Involvement of cross-linked ribosomal protein S19 oligomers and C5a receptor in definitive erythropoiesis.

We performed a series of experiments under a working hypothesis that cross-linked oligomers of ribosomal protein S19 (RP S19) play an essential role in definitive erythropoiesis as a ligand of the C5a receptor of erythroblasts and macrophages. We found molecules functionally and immunologically indistinguishable from RP S19 oligomers in the extracellular fluid of porcine and guinea pig bone marrow. When an increased hematopoietic state was induced in guinea pigs by bloodletting, the bone marrow RP S19 oligomer concentration was concomitantly increased. However, when the RP S19 oligomers were immunologically neutralized or the C5a receptor was pharmacologically antagonized, hyper-erythropoiesis induced by bloodletting was prevented and the anemic state was retarded in guinea pigs. When the RP S19 oligomers were neutralized in mice after bloodletting, the reactive hyper proliferation of erythroblasts in the spleen was prevented. Proerythroblasts and erythroblasts prepared by bone marrow aspiration from healthy individuals were found to express significant levels of the C5a receptor and type 2 transglutaminase genes. Majority of erythroblasts in cord blood of healthy newborns bore the C5a receptor. Taken together, these results support our hypothesis.

Asialoerythropoietin exerts stronger angiogenic activity than erythropoietin via its binding affinity to tissue.

PURPOSE: Although erythropoietin (EPO) is known to express angiogenic and cardioprotective effects, it also induces hypertension, polycythemia, and platelet activation, which may cause serious adverse effects in patients with cardiovascular diseases. We compared the angiogenic effects of EPO and its nonerythropoietic derivative, asialo-EPO (AEPO). METHODS: Lower limb ischemia was induced in ICR and C57/BL mice. Mice were injected intramuscularly with 2 µg/kg of EPO derivatives for 6 or 7 days. To assess biological differences, the tissue affinity of both EPO derivatives was analyzed in vitro using heparin affinity column chromatography. Tissue affinity was also analyzed in vivo using an intramuscular pharmacokinetic study. RESULTS: The survival of ischemic legs was better in the AEPO group than that in the EPO group (5/13 = 38.5 % vs 1/13 = 7.7 %, p < 0.05), and an increase in regenerated vessels was observed in the AEPO group, but not in the EPO group in ICR mice. Vessel/muscle ratios in control, EPO, and AEPO groups were 0.50 ± 0.34, 0.61 ± 0.32, and 2.83 ± 1.13, respectively (p < 0.0001). On the other hand, regenerated vessels were observed in both EPO and AEPO groups (p < 0.001) in C57/BL mice. AEPO, but not EPO, expressed heparin affinity in vitro. Intramuscularly injected EPO gradually decreased in muscle tissue, while AEPO was maintained at 2.5 ng/muscle for 1 day after several hours of a rapid clearance phase in vivo. CONCLUSIONS: AEPO exerts stronger angiogenic effects than those of EPO presumably via its tissue affinity. Administration of AEPO is a promising option for the treatment of patients with critical limb ischemia.

Hypocellularity and insufficient expression of angiogenic factors in implanted autologous bone marrow in patients with chronic critical limb ischemia.

The aim of this study was to identify the clinical parameters of absolutely poor-prognosis patients with chronic critical limb ischemia (AP-CLI). Sixteen no-option CLI patients with arteriosclerosis obliterans: ASO (nine) and non-ASO patients (seven) treated with bone marrow-mononuclear cell implantation (BMI) were analyzed. There were three AP-CLI patients (all ASO). The mRNA expression of several angiogenic factors in the implanted cells was analyzed in comparison with normal donor bone marrow. To observe the response of bone marrow components to hypoxia, normal bone marrow cells were cultured for 24 h in 2.5% O(2), and mRNA expression of angiogenic factors were measured. AP-CLI patients exhibited extraordinary low bone marrow cellularity as well as the percentage of CD34-positive cells. Among angiogenic factors, only VEGF expression was maintained in response to HIF-1, while other factors such as HGF, Ang-1, PLGF, and SDF-1 decreased in the implanted bone marrow cells of the patients with CLI compared to normal bone marrow cells. HIF-1 and all of the five angiogenic factors increased in vitro in response to hypoxia. Thus it is highly likely that angiogenic factors except VEGF do not respond to chronic ischemia in bone marrow in vivo. An organ-protection system against tissue ischemia may be applied for acute hypoxia, but it may be insufficient for chronic ischemia.

Erythropoietin, but not asialoerythropoietin or carbamyl-erythropoietin, attenuates monocrotaline-induced pulmonary hypertension in rats.

Erythropoietin (EPO) has long been utilized for the treatment of renal anemia. The erythropoietin receptor (EPOR) is also expressed in the cardiovascular and central nervous systems in addition to an erythroid lineage, to provide an organoprotective role against several types of cellular stress. Pulmonary hypertension (PH) is a poor prognostic disease caused by primary and secondary pulmonary vascular injury. We observed the effects of EPO derivatives on monocrotaline-induced PH in rats on the supposition that EPO may protect small arteries from injury. Asialoerythropoietin (AEPO) lacks sialic acids in the termini of carbohydrate chains that results in rapid clearance from blood. Carbamyl-erythropoietin (CEPO) interacts with EPOR/ßc heterodimers, but not with EPOR homodimers expressed in erythroid cells. Monocrotaline-injected rats were treated with continuous intravenous injection of 2500 ng/kg/day of EPO, AEPO, or CEPO for 21 days, and lung histology, cardiac function, and mRNA expression in the lungs were examined. Wall thickening of small arteries in the lungs and PH were improved by administration of EPO, but not by its non-hematopoietic derivatives, AEPO, or CEPO. Erythropoietin administration increased mRNA expression of the anti-apoptotic molecule, Bcl-xL, and maintained expression of the CD31 antigen. We conclude that lungs may express EPOR homoreceptors, but not heteroreceptors. Adequate serum erythropoietin levels may be essential for pulmonary protective effects.

Establishment of culturing system for ex-vivo expansion of angiogenic immature erythroid cells, and its application for treatment of patients with chronic severe lower limb ischemia.

Angiogenesis therapy by bone marrow-mononuclear cell implantation (BMI) has been utilized. We found that erythroid cells played an essential role in angiogenesis by BMI. We then tried to establish a novel cell therapy by implantation of ex vivo expanded immature erythroblasts cultured from hematopoietic stem/precursor cells. Immature to mature erythroblasts were purified from human bone marrow, and mRNA expression were analyzed. Strongly expressed VEGF and PLGF in immature erythroid cells decreased according to erythroid maturation. To expand very immature erythroid cells, we established a two-step culturing system, i.e., bone marrow cells were cultured in the presence of Flt-3L, SCF and TPO for 7 days, and the cells were further cultured in the presence of SCF, IGF-I and EPO for an additional 7 days. The in vivo angiogenic effects of implantation of the ex vivo expanded cells were stronger than that of BMI in mouse limb ischemia model. Three patients with severe chronic lower limb ischemia accompanied by Burger's disease or collagen arteritis were enrolled in a pilot clinical trial of the novel cell therapy by transplantation of ex-vivo expanded immature erythroid cells. In the clinical trial, most clinical symptoms such as rest pain and skin ulcers improved in 4 weeks, and did not recur in the one-year follow-up. No adverse events were observed in any of the patients. Moreover this novel cell therapy required only a small amount of bone marrow collection. Further enrollment of patients with chronic severe lower limb ischemia is necessary to confirm the efficacy and safety of this novel cell therapy, and to estimate the necessary amount of bone marrow aspirate.

Locally measured USLE K factor expands sustainable agricultural land in Palau.

Palau is an island in the Micronesia region of the western Pacific Ocean. The island receives heavy rainfall and has steep slopes, so 92% of the land is categorized within the most erodible rank, with a T factor of 5. A recent study reported that the water infiltration rate is proportional to the root mass of the crop soil; therefore, we attempted to evaluate the performance of root mass for preventing soil erosion. We covered parts of the land, with a slope of 15.4° (13.4°-17.3°), with weed control fabric to prevent the growth of grass and roots, then removed the fabric, cultivated the land, planted sweet potatoes, and compared the amount of soil erosion with other areas. Surprisingly, there was no erosion at all in the test plots, although there were 24 rainfall events that caused erosion. For the parameters of the Universal Soil Loss Equation (USLE) equation used in the present study, only the K factor was not actually measured. This means the K factor was larger than the actual value. Land at low risk of soil erosion and suitable for agriculture can be found by measuring K factor locally, even if the area is categorized as unsuitable.

Methane emissions in triple rice cropping: patterns and a method for reduction.

The Mekong Delta paddies are known as hotspots of methane emission, but these emissions are not well studied. We analyzed methane emission patterns based on monitoring data from typical triple rice cropping paddies collected over 5 years. We found that the total emissions in a crop season doubled in the second crop, tripled in the third crop, and reset after the annual natural flood of the Mekong River. The emission peaks occurred around 0 to 3 weeks after starting irrigation, then gradually decreased. In general, the main source of emitted methane is rice-derived carbon by current-season photosynthates and the emission peaks at the rice heading stage. However, the contribution of the rice-derived carbon is negligible in the hotspot paddies because total emission is high. The increase in emission levels from the first to the third crop can be explained by the accumulation of rice residue from the preceding crops, especially rice straw incorporated into the soil. The reset of emission levels after the annual flood means that the rice straw is decomposed without methanogenesis in water with dissolved oxygen. Thus, the annual emission pattern shows that avoiding rice straw incorporating into soil and decomposing rice straw in paddy surface-water reduces methane emissions.

Evaluation of cropping method for perennial ratoon rice: Adaptation of SALIBU to triple-cropping in Vietnam.

Background: Generally, the yield of ratoon rice is at most 50% of the main crop. However, a cropping method "SALIBU" achieved more yield than the main crop and could be used for the cultivation of perennial cropping. Although the SALIBU method is implementing 10 additional management practices to conventional method, the effect of each management practice is unclear. Therefore, we aimed to evaluate the effect size and the robustness of each management practice. Methodology: We evaluated the effect size using an L 16 orthogonal array design pot experiment. For the robustness, we duplicated the experiment under both standard and checked whether the practice shows the same effect size. The bad conditions were low plant density, no fertilization, continuous flooding water management, and late harvesting. Results: The ratoon rice yield was proportional to the number of ratoon tillers used as in conventionally produced ratoon rice. Late cutting was most affected to the number of ratoon tillers. Importantly, this effect was reversed; the effect was positive under bad conditions, but was negative under standard conditions. Furthermore, late irrigation, a recommended management practice, had a robust negative effect on ratoon tillers and yield under both the conditions. Positive large effects were shown in the bad condition only. Discussion: Our results show that the SALIBU cropping method includes practices with unstable, negative, or minimal effects. The practices have unstable effects should be clarifying the interaction with the condition. The practices that have negative effects should exclude. Using practice on small effect size should depend on a cost-benefit analysis. Conclusions: SALIBU will be acceptable to the Mekong Delta triple cropping rice cultivation without the additional practice of original SALIBU cropping method. However, further work is needed to clarify the interaction between late cutting and the cultivation condition, and on the negative effect of late irrigation.

Electron transport in a pi-stacking molecular chain.

We have investigated the electronic structure and transport properties of a pi-stacking molecular chain which is covalently bonded to a H/Si(100) surface, using the first-principles density functional theory approach combined with Green's function method. The highest occupied molecular orbital (HOMO) dispersion is remarkably reduced, but remains noticeable ( approximately 0.1 eV), when a short pi-stacking styrene wire is cut from an infinitely long wire and sandwiched between metal electrodes. We find that the styrene chain's HOMO and lowest unoccupied molecular orbital (LUMO) states are not separated from Si, indicating that it does not work as a wire. By substituting -NO2 or -NH2 for the top -H of styrene, we are able to shift the position of the HOMO and LUMO with respect to the Fermi level. More importantly, we find that the HOMO of styrene-NH2 falls into the band gap of the substrate and is localized in the pi-stacking chain, which is what we need for a wire to be electrically separated from the substrate. The conductance of such an assembly is comparable to that of Au/benzene dithiolate/Au wire based on chemical bonding, and its tunability makes it a promising system for a molecular device.

Imatinib mesilate inhibits neointimal hyperplasia via growth inhibition of vascular smooth muscle cells in a rat model of balloon injury.

Restenosis is a major problem in percutaneous catheter intervention (PCI) for coronary artery stenosis in patients with acute myocardial infarction. Coronary restenosis arises from intimal hyperplasia, i.e., hyperplasia of the vascular smooth muscle cells (SMCs) caused by endothelial cell (EC) damage due to PCI. Drug eluting stent (DES), a novel stent coated with a cell-growth inhibitor, such as rapamycin, has been utilized to block SMC proliferation, but DES also blocks EC repair and thus requires the administration of anti-platelets for a long time to prevent thrombus formation after PCI. Moreover, insufficient prevention of platelet aggregation sometimes induces restenosis after PCI. One of the signal transduction inhibitors, imatinib mesilate, blocks tyrosine kinase activity of platelet-derived growth factor receptor (PDGFR), and therefore it may block the development of neointima through growth inhibition of SMCs without the obstructive effect on EC-repair. We therefore studied the effects of imatinib on neointimal hyperplasia in a balloon injury model of rat carotid arteries. Rats were orally administered with imatinib for 14 days after balloon injury, and sacrificed to analyze the neointimal formation. Intimal hyperplasia was inhibited by imatinib in a dose-dependent manner. Therefore imatinib presumably obstructed the growth of SMCs via interception on growth-signaling of PDGFR. The administration of imatinib after coronary stenting or the use of an imatinib-eluting stent may further reduce the risk of restenosis in patients.

Rice plants reduce methane emissions in high-emitting paddies.

Background: Rice is typically understood to enhance methane emissions from paddy fields. However, rice actually has two separate functions related to methane: i) emission enhancement, such as by providing emission pathways (aerenchyma) and methanogenetic substrates; and ii) emission suppression by providing oxygen pathways, which suppress methanogenesis or enhance methane oxidation. The overall role of rice is thus determined by the balance between its enhancing and suppressing functions. Although existing studies have suggested that rice enhances total methane emissions, we aimed to demonstrate that the balance between rice's emitting and suppressing functions changes according to overall methane emission levels, which have quite a large range (16|-500 kg methane ha -1 crop -1). Methods: Using PVC chambers, we compared methane emissions emitted by rice paddy fields with and without rice plants in rice fields in the Mekong Delta, Vietnam. Samples were analyzed by gas chromatograph. Results: We found high overall methane emission levels and our results indicated that rice in fact suppressed methane emissions under these conditions. Emission reductions increased with the growth of rice, up to 60% of emission rate at the maximum tillering stage, then decreased to 20% after the heading stage, and finally recovering back to 60%.  Discussion: It is known that methane is emitted by ebullition when the emission level is high, and methane emission reductions in rice-planted fields are thought to be due to oxidation and methanogenesis suppression. However, although many studies have found that the contribution of soil organic matter to methanogenesis is small, our results suggested that methanogenesis depended mainly on soil organic matter accumulated from past crops. The higher the methane emission level, the lower the contribution of rice-providing substrate. Conclusion: As a result, during the growing season, rice enhanced methane emissions in low-emission paddy fields but suppressed methane emissions in high-emission paddy fields.

Atrial fibrillation (AF) and complete right bundle branch block (RBBB) are independently associated with increased hemoglobin levels in apparently healthy subjects.

OBJECTIVE: The associations between atrial fibrillation (AF) or complete right bundle branch block (RBBB) and other laboratory tests have not been examined sufficiently in healthy populations. METHODS: These associations were examined with multivariable logistic regression analyses using data from 6,381 apparently healthy subjects including 46 subjects with AF and 100 subjects with RBBB. RESULTS: The mean age, body mass index (BMI), hemoglobin, the prevalence of male sex and a history of coronary heart disease (CHD) were significantly higher in subjects with AF or RBBB than without. The odds ratio (OR) (95% confidence interval (CI)) of AF was 1.62 (1.28-2.05) p<0.0001 for each 1 g/dL increment of hemoglobin and 1.07 (0.36-3.21) p=0.90 and 4.32 (1.29-14.43) p=0.017, respectively for the second and the third tertiles of hemoglobin compared with the first tertile after adjusting for sex, age, BMI, CHD, and other confounding covariates. The OR (95% CI) of RBBB was 1.53 (1.32-1.77) p<0.0001 for each 1 g/dL increment of hemoglobin and 2.53 (1.06-6.00) p=0.036, 3.10 (1.12-8.61) p=0.030, and 4.03 (0.91-17.82) p=0.066, respectively, for the second, third, and fourth quartiles of hemoglobin compared with the first quartile after adjusting for sex, age, BMI, CHD, and other confounding covariates. CONCLUSION: An increased blood levels of hemoglobin was independently associated with AF and RBBB after adjusting for sex, age, BMI, CHD, and other confounding covariates in apparently healthy subjects.

Multiple bilateral coronary-biventricular fistulas with elevated left ventricular end-diastolic pressure.

Arterioventricular fistulas are relatively rare abnormalities and the etiology may be congenital or traumatic. We report a case of a 51-year-old woman in which all three coronary arteries emptied into both ventricles via multiple small fistulas resulting in elevated left ventricular end-diastolic pressure.

Improved cardiac function after sirolimus-eluting stent placement in diabetic patients by pioglitazone: combination therapy with statin.

BACKGROUND: Peroxisome proliferator-activated receptor-gamma (PPAR-gamma) agonists are used as anti-diabetic drugs, and their pleiotrophic action has been reported to improve endothelial function leading to cardioprotective effects. In this study we evaluated the long-term effect of pioglitazone on cardiac function in diabetic patients after percutaneous coronary intervention (PCI) by drug-eluting stent (DES). METHODS AND RESULTS: We investigated 54 diabetic patients who received PCI using a sirolimus-eluting stent. We excluded cases of acute myocardial infarction. They were divided into two groups: Group C received only conventional therapy (n=26) and Group P received additionally pioglitazone 15 mg/day (n=28). The left ventricular ejection fraction (LVEF) was measured by left ventriculography and analyzed before and 8 months after PCI. In Group C, LVEF did not change significantly: 55.6% vs. 56.7%, before and after PCI respectively (p=0.58). However, pioglitazone significantly improved LVEF: 54.4% vs. 60.0% (p=0.014). Multiple linear regression analysis showed that DeltaLVEF was significantly related to pioglitazone therapy (p=0.037). In particular, the combination of pioglitazone and statin improved LVEF (DeltaLVEF 9.6% with vs. 2.2% without statin). CONCLUSIONS: Pioglitazone improved cardiac function after PCI using SES in diabetic patients, especially in combination with a statin.

Serum erythropoietin level as a marker of limb ischemia.

Bone marrow implantation (BMI) has been utilized for the treatment of limb ischemia, however, serum markers have not yet been reported to express the degree of limb ischemia. We analyzed the serum levels of several cytokines including erythropoietin (EPO) in the treated legs and the contralateral ones in 11 patients with limb ischemia treated with BMI. The EPO level in the pre-treated legs in the 5 patients with arteriosclerosis obliterans revealed a good correlation with ankle-brachial pressure index. The EPO level, but not the levels of TNF-alpha, VEGF, and bFGF in the pre-treated legs was significantly higher than that in the contralateral legs in the 11 patients, and the EPO level decreased in 4 weeks after BMI. The serum EPO level may express the degree of limb ischemia presumably through the reactive production of EPO in ischemic tissue.

[Prognostic factors for critical limb ischemia after autologous bone marrow implantation].

OBJECTIVES: Autologous bone marrow implantation (BMI) is effective to treat critical limb ischemia, but the long-term prognosis is not clear. The outcome of BMI treatment for ischemic legs was investigated related to the clinical background of the patient, and short-term effects of BMI. The end event was defined as unexpected lower limb amputation. METHODS AND RESULTS: This study included 21 consecutive patients (mean age 60.0 +/- 13.6 years) with peripheral arterial disease who underwent BMI between December 2001 and March 2005. Twelve patients had arteriosclerosis obliterans (ASO), 5 had Buerger disease (thromboangiitis obliterans), 3 had thromboembolism, and 1 had hypereosinophilic syndrome. The patients with ASO had severe complications such as diabetes and hyperlipidemia. The total number of transplanted CD34-positive cells, ankle-brachial pressure index (ABI), and tissue oxygen pressure (TcO2) were lower in ASO patients than non-ASO patients. Significant risk factors for the event were diagnosis of ASO and low TcO2 (< 30 mmHg) according to the Kaplan-Meier survival curve and log rank test. All 6 patients who required limb amputation had ASO simultaneously with low TcO2 (6 of 9, 67%). In contrast, there was no correlation between the end event and short-term effect of BMI such as improvements in ABI and TcO2. CONCLUSIONS: Treatment with BMI could not save legs in some patients with ASO associated with severe leg ischemia.