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.

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.

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.

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.

Automatic R-wave and impedance testing with the modern patient alert system to reduce inappropriate implantable cardioverter defibrillator shocks due to lead fracture.

A 62-year-old man was afflicted with implantable cardioverter defibrillator (ICD) shocks during sinus rhythm. Stored ICD data revealed that sensing of noise due to fracture of the ventricular lead triggered the delivery of shocks. Since the lead fracture developed suddenly, it is suggested that close, early attention should be paid to the potential of such events during follow-up of ICD leads.

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.

Serial electrocardiographic findings in women with Takotsubo cardiomyopathy.

This study aimed to clarify detailed and serial electrocardiographic findings in patients with Takotsubo cardiomyopathy from onset to recovery. Nine consecutive women aged 65 to 84 years (mean 74) with Takotsubo cardiomyopathy were investigated. Standard 12-lead electrocardiograms were recorded during hospitalization and ST-segment elevation and T-wave inversion were manually measured daily in each patient. All 9 patients had 4 phases found electrocardiographically. Phase 1 was characterized by ST-segment elevation immediately after onset. Subsequently, T-wave inversion was observed from days 1 to 3 (phase 2), then inverted T waves improved transiently from days 2 to 6 (phase 3). After this phase, giant inverted T waves with QT prolongation appeared and persisted > or =2 months until recovery (phase 4). Serum creatine kinase levels were increased only at onset. Left ventricular wall motion abnormalities evaluated using echocardiography improved gradually after phase 3 in all patients. Second T-wave inversions (phase 4) were significantly deeper than those of the first one (phase 2; p <0.05). In conclusion, 4 electrocardiographic phases in patients with Takotsubo cardiomyopathy were shown. This observation may be helpful to understand the pathophysiologic process of Takotsubo cardiomyopathy.