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Innate immune signaling via TLR4 plays critical roles in pathogenesis of metabolic disorders, but the contribution of different lipid species to metabolic disorders and inflammatory diseases is less clear. GM3 ganglioside in human serum is composed of a variety of fatty acids, including long‐chain (LCFA) and very‐long‐chain (VLCFA). Analysis of circulating levels of human serum GM3 species from patients at different stages of insulin resistance and chronic inflammation reveals that levels of VLCFA‐GM3 increase significantly in metabolic disorders, while LCFA‐GM3 serum levels decrease. Specific GM3 species also correlates with disease symptoms. VLCFA‐GM3 levels increase in the adipose tissue of obese mice, and this is blocked in TLR4‐mutant mice. In cultured monocytes, GM3 by itself has no effect on TLR4 activation; however, VLCFA‐GM3 synergistically and selectively enhances TLR4 activation by LPS/HMGB1, while LCFA‐GM3 and unsaturated VLCFA‐GM3 suppresses TLR4 activation. GM3 interacts with the extracellular region of TLR4/MD2 complex to modulate dimerization/oligomerization. Ligand‐molecular docking analysis supports that VLCFA‐GM3 and LCFA‐GM3 act as agonist and antagonist of TLR4 activity, respectively, by differentially binding to the hydrophobic pocket of MD2. Our findings suggest that VLCFA‐GM3 is a risk factor for TLR4‐mediated disease progression., Analysis of GM3 ganglioside composition in human serum under chronic inflammation conditions reveals that the fatty acid chain length of GM3 ganglioside impacts the inflammatory activation of macrophages via direct modulation of TLR4 signaling.

An alternative pathway triggering enhanced platelet release from bone marrow megakaryocytes via a rupture-based mechanism is regulated by IL-1α in response to acute platelet requirements., Intravital visualization of thrombopoiesis revealed that formation of proplatelets, which are cytoplasmic protrusions in bone marrow megakaryocytes (MKs), is dominant in the steady state. However, it was unclear whether this is the only path to platelet biogenesis. We have identified an alternative MK rupture, which entails rapid cytoplasmic fragmentation and release of much larger numbers of platelets, primarily into blood vessels, which is morphologically and temporally different than typical FasL-induced apoptosis. Serum levels of the inflammatory cytokine IL-1α were acutely elevated after platelet loss or administration of an inflammatory stimulus to mice, whereas the MK-regulator thrombopoietin (TPO) was not elevated. Moreover, IL-1α administration rapidly induced MK rupture–dependent thrombopoiesis and increased platelet counts. IL-1α–IL-1R1 signaling activated caspase-3, which reduced plasma membrane stability and appeared to inhibit regulated tubulin expression and proplatelet formation, and ultimately led to MK rupture. Collectively, it appears the balance between TPO and IL-1α determines the MK cellular programming for thrombopoiesis in response to acute and chronic platelet needs.

SummaryDistinct B cell populations, designated regulatory B (Breg) cells, are known to restrain immune responses associated with autoimmune diseases. Additionally, obesity is known to induce local inflammation within adipose tissue that contributes to systemic metabolic abnormalities, but the underlying mechanisms that modulate adipose inflammation remain poorly understood. We identified Breg cells that produce interleukin-10 constitutively within adipose tissue. B cell-specific Il10 deletion enhanced adipose inflammation and insulin resistance in diet-induced obese mice, whereas adoptive transfer of adipose tissue Breg cells ameliorated those effects. Adipose environmental factors, including CXCL12 and free fatty acids, support Breg cell function, and Breg cell fraction and function were reduced in adipose tissue from obese mice and humans. Our findings indicate that adipose tissue Breg cells are a naturally occurring regulatory B cell subset that maintains homeostasis within adipose tissue and that Breg cell dysfunction contributes pivotally to the progression of adipose tissue inflammation in obesity.

in viv in viv in vivo visualization technique was developed. In vivo imaging revealed close spatial and temporal interrelationships between angiogenesis and adipogenesis, which were augmented in obese adipose tissue. In addition, increased leukocyte-platelet-endothelial cell interactions were observed in the microcirculation, a hallmark of inflammation. Upregulated expression of adhesion molecules contribute to local activation of inflammatory processes. We also found that large numbers of CD8+ effector T cells infiltrated into the obese adipose tissue, playing major roles in inflammatory macrophage infiltration into obese adipose tissue, induction and maintenance of inflammation, and systemic insulin resistance. Our results demonstrate the power of our imaging technique to analyze multi-cellular interactions in inflammation in vivo o o o o and to evaluate new therapeutic interventions.

Obese visceral adipose tissue remodeling and dysfunction, based on chronic inflammation and local immunological changes, play major roles in the metabolic syndrome. Therefore, an in vivo visualization technique has been developed to assess the dynamic interplay between multiple cell types in obese adipose. In vivo imaging revealed close spatial and temporal interrelationships between angiogenesis and adipogenesis, which were augmented in obese adipose tissue. In addition, increased leukocyte–platelet–endothelial cell interactions were observed in the microcirculation, a hallmark of inflammation. Upregulated expression of adhesion molecules contribute to the local activation of inflammatory processes. We also found that large numbers of CD8+ effector T cells infiltrated into the obese adipose tissue, playing major roles in inflammatory macrophage infiltration into obese adipose tissue, the induction and maintenance of inflammation, and systemic insulin resistance. Our results demonstrate the power of our imaging technique to analyze multi-cellular interactions in inflammation in vivo and to evaluate new therapeutic interventions.

Inflammation is increasingly regarded as a key process underlying metabolic diseases in obese individuals. In particular, obese adipose tissue shows features characteristic of active local inflammation. At present, however, little is known about the sequence of events that comprises the inflammatory cascade or the mechanism by which inflammation develops. We found that large numbers of CD8(+) effector T cells infiltrated obese epididymal adipose tissue in mice fed a high-fat diet, whereas the numbers of CD4(+) helper and regulatory T cells were diminished. The infiltration by CD8(+) T cells preceded the accumulation of macrophages, and immunological and genetic depletion of CD8(+) T cells lowered macrophage infiltration and adipose tissue inflammation and ameliorated systemic insulin resistance. Conversely, adoptive transfer of CD8(+) T cells to CD8-deficient mice aggravated adipose inflammation. Coculture and other in vitro experiments revealed a vicious cycle of interactions between CD8(+) T cells, macrophages and adipose tissue. Our findings suggest that obese adipose tissue activates CD8(+) T cells, which, in turn, promote the recruitment and activation of macrophages in this tissue. These results support the notion that CD8(+) T cells have an essential role in the initiation and propagation of adipose inflammation.

Obese visceral adipose tissue remodeling and dysfunction, based on chronic inflammation and local immunological changes, play major roles in the metabolic syndrome. Therefore, to assess the dynamic interplay between multiple cell types in obese adipose, an in vivo visualization technique was developed. In vivo imaging revealed close spatial and temporal interrelationships between angiogenesis and adipogenesis, which were augmented in obese adipose tissue. In addition, increased leukocyte-platelet-endothelial cell interactions were observed in the microcirculation, a hallmark of inflammation. Upregulated expression of adhesion molecules contribute to local activation of inflammatory processes. We also found that large numbers of CD8+ effector T cells infiltrated into the obese adipose tissue, playing major roles in inflammatory macrophage infiltration into obese adipose tissue, induction and maintenance of inflammation, and systemic insulin resistance. Our results demonstrate the power of our imaging technique to analyze multi-cellular interactions in inflammation in vivo and to evaluate new therapeutic interventions.

Serum GM3 molecular species were quantified in 125 Japanese residents using tandem mass spectrometry multiple reaction monitoring. Individuals were categorized by the presence or absence of metabolic disease risk factors including visceral fat accumulation, hyperglycemia and dyslipidemia. A total of 23 GM3 molecular species were measured, of these, eight were found to be significantly elevated in individuals with visceral fat accumulation and metabolic disease, defined as the presence of hyperglycemia and dyslipidemia. All of the GM3 molecular species were composed of the sphingoid base sphingosine (d18:1 (Δ4)) and, interestingly, six of the eight elevated GM3 molecular species contained a hydroxylated ceramide moiety. The hydroxylated GM3 species were, in order of decreasing abundance, d18:1-h24:0 ≈ d18:1-h24:1 > d18:1-h22:0 » d18:1-h20:0 > d18:1-h21:0 > d18:1-h18:1. Univariate and multiple linear regression analyses were conducted using a number of clinical health variables associated with obesity, type 2 diabetes, metabolic disease, atherosclerosis and hypertension. GM3(d18:1-h24:1) was identified as the best candidate for metabolic screening, proving to be significantly correlated with intima-media thickness, used for the detection of atherosclerotic disease in humans, and a number of metabolic disease risk factors including autotaxin, LDL-c and homeostatic model assessment insulin resistance (HOMA-IR).

Body weight is tightly regulated by food intake and energy dissipation, and obesity is related to decreased energy expenditure (EE). Herein, we show that nucleotide pyrophosphatase/phosphodiesterase 2 (ENPP2, autotaxin) is an adipose-derived, secreted enzyme that controls adipose expansion, brown adipose tissue (BAT) function, and EE. In mice, Enpp2 was highly expressed in visceral white adipose tissue and BAT and is downregulated in hypertrophied adipocytes/adipose tissue. Enpp2+/− mice and adipocyte-specific Enpp2 knockout mice fed a high-fat diet showed smaller body weight gains and less insulin resistance than control mice fed the same diet. BAT was functionally more active and EE was increased in Enpp2-deficient mice. In humans, ENPP2 expression in subcutaneous fat and ENPP2 levels in serum were reduced in obese subjects. Taken together, our results establish ENPP2 as an adipose-derived, secreted enzyme that regulates adipose obesity and systemic metabolism. They also suggest ENPP2 could be a useful therapeutic target for the treatment of metabolic disease.

inflammation, it is vital to examine the multi-cellular kinetics in living animals. Therefore, we developed in vivo imaging technique based on single- and multi-photon microscopy, and we assessed dynamic cellular interplay in diseased conditions. The mechanism by which thrombotic vessel occlusion occurs independently of plaque development or endothelial cell (EC) disruption remains unclear, largely because of an inability to visualize thrombus formation, especially at the single-platelet level in real time. Here we demonstrate that rapidly developing thrombi composed of discoid platelets can be induced in the mesenteric vessls of living mice, enabling characterization of the kinetics of thrombosis initiation and the multicellular interrelationships during thrombus development. Platelet aggregation without EC disruption was triggered by reactive oxygen species (ROS) photochemically induced by moderate power laser irradiation. Using this technique, we elucidated that Lnk (adapter protein) regulates integrin signaling leading to stabilization of developing thrombus in vivo (Nishimura et at, 2010 JCI). In addition, we established the efficient culture system of human iPS-derived platelets, and we confirmed the functional role. These artificial platelets can circulate, and contribute to their thrombus formation in vivo, indicating the clinical usefulness considering the cell therapy for future (Takayama & Nishimmura et al, 2010 JEM). In addition, we elucidated the contribution of inflammatory cytokines, ROS, and integrin signaling to our thrombosis models (Nishimura et al, 2011 Blood). The inflammatory cytokines TNF-alpha and IL-1 could be key components of the EC response, acting through regulation of von Willebrand factor mobilization to the cell surface. Thrombus formation was then initiated by the binding of platelet GPIb-alpha to endothelial von Willebrand Factor in our model, and this effect was inhibited by the ROS scavenger N-acetylcysteine. Actin linker talin-dependent activation of alphaIIb-beta3 integrin in platelets was required for late phase thrombus stability. Our imaging can be applied to many research fields including metabolic diseases. Our imaging revealed close spatial and temporal interrelationships between angiogenesis and adipogenesis in obese adipose (Nishimura et al, 2007 Diabetes). In addition, increased inflammatory cell interactions were visualized (Nishimura et al, 2008 JCI). We also found that large numbers of CD8+ effector T cells infiltrated into obese adipose, and these cells were essential for the initiation and development of adipose inflammation (Nishimura et al, 2009 Nat Med). In sum, using our imaging system can be a powerful tools to analyze the inflammatory conditions including thrombosis. We clarified the mechanism by which thrombi are rapidly formed by discoid platelets on undisputed endothelium. The initial platelet aggregation subsequently lead to irreversible integrin- and actin-dependent thrombus development. Inflammatory cytokine signaling in ECs played pivotal role in discoid platelet aggregations in vivo.

The mechanism by which thrombotic vessel occlusion occurs independently of plaque development or endothelial cell (EC) disruption remains unclear, largely because of an inability to visualize the formation of thrombus, especially at the single-platelet level in real time. Here we demonstrate that rapidly developing thrombi composed of discoid platelets can be induced in the mesenteric capillaries, arterioles, and large-sized arteries of living mice, enabling characterization of the kinetics of thrombosis initiation and the multicellular interrelationships during thrombus development. Platelet aggregation without EC disruption was triggered by reactive oxygen species (ROS) photochemically induced by moderate power laser irradiation. The inflammatory cytokines TNF-α and IL-1 could be key components of the EC response, acting through regulation of VWF mobilization to the cell surface. Thrombus formation was then initiated by the binding of platelet GPIbα to endothelial VWF in our model, and this effect was inhibited by the ROS scavenger N-acetylcysteine. Actin linker talin-dependent activation of alphaIIb-beta3 integrin or Rac1 in platelets was required for late-phase thrombus stability. Our novel imaging technology illustrates the molecular mechanism underlying inflammation-based thrombus formation by discoid platelets on undisrupted ECs and suggests control of ROS could be a useful therapeutic target for the prevention of thrombotic diseases.

Inflammatory markers such as serum C-reactive protein (CRP), serum amyloid A (SAA), and plasma pentraxin 3 (PTX3), which belong to the pentraxin superfamily, increase due to various inflammatory diseases. Some studies demonstrated that serum CRP and SAA are predictors of cardiovascular diseases, and cardiac rehabilitation (CR) induces anti-inflammatory effects. In the present study, we investigated the effects of CR on pentraxins (serum CRP, SAA, and plasma PTX3) in patients with cardiovascular diseases.Fifty patients with cardiovascular diseases [61 ± 13 (mean ± SD) years old, male/female 44/6] participated. Each subject performed CR using aerobic bicycle exercise two or three times per week for 3-6 months. We measured resting serum high-sensitivity CRP (hsCRP), SAA, and plasma PTX3 before and 3 and 6 months after CR, and compared them with VO(2peak) determined using a standard increment cycle ergometer protocol, B-type natriuretic peptide (BNP), and other biochemical data such as HbA1c.There was a significant positive correlation between hsCRP and SAA (r = 0.92, p 0.001), but no relations between these parameters and PTX3. Plasma PTX3 significantly decreased time dependently during CR (at baseline 3.2 ± 2.0 ng/ml, at 3 months 2.3 ± 0.8 ng/ml, at 6 months 2.1 ± 0.7 ng/ml; all p 0.05). Serum hsCRP tended to decrease, but not statistically significantly. At baseline, plasma PTX3 was negatively correlated with the percentage of the predicted values of VO(2peak) and positively correlated with BNP. CR significantly increased the percentage of the predicted values of VO(2peak) and decreased BNP.Plasma PTX3, an inflammatory marker, which was quite different from CRP and SAA, decreased during cardiac rehabilitation with an improvement of exercise capacity in patients with cardiovascular diseases.

The bioactive lipid lysophosphatidylserine (LPS) is postulated to induce important biological responses and to be produced by phosphatidylserine-specific phospholipase A(1) (PS-PLA(1)). To evaluate the functional roles of LPS in vivo, a facile assay method for PS-PLA(1) has been awaited.Recombinant human PS-PLA(1) was produced using a baculovirus system, and anti-human PS-PLA(1) monoclonal antibodies were generated. Two clones were then selected for a 2-site immunoassay. The resulting PS-PLA(1) assay reagent was applied to a commercial automated immunoassay analyzer.Satisfactory results were obtained for the within-run and between-run precision, interference, detection limit, and linearity of this PS-PLA(1) assay. The mean+/-SD of the serum PS-PLA(1) antigen concentration in the 191 healthy subjects was 33.8+/-16.6microg/l, and the central 95th percentile reference interval for the serum PS-PLA(1) antigen concentration was 13.8-74.1microg/l. The concentration was significantly (p0.001) higher among men (13.8-80.6microg/l) than among women (12.1-68.8microg/l). We did not find a correlation between PS-PLA(1) and existing laboratory tests.The present PS-PLA(1) assay method can be applied to clinical laboratory testing, and further studies are warranted to establish its clinical significance.

Metabolic syndrome is a major risk factor of cardiovascular events, and obese visceral adipose tissue remodeling and malfunctioning based on chronic inflammation plays a central role. To assess dynamic multi-cellular interplay, a novel ex vivo (a–c) and in vivo (d–f) adipose tissue imaging method was developed. We found close spatial and temporal interrelationships between angiogenesis and adipogenesis, and both were augmented in obese adipose (c,arrow). In addition, we found increased leukocyte-platelet-endothelial cell interactions in the micro-circulation of obese visceral adipose that were indicative of activation of the leukocyte adhesion cascade, a hallmark of inflammation (e,f). Both macrophages and endothelial cells showed increased adhesion molecules including ICAM-1 and Selectin families, and P-selectin positive platelets were increased locally in obese adipose. Up-regulated expression of adhesion molecules on multiple cell types suggests their increased interactions contribute to local activation of inflammatory processes within visceral obese adipose tissue. Interestingly, the heightened leukocyte-platelet-endothelial interactions were not observed in obese subcutaneous fat pads. Our results demonstrated the power of our imaging technique to analyze complex inflammatory cellular interplays in vivo and to evaluate new therapeutic interventions against them. Results also indicate that visceral adipose tissue obesity is an inflammatory disease.

Mechano-electrical feedback (MEF) has mainly been studied in isolated single cardiomyocytes using the microelectrode and micropipette techniques, but information regarding its dynamic aspects at the cellular level is limited due to the technical difficulties associated with manipulating single cells and maintaining stable attachment of these devices. To overcome such difficulties, we have combined two experimental methods, namely a carbon fiber technique to hold single myocytes and a ratiometric fluorescence measurement technique to monitor Ca2+ transients or membrane potentials. Following an overview of the experimental technique for stretching myocytes, the results for single rat ventricular myocytes under axial stretching are presented. Ca2+ transients were influenced by the loading conditions and involvement of myofilaments was suspected in regulatory mechanism. Membrane potential measurements during dynamic axial stretching revealed that the action potential duration was prolonged when the stretch was applied during the late phase of twitch contraction, and that depolarization of the resting membrane potential depended on the phase, amplitude and speed of the applied stretch. The amplitude may also modulate the ion selectivity of stretch-activated channels. This combination of the carbon fiber technique with fluorescence measurement could represent a powerful tool for clarifying MEF at the cellular level.

To assess physiological and pathophysiological events that involve dynamic interplay between multiple cell types, real-time, in vivo analysis is necessary. We developed a technique based on confocal laser microscopy that enabled us to analyze and compare the 3-dimensional structures, cellular dynamics, and vascular function within mouse lean and obese adipose tissue in vivo with high spatiotemporal resolution. We found increased leukocyte-EC-platelet interaction in the microcirculation of obese visceral adipose tissue in ob/ob and high-fat diet–induced obese mice. These changes were indicative of activation of the leukocyte adhesion cascade, a hallmark of inflammation. Local platelet activation in obese adipose tissue was indicated by increased P-selectin expression and formation of monocyte-platelet conjugates. We observed upregulated expression of adhesion molecules on macrophages and ECs in obese visceral adipose tissue, suggesting that interactions between these cells contribute to local activation of inflammatory processes. Furthermore, administration of anti–ICAM-1 antibody normalized the cell dynamics seen in obese visceral fat. This imaging technique to analyze the complex cellular interplay within obese adipose tissue allowed us to show that visceral adipose tissue obesity is an inflammatory disease. In addition, this technique may prove to be a valuable tool to evaluate potential therapeutic interventions.

OBJECTIVE—The expansion of adipose tissue mass seen in obesity involves both hyperplasia and hypertrophy of adipocytes. However, little is known about how adipocytes, adipocyte precursors, blood vessels, and stromal cells interact with one another to achieve adipogenesis. RESEARCH DESIGN AND METHODS—We have developed a confocal microscopy-based method of three-dimensional visualization of intact living adipose tissue that enabled us to simultaneously evaluate angiogenesis and adipogenesis in db/db mice. RESULTS—We found that adipocyte differentiation takes place within cell clusters (which we designated adipogenic/angiogenic cell clusters) that contain multiple cell types, including endothelial cells and stromal cells that express CD34 and CD68 and bind lectin. There were close spatial and temporal interrelationships between blood vessel formation and adipogenesis, and the sprouting of new blood vessels from preexisting vasculature was coupled to adipocyte differentiation. CD34+ CD68+ lectin-binding cells could clearly be distinguished from CD34− CD68+ macrophages, which were scattered in the stroma and did not bind lectin. Adipogenic/angiogenic cell clusters can morphologically and immunohistochemically be distinguished from crown-like structures frequently seen in the late stages of adipose tissue obesity. Administration of anti–vascular endothelial growth factor (VEGF) antibodies inhibited not only angiogenesis but also the formation of adipogenic/angiogenic cell clusters, indicating that the coupling of adipogenesis and angiogenesis is essential for differentiation of adipocytes in obesity and that VEGF is a key mediator of that process. CONCLUSIONS—Living tissue imaging techniques provide novel evidence of the dynamic interactions between differentiating adipocytes, stromal cells, and angiogenesis in living obese adipose tissue.

Functional mitral regurgitation (MR) is one of the common and severe complications in patients with dilated cardiomyopathy. The detailed mechanisms that cause functional MR remain to be elucidated. Using two-dimensional transthoracic echocardiography, we investigated the differences in major determinants of MR severity between ischemic cardiomyopathy (ICM) and non-ICM patients.We enrolled 103 patients (91 males; age 64+/-12 years) with significant left ventricular (LV) dilatation. They were divided into ICM group (n=69) with significant coronary disease, and non-ICM (n=34) group without coronary disease. We devised a novel and simple parameter; the short-axis sphericity index (SI), to evaluate global LV remodeling, and used coaptation depth (CD) and tenting area (TA) to evaluate mitral deformity.In all cases, CD, TA and left atrium diameter (LAD) correlated positively with maximum regurgitation area (MRA) (r=0.54, 0.57, 0.57; P0.0001). A negative correlation was observed between MRA and SI (r=-0.33, P=0.0008). There was no significant relationship between MRA and LV ejection fraction (EF). In non-ICM cases, SI tended to be lower with reduced EF. Multivariate stepwise linear regression analysis showed the following equations; ICM: MRA=-9.4+0.81CD+0.21LAD (r2=0.47, P0.0001), non-ICM: MRA=-7.2+0.17LVDs (LV end systolic diameter) -8.7SI+0.27LAD (r2=0.63, P0.0001).The strongest determinants of functional MR severity differ in ICM and non-ICM. While LV diameter and SI (global LV remodeling index) mainly determine the severity in non-ICM, CD that reflects mitral deformity is the major determinant in ICM.

This report presents the first case of an unusual biventricular myocardial infarction caused by pulmonary thromboembolism in a 55-year-old woman who had an anomalous origin of the right coronary artery (RCA) from the left coronary sinus. The RCA consequently courses between the aorta and pulmonary trunk, and dilatation of the pulmonary artery because of elevated pulmonary artery pressure compressed the proximal portion of the RCA. The consequent reduced right coronary oxygen supply and sudden increase in right ventricular afterload contributed to the characteristic right ventricular infarction, in addition to a left ventricular infero-posterior infarction. Her anginal symptoms disappeared following successful anticoagulation therapy and insertion of an inferior vena caval filter, without coronary bypass. This pathophysiologic phenomenon is rare, but can be fatal. (Circ J 2004; 68: 883 - 886)

We experienced a case in which live 3D transoesophageal echocardiography (TEE) was found much more valuable than 2D TEE in assessing mitral lesions in circumferential direction and making surgical plans for mitral valve prolapse.

To elucidate the underlying mechanisms of adult common diseases based on chronic inflammation, it is vital to examine the immune and inflammatory cell kinetics in living animals. Therefore, we developed in vivo imaging technique based on single- and multi-photon microscopy, and we assessed dynamic cellular interplay in diseased conditions. We found that large numbers of CD8+ effector T cells infiltrated into obese adipose, and these cells were essential for the initiation and development of adipose inflammation. In addition, we identified a novel B cell subset that is abundant in adipose tissue. Immunological or genetic B cell depletion, and B cell-specific Il10 deletion enhanced CD8+ T cell activation and adipose inflammation, and exacerbated insulin resistance in obesity. Adipose environmental factors supported B cell survival and IL-10 production. Adipose B cells also exhibited unique surface phenotypes, distinct from those of known regulatory B cell subsets. Our findings indicate that adipose B cells are a naturally occurring regulatory B cell subset that is essential for negative regulation of diet-induced adipose inflammation and maintenance of homeostasis within adipose tissue, and that B cell dysfunction pivotally contributes to the progression of inflammatory processes. Our results also clearly demonstrated the power of our imaging technique to analyze complex cellular interplays in inflammatory diseases, especially parenchymal and stromal cell cross talks, and to evaluate new therapeutic interventions against them.

Background: The mechanism by which thrombotic vessel occlusion occurs independently of plaque development or endothelial cell (EC) disruption remains unclear, largely because of an inability to visualize thrombus formation, especially at the single-platelet level in real time. Methods: Therefore, we developed in vivo imaging technique based on singleand multi-photon microscopy, and we assessed dynamic cellular interplay in thrombosis models. We visualized that rapidly developing thrombi composed of discoid platelets without EC disruptionwas triggered by ROS photochemically inducedbymoderate power laser irradiation. Results: Using this technique, we elucidated that Lnk (adapter protein) regulates integrin signaling leading to stabilization of developing thrombus in vivo (2010 JCI). We established the efficient culture system of human iPS-derived platelets, and we confirmed the functional role. These artificial platelets can circulate, and contribute to their thrombus formation in vivo, indicating the clinical usefulness considering the cell therapy for future (2010 JEM). In addition, we elucidated the contribution of inflammatory cytokines, ROS, and integrin signaling to our thrombosis models (Nishimura et al, 2011 Blood). The inflammatory cytokines TNF-alpha and IL-1 could be key components of the EC response, acting through regulation of von Willebrand factor mobilization to the cell surface. Thrombus formation was then initiated by the binding of platelet GPIbalpha to endothelial vonWillebrand Factor in our model, and this effect was inhibited by the ROS scavenger N-acetylcysteine. Actin linker talindependent activation of alphaIIb-beta3 integrin in platelets was required for late phase thrombus stability. Comment: In sum, using our imaging system can be a powerful tool to analyze thrombus formation. We clarified the mechanism of discoid platelet aggregations on undisputed endothelium. The initial platelet aggregation subsequently leads to irreversible integrinand actindependent thrombus development. Inflammatory cytokine signaling in ECs also played pivotal role.