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The role of cells circulating in the peripheral blood to participate in the natural repair process of osteochondral defects was evaluated in a green fluorescent protein (GFP) transgenic and wild rat parabiosis model. Two weeks after the parabiosis operation, vascular communication between the conjoined rats was confirmed by flow-cytometry analysis. A 1.5 mm diameter and 1.0 mm depth osteochondral defect was made in the patellar groove of each rat femoral bone. Histological examination was performed at 1, 2, 4 and 24 weeks following surgery. In the early postoperative phase (1-4 weeks) there were GFP-negative and -positive cells in the defects of both parabiotic rats. GFP-positive chondrocytes were confirmed partly in the repair tissue of the wild parabiotic rat. In the late postoperative phase (24 weeks), the repaired defects were occupied by cells originating from the adjacent tissue and not from the peripheral blood. The ratio of cells originating from the peripheral blood was approximately 30-40% in the repair tissue at 1 week after surgery, reduced to 0-7% at 24 weeks. From these results it is confirmed that cells circulating in the peripheral blood contributed to the repair of the osteochondral defects, particularly in the early phase of healing. Thus, peripheral blood not only supplies the factors needed for repair but also provides a cell population involved in the wound-healing process. Copyright (c) 2012 John Wiley & Sons, Ltd.

Inflammation plays a crucial role in the pathophysiologicat characteristics of chronic kidney disease; however, the inflammatory mechanisms underlying the chronic kidney disease process remain unclear. Recent evidence indicates that sterile inflammation triggered by tissue injury is mediated through a muttiprotein complex called the inflammasome. Therefore, we investigated the role of the inflammasome in the development of chronic kidney disease using a murine unilateral ureteral obstruction (UUO) model. Inflammasome-related molecules were up-regulated in the kidney after UUO. Apoptosis-associated speck-like protein containing a caspase recruitment domain deficiency significantly reduced inflammatory responses, such as inflammatory cell infiltration and cytokine expression, and improved subsequent renal injury and fibrosis. Furthermore, apoptosis-associated speck-like protein containing a caspase recruitment domain was specifically up-regulated in collecting duct (CD) epithelial cells of the UUO-treated kidney. In vitro experiments showed that extracellular adenosine triphosphate (ATP) induced inflammasome activation in CD epithelial cells through P2X(7)-potassium efflux and reactive oxygen species dependent pathways. These results demonstrate the molecular basis for the inflammatory response in the process of chronic kidney disease and suggest the CD inflammasome as a potential therapeutic target for preventing chronic kidney disease progression.

Inflammation plays a key role in the pathophysiology of hepatic ischemia-reperfusion (I/R) injury. However, the mechanism by which hepatic I/R induces inflammatory responses remains unclear. Recent evidence indicates that a sterile inflammatory response triggered by I/R is mediated through a multiple-protein complex called the inflammasome. Therefore, we investigated the role of the inflammasome in hepatic I/R injury and found that hepatic I/R stimuli upregulated the inflammasome-component molecule, nucleotide-binding oligomerization domain-like receptor (NLR) family pyrin domain-containing 3 (NLRP3), but not apoptosis-associated speck-like protein containing a caspase recruitment domain (ASC). NLRP3(-/-) mice, but not ASC(-/-) and caspase-1(-/-) mice, had significantly less liver injury after hepatic I/R. NLRP3(-/-) mice showed reduced inflammatory responses, reactive oxygen species production, and apoptosis in I/R liver. Notably, infiltration of neutrophils, but not macrophages, was markedly inhibited in the I/R liver of NLRP3(-/-) mice. Bone marrow transplantation experiments showed that NLRP3 not only in bone marrow-derived cells, but also in non-bone marrow-derived cells contributed to liver injury after I/R. In vitro experiments revealed that keratinocyte-derived chemokine-induced activation of heterotrimeric G proteins was markedly diminished. Furthermore, NLRP3(-/-) neutrophils decreased keratinocyte-derived chemokine-induced concentrations of intracellular calcium elevation, Rac activation, and actin assembly formation, thereby resulting in impaired migration activity. Taken together, NLRP3 regulates chemokine-mediated functions and recruitment of neutrophils, and thereby contributes to hepatic I/R injury independently of inflammasomes. These findings identify a novel role of NLRP3 in the pathophysiology of hepatic I/R injury.

Mammalian tribbles homolog 1 (TRIB1) regulates hepatic lipogenesis and is genetically associated with plasma triglyceride (TG) levels and cholesterol, but the molecular mechanisms remain obscure. We explored these mechanisms in mouse livers transfected with a TRIB1 overexpression, a shRNA template or a control (LacZ) adenovirus vector. The overexpression of TRIB1 reduced, whereas induction of the shRNA template increased, plasma glucose, TG, and cholesterol and simultaneously hepatic TG and glycogen levels. The involvement of TRIB1 in hepatic lipid accumulation was supported by the findings of a human SNP association study. A TRIB1 SNP, rs6982502, was identified in an enhancer sequence, modulated enhancer activity in reporter gene assays, and was significantly (P = 9.39 x 10(-7)) associated with ultrasonographically diagnosed nonalcoholic fatty liver disease in a population of 5570 individuals. Transcriptome analyses of mouse livers revealed significant modulation of the gene sets involved in glycogenolysis and lipogenesis. Enforced TRIB1 expression abolished CCAAT/enhancer binding protein A (CEBPA), CEBPB, and MLXIPL proteins, whereas knockdown increased the protein level. Levels of TRIB1 expression simultaneously affected MKK4 (MAP2K4), MEK1 (MAP2K1), and ERK1/2 (MAPK1/3) protein levels and the phosphorylation of JNK, but not of ERK1/2. Pull-down and mammalian two-hybrid analyses revealed novel molecular interaction between TRIB1 and a hepatic lipogenic master regulator, MLXIPL. Co-expression of TRIB1 and CEBPA or MLXIPL reduced their protein levels and proteasome inhibitors attenuated the reduction. These data suggested that the modulation of TRIB1 expression affects hepatic lipogenesis and glycogenesis through multiple molecular interactions.

Inflammasomes are multiple protein complexes that serve as molecular platforms to activate caspase-1 and regulate maturation of a potent proinflammatory cytokine, interleukin (IL)-1 beta, as well as proinflammatory cell death, pyroptosis. Although several types of inflammasomes have been reported so far, recent investigations indicate that the NLRP3 in-flammasome recognizes non-microbial danger signals and leads to sterile inflammatory responses in various disease conditions. Sterile inflammatory responses are also implicated in the development of myocardial infarction (MI). In particular, IL-1 beta is an early and prominent mediator of inflammatory responses in MI, suggesting the pathophysiologic role of NLRP3 inflammasomes in MI. This review highlights the current state of knowledge regarding the role of NLRP3 inflammasomes in MI.

Regeneration of alveolar bone is critical for the successful treatment of periodontal diseases. The periodontal ligament (PDL) has been widely investigated as a source of cells for the regeneration of periodontal tissues. In the present study where we attempted to develop an effective strategy for alveolar bone regeneration, we examined the osteogenic potential of side population (SP) cells, a stem cell-containing population that has been shown to be highly abundant in several kinds of tissues, in PDL cells. Isolated SP cells from the rat PDL exhibited a superior ability to differentiate into osteoblastic cells compared with non-SP (NSP) and unsorted PDL cells in vitro. The mRNA expressions of osteoblast markers and bone morphogenetic protein (BMP) 2 were significantly upregulated in SP cells and were further increased by osteogenic induction. To examine the bone-forming activity of SP cells in vivo, PDL SP cells isolated from green fluorescent protein (GFP)-transgenic rats were transplanted with hydroxyapatite (HA) disks into wild-type animals SP cells exhibited a high ability to induce the mineralized matrix compared with NSP and unsorted PDL cells. At 12 weeks after the implantation, some of the pores in the HA disks with SP cells were filled with mineralized matrices, which were positive for bone matrix proteins, such as osteopontin, bone sialoprotein, and osteocalcin. Furthermore, osteoblast- and osteocyte-like cells on and in the bone-like mineralized matrices were GFP positive, suggesting that the matrices were directly formed by the transplanted cells. These results suggest that PDL SP cells possess enhanced osteogenic potential and could be a potential source for cell-based regenerative therapy for alveolar bone.

Background: Hyaluronan (HA) is a primary component of the extracellular matrix of cells, and it is involved in the pathogenesis of atherosclerosis. The purpose of this study was to investigate the role of HA in neointimal formation after vascular injury and determine its tissue-specific role in vascular smooth muscle cells (VSMCs) by using a cre-lox conditional transgenic (cTg) strategy. Methods and Results: HA was found to be expressed in neointimal lesions in humans with atherosclerosis and after wire-mediated vascular injury in mice. Inhibition of HA synthesis using 4-methylumbelliferone markedly inhibited neointimal formation after injury. In vitro experiments revealed that low-molecular-weight HA (LMW-HA) induced VSMC activation, including migration, proliferation, and production of inflammatory cytokines, and reactive oxygen species (ROS). The migration and proliferation of VSMCs were mediated by the CD44/RhoA and CD44/ERK1/2 pathways, respectively. Because HA synthase 2 (HAS2) is predominantly expressed in injured arteries, we generated cTg mice that overexpress the murine HAS2 gene specifically in VSMCs (cHAS2/CreSM22α mice) and showed that HA overexpression markedly enhanced neointimal formation after cuff-mediated vascular injury. Further, HA-overexpressing VSMCs isolated from cHAS2/CreSM22α mice showed augmented migration, proliferation, and production of inflammatory cytokines and ROS. Conclusion: VSMC-derived HA promotes neointimal formation after vascular injury, and HA may be a potential therapeutic target for cardiovascular disease. © 2013 Kashima et al.

Dental pulp is involved in the formation of bone-like tissue in response to external stimuli. However, the origin of osteoblast-like cells constructing this tissue and the mechanism of their induction remain unknown. We therefore evaluated pulp mineralization induced by transplantation of a green fluorescent protein (GFP)-labeled tooth into a GFP-negative hypodermis of host rats. Five days after the transplantation, the upper pulp cavity became necrotic; however, cell-rich hard tissue was observed adjacent to dentin at the root apex. At 10 days, woven bone-like tissue was formed apart from the dentin in the upper pulp. After 20 days, these hard tissues expanded and became histologically similar to bone. GFP immunoreactivity was detected in the hard tissue-forming cells within the root apex as well as in the upper pulp. Furthermore, immunohistochemical observation of alpha-smooth muscle actin, a marker for undifferentiated cells, showed a positive reaction in cells surrounding this bone-like tissue within the upper pulp but not in those within the root apex. Immunoreactivities of Smad4, Runx2, and Osterix were detected in the hard tissue-forming cells within both areas. These results collectively suggest that the dental pulp contains various types of osteoblast progenitors and that these cells might thus induce bone-like tissue in severely injured pulp. (J Histochem Cytochem 60:861-873, 2012)

Objective: Recent investigations have suggested that the inflammasome plays a role in the development of vascular inflammation and atherosclerosis; however, its precise role remains controversial. We produced double-deficient mice for apolipoprotien E (Apoe) and caspase-1 (Casp1), a key component molecule of the inflammasome, and investigated the effect of caspase-1 deficiency on vascular inflammation and atherosclerosis. Methods and results: Atherosclerotic plaque areas in whole aortas and aortic root of Western diet (WD)-fed Apoe(-/-)Casp1(-/-) mice were significantly reduced compared to those in Apoe(-/-) mice. The amount of macrophages and vascular smooth muscle cells in the plaques was also reduced in Apoe(-/-)Casp1(-/-) mice. No significant differences in plasma lipid profiles and body weight change were observed between these mice. Expression of interleukin (IL)-1 beta in the plaques as well as plasma levels of IL-1 beta, IL-1 alpha, IL-6, CCL2, and TNF-alpha, in Apoe(-/-)Casp1(-/-) mice were lower than those in Apoe(-/-) mice. In vitro experiments showed that calcium phosphate crystals induced caspase-1 activation and secretion of IL-1 beta and IL-1 alpha, in macrophages. Conclusion: Our findings suggest that caspase-1 plays a critical role in vascular inflammation and atherosclerosis, and that modulation of caspase-1 could be a potential target for prevention and treatment of atherosclerosis. (c) 2012 Elsevier Inc. All rights reserved.

Background: Chemokines are involved in multiple aspects of pathogenesis and cellular trafficking in tumorigenesis. In this study, we report that the latest member of the C-X-C-type chemokines, CXCL17 (DMC/VCC-1), recruits immature myeloid-derived cells and enhances early tumor progression. Methodology/Principal Findings: CXCL17 was preferentially expressed in some aggressive types of gastrointestinal, breast, and lung cancer cells. CXCL17 expression did not impart NIH3T3 cells with oncogenic potential in vitro, but CXCL17-expressing NIH3T3 cells could form vasculature-rich tumors in immunodeficient mice. Our data showed that CXCL17-expressing tumor cells increased immature CD11b(+)Gr1(+) myeloid-derived cells at tumor sites in mice and promoted CD31(+) tumor angiogenesis. Extensive chemotactic assays proved that CXCL17-responding cells were CD11b(+)Gr1(high)F4/80(-) cells (similar to 90%) with a neutrophil-like morphology in vitro. Although CXCL17 expression could not increase the number of CD11b(+)Gr1(+) cells in tumor-burdened SCID mice or promote metastases of low metastatic colon cancer cells, the existence of CXCL17-responding myeloid-derived cells caused a striking enhancement of xenograft tumor formation. Conclusions/Significance: These results suggest that aberrant expression of CXCL17 in tumor cells recruits immature myeloid-derived cells and promotes tumor progression through angiogenesis.

The cells of the subodontoblastic cell-rich layer in dental pulp are speculated to contain odontoblast progenitor cells because of their positional relationship with odontoblasts as well as their high alkaline phosphatase (ALP) activity. However, it has yet to be determined whether these cells have the ability to differentiate into odontoblastic cells. In the present study, we firstly found that the majority of cells in the subodontoblastic layer expressed Thy-1, a cell-surface marker of stem and progenitor cells. Then, we evaluated the capacity of Thy-1 high- and low-expressing (Thy-1(high) and Thy-1(low)) cells separated from rat dental pulp cells by use of a fluorescence-activated cell sorter to differentiate into hard tissue-forming cells in vitro and in vivo. Following stimulation with bone morphogenetic protein-2, Thy-1(high) cells in vitro showed accelerated induction of ALP activity and formation of alizarin red-positive mineralized matrix compared with Thy-1(low) cells. Furthermore, subcutaneous implantation of Thy-1(high) cells efficiently induced the formation of bone-like matrix. These results collectively suggest that Thy-1-positive dental pulp cells localized in the subodontoblastic layer had the ability to differentiate into hard tissue-forming cells, and thus these cells may serve as a source of odontoblastic cells.

Background. It has been well-recognized that cancer patients occasionally develop renal disorders independently of direct tumor invasion. However, the clinical entity of paraneoplastic glomerulopathy remains poorly understood, in part due to the lack of an animal model for basic research. In the present study, we investigated whether cancer-bearing rats develop features of glomerulopathy. Methods. RCN-9 rat colon cancer cells (1 x 10(7)) were injected into F344 rats (n = 13) and T cell-deficient F344 rats (nude rats; n = 7) via the portal system. Urinalysis and histological examinations were performed in comparison with control rats (n = 6) that received a vehicle injection. Results. Metastatic growth of RCN-9 cells exclusively in the liver was observed in the cancer-injected F344 rats, whereas direct invasion into the kidney was not evident even microscopically. Two of the cancer-injected F344 rats died within 2 days, but 9 of the 11 that avoided early death showed elevation of urinary protein (up to 158.0 mg/day) compared to controls (mean values: 60.8 +/- 12.9 versus 17.8 +/- 2.1 mg/day, P = 0.0291). Although morphological changes were not evident in light microscopy, abundant IgG in the glomerular tufts of the proteinuric rats was shown immunohistochemically. Ultrastructure analysis revealed electron-dense deposits in the glomerular basement membrane zone and effacement of the podocytic foot process. Interestingly, none of the nude rats showed proteinuria despite of cancer growth, suggesting that a specific immune response was involved. Conclusions. The tumor-bearing rats developed features of glomerulopathy, as expected from the clinical perspective, and this animal model may provide new insights into the development of paraneoplastic glomerulopathies.

There has been little information about metastatic behavior of renal cell carcinoma (RCC) cells because human cancers metastasize only rarely in immunodeficient mice. Moreover, it is difficult to know the effect of host immunity on RCC metastasis due to lack of such RCC cells as transplantable in not only xenograft models but also counterparts with intact immunity. Therefore, we scrutinized in vivo metastasis of RCC cells to seek for the optimal preclinical model to study metastatic behavior. The luciferase-expressing three representative human RCC cell lines (Caki-1, A498, and 786-O) and rat ACI-RCC cell which were established in our laboratory were transplanted into nonobese diabetic/severe combined immunodeficient (NOD/SCID) mice or immunocompetent ACI rats by intracardiac injection as well as orthotopic inoculation. Metastasis was monitored using a bioluminescent imaging technique. Metastasis was rare in the three human RCC cells even when they were directly disseminated into systemic circulation under the condition least susceptible to host immune attack in NOD/SCID mice. In contrast, ACI-RCC cells spontaneously metastasized to pulmonary tissue from orthotopic tumor sites and systemically spread via intracardiac route. Metastases were more extensive when the cells were inoculated into an immunodeficient host, implying suppressive effect of host immunity on colonization of RCC cells. These results suggest that the representative human RCC cells are not adequate resource to study metastasis but that the luciferase-labeled ACI-RCC cell characterized by its luminescent stability, enhanced tumorigenicity, and widespread metastatic potential provides a useful in vivo model for preclinical assessment of cancer progression and potential therapies against RCC.

Bone marrow cell (BMC)-derived myofibroblast-like cells have been reported in various organs, including the pancreas. However, the contribution of these cells to pancreatic fibrosis has not been fully discussed. The present study examined the possible involvement of pancreatic stellate cells (PSCs) originating from BMCs in the development of pancreatic fibrosis in a clinically relevant rat model of acute pancreatitis induced by a choline-deficient/ethionine-supplemented (CDE) diet. BMCs from female transgenic mice ubiquitously expressing green fluorescent protein (GFP) were transplanted into lethally irradiated male rats. Once chimerism was established, acute pancreatitis was induced by a CDE diet. Chronological changes in the number of PSCs originating from the donor BMCs were examined using double immunofluorescence for GFP and markers for PSCs, such as desmin and alpha smooth muscle actin (alpha SMA), 1, 3 and 8 weeks after the initiation of CDE feeding. We also used immunohistochemical staining to evaluate whether the PSCs from the BMCs produce growth factors, such as platelet-derived growth factor (PDGF) and transforming growth factor (TGF) beta 1. The percentage of BMC-derived activated PSCs increased significantly, peaking after 1 week of CDE treatment (accounting for 23.3 +/- 0.9% of the total population of activated PSCs) and then decreasing. These cells produced both PDGF and TGF beta 1 during the early stage of pancreatic fibrosis. Our results suggest that PSCs originating from BMCs contribute mainly to the early stage of pancreatic injury, at least in part, by producing growth factors in a rat CDE diet-induced pancreatitis model. (C) 2012 Elsevier Inc. All rights reserved.

Objective: Several reports describe the role of interleukin (IL)-17 in the development of atherosclerosis; however, its precise role remains controversial. We generated double-deficient mice for apolipoprotein E (apoE) and IL-17 (apoE(-/-)IL-17(-/-) mice) and investigated the effect of IL-17 deficiency on vascular inflammation and atherosclerosis. Methods and results: Atherosclerotic plaque areas in apoE(-/-)IL-17(-/-) mice fed a Western diet (WD) were significantly reduced compared with those in apoE(-/-) mice. No significant differences in plasma lipid profiles were observed between apoE(-/-) and apoE(-/-)IL-17(-/-) mice. The number of infiltrated macrophages in the plaques was significantly decreased in WD-fed apoE(-/-)IL-17(-/-) mice compared with WD-fed apoE(-/-) mice, whereas vascular smooth muscle cell content was not altered by IL-17 deficiency. Expression of inflammatory cytokines (MCP-1, IL-1 beta, IL-6, IFN-gamma, and IL-12 p40) and scavenger receptors (Msr-1, Scarb1, and Olr1) in the plaques was inhibited in WD-fed apoE(-/-)IL-17(-/-) mice. Furthermore, expression of inducible nitric oxide (M1 marker) and arginase-1 (M2 marker) was inhibited in WD-fed apoE(-/-)IL-17(-/-) mice. Conclusion: Our results indicate that IL-17 deficiency reduces vascular inflammation and atherosclerosis and that modulation of IL-17 could be a potential target for prevention and treatment of atherosclerosis. (C) 2012 Elsevier Inc. All rights reserved.

Aims Anthracyclines have profound consequences on the structure and function of the heart, which over time cause a cardiomyopathy that leads to congestive heart failure. Early detection of subclinical left ventricular (LV) dysfunction following a low dose of anthracyclines may be a preventive strategy. The aim of this study was to determine torsion analysis using two-dimensional speckle-tracking imaging (STI), useful for detecting early anthracycline-mediated cardiotoxicity. Methods and results Conventional and Doppler echocardiography images were obtained from 25 patients (mean age 58 +/- 11 years) before chemotherapy and 1 and 3 months after treatment. The cumulative anthracycline doses were 98 +/- 59 and 170 +/- 87 g/m(2) at 1 and 3 months, respectively. After standard echocardiography, LV torsion and twisting velocity profiles from apical and basal short-axis images were analysed using STI. LV dimensions and ejection fraction did not change throughout follow-up. Although isovolumic relaxation time showed prolongation 3 months after chemotherapy, other Doppler indices did not show significant changes. However, significant deteriorations in torsion (P < 0.0001 by ANOVA), twisting rate (P < 0.0001 by ANOVA), and untwisting rate (P < 0.001 by ANOVA) were found 1 month after chemotherapy. A significant negative correlation was observed between cumulative anthracycline doses and torsion (r = 20.524, P < 0.0001). Conclusion LV torsion analysis could be a useful non-invasive approach for early detection of subclinical anthracycline cardiotoxicity.

The targeted delivery of anti-inflammatory agents has great therapeutic potential for treating restenosis following percutaneous coronary intervention. To develop a drug delivery system targeted to injured blood vessels, we examined whether N-acetylglucosamine (GlcNAc)-bearing polymer-coated liposomes (GlcNAc-Ls) are specifically taken up by vascular smooth muscle cells (VSMCs). Flow cytometric analysis revealed that GlcNAc-Ls were taken up by VSMCs in vitro. Furthermore, GlcNAc-Ls were intravenously administered to mice that had undergone wire-mediated vascular injury. GlcNAc-Ls markedly accumulated at the intramural site of the injured vessel walls but not at the contralateral (uninjured) vessel walls. These results demonstrated that GlcNAc-Ls can be specifically taken up by VSMCs both in vitro and in vivo. We propose a novel strategy of using GlcNAc-Ls that has potential for application in drug delivery targeted to injured blood vessels.

The mechanism of selective albuminuria in minimal change nephrotic syndrome, in which glomerular capillaries are diffusely covered by effaced podocyte foot processes with reduced slit diaphragms, is unknown. Podocyte injury is due, in part, to NADPH-induced oxidative stress. Here we studied mechanism of selective albuminuria in puromycin aminonucleoside (PAN) nephrotic rats, a model of minimal change nephrotic syndrome. In these rats, Evans Blue-labeled human albumin was taken up by podocytes and its urinary excretion markedly increased, with retained selectivity for albumin. Immunogold scanning electron micrographic images found increased human albumin in podocyte vesicles and on the apical membrane in nephrotic compared with control rats. Apocynin, an inhibitor of NADPH oxidase, decreased superoxide production in podocytes, and inhibited endocytosis and urinary albumin excretion. Real-time confocal microscopy found an initial delay in the appearance of Evans Blue-labeled human albumin in the tubular lumen, reflecting the time needed for transcellular transport. Immunoprecipitation analysis indicated that FcRn, a receptor for albumin transport, mediated podocyte albumin transport, and treatment with anti-FcRn antibody reduced proteinuria in these nephrotic rats. Thus, podocyte albumin transport was enhanced in PAN nephrotic rats by means of FcRn, which may explain the mechanism of selective proteinuria. This was blocked by apocynin, suggesting a new therapeutic approach.

Regenerative therapies have currently emerged as one of the most promising treatments for repair of the damaged heart. Recently, numerous researchers reported that isolated cell injection treatments can improve heart function in myocardial infarction models. However, significant cell loss due to primary hypoxia or cell wash-out and difficulty to control the location of the grafted cells remains problem. As an attempt to overcome these limitations, we have proposed cell sheet-based tissue engineering, which involves stacking confluently cultured cells (two-dimensional), cell sheets, to construct three-dimensional cell-dense tissues. Cell sheet transplantation has been able to recover damaged heart function. However, no detailed analysis for transplanted cell survival has been previously performed. The present study compared the survival of cardiac cell sheet transplantation to direct cell injection in a rat myocardial infarction model. Luciferase-expressing neonatal rat cardiac cells were harvested as cell sheets from temperature-responsive culture dishes. The transplantation of cell sheets was compared to the direct injection of isolated cells dissociated with trypsin-ethylenediaminetetraacetic acid. These grafts were transplanted to infarcted rat hearts and cardiac function was assessed by echocardiography at 2 and 4 weeks after transplantation. In vivo bioluminescence and histological analyses were performed to examine cell survival. Cell sheet transplantation consistently yielded greater cell survival than cell injection. Immunohistochemistry revealed that cardiac cell sheets existed over the infarcted area as an intact layer. In contrast, the injected cells were scattered with relatively few cardiomyocytes in the infarcted areas. Four weeks after transplantation, cardiac function was also significantly improved in the cell sheet transplantation group compared with the cell injection. Twenty-four hours after cell grafting, significantly greater numbers of mature capillaries were also observed in the cardiac cell sheet transplantation. Additionally, the numbers of apoptotic cells with deterioration of integrin-mediated attachment were significantly lower after cardiac cell sheet transplantation. In accordance with increased cell survival, cardiac function was significantly improved after cardiac cell sheet transplantation in comparison to cell injection. Cell sheet transplantation can repair damaged hearts through improved cell survival and should become a promising therapy in cardiovascular regenerative medicine.

Aims Increasing evidence suggests that CD4(+) T cells contribute to neovascularization in ischaemic tissue. However, the T cell subset responsible for neovascularization after ischaemia remains to be determined. Here, we investigated the role of Th17 cells secreting interleukin (IL)-17, a newly identified subset of CD4(+) T cells, in the neovascularization after murine hindlimb ischaemia. Methods and results Unilateral hindlimb ischaemia was produced in wild-type (WT) C57BL/6 mice. Depletion of CD4(+) T cells resulted in significantly reduced blood flow perfusion in the ischaemic limbs. The expression of IL-17 and retinoic acid receptor-related orphan receptor gamma t (ROR gamma t) was up-regulated in the ischaemic limbs. IL-17-deficient mice showed a significant reduction in blood flow perfusion, inflammatory cell infiltration, and production of angiogenic cytokines in the ischaemic limbs compared with WT mice. In bone marrow transplantation experiments, the absence of IL-17 specifically in bone marrow cells diminished the neovascularization after ischaemia. Furthermore, IL-17-deficient CD4(+) T cells transferred into the ischaemic limbs of T cell-deficient athymic nude mice evoked a significantly limited neovascularization compared with WT CD4(+) T cells. Conclusion These findings identify Th17 cells as a new angiogenic T cell subset and provide new insight into the mechanism by which T cells promote neovascularization after ischaemia.

Triglyceride-rich lipoproteins (TGRLs) and low-density-lipoprotein (LDL) cholesterol are independent risk factors for coronary artery disease. We have previously proposed that the very low-density-lipoprotein (VLDL) receptor is one of the receptors required for foam cell formation by TGRLs in human macrophages. However, the VLDL receptor proteins have not been detected in atherosclerotic lesions of several animal models. Here we showed no VLDL receptor protein was detected in mouse macrophage cell lines (Raw264.7 and J774.2) or in mouse peritoneal macrophages in vitro. Furthermore, no VLDL receptor protein was detected in macrophages in atherosclerotic lesions of chow-fed apolipoprotein E-deficient or cholesterol-fed LDL receptor-deficient mice in vivo. In contrast, macrophage VLDL receptor protein was clearly detected in human macrophages in vitro and in atherosclerotic lesions in myocardial infarction-prone Watanabe-heritable hyperlipidemic (WHHLMI) rabbits in vivo. There are species differences in the localization of VLDL receptor protein in vitro and in vivo. Since VLDL receptor is expressed on macrophages in atheromatous plaques of both rabbit and human but not in mouse models, the mechanisms of atherogenesis and/or growth of atherosclerotic lesions in mouse models may be partly different from those of humans and rabbits. (C) 2011 Elsevier Inc. All rights reserved.

Background: Early diastolic velocity of the mitral annulus and transmitral flow propagation velocity are reported as more reliable determinants of left ventricular diastolic function in patients with atrial fibrillation than are transmitral Doppler indices. This study aimed to test the hypothesis that transmitral flow curve shows pseudorestrictive pattern during rate-controlled atrial fibrillation. Methods: Thirteen paroxysmal atrial fibrillation patients were monitored for three phases: before atrial fibrillation, during atrial fibrillation, and after the recovery of atrial fibrillation to sinus rhythm. Standard two-dimensional, color flow, and tissue Doppler echocardiography were performed. We compared the indices of left ventricular diastolic function among the three phases. Results: The early diastolic velocity of transmitral flow increased significantly during atrial fibrillation (before, 0.76 +/- 0.19 m/sec; during, 0.86 +/- 0.20 m/sec; after recovery to sinus rhythm, 0.73 +/- 0.16 m/sec; P < 0.01). The deceleration time of early transmitral diastolic wave decreased during atrial fibrillation (182.5 +/- 39.6 ms; 149.1 +/- 38.7 ms; 184.0 +/- 44.5 ms, respectively, P < 0.01). The early diastolic velocity of the mitral annulus increased during atrial fibrillation (5.37 +/- 1.31 cm/sec; 7.29 +/- 1.25 cm/sec; 5.37 +/- 1.32 cm/sec; respectively, P < 0.01). The transmitral propagation velocity did not change significantly during atrial fibrillation. Conclusion: Although conventional Doppler indices showed abnormal relaxation pattern, left ventricular diastolic function was preserved during rate-controlled atrial fibrillation, as determined from early diastolic velocity of the mitral annulus and transmitral flow propagation velocity. (Echocardiography 2011;28:289-297).

Inflammation plays a crucial role in the pathophysiology of myocardial infarction (MI). In particular, reperfusion caused by increased thrombolytic activity or revascularization therapy may restore the coronary blood flow and reduce the infarct size, but it also simultaneously enhances the inflammatory response and causes harmful effects on the myocardium a process termed ischemia-reperfusion (I/R) injury. The inflammasome is a large multiprotein complex that is formed in the cytosol in response to danger signals; it drives the proinflammatory cytokine interleukin(IL)-1 beta.Increasing evidence indicates that the inflammasome is a key player in the disease processes of sterile inflammation. In particular, IL-1 beta is a prominent and early mediator of inflammation in I/R injury, suggesting the importance of the inflammasome in myocardial l/R injury. This article reviews the role of the inflammasome in the development of myocardial I/R injury and discusses the potential of the inflammasome as a novel therapeutic target for the treatment of myocardial I/R injury. (Trends Cardiovasc Med 2011;21:37-41) (C) 2011 Elsevier Inc. All rights reserved.

Background-Inflammation plays a key role in the pathophysiology of myocardial ischemia/reperfusion (I/R) injury; however, the mechanism by which myocardial I/R induces inflammation remains unclear. Recent evidence indicates that a sterile inflammatory response triggered by tissue damage is mediated through a multiple-protein complex called the inflammasome. Therefore, we hypothesized that the inflammasome is an initial sensor for danger signal(s) in myocardial I/R injury. Methods and Results-We demonstrate that inflammasome activation in cardiac fibroblasts, but not in cardiomyocytes, is crucially involved in the initial inflammatory response after myocardial I/R injury. We found that inflammasomes are formed by I/R and that its subsequent activation of inflammasomes leads to interleukin-1 beta production, resulting in inflammatory responses such as inflammatory cell infiltration and cytokine expression in the heart. In mice deficient for apoptosis-associated speck-like adaptor protein and caspase-1, these inflammatory responses and subsequent injuries, including infarct development and myocardial fibrosis and dysfunction, were markedly diminished. Bone marrow transplantation experiments with apoptosis-associated speck-like adaptor protein-deficient mice revealed that inflammasome activation in bone marrow cells and myocardial resident cells such as cardiomyocytes or cardiac fibroblasts plays an important role in myocardial I/R injury. In vitro experiments revealed that hypoxia/reoxygenation stimulated inflammasome activation in cardiac fibroblasts, but not in cardiomyocytes, and that hypoxia/reoxygenation-induced activation was mediated through reactive oxygen species production and potassium efflux. Conclusions-Our results demonstrate the molecular basis for the initial inflammatory response after I/R and suggest that the inflammasome is a potential novel therapeutic target for preventing myocardial I/R injury. (Circulation. 2011;123:594-604.)

Background: Bone transplantation is an important procedure often used for bone defect reconstruction after trauma and malignancies. However, the kinetics of free bone graft-derived cells remains unclarified. The authors examined the kinetics of graft-derived cells using transgenic rats systemically expressing firefly luciferase. Methods: Free iliac bone grafts (5 x 5 x 2 mm, n = 10) derived from luciferase transgenic rats were transplanted into the subcutaneous space of the back of wild-type Lewis rats, and the kinetics of graft-derived cells were evaluated over time by determining the level of luminescence emission. Results: Although the luminescence level emitted by luciferase decreased after transplantation, a substantial luminescence level (mean, 1.6 x 10(7) photons/second) was emitted from donor-derived cells even at 180 days after transplantation, suggesting a long-term survival of graft-derived cells. In a computed tomographic image analysis of bone grafts retrieved 180 days after transplantation, high-luminescence grafts with a sufficient number of viable graft-derived cells (mean, 2.6 x 10(7) photons/second; n = 4) showed significantly higher bone graft volume (3.1-fold) and polar moment of inertia of area (7.2-fold) than low-luminescence grafts (mean, 1.0 x 10(7) photons/second; n = 4), indicating that high-luminescence grafts maintain better conditions. Conclusion: These results suggest that bone graft-derived cells can survive for a long time and that the presence of a sufficient number of viable graft-derived cells is essential for graft engraftment and remodeling. (Plast. Reconstr. Surg. 127: 78, 2011.)

Left internal mammary artery (LIMA) bypass conduits undergo gradual longitudinal flow transition from the proximal to distal segments, and the diastolic/systolic (D/S) ratios of the flow indices can diagnose graft patency. However, the influence of graft adaptation on this has not been studied. We examined 46 patients with LIMA graft to the left anterior descending artery using a Doppler-tipped guidewire in the proximal, middle, and distal segments; 34 had patent LIMAs (group A: new LIMAs; <1 month postoperatively; n = 22 and group B: old LIMAs; >= 1 month postoperatively; n = 12), and 12 had new LIMAs with distal stenosis (group C). In diastole, the time-averaged peak velocities, maximum peak velocities, and velocity time integrals in each segment were significantly greater in group A than in groups B or C; however, in systole, they did not differ significantly among the three groups. The D/S ratios of the indices in all segments in group A were significantly greater than those in groups B or C; however, they did not differ between groups B and C in any of the segments. Graft adaptation of a patent LIMA, itself, affects the longitudinal flow transition pattern. The D/S ratio of the three indices in the patent old LIMAs did not differ from those in the LIMAs with distal stenosis early after surgery. The timing of LIMA flow assessment must be considered during assessment of the graft patency from the flow velocity patterns.

Vimentin and desmin are intermediate filament proteins found in various mesenchymal and skeletal muscle cells, respectively. These proteins play an important role in the stabilization of the cytoplasmic architecture. Here, we found, using artificial biomimicking glycopolymers, that vimentin and desmin possess N-acetylglucosamine (GlcNAc)-binding lectin-like properties on the cell surfaces of various vimentin- and desmin-expressing cells such as cardiomyocytes and vascular smooth muscle cells. The rod II domain of these proteins was demonstrated to be localized to the cell surface and to directly bind to the artificial biomimicking GlcNAc-bearing polymer, by confocal laser microscopy and surface plasmon resonance analysis. These glycopolymers strongly interact with lectins and are useful tools for the analysis of lectin-carbohydrate interactions, since glycopolymers binding to lectins can induce the clustering of lectins due to multivalent glycoside ligand binding. Moreover, immunocytochemistry and pull-down assay with His-tagged vimentin-rod II domain protein showed that the vimentin-rod II domain interacts with O-GlcNAc proteins. These results suggest that O-GlcNAc proteins might be one candidate for physiological GlcNAc-bearing ligands with which vimentin and desmin interact. These findings demonstrate a novel function of vimentin and desmin that does not involve stabilization of the cytoplasmic architecture by which these proteins interact with physiological GlcNAc-bearing ligands such as O-GlcNAc proteins on the cell surface through their GlcNAc-binding lectin-like properties.

Myocardial infarction (MI) is accompanied by an inflammatory response, leading to the recruitment of leukocytes and subsequent myocardial injury and healing. Chemokines are potent chemoattractant cytokines that regulate leukocyte trafficking in inflammatory processes. Recent evidence indicates that chemokines play a role not only in leukocyte trafficking but also in angiogenesis and cardioprotection. In particular, stromal cell-derived factor-la (SDF-1 alpha) has generated considerable, interest for its role in the pathophysiology of MI. This review will focus on the role of SDF-1 and its receptor CXC chemokine receptor 4 (CXCR4; ie, the SDF-1/CXCR4 system) in the pathophysiology of MI and discuss their potential as therapeutic targets for MI. (Circ J 2010; 74: 418-423)

The purpose of this study was to determine the relationship between obstructive sleep apnea (OSA) and cardiovascular disorders in a large Japanese population, and to assess the efficacy of continuous positive airway pressure (CPAP) in the treatment of OSA-associated arrhythmias. The study population comprised 1394 Japanese subjects (1086 men and 308 women) who were divided into four groups on the basis of polysomnography (PSG) analysis as follows: the no sleep apnea (N-SA) group (n = 44, apnea-hypopnea index [AHI] < 5), the mild OSA (Mi-OSA) group (n = 197, 5 < AHI < 15), the moderate OSA (Mo) group (n = 368, 15 < AHI < 30), and severe OSA (SOSA) group (n = 785, AHI < 30). The following baseline characteristics were significantly associated with OSA: age (P < 0.001), gender (P < 0.001), body mass index (P < 0.001), hypertension (P < 0.001), diabetes (P = 0.009), and hyperlipidemia (P = 0.013). In the OSA group, PSG revealed the predominance of paroxysmal atrial fibrillation (PAF) (P = 0.051), premature atrial complex short run (P < 0.005), premature ventricular complex (PVC, P = 0.004), sinus bradycardia (P = 0.036), and sinus pause (arrest > 2 s, P < 0.001) during the PSG recording. A total of 316 patients from the group underwent CPAP titration and were then re-evaluated. Continuous positive airway pressure therapy significantly reduced the occurrences of PAF (P < 0.001), PVC (P = 0.016), sinus bradycardia (P = 0.001), and sinus pause (P = 0.004). The results of this study demonstrate a significant relationship between OSA and several cardiac disorders, and also demonstrate the efficacy of CPAP in preventing OSA-associated arrhythmias in a large population of Japanese patients.

Suzuki J, Ueno M, Uno M, Hirose Y, Zenimaru Y, Takahashi S, Osuga J, Ishibashi S, Takahashi M, Hirose M, Yamada M, Kraemer FB, Miyamori I. Effects of hormone-sensitive lipase disruption on cardiac energy metabolism in response to fasting and refeeding. Am J Physiol Endocrinol Metab 297: E1115-E1124, 2009. First published August 25, 2009; doi: 10.1152/ajpendo.91031.2008.-Increased fatty acid (FA) flux and intracellular lipid accumulation (steatosis) give rise to cardiac lipotoxicity in both pathological and physiological conditions. Since hormone-sensitive lipase (HSL) contributes to intracellular lipolysis in adipose tissue and heart, we investigated the impact of HSL disruption on cardiac energy metabolism in response to fasting and refeeding. HSL-knockout (KO) mice and wild-type (WT) littermates were fasted for 24 h, followed by similar to 6 h of refeeding. Plasma FA concentration in WT mice was elevated twofold with fasting, whereas KO mice lacked this elevation, resulting in twofold lower cardiac FA uptake compared with WT mice. Echocardiography showed that fractional shortening was 15% decreased during fasting in WT mice and was associated with steatosis, whereas both of these changes were absent in KO mice. Compared with Langendorff-perfused hearts isolated from fasted WT mice, the isolated KO hearts also displayed higher contractile function and a blunted response to FA. Although cardiac glucose uptake in KO mice was comparable with WT mice under all conditions tested, cardiac VLDL uptake and lipoprotein lipase (LPL) activity were twofold higher in KO mice during fasting. The KO hearts showed undetectable activity of neutral cholesteryl esterase and 40% lower non-LPL triglyceride lipase activity compared with WT hearts in refed conditions accompanied by overt steatosis, normal cardiac function, and increased mRNA expression of adipose differentiation-related protein. Thus, the dissociation between cardiac steatosis and functional sequelae observed in HSL-KO mice suggests that excess FA influx, rather than steatosis per se, appears to play an important role in the pathogenesis of cardiac lipotoxicity.

Vasculopathy in patients with connective tissue diseases (CTDs), including systemic sclerosis (SSc) and systemic lupus erythematosus (SLE), is a serious complication that mainly affects small arteries and capillaries, reduces the blood flow and causes progressive tissue ischemia. Recently, CTD patients have been reported to have abnormalities in circulating endothelial progenitor cells (EPCs); these abnormalities are believed to contribute to the pathophysiology of vasculopathy and to the premature and accelerated development of atherosclerosis in CTD patients. Furthermore, we are currently conducting a clinical pilot study to determine the efficacy of implanting autologous mononuclear cells obtained from the bone marrow and peripheral blood into the ischemic digits or limbs of CTD patients. In this review, we discuss the role of EPCs in the process of neovascularization and in the pathophysiology of CTDs, and we describe a clinical pilot study on the use of autologous cell therapy for treating ischemic digits in patients with CTDs.

We previously created the Alb-DsRed2 transgenic (Tg) rat that specifically expresses the red fluorescent protein, DsRed2, in the liver. Herein, we demonstrate that the DsRed2 expression is sexually dimorphic and exhibits a male-specific pattern. The profiling of sexual dimorphism in DsRed2 expression during pre-pubertal development was investigated using an in vivo fluorescent imaging analysis. The DsRed2 expression decreased gradually in both sexes until 28 days after birth. While DsRed2 expression was not persistent in the female liver, the male hepatic expression increased again at 35 days. Sexual dimorphic DsRed2 expression did not change in gonadectomized male and female Tg-rats. However, female hepatic DsRed2 was induced 72 h after the hypophysectomy. Hepatocytes isolated from the female Tg-rats also revealed DsRed2 induction by 96 h in culture. These results suggest that the pituitary hormone suppresses the female hepatic DsRed2 expression causing the sexual dimorphism of DsRed2 expression. (C) 2009 Elsevier Inc. All rights reserved.

Bone marrow- (BM-) derived cells can differentiate into smooth muscle-like cells (SMLC), resulting in vascular pathogenesis. However, the molecular mechanism of the differentiation remains unknown. We have recently reported that Notch signaling promotes while a Notch target HERP1 inhibit the differentiation of mesenchymal cells to SMC. During the differentiation of BM-derived mononuclear cells into smooth muscle alpha-actin (SMA)-positive cells, expression of Jagged1 and SMC-specific Notch3 was increased. Blocking Notch with gamma-secretase inhibitor prevented the induction of SMA. Wire-mediated vascular injury was produced in femoral arteries in mice transplanted with green fluorescent protein (GFP)-positive cells. Many double-positive cells for GFP/Jagged1 or GFP/Notch3 were detected in the thickened neointima. In contrast, only a few SMA-positive cells were positive for GFP in neointima where HERP1, a suppressor for Notch, were abundantly expressed. In conclusion, Notch-HERP1 pathway plays an important role in differentiation of BM-derived mononuclear cells into SMLC. (C) 2009 Elsevier Inc. All rights reserved

Background. Bone marrow cells (BMCs) are believed to have the ability to generate functional hepatocytes and have some merits as a therapeutic modality for metabolic liver diseases. However, the appearance of BMC-derived hepatocytes (BMDHs) is low. We hypothesized that early BMC injection would be feasible for creating BMDHs for two main reasons: (1) the liver is a hematopoietic organ in neonatal rats and (2) it may allow sufficient time to generate more BMDHs before severe liver injury occurs. Methods. We used Long Evans Cinnamon (LEC) rats as recipients, a model of (1) Wilson disease and (2) liver carcinogenesis. Green fluorescent protein-expressing BMCs were injected into newborn LEC rats through the spleen. The oxidative activity of ceruloplasmin, which is low in LEC rats, was measured to evaluate the treatment. In addition, we performed fluorescence in situ hybridization to clarify the origin of the BMDHs and immunohistochemical analysis to confirm whether these BMDHs had malignant potential. Results. At 18 months after injection, we found some green fluorescent protein-expressing areas macroscopically in the liver of treated LEC rats. The oxidative activity of ceruloplasmin increased in treated LEC rats (n=7) and were much higher than that in untreated LEC rats (P=0.015). Moreover, we confirmed that the BMDHs were generated by cell fusion and was not detected in any of the neoplastic lesions or cholngiofibrotic regions. Conclusion. Our results suggest that this novel strategy for creating BMDHs is effective and safe.

Periodontal ligament (PDL) is a unique connective tissue that not only connects cementum and alveolar bone to support teeth, but also plays an important role in reconstructing periodontal tissues. Previous studies have suggested that PDL cells have osteogenic potential; however, they lack precise histological examinations. Here, we studied bone-like matrix formation by PDL cells in rats using morphological techniques. Rat and human PDL cells exhibited substantial alkaline phosphatase activity and induced mineralization in vitro. RT-PCR analyses showed that PDL cells expressed the osteoblast markers, Runx2, osterix, and osteocalcin. These results suggest that PDL cells share similar phenotypes with osteoblasts. To examine the bone-like matrix formation in vivo, PDL cells isolated from green fluorescent protein (GFP)-transgenic rats were inoculated with hydroxyapatite (HA) disks into wild-type rats. Five weeks after the implantation, the pores in HA disks were occupied by GFP-positive cells. Mineralized matrix formation was also found on the surface of HA pores. At 12 weeks, some of the pores were filled with bone-like mineralized matrices (BLMM), which were positive for the bone matrix proteins, osteopontin, bone sialoprotein, and osteocalcin. Immunohistochemical examination revealed that most of the osteoblast- and osteocyte-like cells on or in the BLMM were GFP-positive, suggesting that the BLMM were directly formed by the inoculated PDL cells. On the pore surfaces, Sharpey's fiber-like structures embedded in cementum-like mineralized layers were also observed. These results collectively suggest that PDL cells have the ability to form periodontal tissues and could be a useful source for regenerative therapies of periodontal diseases.

This study was carried out to compare concentrations of osteopontin (OPN) and osteoprotegerin (OPG) in peripheral arterial disease (PAD). The study population consisted of 200 consecutive subjects in whom both OPN/OPG and ankle-brachial index were measured. It was found that OPN levels, but not OPG levels, were significantly more increased in patients with PAD than those without PAD. Serum OPN levels were significantly lower in subjects with angiotensin converting enzyme inhibitors or angiotensin II receptor blockers than those without these agents. In this study, it has been demonstrated for the first time that serum OPN levels are related to PAD. Inhibition of reninangiotensin system could decrease OPN levels and prevent the progression of PAD.

Bone marrow implantation (BMII) has been performed clinically for the treatment of ischemic cardiovascular diseases. To achieve BMI effectively, accumulation of many bone marrow cells (BMCs) in an infarcted area of the myocardium is important. Previously, we reported that cardiomyocytes show strong interaction with N-acetylglucosamine (GIcNAc) and they can take up GIcNAc-conjugated liposomes. Thus, we examined whether GlcNAc-coated BMCs exhibit Strong interaction with cardiomyocytes. The Cell Surface of BMCs was coated with GIcNAc without causing cell injury by GIcNAc-lipophilic polymers. it was found that the GIcNAc-coated BMCs exhibited strong interaction with cardiomyocytes. At 7 days of coculturing the GlcNAc-coated BMCs with cardiomyocytes, BMC-derived cardiomyocytes were generated. The number of BMC-derived cardiomyocytes was higher following coculture with GlcNAc-coated BMCs than following coculture with uncoated and maltose (MA)-coated BMCs. In this study, we demonstrated that the surface coating of BMCs with GIcNAc can be performed easily by using GIcNAc-lipophilic polymers and that GIcNAc-coated BMCs exhibited strong interaction with cardiomyocytes. Therefore, we think that cell Surface Coating with GIcNAc would help promote accumulation of BMCs in the infarcted area of the myocardium and that this accumulation Would be helpful in the treatment of ischemic cardiovascular diseases with BMI. (C) 2008 Elsevier Ltd. All rights reserved.

Background: The autoantibodies stimulate the beta1-adrenoreceptors on cardiac myocytes similar to norepinephrine, and are associated with reduced cardiac function. Iodine-123 metaiodobenzylguanidine ((123)I-MIBG) is metabolized similarly to norepinephrine. This study was undertaken to investigate the relationship between cardiac stimulation by anti-beta1-adrenoreceptor autoantibodies and myocardial sympathetic nervous activity in patients with chronic heart failure. Methods: We screened for the anti-beta1-adrenoreceptor autoantibodies in 52 patients with chronic heart failure by conducting an enzyme-linked immunosorbent assay, and underwent (123)I-MIBG scintigraphy in 27 of the patients. Anterior planar images of (123)I-MIBG were obtained 15 min and 3 h after the injection. We determined the heart to mediastinum radioactivity ratio (H/M), and calculated the rate of washout of (123)I-MIBG from the heart. Results: Patients with New York Heart Association functional class III or IV had higher levels of anti-beta1-adrenoreceptor autoantibodies than those with class I or II (p<0.01). The autoantibody level was significantly correlated with delayed H/M (r =-0.65, p<0.001) and washout rate (r = 0.65, p<0.001). Sixteen patients with a cardiac event showed higher levels of the autoantibodies (p<0.05). Cardiac event-free survival was poorer in patients with the autoantibody levels >10 U/ml than that <10 U/ml (log-rank= 12.1, p<0.001). Conclusion: The anti-beta1-adrenoreceptor autoantibodies are closely associated with cardiac sympathetic nervous activity assessed by (123)I-MIBG and cardiac event in patients with chronic heart failure. (C) 2008 Elsevier Ireland Ltd. All rights reserved.

The chemokine stromal cell-derived factor-1 (SDF-1) and its receptor (CXCR4, CXC chemokine receptor 4) play a critical role in the process of post-natal neovascularization. Here, we investigated the role of CXCR4(+) bone marrow cells (BMCs) in neovascularization in a murine hindlimb ischaemia model. We found that the expression of CXCR4 in BMCs was specifically upregulated by cultivation; therefore, we used freshly isolated BMCs and cultivated BMCs, designated as BMC(Fr) and BMC(Cul), respectively. The increased CXCR4 expression corresponded to the migratory capacity in response to SDF-1 alpha. Real-time reverse transcription-polymerase chain reaction and immunohistochemical analyses revealed that SDF-1 alpha expression was significantly increased in the ischaemic limbs of mice. Blood flow perfusion and capillary density were significantly accelerated in mice implanted with BMC(Cul) as compared with those in mice implanted with BMC(Fr). The stimulatory effect of BMC(Cul) on neovascularization was significantly impaired when BMC(Cul) derived from CXCR4(+/-) mice were implanted. The implanted BMC(Cul) showed high retention in the ischaemic limbs. Further, the implantation of BMC(Cul) significantly increased the expression of interleukin (IL)-1 beta and vascular endothelial growth factor-A in the ischaemic limbs. The upregulation of CXCR4 expression by cultivation may serve as a useful source of BMCs for accelerating therapeutic angiogenesis in ischaemic cardiovascular diseases.