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Pulmonary arterial hypertension (PAH) is characterized by abnormal outgrowth of pulmonary artery smooth muscle cells (PASMCs) of the media. Abundant expression of endothelin-1 (ET-1) and activated p38 mitogen-activated protein kinase (p38MAPK) has been observed in PAH patients. p38MAPK has been implicated in cell proliferation. An unspecified disturbance in bone morphogenetic protein (BMP) signaling may be involved in the development of PAH. Type I receptors (BMPR1A and BMPR1B) and type II receptor (BMPR2) transduce signals via two distinct pathways, i.e., canonical and non-canonical pathways, activating Smad1/5/8 and p38MAPK, respectively. BMPR1B expression was previously reported to be enhanced in the PASMCs of patients with idiopathic PAH. BMP15 binds specifically to BMPR1B. We assessed the effects of ET-1 on BMP receptor expression and cell proliferation. BMP2 increased BMPR1B expression in human PASMCs after pretreatment with ET-1 in vitro. Although BMP2 alone did not affect PASMC proliferation, BMP2 treatment after ET-1 pretreatment significantly accelerated PASMC proliferation. PH-797804, a selective p38MAPK inhibitor, abrogated this proliferation. Similarly, after ET-1 pretreatment, BMP15 significantly accelerated the proliferation of PASMCs, whereas stimulation with BMP15 alone did not. In conclusion, in PASMCs, ET-1 exposure under pathological conditions alters BMP signaling to activate p38MAPK, resulting in cell proliferation.

Background Cases of giant coronary artery aneurysms (GCAAs) associated with coronary fistula are rarely reported, and they present with various symptoms, including coronary steal syndrome. We report an uncommon case of an asymptomatic giant coronary fistula aneurysm presenting as a progressing left-sided mediastinal mass that has been tracked for years. Case summary A 67-year-old healthy asymptomatic woman was referred to our hospital because of an abnormal shadow on her chest radiography revealing a left-sided mediastinal mass that had progressed in size over the past 4 years. Computed tomography revealed mass progression from 4 to 5 cm in diameter within 2 years. Coronary computed tomography and coronary angiography identified a GCAAs in a coronary fistula originating in the left anterior descending artery and draining into the main pulmonary artery. Transthoracic Doppler echocardiography revealed a unique systolic dominant flow. She underwent coronary artery aneurysmectomy and fistula ligation. The patient has been in good health without any events for 10 months since her discharge. Discussion A GCAAs in a coronary fistula can present as an asymptomatic left-sided mediastinal mass that has progressed in size for years in older adults. Echocardiography can provide clues of the steal phenomenon in coronary artery fistula. A close investigation of mediastinal abnormalities can facilitate the detection of coronary aneurysms.

Upregulated p38 signaling is implicated in the accelerated proliferation of pulmonary artery smooth muscle cells (PA-SMCs) and the pathogenesis of pulmonary artery remodeling observed in pulmonary arterial hypertension (PAH). Previously, we reported that after endothelin-1 (ET-1) pretreatment, bone morphogenetic protein 2 (BMP2) activates p38 signaling and accelerates PA-SMC proliferation. The activity of p38 signaling is tightly regulated by the inactivation of dual-specificity phosphatase 1 (DUSP1). Activated p38 induces DUSP1 expression, forming a negative feedback loop. Prostacyclin IP receptor agonists (prostacyclin and selexipag) are used to treat PAH. In this study, we aimed to verify whether IP receptor agonists affect DUSP1 expression and accelerate the proliferation of PA-SMCs. PA-SMCs were treated with BMP2, ET-1, prostacyclin, and MRE-269, an active metabolite of selexipag, either alone or in combination. We quantified mRNA expressions using real-time quantitative polymerase chain reaction. Pulmonary artery specimens and PA-SMCs were obtained during lung transplantation in patients with PAH. Both prostacyclin and MRE-269 increased DUSP1 expression. Combined treatment with BMP2 and ET-1 induced cyclin D1 and DUSP1 expression and increased PA-SMC proliferation. MRE-269 attenuated BMP2/ET-1-induced cell proliferation. ET-1 increased DUSP1 expression in PA-SMCs from control patients but not in PA-SMCs from patients with PAH. This study showed that the p38/DUSP1 negative feedback loop is impaired in PAH, contributing to unregulated p38 activation and PA-SMC hyperplasia. IP receptor agonist MRE-269 increases DUSP1 expression and inhibit p38-mediated PA-SMC proliferation. Future elucidation of the detailed mechanism underlying reduced DUSP1 expression would be informative for PAH treatment.

Background Endothelial dysfunction is thought to be a major contributor to overall pathogenesis of vasculopathy seen in pulmonary hypertension (PH), which is manifested by the impaired release of nitric oxide (NO) generated through endothelial nitric oxide synthase (eNOS) in endothelial cells. Activation of human eNOS is regulated by phosphorylation at multiple sites including Thr33 and Ser114, which residues are followed by Pro. The peptidyl isomerase Pin1 specifically isomerizes the phospho-protein having Ser/Thr-Pro bond and regulates their activity. Pin1 is involved in proliferation, cell cycle, and apoptosis in cancer, by isomerizing some functional molecules such as JNK, JUN, cyclin D, BAX, etc. However, it is controversial whether direct interaction of Pin1 with eNOS and how eNOS activity is altered by Pin1, especially in PH. Purpose We aimed to clarify whether Pin1 contributes to the PH development using Pin1 knockout mice and Pin1 affects the expression of phosphorylated eNOS (p-eNOS) molecule and pulmonary arterial endothelial cell (PAEC) apoptosis. Methods and results Wild (WT) and Pin1-deficient mice (KO) were exposed to hypoxia (10% O2) or normoxia for 3 weeks to generate hypoxia-induced PH. Hypoxia-inducible factor (HIF1α) expression in lungs was significantly enhanced in WT-hypoxia (WH, n=6) and KO-hypoxia (KH, n=6), suggesting that hypoxic response was certainly occurred in these mice. Pulmonary arterial pressure did not elevate in KH compared with KO-normoxia (KN, n=6) and WT-normoxia (WN, n=6), it was significantly increased only in WH (P Conclusion This study suggests that endogenous Pin1 contributes to the development of PH partly via the dysfunction of PAECs, that is, by the interference with p-eNOS expression and by the increase of apoptosis inducibility to external stimuli. Funding Acknowledgement Type of funding source: Public grant(s) – National budget only. Main funding source(s): JSPS KAKENHI

Background Intestinal flora changes dynamically in early infancy. Association between colonisation of Bifidobacterium and Bacteroides, the dominant bacterial genera in the intestinal flora 6 months after birth, and development of intestinal immunity has been suggested. Antibiotic administration to the mother is an important factor in this colonisation, but few studies have considered antibiotics taken immediately before delivery, including before Caesarean section. The association of the gut flora with the delivery method is also uncertain. Here, we performed a cohort study to determine the influence of antibiotics taken immediately before delivery on the intestinal flora in early infancy, with a focus on Bifidobacterium and Bacteroides, in 130 healthy Japanese infants. Results Faecal samples were collected from infants at 1, 3, and 6 months after birth (383 samples) in a hospital in Japan. The samples were analysed using a next-generation sequencer. Two β-lactam antibiotics were administered to mothers immediately before delivery: Cefazolin was used before Caesarean sections and ampicillin was used for cases with premature rupture of the membranes and in Group B Streptococcus positive cases. At all ages, Bifidobacterium and Bacteroides were dominant, and the mean combined occupancy was 60–70%. Bifidobacterium was the most dominant at all ages but its abundance was significantly lower in infants who were exposed to antibiotics at delivery, and 1 and 3 months after birth, whereas no difference was found between the delivery methods. Bacteroides were the second or third dominant bacteria at all ages but their abundance was significantly lower after Caesarean section compared with that after vaginal delivery, whereas there was no difference due to antibiotic exposure. These effects were confirmed in a linear mixed-effect model. In addition, occupancy by Bifidobacterium at 1 and 3 months and by Bacteroides at 3 months differed between infants with and without siblings. Conclusions Exposure to antibiotics at delivery has an important effect on Bifidobacterium, the dominant bacterial genus of the intestinal flora, at an early age in Japanese infants, whereas Bacteroides is more associated with the delivery method. The influence of siblings is also significant. The clinical significance of these results in later life requires further exploration.

The glucagon-like peptide-1 receptor (GLP-1R) agonist liraglutide is an incretin hormone mimetic used in the treatment of diabetes. However, the effects of liraglutide on pulmonary hypertension (PH) and pulmonary endothelin (ET) system are unknown. Eight-week-old C57BL6/J mice were injected liraglutide or vehicle for 5 weeks. One week after injection, the mice were exposed to either room air (normoxia) or chronic hypoxia (10 % O2) for 4 weeks. The right ventricular systolic pressure (RVSP) was significantly higher in hypoxia + vehicle group than in normoxia + vehicle group. ET-1 mRNA expression in the lungs was comparable among all the groups. ETB mRNA and protein expression in the lungs was significantly lower in hypoxia + vehicle group than in normoxia + vehicle group. The above changes were normalized by liraglutide treatment. The expression of phospho-eNOS and phospho-AMPK proteins in the lungs was significantly higher in hypoxia + liraglutide group than in normoxia + vehicle group. We demonstrated for the first time that liraglutide effectively improved RVSP and RV hypertrophy in hypoxia-induced PH mice by activating eNOS through normalization of impaired ETB pathway and augmentation of AMPK pathway. Therefore, GLP-1R agonists can be promising therapeutic agents for PH.

Idiopathic pulmonary hemosiderosis (IPH) is a rare and life-threatening disorder. Early diagnosis and appropriate management are essential for their better prognosis and patients’ quality of life (QOL). It is considered that Down syndrome patients with IPH have a worse prognosis compared to other IPH cases. A 2-year-old girl with Down syndrome received the diagnosis of IPH after two episodes of massive pulmonary hemorrhage requiring assist ventilation, who suffered from recurrent IPH during tapering period of oral corticosteroid, started liposteroid therapy. We report here a case of successful control of recurrent IPH and improved QOL enormously with tapering dose of corticosteroid after starting liposteroid therapy.

Aims Vascular remodeling results from aberrations in the balance between cell proliferation and death, which is seen in the obstructive vasculature of pulmonary arterial hypertension (PAH). Endothelin (ET)-1 has a potent proliferative activity on vascular smooth muscle cells, and ET receptor inhibitors are used to treat PAH; however, it remains unclear whether ET receptor inhibition contributes to the apoptosis of pulmonary arterial smooth muscle cells (PASMCs), another cause of pulmonary vascular remodeling. Main methods Cultured human PASMCs were treated with the ET A receptor antagonist BQ-123 (100 μM), or the ET B antagonist A-192621 (1 – 100 μM) or BQ-788 (1 – 100 μM) for 48 h. The cells were then incubated for another 24 h with or without doxorubicin (DOX, 1 μM), an anthracyclin antitumor antibiotic that promotes p53-mediated apoptosis. Cell viability and apoptosis were evaluated by MTT assays, caspase-3/7 activity assays, and Western blots for cleaved caspase-3 expression. Key findings The viability of PASMCs was significantly decreased by A-192621 and BQ-788, in a dose-dependent manner. A-192621 and BQ-788 significantly increased the caspase-3/7 activity and cleaved caspase-3 expression in PASMCs. The PASMCs' susceptibility to DOX-induced apoptosis was significantly higher in the presence of A-192621 and BQ-788 than with vehicle. However, BQ-123 did not affect these parameters. Significance Blockade of the ET B receptor increases the extent of apoptosis and susceptibility to DOX-induced apoptosis in PASMCs. Apoptosis caused by ET B receptor blockade in PASMCs may be one of the mechanisms by which vascular remodeling is reduced in ET receptor inhibitor-based PAH treatments.

BACKGROUND: Endothelin receptor antagonists improve pulmonary arterial hypertension (PAH). Mutations in the bone morphogenetic protein (BMP) type 2 receptor (BMPR2) predispose to PAH. Here, we sought to determine whether there might exist interactions between these 2 signaling pathways and their effect on the acquisition of the altered phenotype of pulmonary artery smooth muscle cells (PA-SMCs) observed in PAH. METHODS: Expression of BMPR2, of the BMP agonist BMP4, and of the BMP antagonists gremlin1 and gremlin2 was evaluated in lungs and in PA-SMCs from 6 PAH patients and 14 controls treated with endothelin-1. Endothelin-1 pre-treated PA-SMCs were assessed for proliferation, apoptosis, and downstream signaling activation of Smad1/5/8 and p38 mitogen-activated protein kinase (p38(MAPK)) after BMP2 treatment. RESULTS: In PA-SMCs from PAH patients, expression of BMPR2 and BMP4 decreased, whereas expression of gremlin1 and gremlin2 increased compared with controls. Treatment of control PA-SMCs with endothelin-1 induced a dose-dependent increase in gremlin1 and gremlin2, whereas BMPR2 and BMP4 expression decreased, reaching similar levels as those observed in PAH cells. In control PA-SMCs, endothelin-1 pre-treatment reduced inhibitor of DNA binding 1 (Id1) expression and Smad1/5/8 activation induced by BMP2, whereas it enhanced p38(MAPK) activation. Moreover, BMP2 decreased serum-induced proliferation and increased the pro-apoptotic Bax/Bcl-2 ratio. These effects were attenuated by endothelin-1 pre-treatment. Endothelin-1 did not alter BMPR2 signaling in PA-SMCs from PAH patients. CONCLUSIONS: Endothelin-1 downregulates canonical BMPR2 signaling. This is related to decreased BMPR2 and increased anti-BMP gremlin expression associated with increased activation of p38(MAPK) and results in PA-SMC proliferation.

Objective To investigate factors related to bifidobacterial colonization in early infancy, with a focus on maternal antimicrobial use at delivery. Study design A cross-sectional pilot study was performed. Feces samples of 33 Japanese healthy infants were collected over 10 months and analyzed by next-generation sequencing to examine the diversity and abundance of the gut microbiota. Results The beta diversity index of the gut microbiota differed significantly based on maternal antimicrobial use at delivery (P

Background: Congenital diaphragmatic hernia (CDH) has a high mortality rate mainly due to lung hypoplasia and persistent pulmonary hypertension of the newborn (PPHN). We recently reported that antenatal treatment with sildenafil or simvastatin prevented pulmonary hypoplasia and PPHN in experimental CDH. Objectives. We sought to evaluate the efficacy of antenatal sildenafil-simvastatin combination on pulmonary hypoplasia and PPHN associated to nitrofen-induced CDH. Methods: On embryonic day (E)9.5, nitrofen or vehicle was administered to pregnant rats. Nitrofen-treated rats were randomly assigned to antenatal sildenafil (100 mg/kg/day) and simvastatin (20 mg/kg/day) combination or placebo administration from E11 to E21. On E21, foetuses were delivered by caesarean section, killed and checked for left-sided CDH. Results: In nitrofen-induced CDH, simvastatin-sildenafil totally prevented left-sided CDH in the offspring, but failed to increase body weight and lung-to-body weight ratio. Antenatal simvastatin-sildenafil improved lung parenchyma structure, with decreased alveolar septal thickness and increased radial alveolar count. Antenatal simvastatin-sildenafil restored pulmonary vascular density and external diameter, and reduced pulmonary arteriolar remodeling. This was associated with restored epithelial and vascular activation of apoptotic processes in the lungs. Antenatal combination therapy was more efficacious than antenatal therapy with simvastatin or sildenafil alone. Conclusions: Antenatal simvastatin-sildenafil combination therapy improves pathological features of lung hypoplasia and PPHN in experimental nitrofen-induced CDH.

Article history:Received 21 November 2011Accepted 13 April 2012Keywords:FCP1CTD phosphataseCardiac hypertrophyEndothelin-1RNA polymerase II Aims: Cardiac hypertrophy is associated with the increase of total amount of RNA, which is in accordance withRNA polymerase II (RNAPII) activation via C-terminal domain (CTD) phosphorylation of the largest subunit ofRNAPII. It has been demonstrated that endothelin-1 (ET-1) phosphorylates CTD at the hypertrophic responseincardiomyocytes.However,itisunclearwhetherET-1-inducedhypertrophyisaffectedbytheCTDphosphatase,transcription factor IIF-interacting CTD phosphatase1 (FCP1).Mainmethods: We analyzed whether ET-1-induced cardiomyocyte hypertrophy was affected by overexpressionof FCP1 or dominant-negative form of FCP1 (dnFCP1) in neonatal rat cardiomyocytes.Key findings: The level of ET-1-induced RNAPII CTD phosphorylation was decreased by FCP1 overexpression,whereas it was sustained by dnFCP1. Global RNA synthesis evaluated by [ 3 H]-uridine incorporation showedthat the ET-1-induced increase in RNA synthesis was suppressed by FCP1 and was augmented by dnFCP1. ET-1-induced increase in cell surface area was suppressed by FCP1 and was preserved by dnFCP1. Furthermore, theET-1-induced increase in molecular markers of cardiac hypertrophy, expression of ANP and β-MHC gene, wassuppressed by FCP1 and was not inhibited by dnFCP1.Significance: ET-1-inducedcardiachypertrophyandCTDphosphorylationlevelarefunctionallyregulatedbyFCP1.These findings suggest that FCP1 plays an important role in ET-1-induced cardiac hypertrophy via controllingphosphorylation level of the RNAPII CTD.© 2012 Elsevier Inc. All rights reserved.

BACKGROUND Oxidative stress caused by contrast medium is thought to be one of the main mechanisms of contrast-induced acute kidney injury. A prospective study was conducted to evaluate the relationship between oxidative stress caused by contrast agent administration and long-term renal function. METHODS AND RESULTS Thirty-six consecutive patients who underwent coronary angiography were enrolled. Urinary F2-isoprostane, a marker of oxidative stress, was measured at baseline and 24h after angiography, and serum creatinine was measured at baseline, 24h and 1 year after the procedure. The change in estimated glomerular filtration rate (eGFR) at 1 year after angiography correlated significantly with the change in eGFR at 24h after angiography (r=0.729, P

There is increasing evidence for the presence of cancer stem cells (CSCs) in malignant brain tumors, and these CSCs may play a pivotal role in tumor initiation, growth, and recurrence. Vascular endothelial growth factor (VEGF) promotes the proliferation of vascular endothelial cells (VECs) and the neurogenesis of neural stem cells. Using CSCs derived from human glioblastomas and a retrovirus expressing VEGF, we examined the effects of VEGF on the properties of CSCs in vitro and in vivo. Although VEGF did not affect the property of CSCs in vitro, the injection of mouse brains with VEGF-expressing CSCs led to the massive expansion of vascular-rich GBM, tumor-associated hemorrhage, and high morbidity, suggesting that VEGF promoted tumorigenesis via angiogenesis. These results revealed that VEGF induced the proliferation of VEC in the vascular-rich tumor environment, the so-called stem cell niche.

Aims Pulmonary hypertension (PH) is a common complication of chronic hypoxic lung diseases. Bone morphogenetic protein (BMP) and endothelin-1 signaling pathways have been shown to be altered in hypoxic PH and to play crucial roles in the associated pulmonary artery remodeling. We, therefore, aimed to study the potential link between hypoxia and the alteration of BMP and endothelin-1 signaling observed in pulmonary artery smooth muscle cells (PA-SMCs) in hypoxic PH. Materials and Methods Human PA-SMCs were treated with hypoxia-mimetic agent cobalt chloride (CoCl 2 ; 100 μM), with or without pretreatment with a dual endothelin receptor antagonist bosentan (10 μM). Expressions of preproendothelin-1 (PPET1), BMP type 2 receptor (BMPR-2), and one BMP signaling target gene, the inhibitor of DNA binding 1 (ID1) were evaluated by real time quantitative polymerase chain reaction. BMP2-treated PA-SMCs were assessed for Smad1/5/8 signaling activation by Western Blotting. Key findings Treatment of PA-SMCs with CoCl 2 increased PPET1 gene expression, while it did not alter expressions of endothelin converting enzyme, endothelin receptor type A or type B. Hypoxia-mimetic agent CoCl 2 decreased the expressions of BMPR-2 and ID1 maximally after 3- and 6-hour treatment respectively, while CoCl 2 treatment progressively increased noggin expression. Bosentan pretreatment restored expressions of BMPR-2 and ID1, as well as the activation (by phosphorylation) of Smad1/5/8 signaling induced by BMP2. Significance Hypoxia induces the downregulation of the BMP signaling in PA-SMCs, at least, partly through the endothelin system. In hypoxic PH, increased endothelin-1 production might therefore contribute to the altered BMP signaling and subsequent PA-SMC hyperplasia.

Background: Pulmonary hypertension (PH) is a common complication of chronic hypoxic lung diseases. Alterations in bone morphogenetic protein (BMP) and endothelin signaling have been described in hypoxic PH. We recently reported on endothelin-induced downregulation of BMP signaling in pulmonary artery smooth muscle cells (PA-SMCs), partly through increased expression of BMP antagonist gremlin1. Here, we aimed to study the alterations in BMP signaling in hypoxia. Methods: Lung expressions of BMP antagonists noggin and gremlin1 were evaluated in 4 patients with group3 PH, 6 with idiopathic pulmonary arterial hypertension (iPAH) and 6 controls. Control PA-SMCs were treated with the hypoxia-mimetic agent cobalt chloride (CoCl2; 100 µM), with or without pre-treatment with bosentan (1 µM). Gene expressions of preproendothelin-1 (PPET1), endothelin converting enzyme 1 (ECE1), BMP type 2 receptor (BMPR2), noggin, gremlin1 and inhibitor of DNA binding 1 (Id1) were evaluated by RTQ-PCR. Results: Lung tissue from group3 PH patients presented with increased expression of noggin, gremlin1 and 2, while a less important increase was observed in iPAH lungs. Treatment of PA-SMCs with CoCl2 increased PPET1 expression, while it did not alter ECE1 expression. CoCl2 decreased BMPR2 and Id1 expressions, while it increased noggin and gremlin1 expression. In PA-SMCs, bosentan pretreatment prevented the alteration of Id1 and gremlin1 expressions induced by CoCl2, while it did not alter noggin and BMPR2. Conclusion: Hypoxia induces the downregulation of the BMP signaling in control PA-SMCs, partly through the endothelin system. This may account for the increased expression of gremlin1 observed in hypoxic PH patients.

Congenital diaphragmatic hernia (CDH) has a high mortality rate mainly due to lung hypoplasia and persistent pulmonary hypertension of the newborn (PPHN). Simvastatin has been shown to prevent the development of pulmonary hypertension (PH) in experimental models of PH. We, therefore, hypothesized that antenatal simvastatin would attenuate PPHN in nitrofen-induced CDH in rats. The efficacy of antenatal simvastatin was compared with antenatal sildenafil, which has already been shown to improve pathological features of PPHN in nitrofen-induced CDH. On embryonic day (E) 9.5, nitrofen or vehicle was administered to pregnant Sprague-Dawley rats. On E11, nitrofen-treated rats were randomly assigned to antenatal simvastatin (20 mg·kg−1·day−1orally), antenatal sildenafil (100 mg·kg−1·day−1orally), or placebo administration from E11 to E21. On E21, fetuses were delivered by cesarean section, killed, and checked for left-sided CDH. Lung tissue was then harvested for further pathobiological evaluation. In nitrofen-induced CDH, simvastatin failed to reduce the incidence of nitrofen-induced CDH in the offspring and to increase the body weight, but improved the lung-to-body weight ratio and lung parenchyma structure. Antenatal simvastatin restored the pulmonary vessel density and external diameter, and reduced the pulmonary arteriolar remodeling compared with nitrofen-induced CDH. This was associated with decreased lung expression of endothelin precursor, endothelin type A and B receptors, endothelial and inducible nitric oxide synthase, together with restored lung activation of apoptotic processes mainly in the epithelium. Antenatal simvastatin presented similar effects as antenatal therapy with sildenafil on nitrofen-induced CDH. Antenatal simvastatin improves pathological features of lung hypoplasia and PPHN in experimental nitrofen-induced CDH.

Background: Significantly low serum lipid levels are occasionally seen at the time of diagnosis in children with aplastic anemia (AA). The aim of the present study was to clarify the pathologic and clinical significance of pretreatment serum lipid levels in AA. Methods: A questionnaire seeking precise data about AA in pediatric patients, including the initial laboratory data at the time of onset of AA and the clinical course of these patients, was sent to 18 institutes in Japan; 13 institutes responded to the questionnaire. In this retrospective study, data concerning hematologic examination and serum lipids were available for analysis in 127 children with AA. Serum lipoprotein patterns were analyzed using conventional agarose electrophoresis in eight patients. In order to elucidate the cause of hypolipidemia in AA, we assayed serum macrophage colony stimulating factor (M-CSF), which is well known to have apparent cholesterol-lowering activity, by using an enzyme-linked immunosorbent assay in seven patients with hypocholesterolemia and compared the results with those obtained in patients with iron-deficiency anemia (IDA). Results: We found that pretreatment total cholesterol (TC) and triglyceride levels in the serum correlated well with counts of both nucleated cells and hemopoietic cells in the bone marrow (BM) and were inversely correlated with the lymphocyte ratio in both the BM and peripheral blood. Patients with serum TC lower than 150 mg/dL showed a poor response to any form of therapy except BM transplantation. There was no difference in the serum lipoprotein patterns between the controls and patients examined. The serum M-CSF level was significantly higher in patients with TC levels lower than 150 mg/dL compared with controls. Conclusions: These results indicate that the pretreatment serum lipid level may reflect hematopoietic activity within the BM and can help to predict the therapeutic response of each case of AA to treatment with immunosuppressive drugs, corticosteroids and anabolic steroids. These results also indicate that M-CSF may be one of the contributing causes of the hypocholesterolemia that occurs in both AA and IDA.

Aims Cardiac hypertrophy is elicited by endothelin (ET)-1 as well as other neurohumoral factors, hemodynamic overload, and oxidative stress; HMG-CoA reductase inhibitors (statins) were shown to inhibit cardiac hypertrophy partly via the anti-oxidative stress. One of their common intracellular pathways is the phosphorylation cascade of MEK signaling. Pin1 specifically isomerizes the phosphorylated protein with Ser/Thr-Pro bonds and regulates their activity through conformational changes. There is no report whether the Pin1 activation contributes to ET-1-induced cardiomyocyte hypertrophy and whether the Pin1 inactivation contributes to the inhibitory effect of statins. The aim of this study was to reveal these questions. Main methods We assessed neonatal rat cardiomyocyte hypertrophy using ET-1 and fluvastatin by the cell surface area, ANP mRNA expression, JNK and c-Jun phosphorylation, and [ 3 H]-leucine incorporation. Key findings Fluvastatin inhibited ET-1-induced increase in the cell surface area, ANP expression, and [ 3 H]-leucine incorporation; and it suppressed the signaling cascade from JNK to c-Jun. The phosphorylated Pin1 level, an inactive form, was decreased by ET-1; however, it reached basal level by fluvastatin. Furthermore, Pin1 overexpression clearly elicited cardiomyocyte hypertrophy, which was inhibited by fluvastatin. Significance This is the first report that ET-1-induced cardiomyocyte hypertrophy is mediated through the Pin1 activation and that the inhibitory effect of fluvastatin on cardiomyocyte hypertrophy would partly be attributed to the suppression of the Pin1 function. This study firstly suggests that Pin1 determines the size of hypertrophied cardiomyocyte by regulating the activity of phosphorylated molecules and that statins exert their pleiotropic effects partly via Pin1 inactivation.

Purpose Bone morphogenetic proteins (BMP) have been shown to play crucial roles in not only lung and heart development, but also in the pathogenesis of pulmonary vascular remodeling in pulmonary hypertension (PH). We therefore hypothesized that BMP signaling could be altered in nitrofen-induced congenital diaphragmatic hernia (CDH) and associated PH. Methods Pregnant rats were exposed to either 100 mg nitrofen or vehicle on embryonic day (E) 9.5. On E17 and E21, fetuses were delivered by cesarean section, killed and checked for left-sided CDH. The tissue was then harvested for pathobiological evaluation. Results In nitrofen-induced CDH, pulmonary expressions of BMP4, BMP receptor (BMPR) type 2 and Id1 decreased on E17 and E21. On E17, pulmonary gremlin-1 expression increased, while BMP7 decreased. In the lungs, Id1 expression was correlated to BMP4 and BMPR2 and inversely correlated to gremlin-1 expression. Myocardial expressions of BMPR2, BMPR1A, BMP7 and SERCA-2A decreased, while gremlin-1 and noggin expressions increased on E17. On E21, myocardial expressions of Id1 and SERCA-2A decreased, while gremlin-1 expression increased. Moreover, BMPR2 and BMPR1A expressions were correlated to SERCA-2A expression and inversely correlated to pro-apoptotic Bax/Bcl2 ratio within the myocardium. Conclusion Downregulation of BMP signaling seems to contribute to pulmonary and myocardial anomalies observed in nitrofen-induced CDH.

Obstructive sleep apnea syndrome (OSAS) is associated with recurrent nocturnal hypoxia during sleep; this hypoxia has been implicated in the pathogenesis of cardiovascular complication. However, a useful soluble factor that is sensitively correlated with OSAS severity for the diagnosis remains unidentified. We hypothesized that systemic levels of basic fibroblast growth factor (bFGF), a hypoxia-induced cytokine, were affected by nocturnal hypoxemia in OSAS patients, and we assessed whether the degree of change in the plasma bFGF concentrations before and after nocturnal hypoxia is correlated with the severity of OSAS. Thirty subjects who had suspected OSAS and had been investigated by nocturnal polysomnography (PSG) were enrolled. Plasma bFGF and vascular endothelial growth factor (VEGF) concentrations the night before PSG and the next morning were measured by sandwich enzyme-linked immunosorbent assay. Correlations between the changes in these factors and hypoxia-associated parameters for OSAS severity were analyzed. Patients with OSAS had significantly elevated levels of plasma bFGF but not VEGF and hemoglobin after rising. The degree of change in bFGF concentrations after nocturnal apnea episodes was significantly correlated with diagnostic parameters for OSAS severity. The change in plasma bFGF levels is associated with the degree of hypoxic state in OSAS patients, implying that bFGF might be a useful soluble factor for evaluating OSAS severity.

Background: Contrast-induced nephropathy (CIN) remains a common complication of radiographic procedures. We hypothesized that endothelial dysfunction is the main cause of CIN. To clarify whether contrast medium-induced renal damage is associated with endothelial injury, we measured microparticles derived from endothelial cells as markers of endothelial injury. Circulating microparticles are shed from cell surface respond to cell activation and apoptotic stimuli, reflecting the condition of damaged cells. Methods: Renal function of 35 adult patients was analyzed before and after the use of contrast medium for coronary angiography. Parameters for renal function and urinary 15-isoprostane F2t, a specific marker of oxidative stress were measured before and after radiocontrast administration. Flow cytometry was used to count circulating CD34 + microparticles, which is regarded as one of markers for endothelial damage. Results: The decrease of estimated glomerular filtration rate positively correlated with the amount of contrast medium (r=0.427; P=0.013). Urinary N-acetyl-beta-D-glucosaminidase, a marker of renal tubular injury, was increased after angiography (from 7.6+/− 6.8 to 9.1 +/− 6.0 U/g-CRE, P=0.011). Furthermore, urinary 15-isoprostane F2t positively correlated with the volume of contrast medium (r=0.421; P=0.012). CD34+ microparticle was significantly increased after angiography (1.3-fold increased from basal level, P=0.0017). The increase of CD34+ microparticle was associated with the insult of contrast medium, but not of the amount. Conclusion: Radiocontrast impaired renal function in accordant with the increase of oxidative stress. The release of CD34+ microparticle was also increased by use of radiocontrast. These data suggest that CIN is tightly associated with endothelial injury mediated by radiocontrast-induced oxidative stress.

Background Regeneration of the lung microvasculature and replacing pulmonary artery lesions with functional endothelial cells could be a novel and effective therapeutic strategy for treating advanced pulmonary arterial hypertension (PAH). In the present study it was postulated that granulocyte colony-stimulating factor (G-CFS), which induces the proliferation of endothelial cells, would stimulate endothelial regeneration in situ at sites of impaired lung vasculature and prevent the development of PAH. Methods and Results Daily administration of G-CSF for 48 days did not affect the hemodynamism of normal Fischer 344 rats. PAH was induced with monocrotaline (60 mg/kg) and G-CSF was administered daily (100 μg/kg per day). Echocardiographic findings and an invasive catheter study indicated a significant decrease in the progression of PAH in rats given G-CSF. Furthermore, G-CSF increased Ki-67 positivity in the pulmonary arteries of PAH rats but did not accelerate c-kit positive cell recruitment into peripheral blood. Daily doses of G-CSF at both 2 and 100 μg/kg improved the survival and body weight gain of PAH rats. Conclusions G-CSF improved the progression of PAH in a rat model, possibly by stimulating pulmonary endothelial cells to proliferate at sites of impaired lung vasculature. These findings show that cytokine therapy for PAH is valid based on the concept of vascular regeneration. (Circ J 2007; 71: 138 - 143)

Endothelin-1 (ET-1) is known to be a principal factor in the pathogenesis of primary pulmonary hypertension (PPH). Recently intravenous administration of epoprosterol improved the survival rate in PPH. However, the effect of epoprosterol on ET-1 remains to be investigated. Therefore, we studied a patient with PPH who was treated with a low dose of epoprosterol and examined the serum concentration of ET-1 during the treatment. Epoprosterol greatly decreased the serum concentration of ET-1 in parallel with improvement of the clinical course, suggesting that ET-1 level may be a marker for treatment of PPH.

We have previously observed that an erythroid enhancing activity presents in rat serum in the early stage of drug induced hemolytic anemia. The further studies on biological and physicochemical aspects of this erythroid accelerating factor (EAF) is described in this paper. Hemolytic anemia was induced in rats by single intraperitoneal injection of acetylphenylhydrazine (APH) and serum was obtained from the rats on day 1 after APH injection. It was first fractionated by ultrafiltration on Amicon Diaflo membranes to give a series of fractions lying in the following ranges of molecular weight: 10-30 kDa, 30-50 kDa, 50-100 kDa, and >100 kDa. Among those fractions, largest increase in the number of colony forming unit erythroid CFU-E) colonies was shown in the fraction of >100 kDa that was subsequently fractionated by fast protein liquid chromatography (FPLC) system. EAF activity for CFU-E proliferation was detected in a FPLC fraction corresponding to a molecular weight of about 160 kDa. An addition of EAF significantly increased with dose dependent manner in the number of CFU-E colonies from rat bone marrow mononuclear cells. EAF alone had no burst promoting activity and exhibited no distinct activity to proliferate burst forming unit-erythroid even when interleukin-3 (IL-3) and high concentration (2 U/ml) of erythropoietin (Epo) were added together to the culture. The stimulating effect of EAF on CFU-E was markedly dependent on the presence of adherent cells in the culture. Partially purified protein was relatively heat-unstable (60% at 75 degrees C, 30 minutes) and sensitive to treatment with trypsin and alpha-galactosidase. These results suggest that EAF is a novel factor, possible glycoprotein to reinforce Epo function and is different from various cytokines previously documented because of differences of approximate molecular weight.

Erythroid accelerating factor (EAF) is a peculiar factor because the activity is definitely exhibited in an early stage of hemolytic stress in rats. A presumption can be made that EAF contributes to the acceleration of erythroid differentiation during initial period of sudden loss of erythrocytes. To further assess the relationship between serum EAF and a rapid rise of erythropoiesis in response to acute reduction of red blood cells, We determined the sequential changes of serum EAF and Epo in concomitant with blood parameters, bone marrow (BM) CFU-E and the erythroblastic islets in spleens at short intervals from 0time through 24h after acetylphenyhydrazine (APH) injection to induce hemolysis in rats. Serum EAF was significantly elevated as early as 6h following APH injection, reached a peak at 24h. On the other hand, serum Epo levels were gradually increased from 12h, with peaking on day 3 of the experiment. The number of colonies derived from CFU-E and erythroblastic ratios in BM showed peak increases at 8h after injection of APH. Histologic analysis of spleens revealed that the erythroblastic islets increased from (+) to (3+) at 6h. These results suggest that EAF mediates an early enhance of erythropoietic process during initial period of urgent anemia as acute hemolysis.

Background: Mutations in the bone morphogenetic protein type 2 receptor (BMPR2) are responsible for themajority of cases of heritable pulmonary arterial hypertension (PAH). Low penetrance of BMPR2 mutation in heritable PAH, however, suggests the involvement of second-hit elements in the pathogenesis of PAH. We have previously reported that treatment with endothelin-1 induced in vitro increased expression of noggin, an intrinsic bone morphogenetic protein antagonist, in human pulmonary artery smooth muscle cells (PA-SMCs). Moreover, chronic exposure to hypoxia is a well-known inducer of remodeling in pulmonary arteries. However, the potential link between chronic hypoxia exposure and noggin expression has not been elucidated. Aims: We hypothesized that hypoxia could induce, in PA-SMCs, the expression of endothelin-1 which could secondarily result in the upregulation of noggin. Methods and results: Cultured human PA-SMCs were treated for 3, 6, 24, and 48 h with the hypoxiamimetic agent, cobalt chloride (CoCl2; 100 μM) and gene expressions of preproendothelin-1 (ppET1), endothelin converting enzyme-1 (ECE1) and noggin were then evaluated by QRT-PCR. CoCl2 treatment progressively increased the expressions of ppET1 and noggin, with maximal response after 24 h and 48 h of stimulation respectively. Gene expression of ECE1 was not changed. After pretreatment or not with a non-selective endothelin receptor antagonist (bosentan), we stimulated PA-SMCs with CoCl2 for 5 h. Gene expression of noggin significantly increased after CoCl2 treatment and this reaction was not changed by pretreatment with bosentan. Conclusions: Noggin, an intrinsic bone morphogenetic protein antagonist, was upregulated by CoCl2, independent of hypoxia-induced endothelin-1 pathway at earlier timing (5 h).