Development of an intravital imaging system for the synovial tissue reveals the dynamics of CTLA-4 Ig in vivo

There have been many attempts to visualize the inflamed joints using multiphoton microscopy. However, due to the hypervascular and multilayered structure of the inflamed synovium, intravital imaging of the deep synovial tissue has been difficult. Here, we established original intravital imaging systems to visualize synovial tissue and pathological osteoclasts at the pannus–bone interface using multiphoton microscopy. Combined with fluorescence-labeling of CTLA-4 Ig, a biological agent used for the treatment of rheumatoid arthritis, we identified that CTLA-4 Ig was distributed predominantly within the inflamed synovium and bound to CX3CR1+ macrophages and CD140a+ fibroblasts 6 h after injection, but not to mature osteoclasts. Intravital imaging of blood and lymphatic vessels in the inflamed synovium further showed that extravasated CTLA-4 Ig was immediately drained through lymphatic vessels under acute arthritic conditions, but the drainage activity was retarded under chronic conditions. These results indicate that this intravital synovial imaging system can serve as a platform for exploring the dynamics of immune cells, osteoclasts, and biological agents within the synovial microenvironment in vivo.Hasegawa T., Kikuta J., Sudo T., et al. Development of an intravital imaging system for the synovial tissue reveals the dynamics of CTLA-4 Ig in vivo. Scientific Reports 10, 13480 (2020); https://doi.org/10.1038/s41598-020-70488-y

Group cognitive behavioural therapy (GCBT) versus treatment as usual (TAU) in the treatment of irritable bowel syndrome (IBS): A study protocol for a randomized controlled trial

Background: Irritable bowel syndrome (IBS) is a common disease that affects the quality of life (QOL) and social functioning of sufferers. Visceral anxiety is currently considered a key factor in the onset and exacerbation of IBS, and cognitive-behavioural therapy (CBT) targeting visceral anxiety is thought to be effective. However, access to CBT is limited due to the lack of trained therapists, the substantial time required for therapy and the associated costs. Group CBT (GCBT) may solve some of these problems. We have therefore planned this trial to examine the efficacy of GCBT for IBS. Methods: The trial is a two-armed, parallel group, open label, stratified block randomized superiority trial. The study group will consist of 112 participants (aged 18–75 years) with IBS (Rome-III or IV criteria). Participants will be randomly allocated 1:1 to (i) the intervention group: ten-week GCBT plus treatment as usual (TAU) or (ii) the control group: waiting list (WL) plus TAU. The co-primary outcomes are the change in IBS severity or disease-specific quality of life from baseline to week 13 which is 1 month after the end of treatment. The efficacy of GCBT for IBS will be examined through mixed-effects repeated-measures analysis. Discussion: GCBT, if found effective, can address the issues of the shortage of therapists as well as the time required and the costs associated with individual CBT. Clinically, the findings will help make effective CBT programmes accessible to a large number of distressed IBS patients at lower costs. Theoretically, the results will clarify the relationship between IBS and psychological stress and will help elucidate the underlying mechanisms of IBS. Trial registration: UMIN, CTR-UMIN000031710. Registered on March 13, 2018

Migration arrest of chemoresistant leukemia cells mediated by MRTF-SRF pathway

Background: Dormant chemotherapy-resistant leukemia cells can survive for an extended period before relapse. Nevertheless, the mechanisms underlying the development of chemoresistance in vivo remain unclear. Methods: Using intravital bone imaging, we characterized the behavior of murine acute myeloid leukemia (AML) cells (C1498) in the bone marrow before and after chemotherapy with cytarabine. Results: Proliferative C1498 cells exhibited high motility in the bone marrow. Cytarabine treatment impaired the motility of residual C1498 cells. However, C1498 cells regained their migration potential after relapse. RNA sequencing revealed that cytarabine treatment promoted MRTF-SRF pathway activation. MRTF inhibition using CCG-203971 augmented the anti-tumor effects of chemotherapy in our AML mouse model, as well as suppressed the migration of chemoresistant C1498 cells. Conclusions: These results provide novel insight into the role of cell migration arrest on the development of chemoresistance in AML, as well as provide a strong rationale for the modulation of cellular motility as a therapeutic target for refractory AML.Morimatsu M., Yamashita E., Seno S., et al. Migration arrest of chemoresistant leukemia cells mediated by MRTF-SRF pathway. Inflammation and Regeneration 40, 15 (2020); https://doi.org/10.1186/s41232-020-00127-6

Thrombomodulin induces anti-inflammatory effects by inhibiting the rolling adhesion of leukocytes in vivo

Thrombomodulin (TM) is an integral membrane protein expressed on the surface of vascular endothelial cells that suppresses blood coagulation. Recent studies have shown that TM exhibits anti-inflammatory effects by inhibiting leukocyte recruitment. However, the actual modes of action of TM in vivo remain unclear. Here, we describe the pharmacological effects of recombinant human soluble TM (TM alfa) on leukocyte dynamics in living mice using intravital imaging techniques. Under control conditions, neutrophils exhibited three distinct types of adhesion behavior in vessels: 1) “non-adhesion”, in which cells flowed without vessel adhesion; 2) “rolling adhesion”, in which cells transiently interacted with the endothelium; and 3) “tight binding”, in which cells bound strongly to the endothelial cells. Compared to control conditions, local lipopolysaccharide stimulation resulted in an increased frequency of rolling adhesion that was not homogeneously distributed on vessel walls but occurred at specific endothelial sites. Under inflammatory conditions, TM alfa, particularly the D1 domain which is a lectin-like region of TM, significantly decreased the frequency of rolling adhesion, but did not influence the number of tight bindings. This was the first study to demonstrate that TM alfa exerts anti-inflammatory effects by inhibiting rolling adhesion of neutrophils to vascular endothelial cells in living mice.Nishizawa S., Kikuta J., Seno S., et al. Thrombomodulin induces anti-inflammatory effects by inhibiting the rolling adhesion of leukocytes in vivo. Journal of Pharmacological Sciences 143, 17 (2020); https://doi.org/10.1016/j.jphs.2020.01.001

In vivo dynamic analysis of BMP-2-induced ectopic bone formation

Bone morphogenetic protein (BMP)-2 plays a central role in bone-tissue engineering because of its potent bone-induction ability. However, the process of BMP-induced bone formation in vivo remains poorly elucidated. Here, we aimed to establish a method for intravital imaging of the entire process of BMP-2-induced ectopic bone formation. Using multicolor intravital imaging in transgenic mice, we visualized the spatiotemporal process of bone induction, including appearance and motility of osteoblasts and osteoclasts, angiogenesis, collagen-fiber formation, and bone-mineral deposition. Furthermore, we investigated how PTH1-34 affects BMP-2-induced bone formation, which revealed that PTH1-34 administration accelerated differentiation and increased the motility of osteoblasts, whereas it decreased morphological changes in osteoclasts. This is the first report on visualization of the entire process of BMP-2-induced bone formation using intravital imaging techniques, which, we believe, will contribute to our understanding of ectopic bone formation and provide new parameters for evaluating bone-forming activity.Hashimoto K., Kaito T., Furuya M., et al. In vivo dynamic analysis of BMP-2-induced ectopic bone formation. Scientific Reports 10, 4751 (2020); https://doi.org/10.1038/s41598-020-61825-2

可溶性Flt-1産生低下は、心筋リモデリングおよび心不全増悪に寄与する

Soluble fms-like tyrosine kinase-1 (sFlt-1), an endogenous inhibitor of vascular endothelial growth factor and placental growth factor, is involved in the pathogenesis of cardiovascular disease. However, the significance of sFlt-1 in heart failure has not been fully elucidated. We found that sFlt-1 is decreased in renal failure and serves as a key molecule in atherosclerosis. In this study, we aimed to investigate the role of the decreased sFlt-1 production in heart failure, using sFlt-1 knockout mice. sFlt-1 knockout mice and wild-type mice were subjected to transverse aortic constriction and evaluated after 7 days. The sFlt-1 knockout mice had significantly higher mortality (52% versus 15%; P=0.0002) attributable to heart failure and showed greater cardiac hypertrophy (heart weight to body weight ratio, 8.95±0.45 mg/g in sFlt-1 knockout mice versus 6.60±0.32 mg/g in wild-type mice; P<0.0001) and cardiac dysfunction, which was accompanied by a significant increase in macrophage infiltration and cardiac fibrosis, than wild-type mice after transverse aortic constriction. An anti–placental growth factor–neutralizing antibody prevented pressure overload–induced cardiac hypertrophy, fibrosis, and cardiac dysfunction. Moreover, monocyte chemoattractant protein-1 expression was significantly increased in the hypertrophied hearts of sFlt-1 knockout mice compared with wild-type mice. Monocyte chemoattractant protein-1 inhibition with neutralizing antibody ameliorated maladaptive cardiac remodeling in sFlt-1 knockout mice after transverse aortic constriction. In conclusion, decreased sFlt-1 production plays a key role in the aggravation of cardiac hypertrophy and heart failure through upregulation of monocyte chemoattractant protein-1 expression in pressure-overloaded heart.博士（医学）・乙第1384号・平成28年11月24日© 2016 American Heart Association, Inc.The definitive version is available at " https://doi.org/10.1161/HYPERTENSIONAHA.116.07371

慢性腎臓病では血中可溶型fms様チロシンキナーゼ-1産生の減少が動脈硬化症を悪化させる

Patients with chronic kidney disease (CKD) die of cardiovascular diseases for unknown reasons. Blood vessel formation in plaques and its relationship with plaque stability could be involved with signaling through the Flt-1 receptor and its ligands, vascular endothelial growth factor, and the closely related placental growth factor (PlGF). Flt-1 also exists as a circulating regulatory splice variant short-inhibitory form (sFlt-1) that serves as a decoy receptor, thereby inactivating PlGF. Heparin releases sFlt-1 by displacing the sFlt-1 heparin-binding site from heparin sulfate proteoglycans. Heparin could provide diagnostic inference or could also induce an antiangiogenic state. In the present study, postheparin sFlt-1 levels were lower in CKD patients than in control subjects. More importantly, sFlt-1 levels were inversely related to atherosclerosis in CKD patients, and this correlation was more robust after heparin injection, as verified by subsequent cardiovascular events. Knockout of apolipoprotein E (ApoE) and/or sFlt-1 showed that the absence of sFlt-1 worsened atherogenesis in ApoE-deficient mice. Thus, the relationship between atherosclerosis and PlGF signaling, as regulated by sFlt-1, underscores the underappreciated role of heparin in sFlt-1 release. These clinical and experimental data suggest that novel avenues into CKD-dependent atherosclerosis and its detection are warranted.博士（医学）・甲614号・平成26年3月17

SLPI is a critical mediator that controls PTH-induced bone formation

Osteoclastic bone resorption and osteoblastic bone formation/replenishment are closely coupled in bone metabolism. Anabolic parathyroid hormone (PTH), which is commonly used for treating osteoporosis, shifts the balance from osteoclastic to osteoblastic, although it is unclear how these cells are coordinately regulated by PTH. Here, we identify a serine protease inhibitor, secretory leukocyte protease inhibitor (SLPI), as a critical mediator that is involved in the PTH-mediated shift to the osteoblastic phase. Slpi is highly upregulated in osteoblasts by PTH, while genetic ablation of Slpi severely impairs PTH-induced bone formation. Slpi induction in osteoblasts enhances its differentiation, and increases osteoblast–osteoclast contact, thereby suppressing osteoclastic function. Intravital bone imaging reveals that the PTH-mediated association between osteoblasts and osteoclasts is disrupted in the absence of SLPI. Collectively, these results demonstrate that SLPI regulates the communication between osteoblasts and osteoclasts to promote PTH-induced bone anabolism.Morimoto A., Kikuta J., Nishikawa K., et al. SLPI is a critical mediator that controls PTH-induced bone formation. Nature Communications 12, 2136 (2021); https://doi.org/10.1038/s41467-021-22402-x

Direct cell–cell contact between mature osteoblasts and osteoclasts dynamically controls their functions in vivo

Bone homeostasis is regulated by communication between bone-forming mature osteoblasts (mOBs) and bone-resorptive mature osteoclasts (mOCs). However, the spatial–temporal relationship and mode of interaction in vivo remain elusive. Here we show, by using an intravital imaging technique, that mOB and mOC functions are regulated via direct cell–cell contact between these cell types. The mOBs and mOCs mainly occupy discrete territories in the steady state, although direct cell–cell contact is detected in spatiotemporally limited areas. In addition, a pH-sensing fluorescence probe reveals that mOCs secrete protons for bone resorption when they are not in contact with mOBs, whereas mOCs contacting mOBs are non-resorptive, suggesting that mOBs can inhibit bone resorption by direct contact. Intermittent administration of parathyroid hormone causes bone anabolic effects, which lead to a mixed distribution of mOBs and mOCs, and increase cell–cell contact. This study reveals spatiotemporal intercellular interactions between mOBs and mOCs affecting bone homeostasis in vivo

Osteoclasts adapt to physioxia perturbation through DNA demethylation

Oxygen plays an important role in diverse biological processes. However, since quantitation of the partial pressure of cellular oxygen in vivo is challenging, the extent of oxygen perturbation in situ and its cellular response remains underexplored. Using two-photon phosphorescence lifetime imaging microscopy, we determine the physiological range of oxygen tension in osteoclasts of live mice. We find that oxygen tension ranges from 17.4 to 36.4 mmHg, under hypoxic and normoxic conditions, respectively. Physiological normoxia thus corresponds to 5% and hypoxia to 2% oxygen in osteoclasts. Hypoxia in this range severely limits osteoclastogenesis, independent of energy metabolism and hypoxia-inducible factor activity. We observe that hypoxia decreases ten-eleven translocation (TET) activity. Tet2/3 cooperatively induces Prdm1 expression via oxygen-dependent DNA demethylation, which in turn activates NFATc1 required for osteoclastogenesis. Taken together, our results reveal that TET enzymes, acting as functional oxygen sensors, regulate osteoclastogenesis within the physiological range of oxygen tension, thus opening new avenues for research on in vivo response to oxygen perturbation.Nishikawa K., Seno S., Yoshihara T., et al. Osteoclasts adapt to physioxia perturbation through DNA demethylation. EMBO Reports 22, e53035 (2021); https://doi.org/10.15252/embr.202153035