Seigo Itoh

Increased extracellular and intracellular Ca²⁺ lead to adipocyte accumulation in bone marrow stromal cells by different mechanisms.

Mesenchymal stem cells found in bone marrow stromal cells (BMSCs) are the common progenitors for both adipocyte and osteoblast. An increase in marrow adipogenesis is associated with age-related osteopenia and anemia. Both extracellular and intracellular Ca(2+) ([Ca(2+)]o and [Ca(2+)]i) are versatile signaling molecules that are involved in the regulation of cell functions, including proliferation and differentiation. We have recently reported that upon treatment of BMSCs with insulin and dexamethasone, both high [Ca(2+)]o and high [Ca(2+)]i enhanced adipocyte accumulation, which suggested that increases in [Ca(2+)]o caused by bone resorption may accelerate adipocyte accumulation in aging and diabetic patients. In this study, we used primary mouse BMSCs to investigate the mechanisms by which high [Ca(2+)]o and high [Ca(2+)]i may enhance adipocyte accumulation. In the process of adipocyte accumulation, two important keys are adipocyte differentiation and the proliferation of BMSCs, which have the potential to differentiate into adipocytes. Use of MTT assay and real-time RT-PCR revealed that high [Ca(2+)]i (ionomycin)-dependent adipocyte accumulation is caused by enhanced proliferation of BMSCs but not enhanced differentiation into adipocytes. Using fura-2 fluorescence-based approaches, we showed that high [Ca(2+)]o (addition of CaCl2) leads to increases in [Ca(2+)]i. Flow cytometric methods revealed that high [Ca(2+)]o suppressed the phosphorylation of ERK independently of intracellular Ca(2+). The inhibition of ERK by U0126 and PD0325901 enhanced the differentiation of BMSCs into adipocytes. These data suggest that increased extracellular Ca(2+) provides the differentiation of BMSCs into adipocytes by the suppression of ERK activity independently of increased intracellular Ca(2+), which results in BMSC proliferation.

The role of Notch signaling in diabetic endothelial progenitor cells dysfunction

AIMS To investigate the role of Notch signaling pathway in vasculogenic dysfunction of diabetic EPCs (DM-EPCs). METHODS The study was performed in mice and diabetes was induced with Streptozotocin. The functional consequences of Notch pathway modulation were studied by assessment of colony forming capacity (EPC colony forming assay), EPC differentiation capacity (% of definitive EPC-CFU (dEPC-CFU)), circulating EPCs (EPC culture assay) and migrated cells (migration assay); in the presence of Notch inhibitor (γ-secretase inhibitors (GSI)) compared to control. Notch pathway and VEGF involvement in DM- EPCs were assessed by gene expression (RT-qPCR). RESULTS DM demonstrated to increase Notch pathway expression in bone marrow (BM) EPCs followed by lower EPC-CFU number, EPCs differentiation capacity, number of circulating EPCs, migrated cells and VEGF expression compared to control (p<0.05). Inhibition of Notch pathway by GSI rescued vasculogenic dysfunction in DM-EPCs as represented by increase in EPC-CFU number, differentiation capacity and number of circulating EPCs (p<0.05). CONCLUSION Our findings indicate the involvement of Notch pathway in mediating DM-EPCs dysfunction including less number of EPC-CFU, circulating EPCs and migrated cell number compared to control. Further in vitro inhibition of Notch pathway by GSI rescued DM-EPC dysfunction. Therefore targeting Notch pathway in DM may provide a target to restore DM-EPC dysfunction.

Enhanced accumulation of adipocytes in bone marrow stromal cells in the presence of increased extracellular and intracellular [Ca2+]

The bone marrow stroma contains osteoblasts and adipocytes that have a common precursor: the pluripotent mesenchymal stem cell found in bone marrow stromal cells (BMSCs). Local bone marrow Ca(2+) levels can reach high concentrations due to bone resorption, which is one of the notable features of the bone marrow stroma. Here, we describe the effects of high [Ca(2+)](o) on the accumulation of adipocytes in the bone marrow stroma. Using primary mouse BMSCs, we evaluated the level of adipocyte accumulation by measuring Oil Red O staining and glycerol-3-phosphate dehydrogenase (GPDH) activity. High [Ca(2+)](o) enhanced the accumulation of adipocytes following treatment with both insulin and dexamethasone together but not in the absence of this treatment. This enhanced accumulation was the result of both the accelerated proliferation of BMSCs and their differentiation into adipocytes. Using the fura-2 method, we also showed that high [Ca(2+)](o) induces an increase in [Ca(2+)](i). An intracellular Ca(2+) chelator suppressed the enhancement in adipocyte accumulation due to increased [Ca(2+)](o) in BMSCs. These data suggest a new role for extracellular Ca(2+) in the bone marrow stroma: increased [Ca(2+)](o) induces an increase in [Ca(2+)](i) levels, which in turn enhances the accumulation of adipocytes under certain conditions.

Oxidative stress tolerance of early stage diabetic endothelial progenitor cell

Introduction One of the causes for poor vasculogenesis of diabetes mellitus (DM) is known to rise from the dysfunction of bone marrow-derived endothelial progenitor cells (BM EPCs). However, the origin of its cause is less understood. We aimed to investigate the effect of oxidative stress in early stage of diabetic BM-EPC and whether its vasculogenic dysfunction is caused by oxidative stress. Methods Bone marrow c-Kit+Sca-1+Lin− (BM-KSL) cells were sorted from control and streptozotocin-induced diabetic C57BL6J mice by flow cytometry. BM-KSLs were then assessed for vasculogenic potential (colony forming assay; EPC-CFA), accumulation of intracellular ROS (CM-H2DCFDA), carbonylated protein (ELISA), anti-oxidative enzymes expression (RT-qPCR) and catalase activity (Amplex Red). Results Compared to control, DM BM-KSL had significantly lower EPC-CFUs in both definitive EPC-CFU and total EPC-CFU (p < 0.05). Interestingly, the oxidative stress level of DM BM-KSL was comparable and was not significantly different to control followed by increased in anti-oxidative enzymes expression and catalase activity. Conclusions Primitive BM-EPCs showed vasculogenic dysfunction in early diabetes. However the oxidative stress is not denoted as the major initiating factor of its cause. Our results suggest that primitive BM-KSL cell has the ability to compensate oxidative stress levels in early diabetes by increasing the expression of anti-oxidative enzymes.

High extracellular Ca2+ enhances the adipocyte accumulation of bone marrow stromal cells through a decrease in cAMP

Bone marrow stromal cells (BMSCs) are common progenitors of both adipocytes and osteoblasts. We recently suggested that increased [Ca2+]o caused by bone resorption might accelerate adipocyte accumulation in response to treatment with both insulin and dexamethasone. In this study, we investigated the mechanism by which high [Ca2+]o enhances adipocyte accumulation. We used primary mouse BMSCs and evaluated the levels of adipocyte accumulation by measuring Oil Red O staining. CaSR agonists (both Ca2+ and Sr2+) enhanced the accumulation of adipocytes among BMSCs in response to treatment with both insulin and dexamethasone. We showed that high [Ca2+]o decreases the concentration of cAMP using ELISA. Real-time RT-PCR revealed that increasing the intracellular concentration of cAMP (both chemical inducer (1μM forskolin and 200nM IBMX) and a cAMP analog (10μM pCPT-cAMP)) suppressed the expression of PPARγ and C/EBPα. In addition, forskolin, IBMX, and pCPT-cAMP inhibited the enhancement in adipocyte accumulation under high [Ca2+]o in BMSCs. However, this inhibited effect was not observed in BMSCs that were cultured in a basal concentration of [Ca2+]o. We next observed that the accumulation of adipocytes in the of bone marrow of middle-aged mice (25-40 weeks old) is higher than that of young mice (6 weeks old) based on micro CT. ELISA results revealed that the concentration of cAMP in the bone marrow mononuclear cells of middle-aged mice is lower than that of young mice. These data suggest that increased [Ca2+]o caused by bone resorption might accelerate adipocyte accumulation through CaSR following a decrease in cAMP.

Bradyarrhythmias may induce central sleep apnea in a patient with obstructive sleep apnea

Abstract The relationship between central sleep apnea (CSA) and bradyarrhythmia remains unclear. We report the case of a 70-year-old man with severe obstructive sleep apnea and bradyarrhythmia due to sick sinus syndrome in whom concomitant CSA was alleviated after pacemaker implantation.

Bone Marrow-Derived Regenerated Smooth Muscle Cells Have Ion Channels and Properties Characteristic of Vascular Smooth Muscle Cells

Rationale: Numerous reports, including our own, have recently suggested the presence of putative smooth muscle progenitor cells in the bone marrow (BM) and those smooth muscle-like cells may be differentiated from BM stromal cells (BMSCs). However, few studies have addressed whether the differentiated cells also possess the functional properties of smooth muscle cells (SMCs). Contractility is the primary function of native vascular SMCs. Objective: The aim of this electrophysiological study was to characterize BM-derived SMCs using the patchclamp technique and Ca2+ imaging with fura-2. Methods and results: To investigate whether BM-derived SMCs exhibit functional vascular SMC properties, we measured Ca2+ and K+ currents in BM-derived SMCs using the whole-cell patch-clamp method. The cells showed L-type and T-type Ca2+ channel currents, Ca2+-activated K+ channel (KCa) currents, and delayed rectifier K+ channel (KV) currents. We also measured agonist-evoked [Ca2+]i transients in BM-derived SMCs using fura-2 imaging. Such [Ca2+] i transients were observed in response to the vascular SMC-specific agonists, bradykinin (10-6 M) and angiotensin II (10-7 M). Conclusions: BM-derived SMCs displayed contractile activity and expressed several ion channels critical for contractile behavior in a manner compatible with native vascular SMCs. BMSC-derived cells thus have the potential to differentiate into functional vascular SMCs, suggesting bone marrow stromal tissue as a useful source of cells for the treatment of injured arteries and to construct tissue-engineered grafts for adult arterial revascularization.

Pentraxin 3 Released from Neutrophils Increases Plasma Levels in Patients with Acute Coronary Syndrome

Background. Our recently developed ELISA system for the detection of human pentraxin 3 (PTX3) in plasma has demonstrated that plasma PTX3 levels are increased in patients with unstable angina pectoris. However, the origin of the PTX3 from the ruptured plaque or a systemic process and which cells release PTX3 remain unclear. Methods. Blood samples were taken using an aspiration catheter from the site of the ruptured plaque and from the aorta during acute coronary interventions in 118 patients with acute coronary syndrome. These samples were analyzed for PTX3, and brain natriuretic peptide (BNP) was used as a control. Aspirated thrombi from patients with acute myocardial infarction (AMI) (n = 32) were examined by histological staining. Results. Plasma PTX3 levels were higher in blood samples taken from the site of plaque rupture compared to samples taken from the aorta (5.61±1.91 ng/mL versus 4.72 ± 5.61 ng/mL, 𝑃<0.05). On the other hand, BNP levels, as reference, were not different between the samples (P = 0.45). PTX3-positive neutrophils accounted for 70.4% of cells in harvested thrombi, with the remaining cells consisting of mononuclear cells. Conclusions. Infiltrating neutrophils in thrombi at the plaque rupture site are a diagnostically important source of PTX3 in patients with acute coronary syndrome.

Two Cases of Pilsicainide Intoxication showing the Brugada-type Electrocardiographic Findings and Incessant Wide QRS Tachycardia

We have experienced two patients with the Brugada‐type electrocardiographic abnormalities and incessant wide QRS tachycardia (presumed ventricular tachycardia) induced by intoxication of a class IC antiarrhythmic drug pilsicainide. They were elderly men with impaired renal function. Plasma concentration of pilsicainide was elevated to a toxic level in both patients. After cessation of pilsicainide, incessant wide QRS tachycardia spontaneously subsided and intraventricular conduction delay with coved type ST segment elevation in V1 and V2 disappeared. In the elderly or patients with renal dysfunction, we should be very careful regarding dose adjustment of pilsicainide or it may be better to avoid using this drug.