Effects of D-allose on ATP production and cell viability in neonatal rat cardiomyocytes.

2-Deoxy-d-glucose (2DG) induces anticancer effects through glycolytic inhibition but it may raise the risk of arrhythmia. The rare monosaccharide d-allose also has anticancer properties, but its cardiac effects are unknown. We examined the effects of d-allose on adenosine triphosphate (ATP) production in neonatal rat cardiomyocytes. We showed that 25 mM d-allose selectively reduced glycolytic ATP, but had minimal impact on mitochondrial ATP, while 1 mM 2DG strongly inhibited both. Furthermore, d-allose had less impact on cell viability and was less cytotoxic than 2DG; neither compound caused apoptosis. Thus, d-allose selectively diminished glycolytic ATP production with no apparent effects on cardiomyocytes.


CA9 and PRELID2; hypoxia-responsive potential therapeutic targets for pancreatic ductal adenocarcinoma as per bioinformatics analyses.

A strong hypoxic environment has been observed in pancreatic ductal adenocarcinoma (PDAC) cells, which contributes to drug resistance, tumor progression, and metastasis. Therefore, we performed bioinformatics analyses to investigate potential targets for the treatment of PDAC. To identify potential genes as effective PDAC treatment targets, we selected all genes whose expression level was related to worse overall survival (OS) in The Cancer Genome Atlas (TCGA) database and selected only the genes that matched with the genes upregulated due to hypoxia in pancreatic cancer cells in the dataset obtained from the Gene Expression Omnibus (GEO) database. Although the extracted 107 hypoxia-responsive genes included the genes that were slightly enriched in angiogenic factors, TCGA data analysis revealed that the expression level of endothelial cell (EC) markers did not affect OS. Finally, we selected CA9 and PRELID2 as potential targets for PDAC treatment and elucidated that a CA9 inhibitor, U-104, suppressed pancreatic cancer cell growth more effectively than 5-fluorouracil (5-FU) and PRELID2 siRNA treatment suppressed the cell growth stronger than CA9 siRNA treatment. Thus, we elucidated that specific inhibition of PRELID2 as well as CA9, extracted via exhaustive bioinformatic analyses of clinical datasets, could be a more effective strategy for PDAC treatment.


TJ-17 (Goreisan) mitigates renal fibrosis in a mouse model of folic acid-induced chronic kidney disease.

TJ-17 may have a novel preventive effect against inflammation, oxidative stress, and fibrosis, contributing to innovation in the treatment of CKD.

We the preventive action of TJ-17 against acute kidney injury (AKI) transition to CKD in vivo using a folic acid (FA)-induced mouse model. Mice were treated with food containing TJ-17 at 48 h after FA intraperitoneal injection (AKI phase).


Pemafibrate inhibited renal dysfunction and fibrosis in a mouse model of adenine-induced chronic kidney disease.

These results demonstrated the renoprotective effects of pemafibrate in CKD mice, confirming its potential as a therapeutic agent for renal disorders.

The risks associated with conventional fibrates (fenofibrate, bezafibrate) to the kidneys were evaluated using the Food and Drug Administration Adverse Event Reporting System. Pemafibrate (1 or 0.3 mg/kg/day) was administered daily using an oral sonde. Its renoprotective effects were examined in unilateral ureteral obstruction (UUO)-induced renal fibrosis model mice (UUO mice) and adenine-induced CKD model mice (CKD mice).


Plasma Heparin Cofactor II Activity Is Inversely Associated with Hepatic Fibrosis of Non-Alcoholic Fatty Liver Disease in Patients with Type 2 Diabetes Mellitus.

Plasma HCII activity was inversely associated with clinical hepatic fibrosis indices including FIB-4 index, NFS and APRI and with the prevalence of advanced hepatic fibrosis in patients with T2DM. The results suggest that HCII can serve as a novel biomarker for assessment of hepatic fibrosis of NAFLD in patients with T2DM.

Multiple regression analysis including confounding factors showed that plasma HCII activity independently contributed to decreases in FIB-4 index (p<0.001), NFS (p<0.001) and APRI (p=0.004). In addition, logistic regression analysis for the prevalence of advanced hepatic fibrosis defined by the cutoff points of the clinical scores showed that plasma HCII activity was the sole and common negative factor for prevalence of advanced hepatic fibrosis (FIB-4 index: p=0.002, NFS: p=0.026 and APRI: p=0.012).

Journal of Atherosclerosis and Thrombosis


Effects of high-absorption curcumin for the prevention of hypertensive heart disease: a double-blind, placebo-controlled, randomized clinical study.

' ratio, rather it significantly inhibited the increase in plasma BNP levels in patients with initial signs of hypertensive heart disease.


The novel preventive effect of a Japanese ethical Kampo extract formulation TJ-90 (Seihaito) against cisplatin-induced nephrotoxicity


Role of ferroptosis in cisplatin-induced acute nephrotoxicity in mice

Journal of Trace Elements in Medicine and Biology


Plasma Heparin Cofactor II Activity Is Inversely Associated with Albuminuria and Its Annual Deterioration in Patients with Diabetes.

The plasma HCII activity was inversely and specifically associated with glomerular injury in patients with diabetes. The results suggest that HCII can serve as a novel predictive factor for early-stage DKD development, as represented by albuminuria.


薬剤誘発性大動脈解離易発症モデルマウスを用いた薬効評価

薬剤誘発性大動脈解離易発症モデルマウスを用いた薬効評価

Aortic dissection (or dissecting aortic aneurysm) is a condition in which the aortic wall is separated into two layers at the medial level to form a pseudocavity. The intima crack, called the ``entry'', allows blood to tear through the medial layer and flow in. The location of the ``entry'' and the extent of the dissection can cause a variety of serious complications, including rupture, cardiac tamponade, and obstruction of branched vessels. According to the Guideline on Diagnosis and Treatment of Aortic Aneurysm and Aortic Dissection 2020, it is estimated that 61.4% of the onset of dissection die before arrival at the hospital, and 93% will die within 24 hours after the onset. It has been suggested that the morbidity rate has been increasing in recent years. Since many of them have a fatal prognosis, it is an important issue to prevent the onset itself. However, no effective therapeutic agent or preventive strategy has been established so far. The first reason is that it is extremely difficult to design clinical studies because aortic dissection traced the rapid onset and progression. The second is that the pathophysiology and preventive drug search are not sufficiently conducted even at the basic research level. Epidemiologically, the results of the International Registry of Aortic Dissection (IRAD) revealed that aging, hypertension, atherosclerosis, and hereditary connective tissue diseases are risk factors. The aortic aneurysm also shows similar pathological conditions caused by these risk factors. However, one of the major differences between aneurysm and dissection is the presence of aortic intima rupture. Therefore, we attempted to establish a mouse model developing dissection at a high rate by adding the endothelial dysfunction to a pharmacologically induced aortic aneurysm model mouse. Furthermore, we evaluated the efficacy of pitavastatin and several nutrients using our novel model mice and verified its usefulness as a model animal.


Examination of the antiepileptic effects of valacyclovir using kindling mice- search for novel antiepileptic agents by drug repositioning using a large medical information database.

Despite the availability of more than 20 clinical antiepileptic drugs, approximately 30% of patients with epilepsy do not respond to antiepileptic drug treatment. Therefore, it is important to develop antiepileptic products that function via novel mechanisms. In the present study, we evaluated data from one of the largest global databases to identify drugs with antiepileptic effects, and subsequently attempted to understand the effect of the combination of antiepileptic drugs and valacyclovir in epileptic seizures using a kindling model. To induce kindling in mice, pentylenetetrazol at a dose of 40 mg/kg was administered once every 48 h. Valacyclovir was orally administered 30 min before antiepileptic drug injection in kindled mice, and behavioral seizures were monitored for 20 min following pentylenetetrazol administration. Additionally, c-Fos expression in the hippocampal dentate gyrus was measured in kindled mice. Valacyclovir showed inhibitory effects on pentylenetetrazol-induced kindled seizures. In addition, simultaneous use of levetiracetam and valacyclovir caused more potent inhibition of seizure activity, and neither valproic acid nor diazepam augmented the anti-seizure effect in kindled mice. Furthermore, kindled mice showed increased c-Fos levels in the dentate gyrus. The increase in c-Fos expression was significantly inhibited by the simultaneous use of levetiracetam and valacyclovir. The findings of the present study indicate that a combination of levetiracetam and valacyclovir had possible anticonvulsive effects on pentylenetetrazol-induced kindled epileptic seizures. These results suggest that valacyclovir may have an antiseizure effect in patients with epilepsy.


Dual disruption of eNOS and ApoE gene accelerates kidney fibrosis and senescence after injury.

mice (DKO) were obtained by breeding. Unilateral ureteral obstruction (UUO) was performed on 8-10 week old male mice and the degree of renal tubular injury, fibrosis and kidney senescence were evaluated. DKO manifested elevated blood pressure, higher total cholesterol and lower HDL than WT. DKO showed sustained kidney injury molecule-1 protein expression. Kidney fibrosis was significantly higher in ApoEKO and DKO. mRNA expression of genes related to kidney fibrosis was the highest in DKO. The mRNA expression of Zinc-α2-Glycoprotein and heme oxygenase-1 were significantly decreased in DKO. Furthermore, mRNA expression of p53, p21 and p16 were increased both in ApoEKO and DKO, with DKO being the highest. Senescence associated β-gal positive tubule area was significantly increased in DKO. Increased DNA damage and target of rapamycin-autophagy spatial coupling compartments (TASCCs) formation was found in DKO. Mice with endothelial dysfunction and dyslipidemia developed kidney fibrosis and accelerated senescence even in young mice after injury. These data highlight the fact managing lifestyle-related diseases from a young age is important for CKD prevention.

Biochemical and Biophysical Research Communications


Diphenhydramine may be a preventive medicine against cisplatin-induced kidney toxicity

Cisplatin is widely used as an anti-tumor drug for the treatment of solid tumors. Unfortunately, it causes kidney toxicity as a critical side effect, limiting its use, given that no preventive drug against cisplatin-induced kidney toxicity is currently available. Here, based on a repositioning analysis of the Food and Drug Administration Adverse Events Reporting System, we found that a previously developed drug, diphenhydramine, may provide a novel treatment for cisplatin-induced kidney toxicity. To confirm this, the actual efficacy of diphenhydramine was evaluated in in vitro and in vivo experiments. Diphenhydramine inhibited cisplatin-induced cell death in kidney proximal tubular cells. Mice administered cisplatin developed kidney injury with significant dysfunction (mean plasma creatinine: 0.43 vs 0.15 mg/dl) and showed augmented oxidative stress, increased apoptosis, elevated inflammatory cytokines, and MAPKs activation. However, most of these symptoms were suppressed by treatment with diphenhydramine. Furthermore, the concentration of cisplatin in the kidney was significantly attenuated in diphenhydramine-treated mice (mean platinum content: 70.0 vs 53.4 μg/g dry kidney weight). Importantly, diphenhydramine did not influence or interfere with the anti-tumor effect of cisplatin in any of the in vitro or in vivo experiments. In a selected cohort of 98 1:1 matched patients from a retrospective database of 1467 patients showed that patients with malignant cancer who had used diphenhydramine before cisplatin treatment exhibited significantly less acute kidney injury compared to ones who did not (6.1 % vs 22.4 %, respectively). Thus, diphenhydramine demonstrated efficacy as a novel preventive medicine against cisplatin-induced kidney toxicity.


Effects of Palonosetron on Nausea and Vomiting Induced by Multiple-Day Chemotherapy: A Retrospective Study.

RAs in controlling multiple-day chemotherapy-induced nausea and vomiting.

RAs palonosetron in multiple-day chemotherapy-induced nausea and vomiting. The design of this study was a retrospective case-control study of patients who received a five-day cisplatin-based chemotherapy and were treated with aprepitant, dexamethasone, granisetron, and ramosetron or palonosetron. The patients were divided into two groups: patients given granisetron and ramosetron (the first-generation group), and those given palonosetron (palonosetron group). The percentage of patients with a complete response or total control was assessed. They were divided into three phases: 0-216 h (overall phase), 0-120 h (remedial phase), and 120-216 h (after phase). The remedial phase was further divided into 0-24 h (early phase) and 24-120 h (later phase). Moreover, the nutritional status of each patient was assessed by noting the patients' total calorie-intake per day and total parenteral nutrition. First-generation 5-HT


Investigation of drugs affecting hypertension in bevacizumab-treated patients and examination of the impact on the therapeutic effect.

PPIs prevent hypertension in bevacizumab-treated patients but may reduce bevacizumab's anti-tumoral effects by inducing VEGF expression.


Deletion of H-ferritin in macrophages alleviates obesity and diabetes induced by high-fat diet in mice

Iron accumulation affects obesity and diabetes, both of which are ameliorated by iron reduction. Ferritin, an iron-storage protein, plays a crucial role in iron metabolism. H-ferritin exerts its cytoprotective action by reducing toxicity via its ferroxidase activity. We investigated the role of macrophage H-ferritin in obesity and diabetes. Conditional macrophage-specific H-ferritin (Fth, also known as Fth1) knockout (LysM-Cre Fth KO) mice were used and divided into four groups: wild-type (WT) and LysM-Cre Fth KO mice with normal diet (ND), and WT and LysM-Cre Fth KO mice with high-fat diet (HFD). These mice were analysed for characteristics of obesity and diabetes, tissue iron content, inflammation, oxidative stress, insulin sensitivity and metabolic measurements. RAW264.7 macrophage cells were used for in vitro experiments. Iron concentration reduced, and mRNA expression of ferroportin increased, in macrophages from LysM-Cre Fth KO mice. HFD-induced obesity was lower in LysM-Cre Fth KO mice than in WT mice at 12 weeks (body weight: KO 34.6 ± 5.6 g vs WT 40.1 ± 5.2 g). mRNA expression of inflammatory cytokines and infiltrated macrophages and oxidative stress increased in the adipose tissue of HFD-fed WT mice, but was not elevated in HFD-fed LysM-Cre Fth KO mice. However, WT mice fed an HFD had elevated iron concentration in adipose tissue and spleen, which was not observed in LysM-Cre Fth KO mice fed an HFD (adipose tissue [μmol Fe/g protein]: KO 1496 ± 479 vs WT 2316 ± 866; spleen [μmol Fe/g protein]: KO 218 ± 54 vs WT 334 ± 83). Moreover, HFD administration impaired both glucose tolerance and insulin sensitivity in WT mice, which was ameliorated in LysM-Cre Fth KO mice. In addition, energy expenditure, mRNA expression of thermogenic genes, and body temperature were higher in KO mice with HFD than WT mice with HFD. In vitro experiments showed that iron content was reduced, and lipopolysaccharide-induced Tnf-α (also known as Tnf) mRNA upregulation was inhibited in a macrophage cell line transfected with Fth siRNA. Deletion of macrophage H-ferritin suppresses the inflammatory response by reducing intracellular iron levels, resulting in the prevention of HFD-induced obesity and diabetes. The findings from this study highlight macrophage iron levels as a potential therapeutic target for obesity and diabetes.


Fibroblast-specific ERK5 deficiency changes tumor vasculature and exacerbates tumor progression in a mouse model.

The roles of cancer-associated fibroblasts (CAFs) have been studied in the tumor progression, and CAFs are expected to become the new targets for cancer pharmacotherapies. CAFs contribute to tumor cell survival and proliferation, tumor angiogenesis, immune suppression, tumor inflammation, tumor cell invasion and metastasis, and extracellular matrix remodeling. However, detailed mechanisms of how CAFs function in the living system remain unclear. CAFs include α-smooth muscle actin, expressing activated fibroblasts similar to myofibroblasts, and are highly capable of producing collagen. Several reports have demonstrated the contributions of extracellular-signal-regulated kinase 5 (ERK5) in fibroblasts to the fibrotic processes; however, the roles of CAF-derived ERK5 remain unclear. To investigate the roles of CAF-derived ERK5 in the tumor progression, we created mice lacking the ERK5 gene specifically in fibroblasts. Colon-26 mouse colon cancer cells were implanted into the mice subcutaneously, and the histological analyses of the tumor tissue were performed after 2 weeks. Immunofluorescence analyses showed that recipient-derived fibroblasts existed within the tumor tissue. The present study demonstrated that fibroblast-specific ERK5 deficiency exacerbated tumor progression and it was accompanied with thicker tumor vessel formation and the increase in the number of activated fibroblasts. We combined the results of The Cancer Genome Atlas (TCGA) database analysis with our animal studies, and indicated that regulating ERK5 activity in CAFs or CAF invasion into the tumor tissue can be important strategies for the development of new targets in cancer pharmacotherapies.

Naunyn-Schmiedeberg's Archives of Pharmacology


Novel Hydrophilic Camptothecin Derivatives Conjugated to Branched Glycerol Trimer Suppress Tumor Growth without Causing Diarrhea in Murine Xenograft Models of Human Lung Cancer.

Camptothecin possesses broad antitumor spectra on various cancers. In spite of its marked tumor-suppressing potency, camptothecin is too hydrophobic to be solved in water and therefore not currently in clinical use. CPT-11 (irinotecan) is one of the hydrophilic analogues of camptothecin and widely prescribed. However, its water solubility is still low and furthermore evokes severe diarrhea. Therefore, we designed and synthesized novel highly hydrophilic camptothecin derivatives by conjugating SN38 with branched glycerol trimer (SN38-BGL), which we have been developing as a unique strategy to endow hydrophobic molecule with much hydrophilicity, to maximize the benefit of CPT-11 and minimize the adverse effects. The SN38-BGLs exhibited equivalent or slightly stronger tumor-suppressing effects in murine xenograft human lung cancer models compared to CPT-11. However, neither early- nor late-onset diarrhea was observed when SN38-BGL was administered. Heights of villi in jejunum and ileum were bigger than those from CPT-11-treated mice, indicating that SN38-BGL is less harmful than CPT-11. Ex vivo digestion by liver microsome did not yield SN38 but a couple of other molecules against our expectations, which suggests the involvement of other active metabolites than SN38 and may explain the differences. Hence, SN38-BGLs can be a novel hydrophilic camptothecin derivative without causing severe diarrhea.


Rho-associated protein kinase and cyclophilin a are involved in inorganic phosphate-induced calcification signaling in vascular smooth muscle cells.

Arterial calcification, a risk factor of cardiovascular events, develops with differentiation of vascular smooth muscle cells (VSMCs) into osteoblast-like cells. Cyclophilin A (CypA) is a peptidyl-prolyl isomerase involved in cardiovascular diseases such as atherosclerosis and aortic aneurysms, and rho-associated protein kinase (ROCK) is involved in the pathogenesis of vascular calcification. CypA is secreted in a ROCK activity-dependent manner and works as a mitogen via autocrine or paracrine mechanisms in VSMCs. We examined the involvement of the ROCK-CypA axis in VSMC calcification induced by inorganic phosphate (Pi), a potent cell mineralization initiator. We found that Pi stimulated ROCK activity, CypA secretion, extracellular signal-regulated protein kinase (ERK) 1/2 phosphorylation, and runt-related transcription factor 2 expression, resulting in calcium accumulation in rat aortic smooth muscle cells (RASMCs). The ROCK inhibitor Y-27632 significantly suppressed Pi-induced CypA secretion, ERK1/2 phosphorylation, and calcium accumulation. Recombinant CypA was found to be associated with increased calcium accumulation in RASMCs. Based on these results, we suggest that autocrine CypA is mediated by ROCK activity and is involved in Pi-induced ERK1/2 phosphorylation following calcification signaling in RASMCs.


Proton pump inhibitors block iron absorption through direct regulation of hepcidin via the aryl hydrocarbon receptor-mediated pathway

Proton pump inhibitors (PPIs) have been used worldwide to treat gastrointestinal disorders. A recent study showed that long-term use of PPIs caused iron deficiency; however, it is unclear whether PPIs affect iron metabolism directly. We investigated the effect of PPIs on the peptide hepcidin, an important iron regulatory hormone. First, we used the FDA Adverse Event Reporting System database and analyzed the influence of PPIs. We found that PPIs, as well as H2 blockers, increased the odds ratio of iron-deficient anemia. Next, HepG2 cells were used to examine the action of PPIs and H2 blockers on hepcidin. PPIs augmented hepcidin expression, while H2 blockers did not. In fact, the PPI omeprazole increased hepcidin secretion, and omeprazole-induced hepcidin upregulation was inhibited by gene silencing or the pharmacological inhibition of the aryl hydrocarbon receptor. In mouse experiments, omeprazole also increased hepatic hepcidin mRNA expression and blood hepcidin levels. In mice treated with omeprazole, protein levels of duodenal and splenic ferroportin decreased. Taken together, PPIs directly affect iron metabolism by suppressing iron absorption through the inhibition of duodenal ferroportin via hepcidin upregulation. These findings provide a new insight into the molecular mechanism of PPI-induced iron deficiency.


Skeletal muscle atrophy is caused by disruption in the homeostatic balance of muscle degeneration and regeneration under various pathophysiological conditions. We have previously reported that iron accumulation induces skeletal muscle atrophy a ubiquitin ligase-dependent pathway. However, the potential effect of iron accumulation on muscle regeneration remains unclear. To examine the effect of iron accumulation on myogenesis, we used a mouse model with cardiotoxin (CTX)-induced muscle regeneration and C2C12 mouse myoblast cells . In mice with iron overload, the skeletal muscles exhibited increased oxidative stress and decreased expression of satellite cell markers. Following CTX-induced muscle injury, these mice also displayed delayed muscle regeneration with a decrease in the size of regenerating myofibers, reduced expression of myoblast differentiation markers, and decreased phosphorylation of MAPK signaling pathways. , iron overload also suppressed the differentiation of C2C12 myoblast cells but the suppression could be reversed by superoxide scavenging using tempol. Excess iron inhibits myogenesis oxidative stress, leading to an imbalance in skeletal muscle homeostasis.-Ikeda, Y., Satoh, A., Horinouchi, Y., Hamano, H., Watanabe, H., Imao, M., Imanishi, M., Zamami, Y., Takechi, K., Izawa-Ishizawa, Y., Miyamoto, L., Hirayama, T., Nagasawa, H., Ishizawa, K., Aihara, K.-I., Tsuchiya, K., Tamaki, T. Iron accumulation causes impaired myogenesis correlated with MAPK signaling pathway inhibition by oxidative stress.


Irinotecan-induced neutropenia is reduced by oral alkalization drugs: analysis using retrospective chart reviews and the spontaneous reporting database.

These data indicate that oral alkalization drugs may reduce the frequency of neutropenia caused by irinotecan administration, making it possible to increase the dose safely.


Xanthine Oxidase Inhibition by Febuxostat in Macrophages Suppresses Angiotensin II-induced Aortic Fibrosis.

Our results suggested that FEB ameliorates Ang II-induced aortic fibrosis via suppressing macrophage-derived TGF-1 expression.


Development of a novel aortic dissection mouse model and evaluation of drug efficacy using in-vivo assays and database analyses.

Aortic dissection is a life-threatening disease. At present, the only therapeutic strategies available are surgery and antihypertensive drugs. Moreover, the molecular mechanisms underlying the onset of aortic dissection are still unclear. We established a novel aortic dissection model in mice using pharmacologically induced endothelial dysfunction. We then used the Japanese Adverse Drug Event Report database to investigate the role of pitavastatin in preventing the onset of aortic dissection. To induce endothelial dysfunction, Nω-nitro-L-arginine methyl ester, a nitric oxide synthase inhibitor, was administered to C57BL/6 mice. Three weeks later, angiotensin II (Ang II) and β-aminopropionitrile (BAPN), a lysyl oxidase inhibitor, were administered with osmotic mini-pumps. False lumen formation was used as the pathological determinant of aortic dissection. The incidences of aortic dissection and death from aneurysmal rupture were significantly higher in the Nω-nitro-L-arginine methyl ester, Ang II, and BAPN (LAB) group than they were in the Ang II and BAPN (AB) group.Pitavastatin was administered orally to LAB mice. It significantly lowered the incidences of dissection and rupture. It also decreased inflammation and medial degradation, both of which were exacerbated in the LAB group. The Japanese Adverse Drug Event Report database analysis indicated that there were 113 cases of aortic dissection out of 95 090 patients (0.12%) not receiving statins but only six cases out of 16 668 patients receiving statins (0.04%) (odds ratio: 0.30; P = 0.0043). Our results suggest that endothelial dysfunction is associated with the onset of aortic dissection and pitavastatin can help prevent this condition.


Nitrosonifedipine, a Photodegradation Product of Nifedipine, Suppresses Pharmacologically Induced Aortic Aneurysm Formation.

We have reported that nitrosonifedipine (NO-NIF), a photodegradation product of nifedipine, has strong antioxidant and endothelial protective effects, and can suppress several cardiovascular diseases in animal models. The objective of the present study was to investigate the effects of NO-NIF on aortic aneurysm formation. The mice were infused with β-aminopropionitrile for 2 weeks and angiotensin II for 6 weeks to induce aortic aneurysm formation. The oxidative stress was measured by dihydroethidium staining and nitrotyrosine staining. The expressions of inflammation-related genes were assessed by quantitative real-time PCR and immunohistochemical staining. To clarify the mechanisms of how NO-NIF suppresses vascular cell adhesion molecule (VCAM)-1, endothelial cells were used in in vitro system. NO-NIF suppressed pharmacologically induced the aortic aneurysm formation and aortic expansion without blood pressure changes. NO-NIF suppressed elastin degradation and matrix metalloproteinase-2 mRNA expression. NO-NIF suppressed the reactive oxygen species-cyclophilin A positive feedback loop. Upregulated mRNA expressions of inflammation-related genes and endothelial VCAM-1 were suppressed by NO-NIF co-treatment in aortae. NO-NIF has the potential to be a new, nifedipine-derived therapeutic drug for suppressing aortic aneurysm formation by directly improving aortic structure with its strong ability to reduce oxidative stress and inflammation.


Renoprotective effects of a factor Xa inhibitor: fusion of basic research and a database analysis.

Renal tubulointerstitial injury, an inflammation-associated condition, is a major cause of chronic kidney disease (CKD). Levels of activated factor X (FXa), a blood coagulation factor, are increased in various inflammatory diseases. Therefore, we investigated the protective effects of an FXa inhibitor against renal tubulointerstitial injury using unilateral ureteral obstruction (UUO) mice (a renal tubulointerstitial fibrosis model) and the Food and Drug Administration Adverse Events Reporting System (FAERS) database. The renal expression levels of FX and the FXa receptors protease-activated receptor (PAR)-1 and PAR-2 were significantly higher in UUO mice than in sham-operated mice. UUO-induced tubulointerstitial fibrosis and extracellular matrix expression were suppressed in UUO mice treated with the FXa inhibitor edoxaban. Additionally, edoxaban attenuated UUO-induced macrophage infiltration and inflammatory molecule upregulation. In an analysis of the FAERS database, there were significantly fewer reports of tubulointerstitial nephritis for patients treated with FXa inhibitors than for patients not treated with inhibitors. These results suggest that FXa inhibitors exert protective effects against CKD by inhibiting tubulointerstitial fibrosis.


The uremic toxin indoxyl sulfate interferes with iron metabolism by regulating hepcidin in chronic kidney disease


Methanol extraction fraction from Citrus Sudachi peel exerts lipid reducing effects in cultured cells.

Ectopic fat accumulation is associated with insulin resistance and type 2 diabetes mellitus. Citrus sudachi is an evergreen tree that is found mainly in Tokushima Prefecture in Japan. Previously, it was demonstrated that Citrus sudachi could inhibit the rising trend of blood glucose and fatty acid in human subjects. In the current study, we illustrated the function of methanol extracts from sudachi peel and investigated the mechanism of this effect. We got the five kinds of methanol extracts by using diaion HP-20, and those were named by hydrophobicity from M-F1 to M-F5. Among the 5 kinds of sudachi methanol extracts, only M-F4 significantly decreased the intracellular triglyceride of C2C12 cells. It augmented the AMPK activity and increased the transcription of PPARα and its downstream targets CPT-1b and UCP2. In conclusion, M-F4 improved the lipid metabolism possibly through AMPK, PPARα and their downstream targets like CPT-1b and UCP2. Furthermore, this extract may be useful for preventing obesity and diabetes related diseases. J. Med. Invest. 65:225-230, August, 2018.

The Journal of Medical Investigation : JMI


Mechanisms of the pH- and Oxygen-Dependent Oxidation Activities of Artesunate.

Artemisinin was discovered in 1971 as a constituent of the wormwood genus plant (Artemisia annua). This plant has been used as an herbal medicine to treat malaria since ancient times. The compound artemisinin has a sesquiterpene lactone bearing a peroxide group that offers its biological activity. In addition to anti-malarial activity, artemisinin derivatives have been reported to exert antitumor activity in cancer cells, and have attracted attention as potential anti-cancer drugs. Mechanisms that might explain the antitumor activities of artemisinin derivatives reportedly induction of apoptosis, angiogenesis inhibitory effects, inhibition of hypoxia-inducible factor-1 (HIF-1) activation, and direct DNA injury. Reactive oxygen species (ROS) generation is involved in many cases. However, little is known about the mechanism of ROS formation from artemisinin derivatives and what types of ROS are produced. Therefore, we investigated the iron-induced ROS formation mechanism by using artesunate, a water-soluble artemisinin derivative, which is thought to be the underlying mechanism involved in artesunate-mediated cell death. The ROS generated by the coexistence of iron(II), artesunate, and molecular oxygen was a hydroxyl radical or hydroxyl radical-like ROS. Artesunate can reduce iron(III) to iron(II), which enables generation of ROS irrespective of the iron valence. We found that reduction from iron(III) to iron(II) was activated in the acidic rather than the neutral region and was proportional to the hydrogen ion concentration.


Hydrocortisone administration was associated with improved survival in Japanese patients with cardiac arrest.

There are few reports on hydrocortisone administration after cardiac arrest, and those that have been published included few subjects. This study aimed to evaluate the effect of hydrocortisone administration on the outcomes of patients who experienced cardiac arrest. We investigated the survival discharge rates and the length of hospital stay from cardiac arrest to discharge, stratified by use of hydrocortisone, using a Japanese health-insurance claims dataset that covers approximately 2% of the Japanese population. The study included the data of 2233 subjects who experienced either in-hospital or out-of-hospital cardiac arrest between January 2005 and May 2014. These patients were divided into two groups, based on the administration of hydrocortisone. We adjusted the baseline characteristics, medical treatment, and drug administration data of the two groups using propensity scores obtained via the inverse probability of treatment weighted method. The hydrocortisone group had a significantly higher survival discharge rate (13/61 [21.1%] vs. 240/2172 [11.0%], adjusted odds ratio: 4.2, 95% CI: 1.60-10.98, p = 0.004). In addition, the administration of hydrocortisone was independent predictor of survival to discharge (hazard ratio: 4.6, p < 0.001). The results demonstrate a correlation between hydrocortisone administration and the high rates of survival to discharge.


Dietary iron restriction alleviates renal tubulointerstitial injury induced by protein overload in mice

Increased proteinuria causes tubulointerstitial injury due to inflammation in chronic kidney disease (CKD). Iron restriction exhibits protective effects against renal dysfunction; however, its effects against protein overload-induced tubulointerstitial damage remain unclear. Here, we investigated dietary iron restriction effect on tubulointerstitial damage in mice with protein-overload tubulointerstitial injury. Renal tubulointerstitial injury in animal model was induced by intraperitoneal injection of an overdose of bovine serum albumin (BSA). We divided mice into three groups: normal saline + normal diet (ND), BSA + ND, and BSA + iron-restricted diet (IRD). BSA overload induced renal tubulointerstitial injury in the ND mice, which was ameliorated in the IRD mice. Inflammatory cytokines and extracellular matrix mRNA expression was upregulated in BSA + ND mice kidneys and was inhibited by IRD. BSA-induced increase in renal superoxide production, NADPH oxidase activity, and p22(phox) expression was diminished in the IRD mice. IRD suppression increased BSA-induced renal macrophage infiltration. Moreover, BSA mice exhibited nucleotide-binding oligomerisation domain-like receptor pyrin domain-containing protein (NLRP) inflammasome activation, which was inhibited by IRD. Ferrous iron increased in kidneys with BSA overload and was inhibited by IRD. Thus, iron restriction inhibited oxidative stress and inflammatory changes, contributing to the protective effect against BSA overload-induced tubulointerstitial injury.


Endothelial Nitric Oxide Synthase-Independent Pleiotropic Effects of Pitavastatin Against Atherogenesis and Limb Ischemia in Mice.

Pitavastatin exerts eNOS-independent protective effects against atherogenesis and hind-limb ischemia in mice, which may occur via modifications on key molecules such as AMPK and diverse molecules.

In Study 1, pitavastatin attenuated plaque formation and medial fibrosis of the aortic root with decreased macrophage infiltration in eNOS(-/-) ApoE(-/-) mice. PCR array analysis showed reductions in aortic gene expression of proatherogenic factors, including Ccl2 and Ccr2 in pitavastatin-treated double mutant mice. In addition, pitavastatin activated not only atherogenic p38MAPK and JNK but also anti-atherogenic ERK1/2 and ERK5 in the aorta of the double mutant mice. In Study 2, pitavastatin prolonged hind-limb survival after the surgery with increased BCL2-to-BAX protein ratio and inactivated JNK. Enhanced expression of anti-apoptotic genes, including Vegf, Api5, Atf5, Prdx2, and Dad1, was observed in the ischemic limb of pitavastatin-treated eNOS(-/-) mice. Furthermore, pitavastatin activated both aortic and skeletal muscle AMPK in the eNOS-deficient vascular injury models.

Journal of Atherosclerosis and Thrombosis


The Role of Heparin Cofactor in the Regulation of Insulin Sensitivity and Maintenance of Glucose Homeostasis in Humans and Mice.

The present studies provide evidence to support the idea that HC plays an important role in the maintenance of glucose homeostasis by regulating insulin sensitivity in both humans and mice. Stimulators of HC production may serve as novel therapeutic tools for the treatment of type 2 diabetes.


Iron suppresses erythropoietin expression via oxidative stress-dependent hypoxia-inducible factor-2 alpha inactivation

Renal anemia is a major complication in chronic kidney disease (CKD). Iron supplementation, as well as erythropoiesis-stimulating agents, are widely used for treatment of renal anemia. However, excess iron causes oxidative stress via the Fenton reaction, and iron supplementation might damage remnant renal function including erythropoietin (EPO) production in CKD. EPO gene expression was suppressed in mice following direct iron treatment. Hypoxia-inducible factor-2 alpha (HIF-2α), a positive regulator of the EPO gene, was also diminished in the kidney of mice following iron treatment. Anemia-induced increase in renal EPO and HIF-2α expression was inhibited by iron treatment. In in vitro experiments using EPO-producing HepG2 cells, iron stimulation reduced the expression of the EPO gene, as well as HIF-2α. Moreover, iron treatment augmented oxidative stress, and iron-induced reduction of EPO and HIF-2α expression was restored by tempol, an antioxidant compound. HIF-2α interaction with the Epo promoter was inhibited by iron treatment, and was restored by tempol. These findings suggested that iron supplementation reduced EPO gene expression via an oxidative stress-HIF-2α-dependent signaling pathway.

Laboratory Investigation; a Journal of Technical Methods and Pathology


Topical Application of Nitrosonifedipine, a Novel Radical Scavenger, Ameliorates Ischemic Skin Flap Necrosis in a Mouse Model.

Ischemic skin flap necrosis can occur in random pattern flaps. An excess amount of reactive oxygen species is generated and causes necrosis in the ischemic tissue. Nitrosonifedipine (NO-NIF) has been demonstrated to possess potent radical scavenging ability. However, there has been no study on the effects of NO-NIF on ischemic skin flap necrosis. Therefore, they evaluated the potential of NO-NIF in ameliorating ischemic skin flap necrosis in a mouse model. A random pattern skin flap (1.0 × 3.0 cm) was elevated on the dorsum of C57BL/6 mice. NO-NIF was administered by topical injection immediately after surgery and every 24 hours thereafter. Flap survival was evaluated on postoperative day 7. Tissue samples from the skin flaps were harvested on postoperative days 1 and 3 to analyze oxidative stress, apoptosis and endothelial dysfunction. The viable area of the flap in the NO-NIF group was significantly increased (78.30 ± 7.041%) compared with that of the control group (47.77 ± 6.549%, p < 0.01). NO-NIF reduced oxidative stress, apoptosis and endothelial dysfunction, which were evidenced by the decrease of malondialdehyde, p22phox protein expression, number of apoptotic cells, phosphorylated p38 MAPK protein expression, and vascular cell adhesion molecule-1 protein expression while endothelial nitric oxide synthase protein expression was increased. In conclusion, they demonstrated that NO-NIF ameliorated ischemic skin flap necrosis by reducing oxidative stress, apoptosis, and endothelial dysfunction. NO-NIF is considered to be a candidate for the treatment of ischemic flap necrosis.


Iron-induced skeletal muscle atrophy involves an Akt-forkhead box O3-E3 ubiquitin ligase-dependent pathway

Skeletal muscle wasting or sarcopenia is a critical health problem. Skeletal muscle atrophy is induced by an excess of iron, which is an essential trace metal for all living organisms. Excessive amounts of iron catalyze the formation of highly toxic hydroxyl radicals via the Fenton reaction. However, the molecular mechanism of iron-induced skeletal muscle atrophy has remained unclear. In this study, 8-weeks-old C57BL6/J mice were divided into 2 groups: vehicle-treated group and the iron-injected group (10 mg iron·day-1·mouse-1) during 2 weeks. Mice in the iron-injected group showed an increase in the iron content of the skeletal muscle and serum and ferritin levels in the muscle, along with reduced skeletal muscle mass. The skeletal muscle showed elevated mRNA expression of the muscle atrophy-related E3 ubiquitin ligases, atrogin-1 and muscle ring finger-1(MuRF1), on days 7 and 14 of iron treatment. Moreover, iron-treated mice showed reduced phosphorylation of Akt and forkhead box O3 (FOXO3a) in skeletal muscles. Inhibition of FOXO3a using siRNA in vitro in C2C12 myotube cells inhibited iron-induced upregulation of atrogin-1 and MuRF1 and reversed the reduction in myotube diameters. Iron-load caused oxidative stress, and an oxidative stress inhibitor abrogated iron-induced muscle atrophy by reactivating the Akt-FOXO3 pathway. Iron-induced skeletal muscle atrophy is suggested to involve the E3 ubiquitin ligase mediated by the reduction of Akt-FOXO3a signaling by oxidative stress.

Journal of Trace Elements in Medicine and Biology


Bilirubin exerts pro-angiogenic effects through an Akt-eNOS-dependent pathway

Low serum bilirubin levels are associated with the risk of cardiovascular diseases including peripheral artery disease. Bilirubin is known to exert its property such as antioxidant effect or the enhancement of flow-mediated vasodilation, however, bilirubin action on angiogenesis remains unclear. To investigate the molecular mechanism of bilirubin on angiogenic effect, we first employed C57BL/6J mice with unilateral hindlimb ischemia surgery and divided the mice into two groups (vehicle-treated group and bilirubin-treated group). The analysis of laser speckle blood flow demonstrated the enhancement of blood flow recovery in response to ischemia of mice with bilirubin treatment. The density of capillaries was significantly higher in ischemic-adductor muscles of bilirubin-treated mice. The phosphorylated levels of endothelial nitric oxide synthase (eNOS) and Akt were increased in ischemic skeletal muscles of mice with bilirubin treatment compared with vehicle treatment. In in vitro experiments by using human aortic endothelial cells, bilirubin augmented eNOS and Akt phosphorylation, cell proliferation, cell migration and tube formation. These bilirubin actions on endothelial cell activation were inhibited by LY294002, a phosphatidylinositol 3-kinase inhibitor. In conclusion, bilirubin promotes angiogenesis through endothelial cells activation via Akt-eNOS-dependent manner.


Angiotensin II alters the expression of duodenal iron transporters, hepatic hepcidin, and body iron distribution in mice

Angiotensin II (ANG II) has been shown to affect iron metabolism through alteration of iron transporters, leading to increased cellular and tissue iron contents. Serum ferritin, a marker of body iron storage, is elevated in various cardiovascular diseases, including hypertension. However, the associated changes in iron absorption and the mechanism underlying increased iron content in a hypertensive state remain unclear. The C57BL6/J mice were treated with ANG II to generate a model of hypertension. Mice were divided into three groups: (1) control, (2) ANG II-treated, and (3) ANG II-treated and ANG II receptor blocker (ARB)-administered (ANG II-ARB) groups. Mice treated with ANG II showed increased serum ferritin levels compared to vehicle-treated control mice. In ANG II-treated mice, duodenal divalent metal transporter-1 and ferroportin (FPN) expression levels were increased and hepatic hepcidin mRNA expression and serum hepcidin concentration were reduced. The mRNA expression of bone morphogenetic protein 6 and CCAAT/enhancer-binding protein alpha, which are regulators of hepcidin, was also down-regulated in the livers of ANG II-treated mice. In terms of tissue iron content, macrophage iron content and renal iron content were increased by ANG II treatment, and these increases were associated with reduced expression of transferrin receptor 1 and FPN and increased expression of ferritin. These changes induced by ANG II treatment were ameliorated by the administration of an ARB. Angiotensin II (ANG II) altered the expression of duodenal iron transporters and reduced hepcidin levels, contributing to the alteration of body iron distribution.


Hypoxia decreases glucagon-like peptide-1 secretion from GLUTag cell line

Glucagon-like peptide-1 (GLP-1), an incretin hormone, is secreted from L cells located in the intestinal epithelium. It is known that intestinal oxygen tension is decreased postprandially. In addition, we found that the expression of hypoxia-inducible factor-1α (HIF-1α), which accumulates in cells under hypoxic conditions, was significantly increased in the colons of mice with food intake, indicating that the oxygen concentration is likely reduced in the colon after eating. Therefore, we hypothesized that GLP-1 secretion is affected by oxygen tension. We found that forskolin-stimulated GLP-1 secretion from GLUTag cells, a model of intestinal L cells, is suppressed in hypoxia (1% O2). Forskolin-stimulated elevations of preproglucagon (ppGCG) and proprotein convertase 1/3 (PC1/3) mRNA expression were decreased under hypoxic conditions. The finding that H89, a protein kinase A (PKA) inhibitor, inhibited the forskolin-stimulated increase of ppGCG and PC1/3 indicated that the cAMP-PKA pathway is involved in the hypoxia-induced suppression of the genes. Hypoxia decreased hexokinase 2 mRNA and protein expression and increased lactate dehydrogenase A mRNA and protein expression. Concomitantly, lactate production was increased and ATP production was decreased. Together, the results indicate that hypoxia decreases glucose utilization for ATP production, which probably causes a decrease in cAMP production and in subsequent GLP-1 production. Our findings suggest that the postprandial decrease in oxygen tension in the intestine attenuates GLP-1 secretion.


A Long-Term High-Fat Diet Changes Iron Distribution in Body, Increasing Iron Accumulation Specifically in the Mouse Spleen

Although iron is an essential trace metal, its presence in excess causes oxidative stress in the human body. Recent studies have indicated that iron storage is a risk factor for type 2 diabetes mellitus. Dietary iron restriction or iron chelation ameliorates symptoms of type 2 diabetes in mouse models. However, whether iron content in the body changes with the development of diabetes is unknown. Here, we investigated the dynamics of iron accumulation and changes in iron absorption-related genes in mice that developed obesity and diabetes by consuming a high-fat diet (HFD-fed mice). HFD-fed mice (18-20 wk) were compared with control mice for hematologic features, serum ferritin levels, and iron contents in the gastrocnemius muscle, heart, epididymal fat, testis, liver, duodenum, and spleen. In addition, the spleen was examined histologically. Iron absorption-related gene expression in the liver and duodenum was also examined. Hemoglobin and serum ferritin levels were increased in HFD-fed mice. The HFD-fed mice showed iron accumulation in the spleen, but not in the heart or liver. Increased percentages of the splenic red pulp and macrophages were observed in HFD-fed mice and iron accumulation in the spleen was found mainly in the splenic red pulp. The HFD-fed mice also showed decreased iron content in the duodenum. The mRNA expression of divalent metal transporter-1 (DMT-1), an iron absorption-related gene, was elevated in the duodenum of HFD-fed mice. These results indicate that iron accumulation (specifically accumulation in the spleen) is enhanced by the development of type 2 diabetes induced by HFD.

Journal of Nutritional Science and Vitaminology


Smooth muscle cell specific Hif-1 deficiency suppresses angiotensin II-induced vascular remodeling in mice

Vascular remodelling is mediated by vascular smooth muscle cell (VSMC) proliferation and hypertrophy, both processes of which are linked to medial thickening and fibrosis. Here, we show that hypoxia-inducible factor-1α (Hif-1α) expressed in smooth muscle cells (SMCs) is involved in angiotensin II (Ang II)-induced vascular remodelling in an in vivo model. To clarify the role of Hif-1α in vascular remodelling, we created mice lacking the Hif-1α gene in SMCs (SMKO mice). Ang II infusion induced medial thickening and vascular fibrosis, accompanied by Hif-1α up-regulation, in the aortae of control mice, but not in those of SMKO mice. In accordance with those results, our in vitro studies showed that the deletion of SMC-derived Hif-1α suppressed the Ang II-induced hypertrophy of VSMCs, and our in vivo studies showed that the Ang II-induced expression of fibrosis-related genes in aortae was suppressed by SMC-specific Hif-1α deficiency. In addition, the SMC-specific Hif-1α deficiency suppressed Ang II-induced macrophage infiltration and Ang II-induced expression of inflammation-related genes in aortae. The superoxide production observed in the aortae of control mice with Ang II was suppressed in those of SMKO mice with Ang II, and this finding was consistent with the results of little Ang II-induced c-Src phosphorylation in SMKO mouse aortae. Loss- and gain-of-function analysis in in vitro experiments confirmed that VSMC-derived Hif-1α functions as an intrinsic modulator of vascular remodelling-related gene expression. Our results revealed that SMC-derived Hif-1α is a crucial mediator of Ang II-induced vascular remodelling.


HIF-2α/ARNT complex regulates hair development via induction of p21Waf1/Cip1 and p27Kip1

The hypoxia-inducible factors HIF-1α or HIF-2α form heterodimeric complexes with the aryl hydrocarbon receptor nuclear translocator (ARNT). HIF-1α/ARNT and HIF-2α/ARNT complexes activate hypoxia-inducible genes that play critical roles in angiogenesis, anaerobic metabolism, and other processes in response to O2 deprivation. HIF-2α is known to regulate the function and/or differentiation of stem cells by activating the POU domain transcription factor Oct4; however, the precise underlying mechanism is unknown. This study examined the role of HIF-2α/ARNT in hair development using conditional-knockout mice, in which Arnt was specifically deleted in keratinocytes. In wild-type mice, HIF-2α and ARNT were highly expressed in the precortex above the hair matrix, an area containing differentiating stem cells. An analysis of hair size and type in these mice showed that loss of ARNT decreased the production of zigzag hairs, corresponding to reduced expression of HIF-2α and induction of the mammalian cyclin-dependent kinase inhibitors p21(Waf1/Cip1) and p27 (Kip1). The results suggest that the HIF-2α/ARNT complex regulates hair follicle differentiation via induction of p21(Waf1/Cip1) and possibly p27(Kip1), as p27(Kip1) expression was not altered in ARNT knockout mice. The findings provide insight into a possible mechanism underlying hair growth disorders and can be useful for future studies on hair follicle response to insults, such as chemotherapy and ionizing radiation.-Imamura, Y., Tomita, S., Imanishi, M., Kihira, Y., Ikeda, Y., Ishizawa, K., Tsuchiya, K., Tamaki, T. HIF-2α/ARNT complex regulates hair development via induction of p21(Waf1/Cip1) and p27(Kip1).


Overexpressed HIF-2α in Endothelial Cells Promotes Vascularization and Improves Random Pattern Skin Flap Survival.

Specific overexpression of HIF-2 in ECs promoted vascularization and enhanced skin flap survival in vivo in a mouse model.

Plastic and Reconstructive Surgery. Global Open


Deletion of hypoxia-inducible factor-1α in adipocytes enhances glucagon-like peptide-1 secretion and reduces adipose tissue inflammation.

It is known that obese adipose tissues are hypoxic and express hypoxia-inducible factor (HIF)-1α. Although some studies have shown that the expression of HIF-1α in adipocytes induces glucose intolerance, the mechanisms are still not clear. In this study, we examined its effects on the development of type 2 diabetes by using adipocyte-specific HIF-1α knockout (ahKO) mice. ahKO mice showed improved glucose tolerance compared with wild type (WT) mice. Macrophage infiltration and mRNA levels of monocyte chemotactic protein-1 (MCP-1) and tumor necrosis factor α (TNFα) were decreased in the epididymal adipose tissues of high fat diet induced obese ahKO mice. The results indicated that the obesity-induced adipose tissue inflammation was suppressed in ahKO mice. In addition, in the ahKO mice, serum insulin levels were increased under the free-feeding but not the fasting condition, indicating that postprandial insulin secretion was enhanced. Serum glucagon-like peptide-1 (GLP-1) levels were also increased in the ahKO mice. Interestingly, adiponectin, whose serum levels were increased in the obese ahKO mice compared with the obese WT mice, stimulated GLP-1 secretion from cultured intestinal L cells. Therefore, insulin secretion may have been enhanced through the adiponectin-GLP-1 pathway in the ahKO mice. Our results suggest that the deletion of HIF-1α in adipocytes improves glucose tolerance by enhancing insulin secretion through the GLP-1 pathway and by reducing macrophage infiltration and inflammation in adipose tissue.


Iron chelation by deferoxamine prevents renal interstitial fibrosis in mice with unilateral ureteral obstruction

Renal fibrosis plays an important role in the onset and progression of chronic kidney diseases (CKD). Although several mechanisms underlying renal fibrosis and candidate drugs for its treatment have been identified, the effect of iron chelator on renal fibrosis remains unclear. In the present study, we examined the effect of an iron chelator, deferoxamine (DFO), on renal fibrosis in mice with surgically induced unilateral ureter obstruction (UUO). Mice were divided into 4 groups: UUO with vehicle, UUO with DFO, sham with vehicle, and sham with DFO. One week after surgery, augmented renal tubulointerstitial fibrosis and the expression of collagen I, III, and IV increased in mice with UUO; these changes were suppressed by DFO treatment. Similarly, UUO-induced macrophage infiltration of renal interstitial tubules was reduced in UUO mice treated with DFO. UUO-induced expression of inflammatory cytokines and extracellular matrix proteins was abrogated by DFO treatment. DFO inhibited the activation of the transforming growth factor-β1 (TGF-β1)-Smad3 pathway in UUO mice. UUO-induced NADPH oxidase activity and p22(phox) expression were attenuated by DFO. In the kidneys of UUO mice, divalent metal transporter 1, ferroportin, and ferritin expression was higher and transferrin receptor expression was lower than in sham-operated mice. Increased renal iron content was observed in UUO mice, which was reduced by DFO treatment. These results suggest that iron reduction by DFO prevents renal tubulointerstitial fibrosis by regulating TGF-β-Smad signaling, oxidative stress, and inflammatory responses.


Nitrosonifedipine ameliorates the progression of type 2 diabetic nephropathy by exerting antioxidative effects

Diabetic nephropathy (DN) is the major cause of end-stage renal failure. Oxidative stress is implicated in the pathogenesis of DN. Nitrosonifedipine (NO-NIF) is a weak calcium channel blocker that is converted from nifedipine under light exposure. Recently, we reported that NO-NIF has potential as a novel antioxidant with radical scavenging abilities and has the capacity to treat vascular dysfunction by exerting an endothelial protective effect. In the present study, we extended these findings by evaluating the efficacy of NO-NIF against DN and by clarifying the mechanisms of its antioxidative effect. In a model of type 2 DN (established in KKAy mice), NO-NIF administration reduced albuminuria and proteinuria as well as glomerular expansion without affecting glucose metabolism or systolic blood pressure. NO-NIF also suppressed renal and systemic oxidative stress and decreased the expression of intercellular adhesion molecule (ICAM)-1, a marker of endothelial cell injury, in the glomeruli of the KKAy mice. Similarly, NO-NIF reduced albuminuria, oxidative stress, and ICAM-1 expression in endothelial nitric oxide synthase (eNOS) knockout mice. Moreover, NO-NIF suppressed urinary angiotensinogen (AGT) excretion and intrarenal AGT protein expression in proximal tubular cells in the KKAy mice. On the other hand, hyperglycemia-induced mitochondrial superoxide production was not attenuated by NO-NIF in cultured endothelial cells. These findings suggest that NO-NIF prevents the progression of type 2 DN associated with endothelial dysfunction through selective antioxidative effects.


Androgen receptor promotes sex-independent angiogenesis in response to ischemia and is required for activation of vascular endothelial growth factor receptor signaling.

These results document a physiological role of AR in sex-independent angiogenic potency and provide evidence of novel cross-talk between the androgen/AR signaling and VEGF/kinase insert domain protein receptor signaling pathways.


Dietary iron restriction inhibits progression of diabetic nephropathy in db/db mice.

Excess iron causes oxidative stress through hydroxyl-radical production via Fenton/Haber-Weiss reactions. Recently, body iron reduction has been found to ameliorate diabetes. In the present study, we examined the protective effect of dietary iron restriction against diabetic nephropathy in the db/db mouse model of diabetic nephropathy using db/m mice as controls. The db/db mice were divided into 2 groups and fed a normal diet (ND) or a low iron diet (LID). Increasing urinary albumin excretion was observed in the ND db/db mice, but this was suppressed in db/db mice with LID. Histologically, the db/db mice in the ND group had increased glomerular volume and mesangial area compared to the LID group. Augmented deposition of extracellular matrices was decreased in db/db mice with LID. In terms of oxidative stress, increased superoxide production observed in the kidneys of the ND db/db mice was diminished in the LID group. NADPH oxidase activity and renal expression of NADPH oxidase components p22(phox) and NOX4 were augmented in the ND group, and this was abolished by LID. There were no differences in expression of renal iron importers, transferrin receptor, or divalent metal transporter-1 between db/m mice and db/db mice. The level of ferroportin, an iron exporter, increased in the kidneys of the db/db mice. Urinary iron excretion was significantly higher in ND db/db mice and was reduced in the LID group. These findings suggest that dietary iron restriction exerts a preventive effect on the progression of diabetic nephropathy partly due to the reduction of oxidative stress.

American Journal of Physiology, Renal Physiology


Bovine milk-derived lactoferrin exerts proangiogenic effects in an Src-Akt-eNOS-dependent manner in response to ischemia

Lactoferrin (LF) exerts a variety of biological effects, including the promotion of angiogenesis by increasing the expression of angiogenesis-related genes and reducing blood pressure via a nitric oxide-dependent mechanism. In this study, we investigated the effects of LF on angiogenesis using C57BL/6J mice that received daily unilateral treatment with or without bovine milk-derived LF (bLF) after unilateral hindlimb surgery. The analysis of laser speckle blood flow showed that bLF treatment promoted blood flow recovery in response to ischemic hindlimb. The capillary density of ischemic adductor muscles and the phosphorylation of Src, Akt, and endothelial nitric oxide synthase (eNOS) were also significantly higher in bLF-treated mice than in vehicle-treated mice. Furthermore, bLF increased the phosphorylation levels of Src, Akt, and eNOS in in vitro experiments using human aortic endothelial cells. The action of bLF on eNOS phosphorylation was abolished by both LY294002, a phosphatidylinositol 3-kinase inhibitor, and 4-amino-5-(4-chlorophenyl)-7-(dimethylethyl)pyrazolo [3,4-d]pyrimidine (PP2), an Src inhibitor. Similarly, bLF-induced acceleration of tube formation, cell proliferation, and cell migration in human aortic endothelial cells were inhibited by LY294002 or PP2. Thus, bLF promotes vascular endothelial cell function via an Src Akt eNOS-dependent pathway, thereby contributing to revascularization in response to ischemia.


Nitrosonifedipine ameliorates angiotensin II-induced vascular remodeling via antioxidative effects

Nifedipine is unstable under light and decomposes to a stable nitroso analog, nitrosonifedipine (NO-NIF). The ability of NO-NIF to block calcium channels is quite weak compared with that of nifedipine. Recently, we have demonstrated that NO-NIF reacts with unsaturated fatty acid leading to generate NO-NIF radical, which acquires radical scavenging activity. However, the effects of NO-NIF on the pathogenesis related with oxidative stress, such as atherosclerosis and hypertension, are unclear. In this study, we investigated the effects of NO-NIF on angiotensin II (Ang II)-induced vascular remodeling. Ang II-induced thickening and fibrosis of aorta were inhibited by NO-NIF in mice. NO-NIF decreased reactive oxygen species (ROS) in the aorta and urinary 8-hydroxy-20-deoxyguanosine. Ang II-stimulated mRNA expressions of p22(phox), CD68, F4/80, monocyte chemoattractant protein-1, and collagen I in the aorta were inhibited by NO-NIF. Moreover, NO-NIF inhibited Ang II-induced cell migration and proliferation of vascular smooth muscle cells (VSMCs). NO-NIF reduced Ang II-induced ROS to the control level detected by dihydroethidium staining and lucigenin chemiluminescence assay in VSMCs. NO-NIF suppressed phosphorylations of Akt and epidermal growth factor receptor induced by Ang II. However, NO-NIF had no effects on intracellular Ca(2+) increase and protein kinase C-δ phosphorylation induced by Ang II in VSMCs. The electron paramagnetic resonance spectra indicated the continuous generation of NO-NIF radical of reaction with cultured VSMCs. These findings suggest that NO-NIF improves Ang II-induced vascular remodeling via the attenuation of oxidative stress.

Naunyn-Schmiedeberg's Archives of Pharmacology


Angiotensin II receptor blocker improves tumor necrosis factor-α-induced cytotoxicity via antioxidative effect in human glomerular endothelial cells

Tumor necrosis factor-α (TNF-α) is known to involve the progression of renal dysfunction through its cytotoxicity and proinflammatory effects such as the induction of intercellular adhesion molecule (ICAM)-1 expression in vascular endothelial cells (ECs). Olmesartan, one of the angiotensin II type 1 receptor blockers (ARBs), has been reported to show protective effects on injured ECs by some causal factors of renal disorder other than angiotensin II. However, the effects of olmesartan on TNF-α-induced glomerular EC damage have not been investigated. In the present study, we investigated the effects of RNH-6270, an active metabolite of olmesartan, on TNF-α-induced human glomerular EC (HGEC) damage to clarify the renoprotective mechanisms of ARBs. Cultured HGECs were stimulated by TNF-α, and then cell viability and cytotoxicity were measured by MTT assay and lactate dehydrogenase release assay, respectively. TNF-α-induced oxidative stress was estimated by dihydroethidium assay and lucigenin chemiluminescence assay. ICAM-1 expression and the phosphorylations of mitogen-activated protein kinases were measured using Western blotting assay. RNH-6270 suppressed cell death and the increase in ICAM-1 expression induced by TNF-α via the inhibition of reactive oxygen species in HGECs. Our findings suggested that olmesartan might have protective effects against TNF-α-induced glomerular EC dysfunction.


Heparin cofactor II, a serine protease inhibitor, promotes angiogenesis via activation of the AMP-activated protein kinase-endothelial nitric-oxide synthase signaling pathway

We previously clarified that heparin cofactor II (HCII), a serine proteinase inhibitor, exerts various protective actions on cardiovascular diseases in both experimental and clinical studies. In the present study, we aimed to clarify whether HCII participates in the regulation of angiogenesis. Male heterozygous HCII-deficient (HCII(+/-)) mice and male littermate wild-type (HCII(+/+)) mice at the age of 12-16 weeks were subjected to unilateral hindlimb ligation surgery. Laser speckle blood flow analysis showed that blood flow recovery in response to hindlimb ischemia was delayed in HCII(+/-) mice compared with that in HCII(+/+) mice. Capillary number, arteriole number, and endothelial nitric-oxide synthase (eNOS), AMP-activated protein kinase (AMPK), and liver kinase B1 (LKB1) phosphorylation in ischemic muscles were decreased in HCII(+/-) mice. Human purified HCII (h-HCII) administration almost restored blood flow recovery, capillary density, and arteriole number as well as phosphorylation levels of eNOS, AMPK, and LKB1 in ischemic muscles of HCII(+/-) mice. Although treatment with h-HCII increased phosphorylation levels of eNOS, AMPK, and LKB1 in human aortic endothelial cells (HAECs), the h-HCII-induced eNOS phosphorylation was abolished by compound C, an AMPK inhibitor, and by AMPK siRNA. In a similar fashion, tube formation, proliferation, and migration of HAECs were also promoted by h-HCII treatment and were abrogated by pretreatment with compound C. HCII potentiates the activation of vascular endothelial cells and the promotion of angiogenesis in response to hindlimb ischemia via an AMPK-eNOS signaling pathway. These findings suggest that HCII is a novel therapeutic target for treatment of patients with peripheral circulation insufficiency.


Systemic Preconditioning by a Prolyl Hydroxylase Inhibitor Promotes Prevention of Skin Flap Necrosis via HIF-1-Induced Bone Marrow-Derived Cells.

We demonstrated that transient activation of the HIF signaling pathway by a single systemic DMOG treatment upregulates not only anti-apoptotic pathways but also enhances neovascularization with concomitant increase in the numbers of bone marrow-derived progenitor cells.


Estrogen regulates hepcidin expression via GPR30-BMP6-dependent signaling in hepatocytes

Hepcidin, a liver-derived iron regulatory protein, plays a crucial role in iron metabolism. It is known that gender differences exist with respect to iron storage in the body; however, the effects of sex steroid hormones on iron metabolism are not completely understood. We focused on the effects of the female sex hormone estrogen on hepcidin expression. First, ovariectomized (OVX) and sham-operated mice were employed to investigate the effects of estrogen on hepcidin expression in an in vivo study. Hepcidin expression was decreased in the livers of OVX mice compared to the sham-operated mice. In OVX mice, bone morphologic protein-6 (BMP6), a regulator of hepcidin, was also found to be downregulated in the liver, whereas ferroportin (FPN), an iron export protein, was upregulated in the duodenum. Both serum and liver iron concentrations were elevated in OVX mice relative to their concentrations in sham-operated mice. In in vitro studies, 17β-estradiol (E(2)) increased the mRNA expression of hepcidin in HepG2 cells in a concentration-dependent manner. E(2)-induced hepatic hepcidin upregulation was not inhibited by ICI 182720, an inhibitor of the estrogen receptor; instead, hepcidin expression was increased by ICI 182720. E(2) and ICI 182720 exhibit agonist actions with G-protein coupled receptor 30 (GPR30), the 7-transmembrane estrogen receptor. G1, a GPR30 agonist, upregulated hepcidin expression, and GPR30 siRNA treatment abolished E(2)-induced hepcidin expression. BMP6 expression induced by E(2) was abolished by GPR30 silencing. Finally, both E(2) and G1 supplementation restored reduced hepatic hepcidin and BMP6 expression and reversed the augmentation of duodenal FPN expression in the OVX mice. In contrast, serum hepcidin was elevated in OVX mice, which was reversed in these mice with E(2) and G1. Thus, estrogen is involved in hepcidin expression via a GPR30-BMP6-dependent mechanism, providing new insight into the role of estrogen in iron metabolism.


ロスバスタチンによる脂質低下作用非依存的な形態的および機能的頸動脈硬化改善作用

ロスバスタチンによる脂質低下作用非依存的な形態的および機能的頸動脈硬化改善作用


Iron reduction by deferoxamine leads to amelioration of adiposity via the regulation of oxidative stress and inflammation in obese and type 2 diabetes KKAy mice.

Iron is an essential trace metal for most organisms. However, excess iron causes oxidative stress through production of highly toxic hydroxyl radicals via the Fenton/Haber-Weiss reaction. Iron storage in the body is reported to be associated with fat accumulation and type 2 diabetes mellitus. We investigated the role of iron in adiposity by using KKAy mice and obese and diabetic model mice. Eight-week-old KKAy mice were divided into two groups and treated with deferoxamine (DFO), an iron chelator agent, or a vehicle for 2 wk. DFO treatment diminished fat iron concentration and serum ferritin levels in KKAy mice. Fat weight and adipocyte size were reduced significantly in DFO-treated mice compared with vehicle-treated mice. Macrophage infiltration into fat was also decreased in DFO-treated mice compared with vehicle-treated mice. Superoxide production and NADPH oxidase activity in fat, as well as urinary 8-hydroxy-2'-deoxyguanosine excretion, were decreased in KKAy mice after DFO treatment while p22(phox) expression in adipose tissue was diminished in such mice. Ferritin expression in the fat of DFO-treated KKAy mice was decreased. In addition, F4/80-positive cells also presented through both p22(phox) and ferritin expression. The mRNA expression levels of inflammatory cytokines were also reduced in fat tissue of DFO-treated mice. These findings suggest that reduction of iron levels ameliorates adipocyte hypertrophy via suppression of oxidative stress, inflammatory cytokines, and macrophage infiltration, thereby breaking a vicious cycle in obesity.

American Journal of Physiology, Endocrinology and Metabolism


Basic fibroblast growth factor regulates glucose metabolism through glucose transporter 1 induced by hypoxia-inducible factor-1α in adipocytes

Hypoxia-inducible factor-1 (HIF-1), which is a transcription factor that enhances glycolysis in cells in response to hypoxia, is induced in hypertrophied adipocytes in obesity. Recent studies have shown that growth factors are able to induce HIF-1 by mechanisms independent of hypoxia. Since basic fibroblast growth factor (bFGF), an angiogenic factor, is concentrated in expanding adipose tissue, the possible effects of bFGF on regulation of HIF-1 in adipocytes were investigated. Treatment of differentiated 3T3-L1 adipocytes with bFGF induced HIF-1. Concomitantly, glucose transporter 1 (GLUT1), which is a target gene of HIF-1, was induced at both mRNA and protein levels and was translocated to the plasma membrane. A chromatin immunoprecipitation assay and an RNA interference study indicated that bFGF-induced HIF-1 directly upregulates GLUT1. In addition, it was observed that bFGF increases lactate production of adipocytes. This result indicates that bFGF reprograms the metabolism toward glycolysis. Intraperitoneal injection of bFGF into mice upregulated HIF-1 and GLUT1 in adipose tissues, suggesting that bFGF regulates the metabolism of adipocytes via HIF-1-GLUT1 regulation in vivo. We also found that bFGF inhibits insulin-induced phosphorylation of insulin receptor substrate-1 and Akt, suggesting that bFGF attenuates the insulin signal in adipocytes. Taken together, the findings suggest that bFGF has a harmful effect on the development of type 2 diabetes through metabolism reprogramming and attenuation of the insulin signal.

The International Journal of Biochemistry & Cell Biology


Pathophysiological Response to Hypoxia From the Molecular Mechanisms of Malady to Drug Discovery: Inflammatory Responses of Hypoxia-Inducible Factor 1 (HIF-1) in T Cells Observed in Development of Vascular Remodeling

Recent studies have shown that the cellular immune response to the hypoxic microenvironment constructed by vascular remodeling development modulates the resulting pathologic alterations. A major mechanism mediating adaptive responses to reduced oxygen availability is the regulation of transcription by hypoxia-inducible factor 1 (HIF-1). Impairment of HIF-1-dependent inflammatory responses in T cells causes an augmented vascular remodeling induced by arterial injury, which is shown as prominent neointimal hyperplasia and increase in infiltration of inflammatory cells at the adventitia in mice lacking Hif-1α specifically in T cells. Studies to clarify the mechanism of augmented vascular remodeling in the mutant mice have shown enhanced production of cytokines in activated T cells and augmented antibody production in response to a T-dependent antigen in the mutant mice. This minireview shows that HIF-1α in T cells plays a crucial role in vascular inflammation and remodeling in response to cuff injury as a negative regulator of the T cell-mediated immune response and suggests potential new therapeutic strategies that target HIF-1α.


Pharmacology in health food: Metabolism of quercetin in vivo and its protective effect against arteriosclerosis

Quercetin, a member of the bioflavonoids family, has been proposed to have anti-atherogenic, anti-inflammatory, and anti-hypertensive properties leading to the beneficial effects against cardiovascular diseases. It was recently demonstrated that quercetin 3-O-β-D-glucuronide (Q3GA) is one of the major quercetin conjugates in human plasma, in which the aglycone could not be detected. Although most of the in vitro pharmacological studies have been carried out using only the quercetin aglycone form, experiments using Q3GA would be important to discover the preventive mechanisms of cardiovascular diseases by quercetin in vivo. Therefore we examined the effects of the chemically synthesized Q3GA, as an in vivo form, on vascular smooth muscle cell (VSMC) disorders related to the progression of arteriosclerosis. Platelet-derived growth factor-induced cell migration and proliferation were inhibited by Q3GA in VSMCs. Q3GA attenuated angiotensin II-induced VSMC hypertrophy via its inhibitory effect on JNK and the AP-1 signaling pathway. Q3GA scavenged 1,1-diphenyl-2-picrylhydrazyl radical measured by the electron paramagnetic resonance method. In addition, immunohistochemical studies with monoclonal antibody 14A2 targeting the Q3GA demonstrated that the positive staining specifically accumulates in human atherosclerotic lesions, but not in the normal aorta. These findings suggest Q3GA would be an active metabolite of quercetin in plasma and may have preventative effects on arteriosclerosis relevant to VSMC disorders.


Antioxidant effects of photodegradation product of nifedipine

Recently, increasing evidence suggests that the antihypertensive drug nifedipine acts as a protective agent for endothelial cells, and that the activity is unrelated to its calcium channel blocking. Nifedipine is unstable under light and reportedly decomposes to a stable nitrosonifedipine (NO-NIF). NO-NIF has no antihypertensive effect, and it has been recognized as a contaminant of nifedipine. The present study for the first time demonstrated that NO-NIF changed to a NO-NIF radical in a time-dependent manner when it interacted with human umbilical vein endothelial cells (HUVECs). The electron paramagnetic resonance (EPR) signal of NO-NIF radicals in HUVECs showed an asymmetric pattern suggesting that the radicals were located in the membrane. The NO-NIF radicals had radical scavenging activity for 1,1-diphenyl-2-picrylhydrazyl, whereas neither NO-NIF nor nifedipine did. In addition, the NO-NIF radical more effectively quenched lipid peroxides than NO-NIF or nifedipine. Furthermore, NO-NIF attenuated the superoxide-derived free radicals in HUVECs stimulated with LY83583, and suppressed iron-nitrilotriacetic acid (Fe-NTA)-induced cytotoxicity in rat pheochromocytoma (PC12) cells. Our findings suggest that NO-NIF is a candidate for a new class of antioxidative drugs that protect cells against oxidative stress.


Protective effect of photodegradation product of nifedipine against tumor necrosis factor alpha-induced oxidative stress in human glomerular endothelial cells

Recently, increasing evidence suggests that the antihypertensive drug nifedipine acts as a protective agent for endothelial cells, and that the activity is unrelated to its calcium channel blocking. Nitrosonifedipine (NO-NIF) is metabolically and photochemically produced from nifedipine, and NO-NIF has been recognized as a contaminant of nifedipine because it has no antihypertensive effect. Treatment of tumor necrosis factor-α (TNF-α) suppressed the cell viability and facilitated the expression of Inter-Cellular Adhesion Molecule 1(ICAM-1) in human glomerular endothelial cells (HGECs) though, pretreatment of NO-NIF significantly recovered the TNF-α-induced cell damage to the same extent as Trolox-C did, and suppressed the ICAM-1 expression in a concentration dependent manner. In addition, NO-NIF inhibited the cell toxicity induced by cumene hydroperoxide, which hampers the integrity of cell membrane through oxidative stress, as effective as Trolox-c. These data suggest that NO-NIF is a candidate for a new class of antioxidative drug that protect cells against oxidative stress in glomerular endothelial cells.

The Journal of Medical Investigation : JMI


Deferoxamine promotes angiogenesis via the activation of vascular endothelial cell function.

BACKGROUND: Deferoxamine (DFO), an iron chelator for disorders of excess iron, upregulates the expression of angiogenic factors, such as vascular endothelial growth factor (VEGF) and cyclooxygenase-2 (COX-2), indicating that it affects angiogenesis. Herein, we clarify the effect and mechanism of action of DFO on angiogenesis. METHODS AND RESULTS: In an in vitro study, DFO increased endothelial nitric oxide synthesis (eNOS) phosphorylation in human aortic endothelial cells (HAECs), which were inhibited by the phosphatidylinositol 3-kinase inhibitor LY294002. Tube formation, cell proliferation, and cell migration in HAECs were promoted by DFO, which were significantly reduced by LY294002. In an in vivo study, DFO promoted blood flow recovery in response to the hindlimb ischemia in mice with unilateral hindlimb surgery. The density of capillaries and arterioles in ischemic muscle was higher in DFO-treated mice compared to vehicle-treated mice. Endothelial cell proliferation increased and oxidative stress and apoptosis decreased in ischemic muscles of DFO-treated mice. The phosphorylation of Akt and eNOS on the ischemic side was elevated and urinary nitric oxide/nitric dioxide (NOx) excretion was higher in DFO-treated mice compared to vehicle-treated mice. The effect of DFO on angiogenesis was abolished in eNOS-deficient mice with hindlimb ischemia. CONCLUSION: These findings indicate that DFO promotes revascularization via the activation of vascular endothelial cell function by an Akt-eNOS-dependent mechanism.


Activation of peroxisome proliferator-activated receptor α in megakaryocytes reduces platelet-derived growth factor-BB in platelets.

Platelet-derived growth factor (PDGF)-BB plays a crucial role in atherosclerosis and vascular remodeling by promoting the migration and proliferation of vascular smooth muscle cells. The objective of this study was to clarify the pleiotropic effect of peroxisome proliferator-activated receptor α (PPARα) activators on PDGF-BB expression in megakaryocytes and platelets. The expression of PPARα in a human erythroleukemia (HEL) cells was clearly detected by reverse transcriptase-PCR and immunofluorescence microscopy. The expression level of PPARα in HEL cells was unchanged regardless of differentiation into megakaryocytic cells by treatment with phorbol 12-myristate 13 acetate (TPA). The TPA-induced expression of PDGF-B mRNA and PDGF-BB protein levels in culture media was significantly decreased by treatment with PPARα activators, Wy14643 and fenofibric acid, in a dose-dependent manner. PDGF-BB expression induced by inflammatory cytokines, including interleukin-1β or interleukin-6, was also significantly suppressed by treatment with PPARα activators. Immunohistochemistry of human bone marrow showed the expression of PPARα in both the nucleus and cytoplasm of megakaryocytes. In addition, PDGF-BB levels in platelets were significantly decreased from 1,800±870 to 1,470±840 pg/10(5) platelets (mean±SD, p<0.05) by treatment with 300 mg fenofibrate once daily for 4 weeks in 13 patients with dyslipidemia. Activation of PPARα in megakaryocytes reduces PDGF-BB expression in platelets. PPARα activators may exert vasculo-protective action through suppression of PDGF-BB production in a megakaryocyte/platelet pathway.

Journal of Atherosclerosis and Thrombosis


Plasma heparin cofactor II activity is inversely associated with left atrial volume and diastolic dysfunction in humans with cardiovascular risk factors.

Thrombin has a crucial role in cardiac remodeling through protease-activated receptor-1 activation in cardiac fibroblasts and cardiomyocytes. As heparin cofactor II (HCII) inhibits the action of tissue thrombin in the cardiovascular system, it is possible that HCII counteracts the development of cardiac remodeling. We investigated the relationships between plasma HCII activity and surrogate markers of cardiac geometry, including left atrial volume index (LAVI), relative wall thickness (RWT) and left ventricular mass index, and deceleration time (DcT) and the ratio of peak E velocity to early diastolic mitral annulus velocity (E/e' ratio) as surrogate markers of left ventricular diastolic dysfunction measured using echocardiography in 304 Japanese elderly individuals without systolic heart failure (169 men and 135 women; mean age: 65.4 ± 11.8 years). Mean plasma HCII activity in all participants was 95.8 ± 17.0% and there was no difference between the mean plasma HCII activities in males and females. Multiple regression analysis revealed that there were significant inverse relationships between plasma HCII activity and LAVI (coefficient: -0.2302, P<0.001), between HCII activity and RWT (coefficient: -0.0007, P<0.05), between HCII activity and DcT (coefficient: -0.5189, P<0.05) and between HCII activity and E/e' ratio (coefficient: -0.0558, P<0.01). Plasma HCII activity was independently and inversely associated with the development of cardiac remodeling, including cardiac concentric change, left atrial enlargement and left ventricular diastolic dysfunction. These findings suggest that cardiac tissue thrombin inactivation by HCII is a novel therapeutic target for cardiac remodeling and atherosclerosis.


High plasma aldosterone concentration is a novel risk factor of cognitive impairment in patients with hypertension.

Cognitive impairment leading to dementia is associated with high prevalence of hypertension, decreased quality of life and poor prognosis. Aldosterone is known as a risk factor for cardiovascular and cerebrovascular diseases. In addition, mineral corticoid receptors are abundantly expressed in the hippocampus, which plays a pivotal role in cognitive function; however, it has not been determined whether plasma aldosterone level is associated with cognitive impairment in patients with hypertension. We enrolled 68 patients with essential hypertension and assessed their cardiovascular risk factors, including blood pressure, hyperlipidemia, diabetes mellitus, obesity, smoking, history of cerebral infarction, renal function, parameters of inflammation, oxidative stress and nitric oxide bioavailability, a parameter of cerebral blood flow and carotid plaque by ultrasound examination, plasma renin activity and plasma aldosterone concentration (PAC). The mini-mental state examination (MMSE) was used to evaluate cognitive function. The relevance of cardiovascular risk factors and MMSE score was statistically evaluated. Multiple regression analysis showed that age (P<0.01), PAC (P<0.01) and history of cerebral infarction (P<0.05) were inversely and independently associated with MMSE score. Mineral corticoid receptor antagonists, including spironolactone and eplerenone, increased MMSE score in seven patients with hypertension, but not in the controls. In conclusion, increased PAC is associated with impaired cognitive function and mineral corticoid receptor blockade may protect against not only cardiovascular mortality, but also cognitive impairment in patients with hypertension.Hypertension Research advance online publication, 23 September 2010; doi:10.1038/hr.2010.179.


Heparin cofactor II protects against angiotensin II-induced cardiac remodeling via attenuation of oxidative stress in mice.

Heparin cofactor II (HCII), a serine protease inhibitor, inhibits tissue thrombin action after binding with dermatan sulfate proteoglycans in the extracellular matrix of the vascular system. We previously reported that heterozygous HCII-deficient (HCII(+/-)) humans and mice demonstrate acceleration of vascular remodeling, including atherosclerosis. However, the action of HCII on cardiac remodeling never has been determined. HCII(+/+) and HCII(+/-) mice at age 25 weeks were infused with angiotensin II (Ang II; 2.0 mg/kg/d) for 2 weeks by an osmotic mini-pump. Echocardiography revealed acceleration of cardiac concentric remodeling in HCII(+/-) mice and larger left atrial volume in HCII(+/-) mice than in HCII(+/+) mice. Histopathologic studies showed more prominent interstitial fibrosis in both the left atrium and left ventricle in HCII(+/-) mice than in HCII(+/+) mice. Daily urinary excretion of 8-hydroxy-2'-deoxyguanosine, a parameter of oxidative stress, and dihydroethidium-positive spots, indicating superoxide production in the myocardium, were markedly increased in Ang II-treated HCII(+/-) mice compared to those in HCII(+/+) mice. Cardiac gene expression levels of atrial natriuretic peptides and brain natriuretic peptides, members of the natriuretic peptide family, Nox 4, Rac-1, and p67(phox) as components of NAD(P)H oxidase, and transforming growth factor-beta1 and procollagen III were more augmented in HCII(+/-) mice than in HCII(+/+) mice. However, administration of human HCII protein attenuated all of those abnormalities in Ang II-treated HCII(+/-) mice. Moreover, human HCII protein supplementation almost abolished cardiac fibrosis in Ang II-treated HCII(+/+) mice. The results indicate that HCII has a protective role against Ang II-induced cardiac remodeling through suppression of the NAD(P)H oxidase-transforming growth factor-beta1 pathway.


Dehydroepiandrosterone sulfate is inversely associated with sex-dependent diverse carotid atherosclerosis regardless of endothelial function.

BACKGROUND: Dehydroepiandrosterone sulfate (DHEAS) is thought to be associated with life expectancy and anti-aging. However, its biological significance in atherosclerosis remains controversial. Therefore, the aim of this study was to determine whether DHEAS is associated with development of carotid atherosclerosis in subjects with cardiovascular risk factors. SUBJECTS AND METHODS: A total of 419 Japanese individuals (208 males and 211 females) were recruited from Tokushima University Hospital, Japan. In all subjects, maximum intima-media thickness (max-IMT) in all carotid arteries, and mean-IMT and mean blood flow volume (BFV) in the common carotid arteries (CCA) were measured by ultrasonography; endothelial function was assessed by flow-mediated vasodilation of the brachial artery (%FMD). Serum DHEAS and classical cardiovascular risk factors were also evaluated. Statistical significance was determined by multiple regression analysis to elucidate independent determinants of max-IMT, mean-IMT, mean CCA-BFV, and %FMD. RESULTS: Serum DHEAS levels were higher in males than in females. Multiple regression analysis revealed that DHEAS was an independent negative factor for both max-IMT and mean-IMT in males but not in females. In contrast, DHEAS was the sole positive factor for mean CCA-BFV in females but not in males. In addition, there was no significant relationship between %FMD and DHEAS regardless of sex and other confounding factors. CONCLUSION: Although DHEAS is not involved in endothelial function, DHEAS is inversely associated with sex-dependent diverse carotid atherosclerosis such as increased max-IMT and mean-IMT in males and decreased CCA-BFV in females.


Androgen receptor counteracts Doxorubicin-induced cardiotoxicity in male mice.

Doxorubicin (Dox) has been used as a potent anticancer agent, but serious cardiotoxicity precludes its use in a wide range of patients. We have reported that the androgen-androgen receptor (AR) system plays important roles in cardiac growth and protection from angiotensin II-induced cardiac remodeling. The present study was undertaken to clarify whether the androgen-AR system exerts a cardioprotective effect against Dox-induced cardiotoxicity. Male AR knockout (ARKO) and age-matched littermate male wild-type (WT) mice at 25 wk of age were given ip injections of Dox (20 mg/kg) or a vehicle. The survival rate and left ventricular function in Dox-treated male ARKO mice were reduced compared with those in Dox-treated male WT mice. Electron microscopic study showed prominent vacuole formation of myocardial mitochondria in Dox-treated male ARKO mice. Cardiac oxidative stress and apoptosis of cardiomyocytes were increased more prominently by Dox treatment in male ARKO mice than in male WT mice. In addition, Dox-induced reduction in the expression of cardiac mitochondria transcription factor A (Tfam) and phosphorylation of serine-threonine kinase (Akt) was more pronounced in male ARKO mice than in male WT mice. In cardiac myoblast cells, testosterone up-regulated Akt phosphorylation and Tfam expression and exerted an antiapoptotic effect against Dox-induced cardiotoxicity. Collectively, the results demonstrate that Dox-induced cardiotoxicity is aggravated in male ARKO mice via exacerbation of mitochondrial damage and superoxide generation, leading to enhanced apoptosis of cardiomyocytes. Thus, the androgen-AR system is thought to counteract Dox-induced cardiotoxicity partly through activation of the Akt pathway and up-regulation of Tfam to protect cardiomyocytes from mitochondrial damage and apoptosis.


Effect of angiotensin II on iron-transporting protein expression and subsequent intracellular labile iron concentration in human glomerular endothelial cells

Angiotensin II (Ang II)-induced endothelial injury, which is associated with atherosclerosis, is believed to be mediated by intracellular reactive oxygen species (ROS) through stimulation of nicotinamide adenine dinucleotide phosphate oxidase (NOX). Iron is essential for the amplification of oxidative stress. In this study, we investigated whether Ang II altered iron metabolism and whether the Ang II-induced endothelial injury is attributable to changes in iron metabolism of human glomerular endothelial cells (HGECs). When 90% iron-saturated human transferrin (90% Tf) was applied to HGECs without Ang II, the labile ferrous iron level was same as the effect of control in spite of a significant increase in the total cellular iron concentration. Treatment with Ang II and 30% Tf or 90% Tf significantly (P<0.01) increased the intracellular iron concentration, as well as labile ferrous iron and protein oxidation levels, compared with the effect of separate administration of each compound. Ang II treatment facilitated the protein expression of the Tf receptor, divalent metal transporter 1, and ferroportin 1 in a dose- and time-dependent manner. It was also found that simultaneous exposure of HGECs to Ang II and 90% Tf accelerated hydroxyl radical production, as shown by using an electron paramagnetic resonance spectrometer. These results suggest that Ang II not only induces production of ROS by NOX activation but also iron incorporation followed by an increase in labile iron in HGECs. Both of these events may participate in the progression of oxidative stress because of endothelial cell dysfunction through ferrous iron-mediated ROS generation.


Endothelial nitric oxide synthase-independent protective action of statin against angiotensin II-induced atrial remodeling via reduced oxidant injury.

Activation of the renin-angiotensin system exacerbates atrial remodeling, leading to atrial fibrillation and thrombosis, especially in a condition with decreased NO bioavailability. Recently, it has been reported that statins reduce the incidence of atrial fibrillation through attenuation of atrial remodeling; however, the mechanisms have not been completely elucidated. Therefore, we aimed to clarify the beneficial effect of statin on atrial remodeling in condition with reduced NO bioavailability. Endothelial NO synthase(-/-) mice were sham operated or infused with angiotensin II (Ang II) via an osmotic minipump for 2 weeks, and Ang II-infused mice were divided into 3 treatment groups: pitavastatin, Tempol (a free radical scavenger), or vehicle. Echocardiography and electrocardiography showed that Ang II infusion caused left atrial enlargement and a high incidence of atrial fibrillation, whereas pitavastatin and Tempol prevented these abnormalities. In histological analysis, Ang II-induced atrial interstitial fibrosis, perivascular fibrosis, and cardiomyocyte hypertrophy were all attenuated by pitavastatin and Tempol. Immunohistochemical staining showed that Ang II downregulated thrombomodulin and tissue factor pathway inhibitor and upregulated tissue factor and plasminogen activator inhibitor 1 in the left atrium and that pitavastatin and Tempol corrected the thrombogenic condition. Moreover, pitavastatin and Tempol reduced Ang II-induced atrial superoxide production and atrial transforming growth factor-beta1 expression and Smad 2/3 phosphorylation. Atrial rac1-GTPase activity, known to activate NADPH oxidase, was attenuated by pitavastatin but not by Tempol. In conclusion, pitavastatin exerts endothelial NO synthase-independent protective actions against Ang II-induced atrial remodeling and atrial fibrillation with enhanced thrombogenicity through suppression of oxidant injury.


Angiotensin II receptor blocker attenuates PDGF-induced mesangial cell migration in a receptor-independent manner

Clinical studies have shown that angiotensin II (Ang II) type 1 (AT1) receptor blockers (ARBs) are able to provide renoprotection independent of their blood pressure lowering effects. ARBs also are reported to suppress oxidative stress, inflammation and certain other cellular responses in a receptor-independent manner. We investigated the effects of an ARB, olmesartan, on the cell migration induced by platelet-derived growth factor (PDGF), a major mitogen involved in the pathogenesis of glomerulonephritis in rat mesangial cells (RMCs). Cell migration was determined by a modified Boyden chamber assay. The intracellular signalling pathway was examined by western blotting. AT1 receptor expression was knocked down by small interfering RNAs. The intracellular reactive oxygen species (ROS) was measured by using a fluorescent probe. The O(2)(.-) scavenging activities were studied by the electron paramagnetic resonance-spin trapping method. PDGF-induced cell migration was inhibited by olmesartan in AT1 receptor knockdown RMCs. Olmesartan attenuated big mitogen-activated protein (MAP) kinase 1 (BMK1) and Src activation by PDGF in AT1 receptor knockdown RMCs. PDGF-induced BMK1 activation was suppressed by the Src family tyrosine kinase inhibitors, indicating that Src exists upstream of BMK1. The NADPH oxidase inhibitors inhibited not only PDGF-induced BMK1 and Src activation but also RMC migration. The elevation in ROS generation induced by PDGF was decreased by olmesartan. Olmesartan displayed neither directly ROS scavenging activity nor the inhibition of ROS-mediated intracellular signalling in RMCs. Olmesartan attenuates ROS generation by PDGF, leading to the subsequent inhibition of Src/ BMK1/migration in an AT1 receptor-independent manner in RMCs.