Turbulence Activates Platelet Biogenesis to Enable Clinical Scale Ex Vivo Production.

The ex vivo generation of platelets from human-induced pluripotent cells (hiPSCs) is expected to compensate donor-dependent transfusion systems. However, manufacturing the clinically required number of platelets remains unachieved due to the low platelet release from hiPSC-derived megakaryocytes (hiPSC-MKs). Here, we report turbulence as a physical regulator in thrombopoiesis in vivo and its application to turbulence-controllable bioreactors. The identification of turbulent energy as a determinant parameter allowed scale-up to 8 L for the generation of 100 billion-order platelets from hiPSC-MKs, which satisfies clinical requirements. Turbulent flow promoted the release from megakaryocytes of IGFBP2, MIF, and Nardilysin to facilitate platelet shedding. hiPSC-platelets showed properties of bona fide human platelets, including circulation and hemostasis capacities upon transfusion in two animal models. This study provides a concept in which a coordinated physico-chemical mechanism promotes platelet biogenesis and an innovative strategy for ex vivo platelet manufacturing.

Nardilysin determines hematopoietic stem cell fitness by regulating protein synthesis.

Nardilysin (NRDC) is a multifunctional protein required for maintaining homeostasis in various cellular and tissue contexts. However, its role in hematopoietic stem cells (HSCs) remains unclear. Here, through the conditional deletion of NRDC in hematopoietic cells, we demonstrate that NRDC is required for HSCs expansion in vitro and the reconstitution of hematopoiesis in vivo after transplantation. We found NRDC-deficient HSCs lose their self-renewal ability and display a preferential bias to myeloid differentiation in response to replication stress. Transcriptome data analysis revealed the upregulation of heat shock response-related genes in NRDC-deficient HSCs. Additionally, we observed increased protein synthesis in cultured NRDC-deficient HSCs. Thus, loss of NRDC may cause the inability to control protein synthesis in response to replication induced protein stress, leading to the impaired HSC self-renewal ability. This highlights a novel model of action of NRDC specifically in HSCs.

Nardilysin in vascular smooth muscle cells controls blood pressure via the regulation of calcium dynamics.

Blood pressure is a crucial physiological parameter and its abnormalities can cause a variety of health problems. We have previously reported that mice with systemic deletion of nardilysin (NRDC), an M16 family metalloprotease, exhibit hypotension. In this study, we aimed to clarify the role of NRDC in vascular smooth muscle cell (VSMC) by generating VSMC-specific Nrdc knockout (VSMC-KO) mice. Our findings reveal that VSMC-KO mice also exhibit hypotension. Aortas isolated from VSMC-KO mice exhibited a weakened contractile response to phenylephrine, accompanied by reduced phosphorylation of myosin light chain 2 and decreased rhoA expression. VSMC isolated from VSMC-KO aortas showed a reduced increase in intracellular Ca2+ concentration induced by α-stimulants. These findings suggest that NRDC in VSMC regulates vascular contraction and blood pressure by modulating Ca2+ dynamics.

JMJD6 Autoantibodies as a Potential Biomarker for Inflammation-Related Diseases.

Inflammation is closely associated with cerebrovascular diseases, cardiovascular diseases, diabetes, and cancers, and it is accompanied by the development of autoantibodies in the early stage of inflammation-related diseases. Hence, it is meaningful to discover novel antibody biomarkers targeting inflammation-related diseases. In this study, Jumonji C-domain-containing 6 (JMJD6) was identified by the serological identification of antigens through recombinant cDNA expression cloning. In particular, JMJD6 is an antigen recognized in serum IgG from patients with unstable angina pectoris (a cardiovascular disease). Then, the serum antibody levels were examined using an amplified luminescent proximity homogeneous assay-linked immunosorbent assay and a purified recombinant JMJD6 protein as an antigen. We observed elevated levels of serum anti-JMJD6 antibodies (s-JMJD6-Abs) in patients with inflammation-related diseases such as ischemic stroke, acute myocardial infarction (AMI), diabetes mellitus (DM), and cancers (including esophageal cancer, EC; gastric cancer; lung cancer; and mammary cancer), compared with the levels in healthy donors. The s-JMJD6-Ab levels were closely associated with some inflammation indicators, such as C-reactive protein and intima-media thickness (an atherosclerosis index). A better postoperative survival status of patients with EC was observed in the JMJD6-Ab-positive group than in the negative group. An immunohistochemical analysis showed that JMJD6 was highly expressed in the inflamed mucosa of esophageal tissues, esophageal carcinoma tissues, and atherosclerotic plaques. Hence, JMJD6 autoantibodies may reflect inflammation, thereby serving as a potential biomarker for diagnosing specific inflammation-related diseases, including stroke, AMI, DM, and cancers, and for prediction of the prognosis in patients with EC.

Serum anti-DIDO1, anti-CPSF2, and anti-FOXJ2 antibodies as predictive risk markers for acute ischemic stroke.

BACKGROUND: Acute ischemic stroke (AIS) is a serious cause of mortality and disability. AIS is a serious cause of mortality and disability. Early diagnosis of atherosclerosis, which is the major cause of AIS, allows therapeutic intervention before the onset, leading to prevention of AIS. METHODS: Serological identification by cDNA expression cDNA libraries and the protein array method were used for the screening of antigens recognized by serum IgG antibodies in patients with atherosclerosis. Recombinant proteins or synthetic peptides derived from candidate antigens were used as antigens to compare serum IgG levels between healthy donors (HDs) and patients with atherosclerosis-related disease using the amplified luminescent proximity homogeneous assay-linked immunosorbent assay. RESULTS: The first screening using the protein array method identified death-inducer obliterator 1 (DIDO1), forkhead box J2 (FOXJ2), and cleavage and polyadenylation specificity factor (CPSF2) as the target antigens of serum IgG antibodies in patients with AIS. Then, we prepared various antigens including glutathione S-transferase-fused DIDO1 protein as well as peptides of the amino acids 297-311 of DIDO1, 426-440 of FOXJ2, and 607-621 of CPSF2 to examine serum antibody levels. Compared with HDs, a significant increase in antibody levels of the DIDO1 protein and peptide in patients with AIS, transient ischemic attack (TIA), and chronic kidney disease (CKD) but not in those with acute myocardial infarction and diabetes mellitus (DM). Serum anti-FOXJ2 antibody levels were elevated in most patients with atherosclerosis-related diseases, whereas serum anti-CPSF2 antibody levels were associated with AIS, TIA, and DM. Receiver operating characteristic curves showed that serum DIDO1 antibody levels were highly associated with CKD, and correlation analysis revealed that serum anti-FOXJ2 antibody levels were associated with hypertension. A prospective case-control study on ischemic stroke verified that the serum antibody levels of the DIDO1 protein and DIDO1, FOXJ2, and CPSF2 peptides showed significantly higher odds ratios with a risk of AIS in patients with the highest quartile than in those with the lowest quartile, indicating that these antibody markers are useful as risk factors for AIS. CONCLUSIONS: Serum antibody levels of DIDO1, FOXJ2, and CPSF2 are useful in predicting the onset of atherosclerosis-related AIS caused by kidney failure, hypertension, and DM, respectively.

Nardilysin controls cardiac sympathetic innervation patterning through regulation of p75 neurotrophin receptor.

Cardiac sympathetic innervation is critically involved in the regulation of circulatory dynamics. However, the molecular mechanism for the innervation patterning has remained elusive. Here, we demonstrate that nardilysin (NRDC, Nrdc), an enhancer of ectodomain shedding, regulates cardiac sympathetic innervation. Nardilysin-deficient (Nrdc-/- ) mice show hypoplastic hearts, hypotension, bradycardia, and abnormal sympathetic innervation patterning. While the innervation of left ventricle (LV) of wild-type mice is denser in the subepicardium than in the subendocardium, Nrdc-/- LV lacks such a polarity and is uniformly and more abundantly innervated. At the molecular level, the full-length form of p75 neurotrophin receptor (p75NTR , Ngfr) is increased in Nrdc-/- LV due to the reduced ectodomain shedding of p75NTR . Importantly, the reduction of p75NTR rescued the abnormal innervation phenotype of Nrdc-/- mice. Moreover, sympathetic neuron-specific, but not cardiomyocyte-specific deletion of Nrdc recapitulated the abnormal innervation patterning of Nrdc-/- mice. In conclusion, neuronal nardilysin critically regulates cardiac sympathetic innervation and circulatory dynamics via modulation of p75NTR .

Deficiency of Nardilysin in the Liver Reduces Serum Cholesterol Levels.

Nardilysin (NRDC) has been shown to be involved in post-translational histone modifications, in addition to enhancement in ectodomain shedding of membrane-anchored protein, which play significant roles in various pathophysiology, including glucose homeostasis, inflammatory diseases and cancer. The present study sought to determine roles of NRDC in the liver on lipid and lipoprotein metabolism. We established liver-specific NRDC deficient mice by use of NRD1 floxed mice and albumin promoter-Cre recombinase (Cre) transgenic mice, and found that their serum low-density lipoprotein (LDL) cholesterol levels were significantly lower than those in control littermate mice. In the liver, LDL receptor (LDLR) mRNA expression was significantly upregulated, while inducible degrader of LDLR (IDOL) and microsomal triglyceride transfer protein (MTP) mRNA expression was significantly downregulated, in liver-specific NRDC deficient mice. Hepatic cell-surface LDLR expression levels were significantly elevated and serum pro-protein convertase subtilisin-kexin type 9 (PCSK9) levels were significantly reduced in mice with hepatic NRDC deficiency. In cultured hepatocytes, NRDC deficiency significantly reduced secreted PCSK9 and increased cell-surface LDLR expression. On the other hand, NRDC overexpression in cultured hepatocytes significantly increased secreted PCSK9 and lowered cell-surface LDLR expression. Thus, NRDC in murine hepatocytes appears to play key roles in cholesterol homeostasis, although the precise molecular mechanisms remain to be determined.

Serum anti-LRPAP1 is a common biomarker for digestive organ cancers and atherosclerotic diseases.

Some cancers are related to atherosclerotic diseases; therefore, these two types of disease may share some antibody biomarkers in common. To investigate this, a first screening of sera was performed from patients with esophageal squamous cell carcinoma (ESCC) or acute ischemic stroke (AIS) for serological identification of antigens using recombinant cDNA expression cloning (SEREX). The amplified luminescent proximity homogeneous assay-linked immunosorbent assay (AlphaLISA) method, which incorporates glutathione donor beads and anti-human IgG acceptor beads, was used to evaluate serum antibody levels. SEREX screening identified low-density lipoprotein receptor-related protein-associated protein 1 (LRPAP1) as a target antigen of serum IgG antibodies in the sera of patients with ESCC or AIS. Antigens, including recombinant glutathione S-transferase-fused LRPAP1 protein, were prepared to examine serum antibody levels. AlphaLISA revealed significantly higher antibody levels against the LRPAP1 protein in patients with solid cancers such as ESCC and colorectal carcinoma and some atherosclerosis-related diseases such as AIS and diabetes mellitus compared with healthy donors. Correlation analysis revealed that the elevated serum antibody levels against LRPAP1 were associated with smoking, a well-known risk factor for both cancer and atherosclerosis. Serum LRPAP1 antibody is therefore a common marker for the early diagnosis of some cancers and atherosclerotic diseases and may reflect diseases caused by habitual smoking.

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.

Nardilysin promotes hepatocellular carcinoma through activation of signal transducer and activator of transcription 3.

Nardilysin (NRDC) is a metalloendopeptidase of the M16 family. We previously showed that NRDC activates inflammatory cytokine signaling, including interleukin-6-signal transducer and activator of transcription 3 (STAT3) signaling. NRDC has been implicated in the promotion of breast, gastric and esophageal cancer, as well as the development of liver fibrosis. In this study, we investigated the role of NRDC in the promotion of hepatocellular carcinoma (HCC), both clinically and experimentally. We found that NRDC expression was upregulated threefold in HCC tissue compared to the adjacent non-tumor liver tissue, which was confirmed by immunohistochemistry and western blotting. We also found that high serum NRDC was associated with large tumor size (>3 cm, P = 0.016) and poor prognosis after hepatectomy (median survival time 32.0 vs 73.9 months, P = 0.003) in patients with hepatitis C (n = 120). Diethylnitrosamine-induced hepatocarcinogenesis was suppressed in heterozygous NRDC-deficient mice compared to their wild-type littermates. Gene silencing of NRDC with miRNA diminished the growth of Huh-7 and Hep3B spheroids in vitro. Notably, phosphorylation of STAT3 was decreased in NRDC-depleted Huh-7 spheroids compared to control spheroids. The effect of a STAT3 inhibitor (S3I-201) on the growth of Huh-7 spheroids was reduced in NRDC-depleted cells relative to controls. Our results show that NRDC is a promising prognostic marker for HCC in patients with hepatitis C, and that NRDC promotes tumor growth through activation of STAT3.

Associations among plasma markers for N-methyl-d-aspartate receptor hypofunction, redox dysregulation, and insufficient myelination in patients with schizophrenia.

Background: Several hypotheses regarding the pathomechanisms of schizophrenia have been proposed. If schizophrenia is a unitary disease, then these pathological processes must be linked; however, if such links do not exist, schizophrenia may best be considered a group of disorders. Only a few studies have examined the relationships among these pathomechanisms. Herein, we examined the relationships among deficient myelination, NMDA receptor hypofunction, and metabolic dysregulation by measuring various plasma markers and examining their correlations. Methods: Plasma samples were collected from 90 patients with schizophrenia and 68 healthy controls. Concentrations of nardilysin (N-arginine dibasic convertase, NRDC), a positive regulator of myelination, the NMDA receptor co-agonist d-serine and glycine, various additional amino acids related to NMDA receptor transmission (glutamate, glutamine, and l-serine), and homocysteine (Hcy), were measured. Concentrations were compared using independent samples t-test or logistic regression, and associations were evaluated using Pearson's correlation coefficients. Results: Plasma glycine (t = 2.05, p = 0.042), l-serine (t = 2.25, p = 0.027), and homocysteine (t = 3.71, p < 0.001) concentrations were significantly higher in patients with schizophrenia compared to those in healthy controls. Logistic regression models using age, sex, smoking status, glutamine, glutamate, glycine, l-serine, d-serine, homocysteine, and NRDC as independent variables revealed significantly lower plasma d-serine (p = 0.024) and NRDC (p = 0.028), but significantly higher l-serine (p = 0.024) and homocysteine (p = 0.001) in patients with schizophrenia. Several unique correlations were found between NMDA receptor-related amino acids and NRDC in patients with schizophrenia compared to those in healthy controls, while no correlations were found between plasma homocysteine and other markers. No associations were found between plasma marker concentrations and disease status or cognitive function in patients with schizophrenia, except for a significant correlation between plasma glycine and full intelligence quotient. Conclusion: Reduced myelination and NMDA receptor hypofunction may be related to pathological mechanisms in schizophrenia, while homocysteine dysregulation appears to be an independent pathological process. These results suggest that schizophrenia may be a group of disorders with unique or partially overlapping etiologies.

Subclinical myocardial damage after anthracycline chemotherapy in Japanese patients with breast cancer.

BACKGROUND: Data on the incidence, timing, and severity of myocardial damage after anthracycline-based chemotherapy (AC) in Japanese patients with breast cancer are limited. METHOD: We evaluated cancer therapy-related cardiac dysfunction (CTRCD) in Japanese women with breast cancer (n = 51) after the first AC according to the definitions of the 2022 European Society of Cardiology onco-cardiology guideline, including assessment of high-sensitivity troponin I (TnI) and B-type natriuretic peptide (BNP) levels. RESULTS: CTRCD was detected in 67 % of the patients (3.9 %, 7.8 %, 9.8 %, 43 %, 37 %, 22 %, 20 %, and 9.8 % of patients at 1 week and 1, 2, 3, 6, 9, 12, and 15 months post-AC, respectively) without significant left ventricular ejection fraction reduction (<50 %) and heart failure. Elevated TnI levels (>26 pg/mL) were found in 43 % of patients, and elevated BNP levels (≥35 pg/mL) were observed in 22 % of patients during the follow-up period. CONCLUSIONS: Approximately two-thirds of the Japanese patients in this study experienced CTRCD, which was frequently observed at 3 or 6 months post-AC. However, all patients with CTRCD were diagnosed with mild asymptomatic CTRCD. Although, these patients were diagnosed with mild asymptomatic CTRCD, careful long-term follow-up will be required.

Null mutation of exocyst complex component 3-like does not affect vascular development in mice.

Exocyst is an octameric protein complex implicated in exocytosis. The exocyst complex is highly conserved among mammalian species, but the physiological function of each subunit in exocyst remains unclear. Previously, we identified exocyst complex component 3-like (Exoc3l) as a gene abundantly expressed in embryonic endothelial cells and implicated in the process of angiogenesis in human umbilical cord endothelial cells. Here, to reveal the physiological roles of Exoc3l during development, we generated Exoc3l knockout (KO) mice by genome editing with CRISPR/Cas9. Exoc3l KO mice were viable and showed no significant phenotype in embryonic angiogenesis or postnatal retinal angiogenesis. Exoc3l KO mice also showed no significant alteration in cholesterol homeostasis or insulin secretion, although several reports suggest an association of Exoc3l with these processes. Despite the implied roles, Exoc3l KO mice exhibited no apparent phenotype in vascular development, cholesterol homeostasis, or insulin secretion.

Auxiliary roles of nardilysin in the early diagnosis of acute coronary syndrome: a prospective cohort study, the Nardi-ACS study.

Acute coronary syndrome (ACS) includes myocardial infarction (MI) and unstable angina (UA). MI is defined by elevated necrosis markers, preferably high-sensitivity cardiac troponins (hs-cTn). However, it takes hours for cTn to become elevated after coronary occlusion; therefore, difficulties are associated with diagnosing early post-onset MI or UA. The aim of this prospective cohort study was to examine the diagnostic ability of serum nardilysin (NRDC) for the early detection of ACS. This study consisted of two sequential cohorts, the Phase I cohort, 435 patients presenting to the emergency room (ER) with chest pain, and the Phase II cohort, 486 patients with chest pain who underwent coronary angiography. The final diagnosis was ACS in 155 out of 435 patients (35.6%) in the phase I and 418 out of 486 (86.0%) in the phase II cohort. Among 680 patients who presented within 24 h of onset, 466 patients (68.5%) were diagnosed with ACS. Serum NRDC levels were significantly higher in patients with ACS than in those without ACS. The sensitivity of NRDC in patients who presented within 6 h after the onset was higher than that of hsTnI, and the AUC of NRDC within 1 h of the onset was higher than that of hsTnI (0.718 versus 0.633). Among hsTnI-negative patients (300 of 680 patients: 44.1%), 136 of whom (45.3%) were diagnosed with ACS, the sensitivity and the NPV of NRDC were 73.5 and 65.7%, respectively. When measured in combination with hsTnI, NRDC plays auxiliary roles in the early diagnosis of ACS.

Identification and characterization of nardilysin as a novel dimethyl H3K4-binding protein involved in transcriptional regulation.

Histone methylation on lysine residues is believed to function primarily as docking sites to recruit specific proteins termed as histone code "readers" or "effectors." Each lysine residue can be mono-, di, and tri-methylated and different methylation states can have different effect on chromatin function. While an increasing number of proteins have been identified and characterized as specific effectors for methylated histones, very few of the proteins are known to recognize a particular state of methylation. In this study, we identified nardilysin (NRDc), a member of M16 family metalloendopeptidases, as a novel dimethyl-H3K4 (H3K4me2)-binding protein. Among three methylated states, NRDc binds preferentially H3K4me2 both in vitro and in vivo. Biochemical purification demonstrated that NRDc interacts with the NCoR/SMRT corepressor complex. We identified target genes repressed by NRDc through microarray. We showed that NRDc is physically associated with and recruits the NCoR complex to some of the repressed genes and this association correlates with binding of H3K4me2. Thus, our study has identified a novel H3K4me2-binding protein and revealed a role of NRDc in transcriptional regulation.

LSR defines cell corners for tricellular tight junction formation in epithelial cells.

Epithelial cell contacts consist of not only bicellular contacts but also tricellular contacts, where the corners of three cells meet. At tricellular contacts, tight junctions (TJs) generate specialized structures termed tricellular TJs (tTJs) to seal the intercellular space. Tricellulin is the only known molecular component of tTJs and is involved in the formation of tTJs, as well as in the normal epithelial barrier function. However, the detailed molecular mechanism of how tTJs are formed and maintained remains elusive. Using a localization-based expression cloning method, we identified a novel tTJ-associated protein known as lipolysis-stimulated lipoprotein receptor (LSR). Upon LSR knockdown in epithelial cells, tTJ formation was affected and the epithelial barrier function was diminished. Tricellulin accumulation at the tricellular contacts was also diminished in these cells. By contrast, LSR still accumulated at the tricellular contacts upon tricellulin knockdown. Analyses of deletion mutants revealed that the cytoplasmic domain of LSR was responsible for the recruitment of tricellulin. On the basis of these observations, we propose that LSR defines tricellular contacts in epithelial cellular sheets by acting as a landmark to recruit tricellulin for tTJ formation.

Immunohistochemical and clinicopathological study regarding nardilysin on extramammary Paget's disease.

It has been reported that nardilysin (NRDC), a metalloendopeptidase which regulates various growth factors and cytokines, is associated with malignancies in a conflicting manner, in which it promoted gastric, hepatocellular, and colorectal cancers and suppressed pancreatic ductal adenocarcinoma. However, it has not been investigated how NRDC is associated with cutaneous malignancies for now. Immunohistochemical staining has revealed that NRDC expression is observed in all extramammary Paget's disease (EMPD) cases. Notably, other cutaneous malignancies including basal cell carcinoma, squamous cell carcinoma, and eccrine porocarcinoma, did not show increased NRDC expression in immunohistochemistry. EMPD typically presents several types of lesions including nodules, and positive staining of NRDC on EMPD was observed regardless of the type of lesions. Examination using samples taken from nodular lesions showed that some cases showed heterogenous NRDC expression within each lesion. We also found that NRDC staining was weaker in the marginal parts of EMPD lesion than in the central parts in several cases, and tumor cells tend to be distributed beyond the macroscopic skin lesions in these cases. It was speculated that decreased NRDC expression in the marginal zones of the skin lesions may be associated with the ability of tumor cells to produce the cutaneous manifestation of EMPD. This study suggests that NRDC may be associated with EMPD like other malignancies reported previously.

Ketone bodies: A double-edged sword for mammalian life span.

Accumulating evidence suggests health benefits of ketone bodies, and especially for longevity. However, the precise role of endogenous ketogenesis in mammalian life span, and the safety and efficacy of the long-term exogenous supplementation of ketone bodies remain unclear. In the present study, we show that a deficiency in endogenous ketogenesis, induced by whole-body Hmgcs2 deletion, shortens life span in mice, and that this is prevented by daily ketone body supplementation using a diet containing 1,3-butanediol, a precursor of ß-hydroxybutyrate. Furthermore, feeding the 1,3-butanediol-containing diet from early in life increases midlife mortality in normal mice, but in aged mice it extends life span and prevents the high mortality associated with atherosclerosis in ApoE-deficient mice. By contrast, an ad libitum low-carbohydrate ketogenic diet markedly increases mortality. In conclusion, endogenous ketogenesis affects mammalian survival, and ketone body supplementation may represent a double-edged sword with respect to survival, depending on the method of administration and health status.

Utility of atherosclerosis-associated serum antibodies against colony-stimulating factor 2 in predicting the onset of acute ischemic stroke and prognosis of colorectal cancer.

Introduction: Autoantibodies against inflammatory cytokines may be used for the prevention of atherosclerosis. Preclinical studies consider colony-stimulating factor 2 (CSF2) as an essential cytokine with a causal relationship to atherosclerosis and cancer. We examined the serum anti-CSF2 antibody levels in patients with atherosclerosis or solid cancer. Methods: We measured the serum anti-CSF2 antibody levels via amplified luminescent proximity homogeneous assay-linked immunosorbent assay based on the recognition of recombinant glutathione S-transferase-fused CSF2 protein or a CSF2-derived peptide as the antigen. Results: The serum anti-CSF2 antibody (s-CSF2-Ab) levels were significantly higher in patients with acute ischemic stroke (AIS), acute myocardial infarction (AMI), diabetes mellitus (DM), and chronic kidney disease (CKD) compared with healthy donors (HDs). In addition, the s-CSF2-Ab levels were associated with intima-media thickness and hypertension. The analyzes of samples obtained from a Japan Public Health Center-based prospective study suggested the utility of s-CSF2-Ab as a risk factor for AIS. Furthermore, the s-CSF2-Ab levels were higher in patients with esophageal, colorectal, gastric, and lung cancer than in HDs but not in those with mammary cancer. In addition, the s-CSF2-Ab levels were associated with unfavorable postoperative prognosis in colorectal cancer (CRC). In CRC, the s-CSF2-Ab levels were more closely associated with poor prognosis in patients with p53-Ab-negative CRC despite the lack of significant association of the anti-p53 antibody (p53-Ab) levels with the overall survival. Conclusion: S-CSF2-Ab was useful for the diagnosis of atherosclerosis-related AIS, AMI, DM, and CKD and could discriminate poor prognosis, especially in p53-Ab-negative CRC.

Nardilysin in adipocytes regulates UCP1 expression and body temperature homeostasis.

Brown adipose tissue (BAT) dissipates chemical energy as heat through uncoupling protein 1 (UCP1). The induction of mitochondrial reactive oxygen species (ROS) in BAT was recently identified as a mechanism that supports UCP1-dependent thermogenesis. We previously demonstrated that nardilysin (NRDC) plays critical roles in body temperature homeostasis. Global NRDC-deficient (Nrdc-/-) mice show hypothermia due to a lower set point for body temperature, whereas BAT thermogenesis at room temperature (RT) is enhanced mainly to compensate for poor thermal insulation. To examine the primary role of NRDC in BAT thermogenesis, we generated adipocyte-specific NRDC-deficient (Adipo-KO) mice by mating Nrdc floxed (Nrdcflox/flox) mice with adiponectin-Cre mice. Adipo-KO mice showed hyperthermia at both RT and thermoneutrality. They were also more cold-tolerant than Nrdcflox/flox mice. However, UCP1 mRNA levels were significantly lower in Adipo-KO BAT at RT, thermoneutrality, and 4 °C, whereas no significant differences were observed in UCP1 protein levels at RT and 4 °C. We examined the protein stability of UCP1 using the cycloheximide chase assay and found that NRDC negatively regulated its stability via the ubiquitin-proteasome pathway. NRDC may be also involved in ROS-mediated in vivo thermogenesis because the inhibitory effects of N-acetyl cysteine, an ROS scavenger, on ß3 agonist-induced thermogenesis were stronger in Adipo-KO mice. Collectively, the present results demonstrate that NRDC in BAT controls adaptive thermogenesis and body temperature homeostasis possibly via the regulation of UCP1 protein stability and ROS levels.