The evidence to date: implications of l-ascorbic acid in the pathophysiology of aging.

L-Ascorbic acid, commonly known as vitamin C, has been used not only for disease prevention and in complementary and alternative medicine, but also for anti-aging purposes. However, the scientific evidence is not yet sufficient. Here, we review the physiological functions of vitamin C and its relationship with various pathological conditions, including our previous findings, and discuss the prospects of its application in healthy longevity. In summary, vitamin C levels are associated with lifespan in several animal models. Furthermore, clinical studies have shown that the blood vitamin C levels are lower in middle-aged and older adults than in younger adults. Lower blood vitamin C levels have also been observed in various pathological conditions such as chronic kidney disease and chronic obstructive pulmonary disease in the elderly. These observations suggest the implications of vitamin C in age-related pathological mechanisms owing to its physiological functions.

Essential role of ROS - 8-Nitro-cGMP signaling in long-term memory of motor learning and cerebellar synaptic plasticity.

Although reactive oxygen species (ROS) are known to have harmful effects in organisms, recent studies have demonstrated expression of ROS synthases at various parts of the organisms and the controlled ROS generation, suggesting possible involvement of ROS signaling in physiological events of individuals. However, physiological roles of ROS in the CNS, including functional roles in higher brain functions or neuronal activity-dependent ROS production, remain to be elucidated. Here, we demonstrated involvement of ROS - 8-NO2-cGMP signaling in motor learning and synaptic plasticity in the cerebellum. In the presence of inhibitors of ROS signal or ROS synthases, cerebellar motor learning was impaired, and the stimulus inducing long-term depression (LTD), cellular basis for the motor learning, failed to induce LTD but induced long-term potentiation (LTP)-like change at cerebellar synapses. Furthermore, ROS was produced by LTD-inducing stimulus in enzyme-dependent manner, and excess administration of the antioxidant vitamin E impaired cerebellar motor learning, suggesting beneficial roles of endogenous ROS in the learning. As a downstream signal, involvement of 8-NO2-cGMP in motor learning and cerebellar LTD were also revealed. These findings indicate that ROS - 8-NO2-cGMP signal is activated by neuronal activity and is essential for cerebellum-dependent motor learning and synaptic plasticity, demonstrating involvement of the signal in physiological function of brain systems.

Role of inter-alpha-trypsin inhibitor heavy chain 4 and its citrullinated form in experimental arthritis murine models.

Inter-α-trypsin inhibitor heavy chain 4 (ITIH4) is a major protein in serum and reported to be upregulated at the onset of rheumatoid arthritis (RA). Its citrullinated form, cit-ITIH4, is specifically found in the serum and synovial fluid of patients with RA. However, the detailed function of ITIH4 in arthritis remains unknown. The aim of this study was to clarify the role of ITIH4 and cit-ITIH4 using experimental arthritis models. ITIH4 and cit-ITIH4 expression was examined in steady-state mice and two different arthritis models, and their pathological effects were examined in Itih4-deficient mice. In naïve C57BL/6 (WT) mice, ITIH4 was expressed as mRNA in the liver and the lung and was expressed as protein in serum and hepatocytes. In K/BxN serum transferred arthritis (K/BxN-STA) and collagen-induced arthritis (CIA), ITIH4 and cit-ITIH4 in sera were increased before the onset of arthritis, and cit-ITIH4 was further increased at the peak of arthritis. In Itih4-deficient mice, citrullinated proteins in serum and joints, especially 120 kDa protein, were clearly diminished; however, there was no significant difference in arthritis severity between WT and itih-/- mice either in the K/BxN-STA or CIA model. CIA mice also exhibited pulmonary lesions and itih4-/- mice tended to show enhanced inflammatory cell aggregation compared to WT mice. Neutrophils in the lungs of itih4-/- mice were significantly increased compared to WT mice. In summary, ITIH4 itself did not alter the severity of arthritis but may inhibit autoimmune inflammation via suppression of neutrophil recruitment.

Amelioration of Astrocyte-Mediated Neuroinflammation by EI-16004 Confers Neuroprotection in an MPTP-induced Parkinson's Disease Model.

Parkinson's disease (PD) is a neurodegenerative disorder that results in motor impairment due to dopaminergic neuronal loss. The pathology of PD is closely associated with neuroinflammation, which can be characterized by astrocyte activation. Thus, targeting the inflammatory response in astrocytes might provide a novel therapeutic approach. We conducted a luciferase assay on an in-house chemical library to identify compounds with anti-inflammatory effects capable of reducing MPP+-induced NF-κB activity in astrocytes. Among the compounds identified, EI-16004, a novel 3-benzyl-N-phenyl-1H-pyrazole-5-carboxamides, exhibited a significant anti-inflammatory effect by significantly reducing MPP+-induced astrocyte activation. Biochemical analysis and docking simulation indicated that EI-16004 inhibited the MPP+-induced phosphorylation of p65 by attenuating ERK phosphorylation, and EI-16004 reduced pro-inflammatory cytokine and chemokine levels in astrocytes. In vivo studies on the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced PD model in male C57BL/6 mice showed that EI-16004 ameliorated motor impairment and protected against dopaminergic neuronal loss, and EI-16004 effectively mitigated the MPTP-induced astrocyte activation in striatum (STR) and substantia nigra (SN). These results indicate EI-16004 is a potential neuroprotective agent for the prevention and treatment of astrocyte-mediated neuroinflammatory conditions in PD.

Glutamate oxidase sheets-Prussian blue grafted amperometric biosensor for the real time monitoring of glutamate release from primary cortical neurons.

Glutamate (GLU) is a primary excitatory neurotransmitter, and its dysregulation is associated with several neurodegenerative disorders. A major challenge in GLU estimation is the existence of other biomolecules in the brain that could directly get oxidized at the electrode. Hence, highly selective electroenzymatic biosensors that enable rapid estimation of GLU are needed. Initially, a copolymer, poly(2-dimethylaminoethyl methacrylate- styrene) was synthesized through reversible addition-fragmentation chain transfer polymerization to noncovalently functionalize reduced graphene oxide (rGO), named DS-rGO. Glutamate oxidase macromolecule immobilized DS-rGO formed enzyme nanosheets, which was drop-coated over Prussian blue electrodeposited disposable electrodes to fabricate the GLU biosensor. The interconnectivity between the enzyme nanosheets and the Prussian blue endows the biosensor with enhanced conductivity and electrochemical activity. The biosensor exhibited a linearity: 3.25-250 µM; sensitivity: 3.96 µA mM-1 cm-2, and a limit of detection: 0.96 µM for GLU in the Neurobasal Medium. The biosensor was applied to an in vitro primary rat cortical model to discriminate GLU levels in Neurobasal Medium, before and after KCl mediated depolarization, which provides new insights for elucidating neuronal functioning in the brain.

Resveratrol Upregulates Senescence Marker Protein 30 by Activating AMPK/Sirt1-Foxo1 Signals and Attenuating H2O2-Induced Damage in FAO Rat Liver Cells.

Resveratrol (RSV) is a polyphenol with numerous biological functions, including anti-inflammatory, antioxidant, and anti-aging activities. The novel senescence marker protein-30 (SMP30) indicates aging, and it suppresses hepatic oxidative stress. However, the effects of RSV on SMP30 expression regulation remain unclear. We observed that RSV positively regulates SMP30 expression in rat hepatoma-derived FAO cells. However, this was abolished by Compound C and EX-527 that specifically inhibit AMP-activated protein kinase (AMPK) and Silent Information Regulator T1 (Sirt1), respectively. We predicted binding sites for AMPK, forkhead box protein O1 (Foxo1), and Sirt1 downstream molecules as possible SMP30 promoters using the JASPAR and UniProtKB databases. We identified a Foxo1 binding site in the promoter region of SMP30. Inhibiting Foxo1 with AS1842527 also decreased the RSV-induced upregulation of SMP30 expression. Moreover, RSV suppressed the substantial downregulation of SMP30 expression caused by oxidative stress and hydrogen peroxide (H2O2) and released accumulated lactate dehydrogenase. These results demonstrate that, as a novel food factor, RSV-induced upregulation of SMP30 by activating AMPK/Sirt1-Foxo1 signaling and may attenuates H2O2-induced oxidative damage. The findings of this study offer new perspectives of the anti-ageing properties of RSV.

Single-cell RNA sequencing to detect age-associated genes that identify senescent cells in the liver of aged mice.

Senescent cells are predicted to occur and increase in animal tissues with aging. However, senescent cells in the tissues of aged animals remain to be identified. We refer to the marker genes to identify senescent cells in tissues as "age-associated genes". In this study, we searched for age-associated genes to identify senescent cells in the livers of aged animals. We performed single-cell RNA sequencing (scRNA-seq) to screen candidates for age-associated genes using young and aged rat primary hepatocytes. To remove animal species specificity, gene expression analyses in mouse livers were performed, confirming age-associated increases in the mRNA expression levels of Glipr1, Clec12a, and Phlda3. Moreover, the mRNA expression levels of Glipr1 and Phlda3 were increased by stress-induced premature senescence using doxorubicin in primary hepatocytes and livers of young mice. Transcriptome data of aged rat hepatocytes suggested that Glipr1, Clec12a, and Phlda3 were expressed in almost identical cells. Fluorescence in situ hybridization (FISH) confirmed the presence of cells with abundant Glipr1, Clec12a, and Phlda3 mRNA in 27-month-old mouse primary hepatocytes, which are considered to be senescent cells. This study is the first to identify Glipr1, Clec12a, and Phlda3 as age-associated genes in the mouse liver.

Effects of heated tobacco product aerosol extracts on DNA methylation and gene transcription in lung epithelial cells.

AIMS: Smoking causes DNA methylation (DNAm) alterations that lead to lung cancer development. Although the use of heated tobacco products (HTPs) has recently increased, their impact on health remains unclear. This study aimed to evaluate the effects of HTPs on DNAm and gene transcription in human lung epithelial cells in vitro. MAIN METHODS: Human lung adenocarcinoma (A549) cells with type II alveolar epithelial characteristics were treated with aerosol extracts of two HTPs or a smoke extract of combustible reference cigarette (RC). Global 5-methylcytosine (5-mC) and 5-hydroxymethylcytosine (5-hmC) levels were quantified using dot blot analysis. Furthermore, reduced representation of bisulfite sequencing, DNA microarray, and quantitative PCR analyses were performed to determine CpG methylation and gene transcription changes induced by HTP and RC. KEY FINDINGS: Global 5-mC and 5-hmC levels were decreased by the RC extract but not the HTP extracts. However, an HTP extract altered the CpG methylation pattern, and Gene Ontology enrichment analysis of the differentially methylated regions of the RC and HTP groups showed a similar pattern. The HTP extract affected gene expression, albeit to a lesser extent than the RC extract. In particular, the HTP extract markedly affected the mRNA expression and promoter methylation of cytochrome P450 family 1 subfamily A member 1 (CYP1A1), which is associated with carcinogenic risk. SIGNIFICANCE: The study results suggest that HTPs as well as conventional combustible cigarettes can alter CpG methylation and gene transcription in lung epithelial cells.

Accuracy of Therapeutic Drug Monitoring of Teicoplanin at the Onset of Febrile Neutropenia.

Background and Objectives: Teicoplanin (TEIC) is an effective drug for patients with febrile neutropenia (FN); however, it has been reported that these patients may have increased TEIC clearance compared with patients who do not have FN. The purpose of this study was to study therapeutic drug monitoring in patients with FN when the TEIC dosing design was based on the population mean method. Materials and Methods: Thirty-nine FN patients with hematological malignancy were included in the study. To calculate the predicted blood concentration of TEIC, we used the two population pharmacokinetic (population PK) parameters (parameters 1 and 2) reported by Nakayama et al. and parameter 3, which is a modification of the population PK of Nakayama et al. We calculated the mean prediction error (ME), an indicator of prediction bias, and the mean absolute prediction error (MAE), an indicator of accuracy. Furthermore, the percentage of predicted TEIC blood concentration within 25% and 50% of the measured TEIC blood concentration was calculated. Results: The ME values were -0.54, -0.25, and -0.30 and the MAE values were 2.29, 2.19, and 2.22 for parameters 1, 2, and 3, respectively. For all of the three parameters, the ME values were calculated as minus values, and the predicted concentrations tended to be biased toward smaller values relative to the measured concentrations. Patients with serum creatinine (Scr) < 0.6 mg/dL and neutrophil counts < 100/µL had greater ME and MAE values and a smaller percentage of predicted TEIC blood concentration within 25% of measured TEIC blood concentrations compared with other patients. Conclusions: In patients with FN, the accuracy of predicting TEIC blood concentrations was good, with no significant differences between each parameter. However, patients with a Scr < 0.6 mg/dL and a neutrophil count < 100/µL showed slightly inferior prediction accuracy.

Moderate protein intake percentage in mice for maintaining metabolic health during approach to old age.

Nutritional requirements for maintaining metabolic health may vary with each life stage, such as young, middle, and old age. To investigate the appropriate ratio of nutrients, particularly proteins, for maintaining metabolic health while approaching old age, young (6-month-old) and middle-aged (16-month-old) mice were fed isocaloric diets with varying protein percentages (5%, 15%, 25%, 35%, and 45% by calorie ratio) for two months. The low-protein diet developed mild fatty liver, with middle-aged mice showing more lipids than young mice, whereas the moderate-protein diet suppressed lipid contents and lowered the levels of blood glucose and lipids. Self-organizing map (SOM) analysis revealed that plasma amino acid profiles differed depending on age and difference in protein diet and were associated with hepatic triglyceride and cholesterol levels. Results indicate that the moderate protein intake percentages (25% and 35%) are required for maintaining metabolic health in middle-aged mice, which is similar to that in young mice.

Renal Function in Aged C57BL/6J Mice Is Impaired by Deposition of Age-Related Apolipoprotein A-II Amyloid Independent of Kidney Aging.

Spontaneous and age-related amyloidosis has been reported in C57BL/6J mice. However, the biochemical characteristics of age-related amyloidosis remain unclear. Herein, the age-related prevalence of amyloidosis, the types of amyloid fibril proteins, and the effects of amyloid deposition were investigated in renal function in C57BL/6J mice. The results obtained revealed a high incidence of amyloidosis in C57BL/6J mice originating from The Jackson Laboratory as well as the deposition of large amounts of amyloid in the glomeruli of aged mice. The amyloid fibril protein was identified as wild-type apolipoprotein A-II (ApoA-II). Induction of amyloid deposition in 40-week-old mice, equivalent to that of spontaneous development in 80-week-old mice, to rule out the effects of aging, revealed subsequent damage to kidney function by amyloid deposits. Furthermore, amyloid deposition in the mesangial region decreased podocyte density, compromised foot processes, and led to the accumulation of fibroblast growth factor 2 in glomeruli. Collectively, these results suggest that ApoA-II deposition is a general pathology in aged C57BL/6J mice and is dependent on supplier colonies. Therefore, the effects of age-related amyloid deposition need to be considered in research on aging in mice.

Time to Onset of Gemcitabine-induced Thrombotic Microangiopathy in a Japanese Population: A Case Series and Large-scale Pharmacovigilance Analysis.

BACKGROUND/AIM: Gemcitabine-induced thrombotic microangiopathy (G-TMA) is associated with a high mortality rate. However, owing to its low incidence, data on G-TMA remain limited. Therefore, a detailed review of G-TMA cases is critical to understand this adverse event. In addition, reviewing literature and pharmacovigilance analytics may be useful to characterise G-TMA. Here, time to onset of G-TMA was analysed based on available data. PATIENTS AND METHODS: We collected data for a case of TMA following gemcitabine administration at the Tokyo Metropolitan Geriatric Hospital. We also reviewed the literature on G-TMA cases in Japan from April 2000 to March 2022 to provide a case series. Moreover, we performed time-to-onset analysis of G-TMA using the data from the Japanese Adverse Drug Event Report (JADER) database. RESULTS: Our case involved a patient with pancreatic cancer who developed thrombotic thrombocytopenic purpura 13 months after starting gemcitabine treatment. From the literature reviewed, in 14 out of 17 cases, G-TMA occurred 5-8 months after treatment initiation. The analysis of data from the JADER database showed that the median time to onset of G-TMA was 161 days. Weibull shape parameter analysis showed that the pattern of onset of G-TMA represented a random failure. CONCLUSION: This study elucidated the time to onset of G-TMA in a Japanese population. Weibull shape parameter analysis showed that G-TMA may not necessarily develop in a dose-dependent manner. These results may be useful for monitoring G-TMA in the clinical setting.

Release of SMP30 in Extracellular Vesicles under Conditions of Ascorbic Acid Deficiency Is Involved with Acute Phase Response in ODS Rat.

Senescence marker protein-30 (SMP30) is a senescence marker molecule that exhibits lactonase activity in the ascorbic acid (AsA) biosynthesis pathway, except in primate mammals, including humans. Although numerous studies have shown that hepatic AsA deficiency causes acute-phase responses, details of the relationship between SMP30 expression and acute-phase responses in AsA-deficient conditions remain to be elucidated. Here, we investigated the effects of AsA deficiency on the relationship between SMP30 and acute liver injury in osteogenic disorder Shionogi (ODS) rats, which have a hereditary defect in AsA biosynthesis. Male-ODS rats (4 wk old) were pair-fed an AsA-free diet with distilled or 0.1% AsA-dissolved water for 14 d. Under AsA-deficient conditions, hepatic SMP30 protein level was decreased and liver injury markers, the serum aspartate aminotransferase/alanine transaminase ratio and cytokine-induced neutrophil chemoattractant-1 (CINC-1) concentration, were elevated. In contrast, SMP30 protein level in extracellular vesicles (EVs) was significantly increased in addition to the positive acute proteins haptoglobin and asialoglycoprotein receptor 1 (ASGPR1), hepatic-derived specific markers expression under AsA-deficient conditions. AsA deficiency also activated signal transducer and activator of transcription 3 (STAT3) which is linked to EVs release in the liver. These results suggest that the release of SMP30 in EVs by AsA deficiency is involved with acute-phase responses.

Vitamin C Is Essential for the Maintenance of Skeletal Muscle Functions.

Vitamin C (L-ascorbic acid, VC) is a water-soluble antioxidant essential for collagen polymerization. Previously, we reported that long-term VC deficiency causes muscle atrophy and deterioration in physical ability using female senescence marker protein-30 (SMP30)-deficient mice with a lack of VC synthesis, which is similar to that observed in humans. To determine whether these findings also hold true for male SMP30-deficient mice, two-month-old male SMP30-deficient mice were divided into two groups: the VC-treated group (VC(+)) was administered 1.5 g/L VC, and the VC-untreated group (VC(-)) was supplied water without VC. The VC level at four weeks in the gastrocnemius muscles from the VC(+) and VC(-) groups was 205.7 ± 8.5 nmol/g tissue and 13.1 ± 0.6 nmol/g tissue, respectively. Thus, four weeks was enough to reduce the VC level in the skeletal muscle in the VC-untreated group. On the other hand, muscle weights of the gastrocnemius, soleus, plantaris, tibialis anterior, and extensor digitorum longus in the VC(-) group were significantly reduced by VC deficiency after twelve weeks. The physical endurance of the VC(-) group at eight weeks was markedly lower than that of the VC(+) group. The grasping strength and activity in the cage in the nocturnal phases of the VC(-) group were markedly lower at twelve and sixteen weeks than those of the VC(+) group. Interestingly, muscle atrophy and declined physical ability were completely restored with VC supplementation for twelve weeks after VC deficiency. Thus, VC is essential for maintaining skeletal muscle function in both male and female SMP30-deficient mice with a lack of VC synthesis.

Influence of oestrogen on satellite cells and myonuclear domain size in skeletal muscles following resistance exercise.

BACKGROUND: Oestrogen deficiency reduces skeletal muscle mass and force generation in postmenopausal women. Muscle mass is maintained by satellite cells, which are regulated by oestrogen. Although oestrogen therapy enhances muscle hypertrophy induced by resistance training in postmenopausal women, the molecular mechanism is unclear. METHODS: Adult female rats (10 weeks old) were divided into six groups: sham sedentary (Sham-Sed), sham climbing training (Sham-CT), ovariectomy sedentary (OVX-Sed), ovariectomy climbing training (OVX-CT), ovariectomy plus oestrogen treatment sedentary (OVX+E-Sed), and ovariectomy plus oestrogen treatment climbing training (OVX+E-CT). At 8 weeks after ovariectomy, rats in the training group were trained (one session every 3 days for 8 weeks) to climb a ladder while bearing a load. Oestrogen treatment involved subcutaneous insertion of a 17ß-oestradiol pellet. After 8 weeks, the flexor hallucis longus muscle was collected and analysed. RESULTS: Following climbing training, the flexor hallucis longus muscle mass and muscle-to-body weight ratios were dramatically increased by training (main effect of training, P < 0.01); the OVX+E-CT group showed the highest values (main effect of group, P < 0.01). The cross-sectional area of all muscle fibre types was increased by training (main effect of training, P < 0.01). Particularly, the cross-sectional area of MHC IIa in the OVX+E-CT group was significantly larger than that in the Sham-CT and OVX-CT groups. Satellite cell numbers were increased in all training groups (main effect of training, P < 0.05), and the myonuclear number was increased by training (main effect of training, P < 0.01), but there was no main group effect. The myonuclear domain size of all muscle fibre types and MHC IIa was increased in all training groups (main effect of training, P < 0.01) and showed a main group effect (P < 0.01). The myonuclear domain sizes of all muscle fibre types and MHC IIa in the OVX+E-CT group were significantly larger than those in the Sham-CT and OVX-CT groups. The total RNA contents revealed main effects of training and the group (P < 0.01); the OVX+E-CT group showed the highest contents (main effect of group, P < 0.01). The mRNA and protein levels of rpS6 were increased in the OVX+E-Sed and CT groups (main effects of group, P < 0.05). Particularly, the 28S ribosomal RNA content in OVX+E-Sed group was significantly higher than that in the OVX-Sed group. CONCLUSIONS: Oestrogen enhanced the resistance training-induced increase in myonuclear domain size but did not affect satellite cells and ribosome biogenesis.

Compartmentalized citrullination in Muller glial endfeet during retinal degeneration.

Muller glia (MG) play a central role in reactive gliosis, a stress response associated with rare and common retinal degenerative diseases, including age-related macular degeneration (AMD). The posttranslational modification citrullination​ targeting glial fibrillary acidic protein (GFAP) in MG was initially discovered in a panocular chemical injury model. Here, we report in the paradigms of retinal laser injury, a genetic model of spontaneous retinal degeneration (JR5558 mice) and human wet-AMD tissues that MG citrullination is broadly conserved. After laser injury, GFAP polymers that accumulate in reactive MG are citrullinated in MG endfeet and glial cell processes. The enzyme responsible for citrullination, peptidyl arginine deiminase-4 (PAD4), localizes to endfeet and associates with GFAP polymers. Glial cell-specific PAD4 deficiency attenuates retinal hypercitrullination in injured retinas, indicating PAD4 requirement for MG citrullination. In retinas of 1-mo-old JR5558 mice, hypercitrullinated GFAP and PAD4 accumulate in MG endfeet/cell processes in a lesion-specific manner. Finally, we show that human donor maculae from patients with wet-AMD also feature the canonical endfeet localization of hypercitrullinated GFAP. Thus, we propose that endfeet are a "citrullination bunker" that initiates and sustains citrullination in retinal degeneration.

Determination of tissue-specific interaction between vitamin C and vitamin E in vivo using senescence marker protein-30 knockout mice as a vitamin C synthesis deficiency model.

Vitamin E (α-tocopherol; VE) is known to be regenerated from VE radicals by vitamin C (L-ascorbic acid; VC) in vitro. However, their in vivo interaction in various tissues is still unclear. Therefore, we alternatively examined the in vivo interaction of VC and VE by measurement of their concentrations in various tissues of senescence marker protein-30 (SMP30) knockout (KO) mice as a VC synthesis deficiency model. Male SMP30-KO mice were divided into four groups (VC+/VE+, VC+/VE-, VC-/VE+ and VC-/VE-), fed diets with or without 500 mg/kg VE and given water with or without 1·5 g/l VC ad libitum. Then, VC and VE concentrations in the plasma and various tissues were determined. Further, gene expression levels of transporters associated with VC and VE, such as α-tocopherol transfer protein (α-TTP) and sodium-dependent vitamin C transporters (SVCTs), were examined. These results showed that the VE levels in the VC-depleted (VC-/VE+) group were significantly lower than those in the VC+/VE+ group in the liver and heart; the VC levels in the VE-depleted (VC+/VE-) group were significantly lower than those in the VC+/VE+ group in the kidneys. The α-TTP gene expression in the liver and kidneys was decreased by VC and/or VE depletion. Moreover, SVCT1 gene expression in the liver was decreased by both VC and VE depletion. In conclusion, these results indicate that VC spares VE mainly in the liver and heart and that VE spares VC in the kidneys of SMP30-KO mice. Thus, interaction between VC and VE is likely to be tissue specific.

CXCL1-Triggered PAD4 Cytoplasmic Translocation Enhances Neutrophil Adhesion through Citrullination of PDIA1.

AIMS: Vascular inflammation is critical for the development and progression of atherosclerosis. Previously, we reported that neutrophils adhere to the vascular endothelium in low-density lipoprotein receptor null mice fed a high-fat diet through hypercitrullination of histone H3 by peptidylarginine deiminase 4 (PAD4) in neutrophils. However, the involvement of PAD4 and citrullination of proteins other than histone H3 in neutrophil adhesion is not well known. In this study, we investigated the function of PAD4 and identified citrullinated proteins during vascular inflammation. METHODS: We pefformed flow assay under physiological flow conditions using differentiated HL-60 (dHL-60) cells stimulated with CXCL1 and human umbilical vein endothelial cells (HUVECs). Furthermore, phalloidin stain for dHL-60 stimulated with CXCL1 to observe F-actin polymerization and immunohistochemistry for the activated ß2-integrin was conducted. To identify a target of citrullination in the cytoplasm of dHL-60 cells, liquid chromatography-mass spectrometry (LC-MS/MS) for dHL-60 stimulated with CXCL1 was performed. RESULTS: Inhibition or knockdown of PAD4 significantly decreased adhesion of under physiological flow conditions. Thr-Asp-F-amidine trifluoroacetate salt (TDFA), a PAD4 inhibitor, inhibited cytoplasmic translocation of PAD4 by CXCL1. TDFA or knockdown of PAD4 significantly decreased expression of ß2-integrin and F-actin polymerization activated by CXCL1. Moreover, LC-MS/MS identified protein disulfide isomerase A1 (PDIA1) as a target of citrullination in the cytoplasm of dHL-60 cells. Knockdown of PDIA1 significantly decreased adhesion of dHL-60 cells to HUVECs, expression of ß2-integrin, and F-actin polymerization. CONCLUSIONS: Cytoplasmic translocation of PAD4 by CXCL1 induces neutrophil adhesion to vascular endothelial cells and citrullination of PDIA1.

Effects of functional variants of vitamin C transporter genes on apolipoprotein E E4-associated risk of cognitive decline: The Nakajima study.

Apolipoprotein E E4 (APOE4) is a risk factor for cognitive decline. A high blood vitamin C (VC) level reduces APOE4-associated risk of developing cognitive decline in women. In the present study, we aimed to examine the effects of functional variants of VC transporter genes expressed in the brain (SLC2A1, SLC2A3, and SLC23A2) on APOE4-associated risk of developing cognitive decline. This case-control study involved 393 Japanese subjects: 252 cognitively normal and 141 cognitively impaired individuals (87 mild cognitive impairment and 54 dementia). Database searches revealed that rs1279683 of SLC23A2, and rs710218 and rs841851 of SLC2A1 are functional variants that are significantly associated with the altered expression of the respective genes and genotyped as three single nucleotide variants (SNVs). When stratified by SNV genotype, we found a significant association between APOE4 and cognitive decline in minor allele carriers of rs1279683 (odds ratio [OR] 2.02, 95% CI, 1.05-3.87, p = 0.035) but not in the homozygote carriers of the major allele. Significant associations between APOE4 and cognitive decline were also observed in participants with major allele homozygotes of rs710218 (OR 2.35, 95% CI, 1.05-5.23, p = 0.037) and rs841851 (OR 3.2, 95% CI, 1.58-6.46, p = 0.0012), but not in minor allele carriers of the respective SNVs. In contrast, the three functional SNVs showed no significant effect on cognitive decline. Our results imply that functional SNVs of VC transporter genes can affect APOE4-associated risk of developing cognitive decline via altered VC levels in the brain.

Reduced Plasma Ascorbate and Increased Proportion of Dehydroascorbic Acid Levels in Patients Undergoing Hemodialysis.

Ascorbate functions as an electron donor and scavenges free radicals. Dehydroascorbic acid (DHA), the oxidized form of ascorbate, is generated as a result of these reactions. While low plasma ascorbate levels have been reported in hemodialysis patients worldwide, no studies have measured DHA because it is not generalized. In this study, we aimed to clarify whether plasma ascorbate levels are low in dialysis patients and whether plasma ascorbate levels fluctuate before and after dialysis. Moreover, we applied our previously established method to measure the plasma ascorbate and DHA levels in chronic kidney disease (CKD) stage G3-G5 non-hemodialysis-dependent patients, and pre- and post-dialysis plasma ascorbate and DHA levels in CKD stage G5D hemodialysis patients. The sample size was calculated using G-power software. The pre-dialysis plasma total ascorbate levels, including DHA, were significantly (56%) lower in hemodialysis patients than in non-hemodialysis-dependent CKD patients. After dialysis, there was a 40% reduction in the plasma total ascorbate levels. Hemodialysis increased the post-dialysis plasma proportions of DHA from 37% to 55%. The study results demonstrated lower plasma total ascorbate levels in hemodialysis patients compared with in non-hemodialysis-dependent CKD patients; these low levels in hemodialysis patients were further reduced by hemodialysis and increased DHA proportion.