Mitsuhiro Fukata

Chemotherapy-resistant human AML stem cells home to and engraft within the bone-marrow endosteal region

Acute myelogenous leukemia (AML) is the most common adult leukemia, characterized by the clonal expansion of immature myeloblasts initiating from rare leukemic stem (LS) cells. To understand the functional properties of human LS cells, we developed a primary human AML xenotransplantation model using newborn nonobese diabetic/severe combined immunodeficient/interleukin (NOD/SCID/IL)2rγnull mice carrying a complete null mutation of the cytokine γc upon the SCID background. Using this model, we demonstrated that LS cells exclusively recapitulate AML and retain self-renewal capacity in vivo. They home to and engraft within the osteoblast-rich area of the bone marrow, where AML cells are protected from chemotherapy-induced apoptosis. Quiescence of human LS cells may be a mechanism underlying resistance to cell cycle–dependent cytotoxic therapy. Global transcriptional profiling identified LS cell–specific transcripts that are stable through serial transplantation. These results indicate the potential utility of this AML xenograft model in the development of novel therapeutic strategies targeted at LS cells.

Purified human hematopoietic stem cells contribute to the generation of cardiomyocytes through cell fusion

To obtain insights into the cardiomyogenic potential of hematopoietic tissue, we intravenously (i.v.) injected purified hematopoietic stem/progenitor cells into newborn recipients that may fully potentiate the developmental plasticity of stem cells. Transplantation of mouse bone marrow (BM) lineage antigen‐negative (Lin−) cells resulted in the generation of the cells that displayed cardiomyocyte‐specific antigenic profiles and contractile function when transplanted into syngeneic newborn recipients. To clarify the mechanism underlying the cardiomyogenic potential, green fluorescent protein (GFP)‐labeled BM Lin−ScaI+ hematopoietic progenitors were transplanted into neonatal mice constitutively expressing cyan fluorescence protein (CFP). Lambda image acquisition and linear unmixing analysis using confocal microscopy successfully separated GFP and CFP, and revealed that donor GFP+ cardiomyocytes coexpressed host‐derived CFP. We further reconstituted human hemopoietic‐ and immune systems in mice by injecting human cord blood (CB)‐derived Lin−CD34+CD38− hematopoietic stem cells (HSCs) into neonatal T cell−B cell−NK cell− immune‐deficient NOD/SCID/IL2rγnull mice. Fluoroescence in situ hybridization analysis of recipient cardiac tissues demonstrated that human and murine chromosomes were colocalized in the same cardiomyocytes, indicating that cell fusion occurred between human hematopoietic progeny and mouse cardiomyocytes. These syngeneic‐ and xenogeneic neonatal transplantations provide compelling evidence that hematopoietic stem/progenitor cells contribute to the postnatal generation of cardiomyocytes through cell fusion, not through transdifferentiation.— Ishikawa, F., Shimazu, H., Shultz, L. D., Fukata, M., Nakamura, R., Lyons, B., Shimoda, K., Shimoda, S., Kanemaru, T., Nakamura, K‐i., Ito, H., Kaji, Y., Perry, A. C. F., Harada, M. Purified human hematopoietic stem cells contribute to the generation of cardiomyocytes through cell fusion. FASEB J. 20, E11–E17 (2006)

Contribution of bone marrow-derived hematopoietic stem/progenitor cells to the generation of donor-marker⁺ cardiomyocytes in vivo.

Background Definite identification of the cell types and the mechanism relevant to cardiomyogenesis is essential for effective cardiac regenerative medicine. We aimed to identify the cell populations that can generate cardiomyocytes and to clarify whether generation of donor-marker+ cardiomyocytes requires cell fusion between BM-derived cells and recipient cardiomyocytes. Methodology/Principal Findings Purified BM stem/progenitor cells from green fluorescence protein (GFP) mice were transplanted into C57BL/6 mice or cyan fluorescence protein (CFP)-transgenic mice. Purified human hematopoietic stem cells (HSCs) from cord blood were transplanted into immune-compromised NOD/SCID/IL2rγnull mice. GFP+ cells in the cardiac tissue were analyzed for the antigenecity of a cardiomyocyte by confocal microscopy following immunofluorescence staining. GFP+ donor-derived cells, GFP+CFP+ fused cells, and CFP+ recipient-derived cells were distinguished by linear unmixing analysis. Hearts of xenogeneic recipients were evaluated for the expression of human cardiomyocyte genes by real-time quantitative polymerase chain reaction. In C57BL/6 recipients, Lin−/lowCD45+ hematopoietic cells generated greater number of GFP+ cardiomyocytes than Lin−/lowCD45− mesenchymal cells (37.0+/−23.9 vs 0.00+/−0.00 GFP+ cardiomyocytes per a recipient, P = 0.0095). The number of transplanted purified HSCs (Lin−/lowSca-1+ or Lin−Sca-1+c-Kit+ or CD34−Lin−Sca-1+c-Kit+) showed correlation to the number of GFP+ cardiomyocytes (P<0.05 in each cell fraction), and the incidence of GFP+ cardiomyocytes per injected cell dose was greatest in CD34−Lin−Sca-1+c-Kit+ recipients. Of the hematopoietic progenitors, total myeloid progenitors generated greater number of GFP+ cardiomyocytes than common lymphoid progenitors (12.8+/−10.7 vs 0.67+/−1.00 GFP+ cardiomyocytes per a recipient, P = 0.0021). In CFP recipients, all GFP+ cardiomyocytes examined coexpressed CFP. Human troponin C and myosin heavy chain 6 transcripts were detected in the cardiac tissue of some of the xenogeneic recipients. Conclusions/Significance Our results indicate that HSCs resulted in the generation of cardiomyocytes via myeloid intermediates by fusion-dependent mechanism. The use of myeloid derivatives as donor cells could potentially allow more effective cell-based therapy for cardiac repair.

Relationship between atrial fibrillation and gastroesophageal reflux disease: a multicenter questionnaire survey.

Objectives: The relationship between atrial fibrillation (AF) and gastroesophageal reflux disease (GERD) remains controversial, and investigations into this relationship have been based on small series. This multicenter survey evaluated the relationship between these diseases. Methods: The study enrolled 188 consecutive subjects (110 males and 78 females, mean age 60.4 ± 0.9 years) treated as outpatients. Patients were classified by the frequency scale for symptoms of GERD (F-scale) after obtaining informed consent for screening for GERD. Scores on this questionnaire were correlated to baseline characteristics obtained from medical records. The cutoff value for a diagnosis of GERD was set at 8.0 points. Results: Total scores on the F-scale were significantly greater in female subjects (p = 0.004) and in patients with AF (p = 0.019) compared to the other subjects. Univariate and multivariate analysis of the prevalence of GERD demonstrated that GERD was not related to gender, hypertension, dyslipidemia or coronary artery disease and that AF alone showed a significant (p < 0.001) correlation with GERD. Conclusions: This multicenter questionnaire survey demonstrated that among traditional cardiovascular risk factors, AF was an independent risk factor for GERD. A large cohort study to assess the potential relationship between GERD and AF is warranted.

Identification of resident and inflammatory bone marrow derived cells in the sclera by bone marrow and haematopoietic stem cell transplantation

Aims: To characterise bone marrow derived cells in the sclera under normal and inflammatory conditions, we examined their differentiation after transplantation from two different sources, bone marrow and haematopoietic stem cells (HSC). Methods: Bone marrow and HSC from green fluorescent protein (GFP) transgenic mice were transplanted into irradiated wild-type mice. At 1 month after transplantation, mice were sacrificed and their sclera examined by histology, immunohistochemistry (CD11b, CD11c, CD45), and transmission and scanning electron microscopy. To investigate bone marrow derived cell recruitment under inflammatory conditions, experimental autoimmune uveitis (EAU) was induced in transplanted mice. Results: GFP positive cells were distributed in the entire sclera and comprised 22.4 (2.8)% (bone marrow) and 28.4 (10.9)% (HSC) of the total cells in the limbal zone and 18.1 (6.7)% (bone marrow) and 26.3 (3.4)% (HSC) in the peripapillary zone. Immunohistochemistry showed that GFP (+) CD11c (+), GFP (+) CD11b (+) cells migrated in the sclera after bone marrow and HSC transplantation. Transmission and scanning electron microscopy revealed antigen presenting cells among the scleral fibroblasts. In EAU mice, vast infiltration of GFP (+) cells developed into the sclera. Conclusion: We have provided direct and novel evidence for the migration of bone marrow and HSC cells into the sclera differentiating into macrophages and dendritic cells. Vast infiltration of bone marrow and HSC cells was found to be part of the inflammatory process in EAU.

Reduced Plasma Eicosapentaenoic Acid–Arachidonic Acid Ratio in Peripheral Artery Disease

A reduced ratio of plasma eicosapentaenoic acid–arachidonic acid (EPA-AA) is a newly recognized atherosclerotic risk factor. This ratio has not been fully investigated in peripheral artery disease (PAD). Seventy Japanese patients with atherosclerotic risk factors were enrolled and divided into 2 groups, those with PAD (group A: n = 38) and those without PAD (group B: n = 32). The EPA-AA ratio (P = .001) and ankle–brachial index (ABI: P < .001) in group A were significantly lower than those in group B. Univariate and multivariate analyses demonstrated that EPA-AA, ABI, and prescription of clopidogrel had significant correlation with PAD. Given the appropriate cutoff values, EPA-AA (odds ratio [OR] = 11.7, 95% confidence interval [CI] = 3.0-45.8; P < .001) and ABI (OR = 44.0, 95% CI = 5.4-358.5; P < .001) are factors independently associated with PAD. In conclusion, this study demonstrated that reduced plasma EPA/AA may underlie PAD at least in Japanese.

Influence of common cardiac drugs on gastroesophageal reflux disease: Multicenter questionnaire survey

BACKGROUND Although gastroesophageal reflux disease (GERD) causes noncardiac chest pain mimicking angina pectoris, systemic studies surveying the effects of common cardiac drugs on symptomatic GERD are rare. METHODS To investigate the drugrelated GERD, this multicenter trial enrolled 201 consecutive cardiac outpatients (69.7 ± 10.5 y) after obtaining written informed consent. They were assessed using the Frequency Scale for Symptoms of GERD (F-scale) to screen for GERD with a cut-off value of 8.0. Clinical background was obtained from medical records. Gastric medicine was empirically administered at the discretion of the attending physician. F-scale score and incidence of GERD were analyzed individually in relation to background and prescription. RESULTS The average F-scale score did not correlate with gender, age or underlying diseases. F-scale score was elevated significantly (p = 0.006) by administration of calcium channel blockers to the patients treated with gastric medicine, suggesting that calcium channel blockers exacerbate the possibly preexisting GERD. Incidence of GERD within 2 months after starting warfarin tended to be greater than that at other durations (p = 0.087). Patients showing a high score (≥ 8.0) suggestive of GERD showed a correlation with the combined administration of calcium channel blockers (OR = 3.19; 95% CI of 1.01 - 10.11; p = 0.049) and warfarin (OR = 3.05; 95% CI of 1.00 - 9.27; p = 0.049) in the best logistic model. CONCLUSION Although larger cohort is required, this survey demonstrates that the combination of calcium channel blockers and warfarin is an independent risk factor for GERD.

Anticoagulation Stability Depends on CHADS2 Score and Hepatorenal Function in Warfarin-treated Patients, Including Those with Atrial Fibrillation

Aim: Although warfarin remains important despite the widespread use of nonvitamin K antagonist oral anticoagulants (NOACs), to date, the reality of warfarin use in the “NOACs era” is unclear. This multicenter observational study aimed to clarify the key factors contributing to warfarin treatment stability. Methods: The practical use of warfarin, stability of warfarin therapy, and factors contributing to this stability were investigated in community-based hospitals through a real-world study. Clinical data were retrospectively extracted from the medical records of warfarin-treated Japanese patients (age, 71.3 ± 5.5 years) with atrial fibrillation (AF), prosthetic heart valve, or other concerns requiring anticoagulation. Treatment stability was considered as time in therapeutic range of international normalized ratio of prothrombin time (TTR: %). The factors contributing to TTR were investigated, including CHADS2 score components. Results: Mean CHADS2 score was highest (1.38 ± 0.88, p < 0.001), and most CHADS2 score components in addition to hepatorenal dysfunction were factors contributing to the low TTR in patients with AF (n = 176). The similarity was found in overall patients who were prescribed warfarin (n = 518). TTR decreased according to the CHADS2 score component accumulation. Gender, dose and prescription interval of warfarin, and co-administration of antiplatelet agents did not correlate with the low TTR. Conclusions: This retrospective study demonstrated that the CHADS2 score component accumulation and hepatorenal dysfunction are factors significantly contributing to the low TTR, which is indicative of poor warfarin treatment stability, in patients such as those with AF.

Increased Coupling Interval Variability – Mechanistic, Diagnostic and Prognostic Implication of Premature Ventricular Contractions and Underlying Heart Diseases –

constant. Therefore, CI variability indicates different mechanisms of PVC occurrence or modulation of PVC propagation. Three major mechanisms of tachyarrhythmia including PVC development are reentry, triggered activity and abnormal automaticity. In the case of ordered reentry, the impulse propagates around an anatomical obstacle. Altered conduction velocity within the circuit or switching exit causes variable CI (Figure B). In the case of functional reentry, there is no fixed obstacle. When the excitation travels within damaged tissue, an impulse with a short wavelength is fragmented, functionally blocked, pursuing the tail of relative refractory tissue. Therefore, the CI of PVC exiting from multiple reentrant circuits shows wide variation (Figure C). CI variability is also implicated in the case of nonreentrant mechanisms. Diseased myocardium with fibrosis, stretch or dilatation is associated with focal arrhythmogenic mechanisms. Triggered activity is observed in damaged tissue with less Ca2+ handling capacity. Intracellular Ca2+ overloading evokes delayed afterdepolarization at the end of a regular action potential, leading to triggered PVC. Inhomogeneous Ca2+ overloading causes focal PVC with different CI (Figure D). The same is true in the case of abnormal enhanced automaticity in depolarized tissue. Resting membrane potential is less negative in the diseased ventricle, where the Na current is inactivated, and the Ca current is counterbalanced by a hyperpolarizing background K current. Variable extent of the counterbalance causes abnormal automaticity and PVC with different CI. These focal arrhythmogenic mechanisms are likely based on the lack of electrotonic effect in damaged but viable myocardium.9 Intracellular Ca2+ overloading causes degradation of gap junctional proteins. Considering that myocardial cells are coupled to each other through gap junctions, which play a pivotal role in maintaining electrotonus and preventing membrane potential fluctuation, focal mechanisms may be related to gap junctional impairment characterized by the ischemic or failing heart.10 Contrary to the outstanding new findings, this article has a few limitations (ie, CI variability cannot be evaluated by routine clinical ambulatory monitoring, and the reproducibility of measurement of this unique parameter remains unanswered). Furthermore, the strict relationship between PVC morphology and CI variability was not tested in the study, in which the number of PVC morphologies showed marginal significance (P=0.051) as a determinant of SDNV in their multiple linear number of studies have investigated premature ventricular contractions (PVC) to reflect mortality of patients with heart failure (HF) caused by ischemic or dilated cardiomyopathy.1,2 The origin, morphology and frequency of the PVC are the main interest, with the coupling interval (CI) drawing less attention because it is believed to be constant under the stable basic cycle length.3 In the last century, Thanavaro et al4 reported the relationship between the CI and type of PVC (ie, repetitive PVC tends to occur at the optimal CI range in myocardial infarct patients). On the other hand, Lowery et al indicated that a variable CI leads to multiform and repetitive PVC.5 In this century, advances in ambulatory data analysis have clarified the CI dynamics as they relate to the following ventricular arrhythmia mechanism and underlying heart disease severity. However, the relationship between CI variability and fatal ventricular arrhythmia occurrence and prognosis are complicated (ie, Sosnowski et al6 demonstrated high CI variability, whereas Lerma et al7 concluded low CI variability as a new risk factor of fatal ventricular arrhythmias). Therefore, the role of CI variability in mortality is still in dispute.

Open-loop, clockwise QT-RR hysteresis immediately before the onset of torsades de pointes in type 2 long QT syndrome

Delay of QT interval adaptation to sudden heart rate change causes hysteresis in dynamic QT-RR relationship. We analyzed QT-RR plotting during and after exercise in a patient with genetically identified type 2 long QT syndrome before and after starting oral propranolol. Blunted QT shortening by exercise and augmented postexercise QT prolongation resulted in an open-loop, clockwise QT-RR hysteresis immediately before the onset of torsades de pointes before propranolol. However, this hysteresis was eliminated by propranolol. QT-RR analysis provided insight into the mechanisms of the onset of torsades de pointes at least in this case of type 2 long QT syndrome.