Kiyoto Nishi

Critical roles of nardilysin in the maintenance of body temperature homoeostasis

Body temperature homoeostasis in mammals is governed centrally through the regulation of shivering and non-shivering thermogenesis and cutaneous vasomotion. Non-shivering thermogenesis in brown adipose tissue (BAT) is mediated by sympathetic activation, followed by PGC-1α induction, which drives UCP1. Here we identify nardilysin (Nrd1 and NRDc) as a critical regulator of body temperature homoeostasis. Nrd1−/− mice show increased energy expenditure owing to enhanced BAT thermogenesis and hyperactivity. Despite these findings, Nrd1−/− mice show hypothermia and cold intolerance that are attributed to the lowered set point of body temperature, poor insulation and impaired cold-induced thermogenesis. Induction of β3-adrenergic receptor, PGC-1α and UCP1 in response to cold is severely impaired in the absence of NRDc. At the molecular level, NRDc and PGC-1α interact and co-localize at the UCP1 enhancer, where NRDc represses PGC-1α activity. These findings reveal a novel nuclear function of NRDc and provide important insights into the mechanism of thermoregulation.

Nardilysin prevents amyloid plaque formation by enhancing α-secretase activity in an Alzheimer's disease mouse model

Amyloid beta (Aβ) peptide, the main component of senile plaques in patients with Alzheimers disease (AD), is derived from proteolytic cleavage of amyloid precursor protein (APP) by β- and γ-secretases. Alpha-cleavage of APP by α-secretase has a potential to preclude the generation of Aβ because it occurs within the Aβ domain. We previously reported that a metalloendopeptidase, nardilysin (N-arginine dibasic convertase; NRDc) enhances α-cleavage of APP, which results in the decreased generation of Aβ in vitro. To clarify the in vivo role of NRDc in AD, we intercrossed transgenic mice expressing NRDc in the forebrain with an AD mouse model. Here we demonstrate that the neuron-specific overexpression of NRDc prevents Aβ deposition in the AD mouse model. The activity of α-secretase in the mouse brain was enhanced by the overexpression of NRDc, and was reduced by the deletion of NRDc. However, reactive gliosis adjacent to the Aβ plaques, one of the pathological features of AD, was not affected by the overexpression of NRDc. Taken together, our results indicate that NRDc controls Aβ formation through the regulation of α-secretase.

Intermittent infusions of carperitide or inotoropes in out-patients with advanced heart failure

BACKGROUND The ambulatory treatment of advanced heart failure (HF) with intermittent infusions of inotropes or natriuretic peptide chosen immediately before each infusion has not been described. METHODS Between May 2005 and July 2009, we treated 11 patients presenting with advanced HF, who received a total of 369 infusions of carperitide, olprinone, dopamine, or dobutamine, once or twice weekly. The pharmaceutical was selected before each infusion based on the systolic blood pressure (BP). RESULTS Carperitide, olprinone, and catecholamines were administered to 8 (73 infusions of 0.030±0.004μg/kg/min for 3.3±0.8h), 4 (18 infusions of 0.070±0.017μg/kg/min for 3.3±0.5h), and 6 patients (278 infusions of 3.6±1.9μg/kg/min for 2.8±1.0h), respectively. No adverse effect requiring cessation of infusion was observed. Over a mean follow-up of 29.3±28.8months (range 2-104), 4 patients died, all from cardiac causes. The Kaplan-Meier cumulative survival rate was 69.3% at 20 months (median follow-up). Compared with the pre-infusion period, the duration and number of hospitalizations for management of HF were decreased by 73.9% (p=0.017), and 51.9% (p=0.007), respectively, during the treatment period, and the overall medical costs by 56.9% (p=0.021). CONCLUSIONS In this study population, intermittent drug infusions selected from inotropes or natriuretic peptide based on the baseline systolic BP significantly decreased the length and number of hospitalizations and costs, without increasing mortality. These results indicate that intermittent infusions might be one of the therapeutic options in advanced HF.

The clinical and hemodynamic factors that influence the concentrations of biomarkers of myocyte injury measured by high sensitive assay PATHFAST

BACKGROUND Subclinical myocyte injury plays an important role in the progression of congestive heart failure. However, the clinical and hemodynamic factors that influence the concentrations of biomarkers of myocyte injury have not been clarified. METHODS Blood was sampled during diagnostic cardiac catheterization from 108 consecutive patients without acute coronary syndrome and acute cardiac decompensation. The serum concentrations of B-type natriuretic peptide (BNP), cardiac troponin I (cTnI), creatine kinase (CK)-MB, and myoglobin were measured simultaneously by high sensitive PATHFAST assay. Single and multiple variable regression analyses were carried out in search of correlations between clinical and hemodynamic variables and concentrations of biomarkers. RESULTS By multiple variable analysis, hemoglobin concentration, pulmonary capillary wedge pressure (PCWP), left ventricular (LV) ejection fraction, and estimated glomerular filtration rate (GFR) were independently correlated with a BNP concentration ≥ median 72.1 pg/ml. The only factors independently correlated with a concentration of cTnI ≥ median 0.01 ng/ml were PCWP and estimated GFR. Cardiac index emerged as a single, powerful, independent correlate of CK-MB concentration ≥ median 0.66 ng/ml, and estimated GFR emerged as a single independent correlate of myoglobin concentration ≥ median 40.1 ng/ml. CONCLUSIONS Clinical and hemodynamic factors influence the concentrations of BNP, cTnI, CK-MB, and myoglobin. These factors should be considered when interpreting the concentrations of these biochemical markers.

Nardilysin Is Required for Maintaining Pancreatic β-Cell Function.

Type 2 diabetes (T2D) is associated with pancreatic β-cell dysfunction, manifested by reduced glucose-stimulated insulin secretion (GSIS). Several transcription factors enriched in β-cells, such as MafA, control β-cell function by organizing genes involved in GSIS. Here we demonstrate that nardilysin (N-arginine dibasic convertase; Nrd1 and NRDc) critically regulates β-cell function through MafA. Nrd1−/− mice showed glucose intolerance and severely decreased GSIS. Islets isolated from Nrd1−/− mice exhibited reduced insulin content and impaired GSIS in vitro. Moreover, β-cell-specific NRDc-deficient (Nrd1delβ) mice showed a diabetic phenotype with markedly reduced GSIS. MafA was specifically downregulated in islets from Nrd1delβ mice, whereas overexpression of NRDc upregulated MafA and insulin expression in INS832/13 cells. Chromatin immunoprecipitation assay revealed that NRDc is associated with Islet-1 in the enhancer region of MafA, where NRDc controls the recruitment of Islet-1 and MafA transcription. Our findings demonstrate that NRDc controls β-cell function via regulation of the Islet-1–MafA pathway.

Serial measurements of high sensitive cardiac troponin I in patients with acutely decompensated heart failure treated with carperitide or nitrates.

BACKGROUND In patients with acutely decompensated heart failure (ADHF), elevated serum concentration of cardiac troponin is an independent predictor of adverse cardiac events. In ADHF with a preserved systolic blood pressure, treatment with intravenous vasodilator is recommended. However, the effect of vasodilators on troponin concentrations has not been elucidated well. METHODS AND RESULTS Serial high sensitive cardiac troponin I (hs-TnI) was measured in 36 patients presenting with ADHF and preserved systolic blood pressure, of whom 20 were treated with atrial natriuretic peptide (ANP) and 16 with nitrates. The concentrations of hs-TnI ranged from 0.069+/-0.114ng/ml at baseline to 0.076+/-0.121ng/ml at 5h, 0.062+/-0.106ng/ml at 1 day, and 0.056+/-0.089ng/ml at day 7 (n=36,ns). The relative change in hs-TnI between baseline and at 5h, day 1 and day 7 were 1.13+/-0.43, 0.95+/-0.44 and 0.93+/-0.64 in patients treated with ANP, and 1.02+/-0.19, 0.95+/-0.31 and 1.19+/-1.38 in patients treated with nitrates (ns; ANP versus nitrates). On day 7, a hs-TnI change, >20% decrease from baseline, was observed in 55% patients with ANP versus 56% patients with nitrates (ns). The cardiac event rates were similar in both groups. CONCLUSIONS In ADHF patients with preserved systolic blood pressure, the administration of intravenous vasodilators did not decrease hs-TnI over the first 7 days. Treatments with ANP and nitrates were associated with similar short-term decreases in hs-TnI and long-term adverse cardiac events.

Nardilysin is a promising biomarker for the early diagnosis of acute coronary syndrome

BACKGROUND Biomarkers for detection of transient myocardial ischemia in patients with unstable angina (UA) or for very early diagnosis of acute myocardial infarction (AMI) are not currently available. METHODS AND RESULTS We performed two sequential screenings of autoantibodies elevated shortly after the onset of acute coronary syndrome (ACS), and focused on metalloendopeptidase nardilysin (NRDC) among 19 identified candidate antigens. In a retrospective analysis among 93 ACS and 117 non-ACS patients, the serum level of NRDC was significantly increased in patients with ACS compared with that in patients with non-ACS (2073.5±189.8pg/ml versus 775.7±63.4pg/ml, P<0.0001). The area under the curve of NRDC for the diagnosis of ACS was 0.822 by the receiver operating characteristic curves analysis. In the time course analysis in 43 consecutive ACS patients (AMI: N=35 and UA: N=8), serum concentration of NRDC was significantly increased even in UA patients with a peak serum NRDC levels reached at admission both in AMI and UA patients. In a mouse model of AMI, we found an acute increase in serum NRDC and reduced NRDC expression in ischemic regions shortly after coronary artery ligation. NRDC expression was also reduced in infarcted regions in human autopsy samples from AMI patients. Moreover, the short treatment of primary culture of rat cardiomyocytes with H2O2 or A23187 induced NRDC secretion without cell toxicity. CONCLUSION NRDC is a promising biomarker for the early detection of ACS, even in UA patients without elevation of necrosis markers.

Nardilysin is involved in autoimmune arthritis via the regulation of tumour necrosis factor alpha secretion

Objective Tumour necrosis factor alpha (TNF-α) plays an important role in rheumatoid arthritis (RA). TNF-α is synthesised as a membrane-anchored precursor and is fully activated by a disintegrin and metalloproteinase 17 (ADAM17)-mediated ectodomain shedding. Nardilysin (NRDC) facilitates ectodomain shedding via activation of ADAM17. This study was undertaken to elucidate the role of NRDC in RA. Methods NRDC-deficient (Nrdc–/– ) mice and macrophage-specific NRDC-deficient (NrdcdelM ) mice were examined in murine RA models, collagen antibody-induced arthritis (CAIA) and K/BxN serum transfer arthritis (K/BxN STA). We evaluated the effect of gene deletion or silencing of Nrdc on ectodomain shedding of TNF-α in macrophages or monocytes. NRDC concentration in synovial fluid from patients with RA and osteoarthritis (OA) were measured. We also examined whether local gene silencing of Nrdc ameliorated CAIA. Results CAIA and K/BxN STA were significantly attenuated in Nrdc–/– mice and NrdcdelM mice. Gene deletion or silencing of Nrdc in macrophages or THP-1 cells resulted in the reduction of TNF-α shedding. The level of NRDC is higher in synovial fluid from RA patients compared with that from OA patients. Intra-articular injection of anti-Nrdcsmall interfering RNA ameliorated CAIA. Conclusion These data indicate that NRDC plays crucial roles in the pathogenesis of autoimmune arthritis and could be a new therapeutic target for RA treatment.

Nardilysin controls intestinal tumorigenesis through HDAC1/p53–dependent transcriptional regulation

Colon cancer is a complex disease affected by a combination of genetic and epigenetic factors. Here we demonstrate that nardilysin (N-arginine dibasic convertase; NRDC), a metalloendopeptidase of the M16 family, regulates intestinal tumorigenesis via its nuclear functions. NRDC is highly expressed in human colorectal cancers. Deletion of the Nrdc gene in ApcMin mice crucially suppressed intestinal tumor development. In ApcMin mice, epithelial cell-specific deletion of Nrdc recapitulated the tumor suppression observed in Nrdc-null mice. Moreover, epithelial cell-specific overexpression of Nrdc significantly enhanced tumor formation in ApcMin mice. Notably, epithelial NRDC controlled cell apoptosis in a gene dosage-dependent manner. In human colon cancer cells, nuclear NRDC directly associated with HDAC1, and controlled both acetylation and stabilization of p53, with alterations of p53 target apoptotic factors. These findings demonstrate that NRDC is critically involved in intestinal tumorigenesis through its epigenetic regulatory function, and targeting NRDC may lead to a novel prevention or therapeutic strategy against colon cancer.

Nardilysin inhibits pancreatitis and suppresses pancreatic ductal adenocarcinoma initiation in mice

Objective Nardilysin (NRDC), a zinc peptidase, exhibits multiple localisation-dependent functions including as an enhancer of ectodomain shedding in the extracellular space and a transcriptional coregulator in the nucleus. In this study, we investigated its functional role in exocrine pancreatic development, homeostasis and the formation of pancreatic ductal adenocarcinoma (PDA). Design We analysed Ptf1a-Cre; Nrdcflox/flox mice to investigate the impact of Nrdc deletion. Pancreatic acinar cells were isolated from Nrdcflox/flox mice and infected with adenovirus expressing Cre recombinase to examine the impact of Nrdc inactivation. Global gene expression in Nrdc-cKO pancreas was analysed compared with wild-type pancreas by microarray analysis. We also analysed Ptf1a-Cre; KrasG12D; Nrdcflox/flox mice to investigate the impact of Nrdc deletion in the context of oncogenic Kras. A total of 51 human samples of pancreatic intraepithelial lesions (PanIN) and PDA were examined by immunohistochemistry for NRDC. Results We found that pancreatic deletion of Nrdc leads to spontaneous chronic pancreatitis concomitant with acinar-to-ductal conversion, increased apoptosis and atrophic pancreas in mice. Acinar-to-ductal conversion was observed mainly through a non-cell autonomous mechanism, and the expression of several chemokines was significantly increased in Nrdc-null pancreatic acinar cells. Furthermore, pancreatic deletion of Nrdc dramatically accelerated KrasG12D -driven PanIN and subsequent PDA formation in mice. These data demonstrate a previously unappreciated anti-inflammatory and tumour suppressive functions of Nrdc in the pancreas in mice. Finally, absence of NRDC expression was observed in a subset of human PanIN and PDA. Conclusion Nrdc inhibits pancreatitis and suppresses PDA initiation in mice.