but how its downstream region undergoes proteolytic processing remains poorly explored. Here, we provide the first snapshot of endogenous peptides from the ghrelin precursor by profiling the secretopeptidome of cultured mouse ghrelin-producing cells during exocytosis. Mapping of MS/MS sequenced peptides to the precursor highlighted three atypical monobasic processing sites, including the established C-terminus of ghrelin and the N-terminal cleavage site for obestatin, a putative 23-amino-acid C-terminally amidated peptide. However, we found that mouse obestatin does not occur in the form originally reported, but that a different amidation site is used to generate a shorter peptide. These data can be extended to study and characterize the precursor-derived peptides located downstream of ghrelin in different biological contexts.

NERP-2 regulates gastric acid secretion and gastric emptying via the orexin pathway.

Neuroendocrine regulatory peptide (NERP)-2 is derived from a distinct region of VGF, a neurosecretory protein originally identified as a product of a nerve growth factor-responsive gene in rat PC12 cells. Colocalization of NERP-2 with orexin-A in the lateral hypothalamus increases orexin-A-induced feeding and energy expenditure in both rats and mice. Orexigenic and anorectic peptides in the hypothalamus modulate gastric function. In this study, we investigated the effect of NERP-2 on gastric function in rats. Intracerebroventricular administration of NERP-2 to rats increased gastric acid secretion and gastric emptying, whereas peripheral administration did not affect gastric function. NERP-2-induced gastric acid secretion and gastric emptying were blocked by an orexin 1 receptor antagonist, SB334867. NERP-2 also induced Fos expression in the lateral hypothalamus and the dorsomotor nucleus of the vagus X, which are key sites in the central nervous system for regulation of gastric function. Atropine, a blocker of vagal efferent signal transduction, completely blocked NERP-2-induced gastric acid secretion. These results demonstrate that central administration of NERP-2 activates the orexin pathway, resulting in elevated gastric acid secretion and gastric emptying.

Three molecular forms of atrial natriuretic peptides: quantitative analysis and biological characterization.

Atrial natriuretic peptide (ANP) is primarily produced in the heart tissue and plays a pivotal role in maintaining cardiovascular homeostasis in endocrine and autocrine/paracrine systems and has clinical applications as a biomarker and a therapeutic agent for cardiac diseases. ANP is synthesized by atrial cardiomyocytes as a preprohormone that is processed by a signal peptidase and stored in secretory granules as a prohormone. Subsequent proteolytic processing of ANP by corin during the secretion process results in a bioactive form consisting of 28 amino acid residues. Mechanical stretch of the atrial wall and multiple humoral factors directly stimulates the transcription and secretion of ANP. Secreted ANP elicits natriuretic and diuretic effects via cyclic guanosine monophosphate produced through binding to the guanylyl cyclase-A/natriuretic peptide receptor-A. Circulating ANP is subjected to rapid clearance by a natriuretic peptide receptor-C-mediated mechanism and proteolytic degradation by neutral endopeptidase. In humans, ANP is present as three endogenous molecular forms: bioactive α-ANP, a homodimer of α-ANP designated as ß-ANP, and an ANP precursor designated as proANP (also referred to as γ-ANP). The proANP and especially ß-ANP, as minor forms in circulation, are notably increased in patients with cardiac diseases, suggesting the utility of monitoring the pathophysiological conditions that result in abnormal proANP processing that cannot be monitored by inactive N-terminal proANP-related fragments. Emerging plate-based sandwich immunoassays for individual quantitation of the three ANP forms enables evaluation of diagnostic implications and net ANP bioactivity. This new tool may provide further understanding in the pathophysiology of cardiac diseases. Copyright © 2017 European Peptide Society and John Wiley & Sons, Ltd.

Multiomics approach to identify novel biomarkers for dilated cardiomyopathy: Proteome and transcriptome analyses of 4C30 dilated cardiomyopathy mouse model.

Dilated cardiomyopathy (DCM) is an intractable disease, without any radical treatment option other than cardiac transplantation. Additionally, biomarkers to determine progressive staging are not yet available. Irrespective of the diversity of causative gene mutations, the phenotype of DCM is rather common. Therefore, it is plausible to determine DCM staging in terms of variations in protein and mRNA levels. In this study, we performed proteome and transcriptome analysis of the left ventricle of 4C30 DCM model mice showing mild and severe phenotypes at 12 and 24 weeks (wk) after birth, respectively. Proteomic analyses results showed 109 proteins that increased and 133 others that decreased among 1874 detected proteins. We selected biomarker candidates by confirming consistent alterations in protein levels at 12 and 24 wk, and mRNA levels at 12 wk, and narrowed these down based on the requirement that they should be detectable in blood. Finally, we selected six biomarker candidates based on sustained or augmented alteration at 24 wk and confirmed their definite alterations in the left ventricle by quantitative polymerase chain reaction, western blot analysis, and multiple reaction monitoring (MRM). To assess the validity of this strategy, we measured plasma concentrations of the six candidates by MRM method and identified two proteins (FTL1 and GRP78) that demonstrated significant elevation in the 4C30 mice plasma. Taken together, a multiomics strategy comparing tissue expression levels of proteins and mRNAs between diseased and control groups, with appropriate confirmation, is a promising approach for the discovery of new biomarkers. © 2016 Wiley Periodicals, Inc. Biopolymers (Pept Sci) 106: 491-502, 2016.

Peptidomics for Studying Limited Proteolysis.

Limited proteolysis is a pivotal mechanism regulating protein functions. Identifying physiologically or pathophysiologically relevant cleavage sites helps to develop molecular tools that can be used for diagnostics or therapeutics. During proteolysis of secretory and membrane proteins, part of the cleaved protein is liberated and destined to undergo degradation but should retain original cleavage sites created by proteolytic enzymes. We profiled endogenous peptides accumulated for 4 h in media conditioned by primary cultured rat cardiac fibroblasts. A total of 3916 redundant peptide sequences from 94 secretory proteins and membrane proteins served to identify limited cleavage sites, both annotated and unannotated, for signal peptide or propeptide removal, peptide hormone processing, ectodomain shedding, and regulated intramembrane proteolysis. Incorrectly predicted signal cleavage sites are found in typical proteins such as extracellular matrix proteins and the peptide hormone precursor adrenomedullin ADM. The revealed signal peptide cleavage site for ADM was experimentally verified by identifying the major molecular form of flanking proadrenomedullin N-terminal peptide. We suggest that profiling of endogenous peptides, like transcriptome sequence reads, makes sense in regular cells such as fibroblasts and that peptidomics provides insight into proteolysis-regulated protein functions.

Free Thiol of Transthyretin in Human Plasma Most Accessible to Modification/Oxidation.

Free-thiol(s) in proteins, especially, when located on the surface of the molecule, are susceptible to oxidation/modification, which may cause loss of function or an alteration in the ternary structure. This suggests that the status of thiol group(s) of cysteine residue(s) in a protein, i.e., free-thiol versus an oxidized/modified form, in vivo, could reflect the physiological state of the molecule with respect to susceptibility to oxidative stress. To address this issue, we established an efficient method for isolating proteins that contain free thiol groups from a complex mixture, which permits the amount of free-thiol form(s) to modified/oxidized forms to be estimated. Albumin, which accounts for 55% of the total plasma proteins and has such a free thiol and has been reported to scavenge various reactive oxygen species (ROS) in vivo. The developed method was used to isolate the free form of albumin from fresh plasma. However, contrary to our expectations, transthyretin (TTR), which also has a single free thiol, was found to be the major protein that was the most susceptible to modification/oxidation. In addition, the free-thiol form could be separated from oxidized or modified molecules, permitting the relative abundance of the free-thiol form to be estimated. The findings show that the levels of the free-thiol form of TTR in plasma was significantly lowered after a hydrogen peroxide treatment, even at low concentrations (0.1 mM), suggesting that TTR could be a useful biomarker for monitoring a ROS imbalance in relation to various oxidative stress conditions.

Chronic effects of neuroendocrine regulatory peptide (NERP-1 and -2) on insulin secretion and gene expression in pancreatic ß-cells.

Neuroendocrine regulatory peptides (NERP-1 and -2) are novel amidated peptides derived from VGF, a polypeptide secreted from neurons and endocrine cells through a regulated pathway. Dr. Nakazato Masamitsu reported that NERP-1 and -2 may have a local modulator function on the human endocrine system, and clearly showed expression of NERP-1 and -2 in human pancreas islets. Based on these data, we investigated the alteration of insulin secretion, insulin granule-related protein, and pancreas-specific transcription factors in response to NERPs expression. We confirmed the expression of NERP-1 and -2 in the pancreas of a human diabetes patient, in addition to diabetic animal models. When INS1 cells and primary rat islets were incubated with 10nM NERPs for 3 days, glucose-stimulated insulin secretion levels were blunted by NERP-1 and -2. The number of insulin granules released from the readily releasable pool, which is associated with the first phase of glucose-stimulated insulin release, was decreased by NERP-1 and -2. Insulin granule-related proteins and mRNAs were down-regulated by NERP-2 treatment. NERP-2 decreased the expression of BETA2/NeuroD and insulin and controlled the nucleo-cytoplasmic translocation of FOXO1 and Pdx-1. We observed that NERP-2 levels were dramatically increased in diabetic pancreas. In conclusion, NERP-2 may play an important role in insulin secretion through the regulation of insulin secretory granules and ß-cell transcription factors. In addition, NERP-2 expression is increased in diabetic conditions. Therefore, we suggest that NERPs may be potent endogenous suppressors of glucose-dependent insulin secretion.