Tamami Nakamura

Heat Shock Transcription Factor 1 Opens Chromatin Structure of Interleukin-6 Promoter to Facilitate Binding of an Activator or a Repressor

Heat shock transcription factor 1 (HSF1) not only regulates expression of heat shock genes in response to elevated temperature, but is also involved in developmental processes by regulating genes such as cytokine genes. However, we did not know how HSF1 regulates non-heat shock genes. Here, we show that constitutive HSF1 binding to the interleukin (IL)-6 promoter is necessary for its maximal induction by lipopolysaccharide (LPS) stimulation in mouse embryo fibroblasts and peritoneal macrophages. Lack of HSF1 inhibited LPS-induced in vivo binding of an activator NF-κB and a repressor ATF3 to IL-6 promoter. Neither NF-κB nor ATF3 binds to the IL-6 promoter in unstimulated HSF1-null cells even if they were overexpressed. Treatment with histone deacetylase inhibitor or a DNA methylation inhibitor restored LPS-induced IL-6 expression in HSF1-null cells, and histone modification enzymes were recruited on the IL-6 promoter in the presence of HSF1. Consistently, chromatin structure of the IL-6 promoter in the presence of HSF1 was more open than that in its absence. These results indicate that HSF1 partially opens the chromatin structure of the IL-6 promoter for an activator or a repressor to bind to it, and provides a novel mechanism of gene regulation by HSF1.

Heat Shock Transcription Factor 1 Inhibits Expression of IL-6 through Activating Transcription Factor 3

The febrile response is a complex physiological reaction to disease, including a cytokine-mediated increase in body temperature and the activation of inflammatory systems. Fever has beneficial roles in terms of disease prognosis, partly by suppressing the expression of inflammatory cytokines. However, the molecular mechanisms underlining the fever-mediated suppression of inflammatory gene expression have not been clarified. In this study, we showed that heat shock suppresses LPS-induced expression of IL-6, a major pyrogenic cytokine, in mouse embryonic fibroblasts and macrophages. Heat shock transcription factor 1 (HSF1) activated by heat shock induced the expression of activating transcription factor (ATF) 3, a negative regulator of IL-6, and ATF3 was necessary for heat-mediated suppression of IL-6, indicating a fever-mediated feedback loop consisting of HSF1 and ATF3. A comprehensive analysis of inflammatory gene expression revealed that heat pretreatment suppresses LPS-induced expression of most genes (86%), in part (67%) via ATF3. When HSF1-null and ATF3-null mice were injected with LPS, they expressed much higher levels of IL-6 than wild-type mice, resulting in an exaggerated febrile response. These results demonstrate a novel inhibitory pathway for inflammatory cytokines.

Influence of aging on the quantity and quality of human cardiac stem cells.

Advanced age affects various tissue-specific stem cells and decreases their regenerative ability. We therefore examined whether aging affected the quantity and quality of cardiac stem cells using cells obtained from 26 patients of various ages (from 2 to 83 years old). We collected fresh right atria and cultured cardiosphere-derived cells (CDCs), which are a type of cardiac stem cell. Then we investigated growth rate, senescence, DNA damage, and the growth factor production of CDCs. All samples yielded a sufficient number of CDCs for experiments and the cellular growth rate was not obviously associated with age. The expression of senescence-associated b-galactosidase and the DNA damage marker, gH2AX, showed a slightly higher trend in CDCs from older patients (≥65 years). The expression of VEGF, HGF, IGF-1, SDF-1, and TGF-b varied among samples, and the expression of these beneficial factors did not decrease with age. An in vitro angiogenesis assay also showed that the angiogenic potency of CDCs was not impaired, even in those from older patients. Our data suggest that the impact of age on the quantity and quality of CDCs is quite limited. These findings have important clinical implications for autologous stem cell transplantation in elderly patients.

Increased plasma VEGF levels following ischemic preconditioning are associated with downregulation of miRNA-762 and miR-3072-5p

Ischemic preconditioning (IPC) has protective effects against ischemia-perfusion injury of organs. In the present study, we investigated the associated mechanisms after performing remote IPC (rIPC) of lower limbs by clamping abdominal aorta in mice. Subsequent experiments showed decreased damage and paralysis of lower limbs following spinal cord injury (SCI). Concomitantly, plasma vascular endothelial growth factor (VEGF) levels were increased 24 h after rIPC compared with those in sham-operated animals. In subsequent microRNA analyses, thirteen microRNAs were downregulated in exosomes 24 h after rIPC. Further studies of femoral CD34-positive bone marrow (BM) cells confirmed downregulation of these seven microRNAs 24 h after rIPC compared with those in sham-operated controls. Subsequent algorithm-based database searches suggested that two of the seven microRNAs bind to the 3′ UTR of VEGF mRNA, and following transfection into CD34-positive BM cells, anti-miR-762, and anti-miR-3072-5p inhibitors led to increased VEGF concentrations. The present data suggest that rIPC transiently increases plasma VEGF levels by downregulating miR-762 and miR-3072-5p in CD34-positive BM cells, leading to protection against organ ischemia.

Combinatorial Treatment with Apelin-13 Enhances the Therapeutic Efficacy of a Preconditioned Cell-Based Therapy for Peripheral Ischemia

Hypoxic pretreatment of peripheral blood mononuclear cells (PBMNCs) enhances therapeutic angiogenesis in ischemic tissues after cell transplantation. However, newly formed vessels generated using this approach are immature and insufficient for promoting functional recovery from severe ischemia. In this study, we examined whether apelin-13, a regulator of vessel maturation, could be an effective promoter of therapeutic angiogenesis, following severe limb ischemia. Combinatorial treatment of hypoxic preconditioned PBMNCs with apelin-13 resulted in increased blood perfusion and vascular reactivity in ischemic mouse hindlimbs compared with a monotherapy comprising each factor. Apelin-13 upregulated expression of PDGF-BB and TGF-β1 in hypoxic PBMNCs, as well as that of PDGFR-β in vascular smooth muscle cells (VSMCs). Proliferation and migration of VSMCs treated with apelin-13 was accelerated in the presence of PDGF-BB. Interestingly, expression of an apelin receptor, APJ, in PBMNC was increased under hypoxia but not under normoxia. In addition, an in vitro angiogenesis assay using a co-culture model comprising mouse thoracic aorta, hypoxic PBMNCs, and apelin-13 demonstrated that combinatorial treatment recruited mural cells to sprouted vessel outgrowths from the aortic ring, thereby promoting neovessel maturation. Thus, combinatorial injection of hypoxic PBMNCs and apelin-13 could be an effective therapeutic strategy for patients with severe ischemic diseases.

Treatment of refractory cutaneous ulcers with mixed sheets consisting of peripheral blood mononuclear cells and fibroblasts.

The purpose of this study was to confirm the therapeutic effects of mixed sheets consisting of peripheral blood mononuclear cells (PBMNCs) and fibroblasts on cutaneous skin ulcers. Vascular endothelial growth factor (VEGF) secretion in mixed cell sheets was much higher than in PBMNCs and fibroblasts. Concerning the mechanism, transforming growth factor beta 1 and platelet-derived growth factor BB secreted from PBMNCs enhanced VEGF production in fibroblasts. In wounds created on the backs of diabetic mice, the therapeutic effect of mixed cell sheets was similar to that of daily treatment with trafermin, a recombinant human basic fibroblast growth factor. Although abnormal granulation tissue and inflammatory cell infiltration were observed in trafermin-treated wounds, the transplantation of mixed cell sheets resulted in the natural anatomy of subcutaneous tissues. The expression patterns of identical wound-healing factors in wounds were different between mixed sheet-transfected and trafermin-treated animals. Because mixed cell sheets transplanted into full-thickness skin defects were eliminated in hosts by day 21 in syngeneic transplantation models, allogeneic transplantation was performed using mice with different genetic backgrounds. The wound-healing rates were similar between the mixed cell sheet and trafermin groups. Our data indicated that mixed cell sheets represent a promising therapeutic material for cutaneous ulcers.

Age-related increase in Wnt inhibitor causes a senescence-like phenotype in human cardiac stem cells

Aging of cardiac stem/progenitor cells (CSCs) impairs heart regeneration and leads to unsatisfactory outcomes of cell-based therapies. As the precise mechanisms underlying CSC aging remain unclear, the use of therapeutic strategies for elderly patients with heart failure is severely delayed. In this study, we used human cardiosphere-derived cells (CDCs), a subtype of CSC found in the postnatal heart, to identify secreted factor(s) associated with CSC aging. Human CDCs were isolated from heart failure patients of various ages (2-83 years old). Gene expression of key soluble factors was compared between CDCs derived from young and elderly patients. Among these factors, SFRP1, a gene encoding a Wnt antagonist, was significantly up-regulated in CDCs from elderly patients (≥65 years old). sFRP1 levels was increased significantly also in CDCs, whose senescent phenotype was induced by anti-cancer drug treatment. These results suggest the participation of sFRP1 in CSC aging. We show that the administration of recombinant sFRP1 induced cellular senescence in CDCs derived from young patients, as indicated by increased levels of markers such as p16, and a senescence-associated secretory phenotype. In addition, co-administration of recombinant sFRP1 could abrogate the accelerated CDC proliferation induced by Wnt3A. Taken together, our results suggest that canonical Wnt signaling and its antagonist, sFRP1, regulate proliferation of human CSCs. Furthermore, excess sFRP1 in elderly patients causes CSC aging.

Short- and long-term outcomes of intramyocardial implantation of autologous bone marrow-derived cells for the treatment of ischaemic heart disease

Objectives Ischaemic heart disease remains a major cause of death in Japan. We have implanted autologous bone marrow-derived cells locally into the ischaemic region as a therapy in addition to coronary artery bypass grafting since 1999. We describe the outcomes of our cell therapy for ischaemic heart disease. Methods Eleven patients underwent local implantation of bone marrow-derived cells into the ischaemic region during coronary artery bypass grafting. Clinical outcomes during the acute and chronic phases were recorded. Results In the acute phase, no adverse effects were observed. Left ventricular ejection fraction values were not significantly different before and after treatment. Seven of the 11 patients showed improved blood perfusion in the area of cell therapy 1 month after treatment. In the chronic phase, 5 of 11 patients exhibited improved regional blood flow 1 year after treatment. Overall survival at 1, 5 and 10 years was 100%, 83.3% and 83.3%, respectively. Freedom from major adverse cardiac and cerebrovascular events at 1, 5 and 10 years was 100%, 80.8% and 80.8%, respectively. Death from all causes or freedom from major adverse cardiac and cerebrovascular events at 1, 5 and 10 years was 100%, 64.6% and 64.6%, respectively. Conclusions Local implantation of bone marrow-derived cells in patients with ischaemic heart disease is safe and feasible. Cell therapy is a therapeutic option for otherwise untreatable ischaemic heart disease.

Rapidly progressing left atrial hemangiopericytoma.

Cardiac hemangiopericytoma is a rare soft tissue tumor. We describe a case of hemangiopericytoma in the left atrium, which was diagnosed as myxoma preoperatively. A 70-year-old woman was admitted with heart failure. An echocardiogram showed a large myxoma-like mass in the left atrium, herniating into the left ventricle; therefore, an emergency operation was performed. Histological examination revealed a malignant hemangiopericytoma. The patient’s postoperative course was uneventful, but she died due to a local recurrence 4 months after the operation.