👤 Michiaki Mukai

Epstein-Barr virus-induced 3 (EBI3) functions as a component of the heterodimer cytokine IL-27, which regulates innate and acquired immune responses. The expression of EBI3 gene is induced by Toll-like receptors (TLRs). Repeated treatment with imiquimod (IMQ), a TLR7 agonist, induces splenomegaly and cytopaenia due to increased splenic function. Although immune cell activation is speculated to play a role in chronic infection-mediated splenomegaly, the detailed mechanisms remain unknown. This study shows that IMQ treatment induces marked splenomegaly and severe bicytopaenia (anaemia and thrombocytopaenia) in wild-type mice. In IMQ-treated mice, myeloid cell populations in the spleen increased, and extramedullary haematopoiesis was observed. RNA-seq analysis revealed the upregulation of type I interferon (IFN)-related genes in the spleens of IMQ-treated mice. IMQ-induced pathological changes were partially mitigated by EBI3 deficiency. To investigate the mechanism of the improved phenotypes in the Ebi3 KO mice, we examined the involvement of IL-27, a heterodimer of EBI3 and IL-27p28. The expression of Il27a, which encodes IL-27p28, was increased in the spleen and peripheral blood by IMQ treatment. Furthermore, IL-27 stimulation upregulated type I IFN-related genes in bone marrow-derived macrophage cultures without type I IFN. These findings suggest that EBI3 deficiency mitigated IMQ-mediated pathological changes, presumably via a lack of IL-27 formation. Our study thus provides insights into the molecular mechanisms underlying chronic infection-mediated splenomegaly. Show less

Platelet-rich plasma (PRP), which is prepared by concentrating platelets in autologous blood, shows efficacy in chronic skin wounds via multiple growth factors. However, it exhibits heterogeneity across patients, leading to unstable therapeutic efficacy. Human induced pluripotent stem cell (iPSC)-derived megakaryocytes and platelets (iMPs) are capable of providing a stable supply, holding promise as materials for novel platelet concentrate-based therapies. In this context, we evaluated the effect of iMPs on wound healing and validated lyophilization for clinical applications. The growth factors released by activated iMPs were measured. The effect of the administration of iMPs on human fibroblasts and human umbilical vein endothelial cells (HUVECs) was investigated in vitro. iMPs were applied to dorsal skin defects of diabetic mice to assess the wound closure rate and quantify collagen deposition and angiogenesis. Following the storage of freeze-dried iMPs (FD-iMPs) for three months, the stability of growth factors and their efficacy in animal models were determined. Multiple growth factors that promote wound healing were detected in activated iMPs. iMPs specifically released FGF2 and exhibited a superior enhancement of HUVEC proliferation compared to PRP. Moreover, an RNA-seq analysis revealed that iMPs induce polarization to stalk cells and enhance ANGPTL4 gene expression in HUVECs. Animal studies demonstrated that iMPs promoted wound closure and angiogenesis in chronic wounds caused by diabetes. We also confirmed the long-term stability of growth factors in FD-iMPs and their comparable effects to those of original iMPs in the animal model. Our study demonstrates that iMPs promote angiogenesis and wound healing through the activation of vascular endothelial cells. iMPs exhibited more effectiveness than PRP, an effect attributed to the exclusive presence of specific factors including FGF2. Lyophilization enabled the long-term maintenance of the composition of the growth factors and efficacy of the iMPs, therefore contributing to stable supply for clinical application. These findings suggest that iMPs provide a novel treatment for chronic wounds. Show less

The epithelial-to-mesenchymal transition (EMT) is a critical process by which cancer cells acquire malignant features. However, the molecular mechanism and functional implications of EMT and the mesenchymal-to-epithelial transition (MET) in tumor progression remain elusive. In this study, we established two aggressive cancer cell lines from the human oral cancer cell line SAS, mesenchymal-like SAS-m4 and epithelial-like SAS-δ. SAS-δ is a revertant cell obtained by inducing MET in SAS-m4. SAS-δ, but not SAS-m4, exhibited abnormal cell growth, including piled-up overgrowth and invasive tumor formation in the tongues of nude mice, suggesting that SAS-δ represented more advanced cancer cells than the parental SAS cells. EMT-related transcriptional factor SLUG is phosphorylated at T208 and partly stabilized by the Hippo pathway kinases, LATS1 and LATS2. Depletion of SLUG promoted the invasive activity of SAS-δ by increasing the protein levels of LATS1/2 and the proportion of the phosphorylated form among total SLUG protein. Our results suggest that the LATS1/2-SLUG axis regulates the transition of SAS cells to the advanced stage via repeated switching between EMT and MET. Therefore, an anti-SLUG-pT208 antibody would be valuable not alone as a malignant tumor marker antibody but also as a prognostic tool for patients with malignant disease. Show less

Concentrations of circulating apolipoproteins are strongly linked to risk for coronary artery disease (CAD). The relative importance of the additional knowledge of apolipoprotein concentrations within specific lipoprotein species for CAD risk prediction is limited. This study sought to evaluate the performance of a high-density lipoprotein (HDL) apolipoproteomic score, based on targeted mass spectrometry of HDL-associated apolipoproteins, for the detection of angiographic CAD and outcomes. HDL-associated apolipoprotein (apo) A-1, apoC-1, apoC-2, apoC-3, and apoC-4 were measured in 943 participants without prevalent myocardial infarction (MI) referred for coronary angiography in the CASABLANCA (Catheter Sampled Blood Archive in Cardiovascular Diseases) study. A composite HDL apolipoproteomic score (pCAD) was associated with likelihood of obstructive CAD (≥70% lesion in ≥1 vessel) and with incident cardiovascular outcomes over 4-year follow-up. There were 587 (62.2%) patients with coronary stenosis. The pCAD score was associated with the presence of obstructive CAD (odds ratio: 1.39; 95% confidence interval [CI]: 1.14 to 1.69; p < 0.001), independently of conventional cardiovascular risk factors including circulating plasma apoA-1 and apoB. The C-index for pCAD was 0.63 (95% CI: 0.59 to 0.67) for the presence of obstructive CAD. Although pCAD was not associated with cardiovascular mortality among all individuals (hazard ratio: 1.24; 95% CI: 0.93 to 1.66; p = 0.15), there was evidence of association for individuals with obstructive CAD (hazard ratio: 1.48; 95% CI: 1.07 to 2.05; p = 0.019). An HDL apolipoproteomic score is associated with the presence of CAD, independent of circulating apoA-1 and apoB concentrations and other conventional cardiovascular risk factors. Among individuals with CAD, this score may be independently associated cardiovascular death. (The CASABLANCA Study: Catheter Sampled Blood Archive in Cardiovascular Diseases [CASABLANCA]; NCT00842868). Show less

The cytokine, interleukin-18 (IL-18), was originally identified as an interferon-γ-inducing proinflammatory factor; however, there is increasing evidence suggesting that it has non-immunological effects on physiological functions. We have previously investigated the potential pathophysiological relationship between IL-18 and dyslipidemia, non-alcoholic fatty liver disease and non-alcoholic steatohepatitis, which were mediated by lipid energy imbalance. Therefore, herein we focused on brown adipocytes (BAs) and brown adipose tissue (BAT) related to energy consumption as non-shivering thermogenesis. Il18 Compared with Il18 This study demonstrated the critical function of IL-18 in differentiation and lipid metabolism in BAs. Furthermore, IL-18 may contribute to novel treatments by improving the energy imbalance. Show less

Notch1 regulates binary cell fate determination and is critical for angiogenesis and cardiovascular development. However, the pathophysiological role of Notch1 in the postnatal period is not known. We hypothesize that Notch1 signaling in vascular smooth muscle cells (SMCs) may contribute to neointimal formation after vascular injury. We performed carotid artery ligation in wild-type, control (SMC-specific Cre recombinase transgenic [smCre-Tg]), general Notch1 heterozygous deficient (N1+/-), SMC-specific Notch1 heterozygous deficient (smN1+/-), and general Notch3 homozygous deficient (N3-/-) mice. Compared with wild-type or control mice, N1+/- and smN1+/- mice showed a 70% decrease in neointimal formation after carotid artery ligation. However, neointimal formation was similar between wild-type and N3-/- mice. Indeed, SMCs derived from explanted aortas of either N1(+/-)- or smN1+/- mice showed decreased chemotaxis and proliferation and increased apoptosis compared with control or N3-/- mice. This correlated with decreased staining of proliferating cell nuclear antigen-positive cells and increased staining of cleaved caspase-3 in the intima of N1(+/-)- or smN1+/- mice. In SMCs derived from CHF1/Hey2-/- mice, activation of Notch signaling did not lead to increased SMC proliferation or migration. These findings indicate that Notch1, rather than Notch3, mediates SMC proliferation and neointimal formation after vascular injury through CHF1/Hey2 and suggest that therapies that target Notch1/CHF1/Hey2 in SMCs may be beneficial in preventing vascular proliferative diseases. Show less

Gastric inhibitory polypeptide (GIP) is an incretin that potentiates insulin secretion from pancreatic beta-cells by binding to GIP receptor (GIPR) and subsequently increasing the level of intracellular adenosine 3',5'-cyclic monophosphate (cAMP). We have identified a novel GIPR splice variant in mouse beta-cells that retains intron 8, resulting in a COOH-terminal truncated form (truncated GIPR). This isoform was coexpressed with full-length GIPR (wild-type GIPR) in normal GIPR-expressing tissues. In an experiment using cells transfected with both GIPRs, truncated GIPR did not lead to cAMP production induced by GIP but inhibited GIP-induced cAMP production through wild-type GIPR (n = 3-4, P < 0.05). Wild-type GIPR was normally located on the cell surface, but its expression was decreased in the presence of truncated GIPR, suggesting a dominant negative effect of truncated GIPR against wild-type GIPR. The functional relevance of truncated GIPR in vivo was investigated. In high-fat diet-fed obese mice (HFD mice), blood glucose levels were maintained by compensatory increased insulin secretion (n = 8, P < 0.05), and cAMP production (n = 6, P < 0.01) and insulin secretion (n = 10, P < 0.05) induced by GIP were significantly increased in isolated islets, suggesting hypersensitivity of the GIPR. Total GIPR mRNA expression was not increased in the islets of HFD mice, but the expression ratio of truncated GIPR to total GIPR was reduced by 32% compared with that of control mice (n = 6, P < 0.05). These results indicate that a relative reduction of truncated GIPR expression may be involved in hypersensitivity of GIPR and hyperinsulinemia in diet-induced obese mice. Show less