Youichi Katoh

Isolation of Bone Marrow Stromal Cell–Derived Smooth Muscle Cells by a Human SM22α Promoter In Vitro Differentiation of Putative Smooth Muscle Progenitor Cells of Bone Marrow

Background—Bone marrow stromal cells (BMSCs) have many characteristics of mesenchymal stem cells that can differentiate into smooth muscle cells (SMCs). However, there have been few studies closely following the cell development of smooth muscle lineage among BMSCs. Methods and Results—To investigate the possible existence of a cell population committed to the SMC lineage among bone marrow adhesion cells, we tried to detect and follow the in vitro differentiation of such a cell type by using a promoter-sorting method with a human SM22&agr; promoter (−480 bp)/green fluorescent protein (GFP) construct. The construct was transfected to adhesion cells that appeared 5 days after the seeding of mononuclear cells from bone marrow. GFP was first detectable 5 days after the transfection in a cell population [Ad(G) cells], which expressed PDGF-&bgr; but neither mature (calponin) nor immature (SMemb) SMC-specific proteins at that time. However, the cells were eventually grown into individual clones that expressed SMC-specific proteins (&agr;-smooth muscle actin, calponin, and SM-1), suggesting that Ad(G) cells have partly at least progenitor properties. Because early studies have reported that PDGF-&bgr; signaling plays pivotal roles in the differentiation of mesenchymal smooth muscle progenitor cells, Ad(G) cells might be putative mesenchymal smooth muscle progenitors expressing PDGF-&bgr;. Conclusions—We demonstrated the presence of a cell population fated to become SMCs and followed their differentiation into SMCs among BMSCs.

MEF2B IS A COMPONENT OF A SMOOTH MUSCLE-SPECIFIC COMPLEX THAT BINDS AN A/T-RICH ELEMENT IMPORTANT FOR SMOOTH MUSCLE MYOSIN HEAVY CHAIN GENE EXPRESSION

To understand smooth muscle-specific gene expression, we have focused our studies on the smooth muscle myosin heavy chain (SMHC) gene, a smooth muscle-specific marker. In this study, we demonstrate that the SMHC promoter region (−1594 to −1462 base pairs) containing the A/T-rich element can activate the heterologous thymidine kinase promoter in smooth muscle cells, but not in fibroblasts. Mutations of this A/T-rich element decreased SMHC promoter activity significantly. Both gel mobility shift assays and DNase I footprinting revealed that this region binds to specific protein complexes from smooth muscle nuclear extracts, whereas nuclear extracts from skeletal muscle and fibroblasts produced a different binding pattern. We also demonstrate that the protein complex obtained from smooth muscle nuclear extract reacts with MEF2B-specific antibody, but not with antibodies specific to MEF2A, MEF2C, or MEF2D, suggesting that only MEF2B protein binds to the A/T-rich element. Furthermore, MEF2B overexpression in smooth muscle cells up-regulated the SMHC promoter, suggesting that MEF2B is important for SMHC gene regulation. This is the first report demonstrating a role for MEF2 factors in smooth muscle-specific gene expression.

Growth and differentiation of smooth muscle cells during vascular development

Vascular smooth muscle cells have been the subject of intense study because of their importance in vascular disease process. Although significant progress has been made toward defining the various growth factors affecting the smooth muscle phenotype, very little is known about factors that promote smooth muscle differentiation. In the past few years, a number of key advances have been made in our understanding of skeletal muscle myogenesis, with the discovery of myogenic determination genes. In spite of these advances, the development and differentiation of smooth muscle cells remain vastly underexplored. This review is an attempt to understand the current status of the field and to highlight some of the recent progress made toward this goal.

Messenger RNA levels of guanine nucleotide-binding proteins are reduced in the ventricle of cardiomyopathic hamsters.

The expression of guanine nucleotide-binding protein (G-protein) genes (Gs alpha, Go alpha, Gi alpha 1, Gi alpha 2, and Gi alpha 3) was examined in the ventricle of cardiomyopathic Syrian hamsters of the Bio14.6 strain (10-35 weeks old). Northern blot analysis of total cellular RNA revealed that all G-protein genes except Gi alpha 1 were expressed in the ventricle of Syrian hamsters. Gs alpha and Gi alpha 2 genes were abundantly expressed. The expression levels of the Gs alpha and Gi alpha 2 messenger RNAs in Bio14.6 ventricles were lower than the levels in ventricles of the F1B hamster strain; the abundance of Go alpha and Gi alpha 3 messenger RNAs did not change markedly. Moreover, the messenger RNA levels of Gs alpha and Gi alpha 2 decreased as the stage of cardiomyopathy progressed. Since G-proteins are linked to adenylate cyclase, these alterations of G-protein messenger RNA levels may be related to reduced contractility of cardiomyopathic heart.

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.

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.

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.

Watching National Team Matches in World Cup Soccer 2014 on Television was Associated with Increasing Frequency of Premature Ventricular Contractions

Objective: Psychological triggers, such as emotional stress, increase the incidence of acute cardiovascular events. The association between soccer championships and risk of cardiovascular events remains controversial. A World Cup Soccer (WCS) match involving a national team might be a strong enough trigger to induce cardiovascular events. However, there are no reports of a multicenter study that has investigated the relationship between watching WCS and cardiac arrhythmia. Methods: We assessed 25 patients who were evaluated for ischemic changes and/or arrhythmia using 24-h Holter electrocardiography in four cardiology divisions during WCS 2014. The patients were divided into two groups: the watching (W) group consisted of 7 patients who watched WCS on live television and the Non-Watching (NW) group consisted of 18 patients who did not watch WCS. Heart rates, arrhythmia, and ischemic changes were evaluated. Results: There were no differences in the clinical characteristics, heart rates, premature atrial contraction frequencies, and ischemic changes between the two groups. Although there were no differences in total Premature Ventricular Contractions (PVCs), the frequency of PVCs during matches (61 ± 101 vs. 7 ± 8, P = 0.03) and 1 hour before matches (15 ± 17 vs. 3 ± 5, P = 0.01) were significantly higher in the W group than in the NW group. No sustained ventricular tachycardia or fibrillation was observed. Conclusions: A significant association between watching WCS and frequency of PVCs was observed in patients with/or suspected of having cardiovascular disease. Received Date: February 13, 2016 Accepted Date: March 28, 2016 Published Date: April 01, 2016 Citation: Shimada, K., et al. Watching National Team Matches in World Cup Soccer 2014 on Television was Associated with Increasing Frequency of Premature Ventricular Contractions. (2016) J Heart Cardiol 2(1): 17-21. DOI: 10.15436/2378-6914.16.022 Journal of Heart and Cardiology Open Access Review Article Copyrights:

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.