👤 Seiichi Yoshimoto

Cholesterol efflux capacity (CEC) is robust biomarker for atherosclerotic cardiovascular disease (ASCVD). However, cell-based CEC assays require complex procedures that limit clinical use. The immobilized liposome-bound gel beads (ILG) method, a newly developed cell-free CEC assay, demonstrates sufficient performance for clinical application. This study investigated the clinical significance of CEC measured by the ILG method in relation to HDL subclasses and coronary artery plaque characteristics. We analyzed CEC and HDL parameters, including the ratio of apolipoprotein E (apoE)-HDL-C to HDL-C (%apoE) and HDL CEC correlated positively with HDL-C and %apoE. Among the patients, 26 (42.6%) exhibited large lipid-rich plaques on OCT. Univariable analysis showed that CEC was significantly lower in patients with large lipid-rich plaques compared to those without. While this association did not reach statistical significance after multivariable adjustment (p = 0.109), the addition of CEC to traditional risk factors improved the model's explanatory power (Nagelkerke R CEC measured using the ILG method reflects HDL subclass features and is associated with the burden of lipid-rich coronary artery plaques. These findings suggest the significance of CEC evaluated using the ILG method, supporting its potential for enhanced ASCVD risk assessment and further clinical applications. Show less

Alternative RNA splicing adds diverse variations to gene function, and its abnormalities are occasionally associated with the etiology of disease. We examined this possibility in pre-eclampsia. We performed transcriptome analysis of placentas from pre-eclamptic and normotensive pregnancies and screened for disease-specific aberrant splicing. We identified aberrant splicing at exon 14 in the ZC3H4 gene. This in-frame exon is generally skipped in placentas from normal pregnancies but often observed in those from pre-eclampsia patients. The level of exon inclusion did not correlate with disease severity, such as blood pressure or fetal weight, but showed an association with the decrease in placental weight. Significantly, placental blood flow resistance measured by Doppler ultrasound correlated with the level of ZC3H4 exon 14 inclusion, suggesting that this retention leads to the onset and/or symptoms of pre-eclampsia. ZC3H4 is known to act on transcriptional regulation via suppression of lncRNA expression. Moreover, the SOD1 gene, encoding superoxide dismutase that eliminates toxic free superoxide radicals, was identified in the downstream gene group for ZC3H4. Indeed, the expression of SOD1 was found in this current study to be decreased in the pre-eclamptic placenta in correlation with the levels of ZC3H4 exon 14 retention. Aberrant splicing of ZC3H4 gene may induce excessive oxidative stress in the placenta via the downregulation of downstream SOD1 expression thereby leading to the onset and development of pre-eclampsia. Show less

Perivascular epithelioid cell tumors (PEComas) rarely appear in the head and neck region. This case report describes two transcription factor E3 (TFE3)-rearranged PEComa cases, consisting of one in the orbit and one in the nasal cavity. Both cases demonstrated sheet-like or focal nested architecture and comprised epithelioid cells with abundant clear to eosinophilic cytoplasm and vascular stroma. The first case exhibited partial pleomorphism, a small necrosis area, and slightly increased mitosis and was classified as malignant. The second case demonstrated mild atypia and no mitosis or necrosis and was categorized as benign. The nasal tumor was initially considered a TFE3-rearranged renal cell carcinoma metastasis. However, a subsequent renal tumor biopsy revealed angiomyolipoma. The RNA sequence revealed ZC3H4::TFE3 and PRCC::TFE3 fusions in the first and second cases, respectively. The fusion partner gene ZC3H4 is uncommon, and this is the third reported PEComa case. The fusion partner gene PRCC is often reported in TFE3-rearranged renal cell carcinoma, and this PEComa case is the second reported in the head and neck region. The initially reported cases with the fusion partner genes ZC3H4 and PRCC were categorized as malignant. These cases were discussed with a literature review. Show less

Most organs are maintained lifelong by resident stem/progenitor cells. During development and regeneration, lineage-specific stem/progenitor cells can contribute to the growth or maintenance of different organs, whereas fully differentiated mature cells have less regenerative potential. However, it is unclear whether vascular endothelial cells (ECs) are also replenished by stem/progenitor cells with EC-repopulating potential residing in blood vessels. It has been reported recently that some EC populations possess higher clonal proliferative potential and vessel-forming capacity compared with mature ECs. Nevertheless, a marker to identify vascular clonal repopulating ECs (CRECs) in murine and human individuals is lacking, and, hence, the mechanism for the proliferative, self-renewal, and vessel-forming potential of CRECs is elusive. We analyzed colony-forming, self-renewal, and vessel-forming potential of ABCG2 (ATP binding cassette subfamily G member 2)-expressing ECs in human umbilical vessels. To study the contribution of In human and mouse vessels, ECs with higher These results are the first to establish that a single prospective marker identifies CRECs in mice and human individuals, which holds promise to provide new cell therapies for repair of damaged vessels in patients with endothelial dysfunction. Show less

It is thought that systemic sclerosis (SSc) might be a T helper 17 (Th17) cell-driven autoimmune disease. Noticeably, pulmonary arterial hypertension (PAH) is a leading cause of death in patients with SSc. Here, we investigated the association between serum Th17-related cytokines and prevalence of PAH in SSc patients. This study included 72 SSc patients and 51 healthy controls (HC). We determined clinical manifestations, immunophenotypes including Th subsets in peripheral blood lymphocytes, and the serum levels of interleukin (IL)-17A, IL-17A/F, IL-17B. IL-17C, IL-17D. IL-1β, IL-6, IL-21, IL-22, and IL-23. The frequency of Th17 cells was significantly increased in SSc patients compared to HC and was positively correlated with the modified Rodnan skin scores. Furthermore, the serum levels of IL-17A, IL-17D, IL-1β, and IL-6 were significantly increased in SSc patients compared to HC. SSc patients with detected IL-17A showed high levels of IL-17A/F, IL-1β, IL-6, and IL-22, and high frequency of Th17 cells. Interestingly, these patients exhibited the reduced lung functions and increased prevalence of PAH significantly compared to patients with undetected IL-17A. Similarly, SSc patients with detected IL-17A and high IL-6 (≥1.2 pg/mL) exhibited the decreased lung functions and increased prevalence of PAH compared to patients with undetected IL-17A and low IL-6. We found that SSc patients with high levels of serum IL-17A or both IL-17A and IL-6 show reduced lung functions and high prevalence of PAH. Consequently, it is highly probable that Th17/IL-17A axis is critical for the prevalence of PAH in SSc patients. Show less

Cancer cell resistance arises when tyrosine kinase inhibitor (TKI)-targeted therapies induce a drug-tolerant persister (DTP) state with growth via genetic aberrations, making DTP cells potential therapeutic targets. We screened an anti-cancer compound library and identified fibroblast growth factor receptor 1 (FGFR1) promoting alectinib-induced anaplastic lymphoma kinase (ALK) fusion-positive DTP cell's survival. FGFR1 signaling promoted DTP cell survival generated from basal FGFR1- and fibroblast growth factor 2 (FGF2)-high protein expressing cells, following alectinib treatment, which is blocked by FGFR inhibition. The hazard ratio for progression-free survival of ALK-TKIs increased in patients with ALK fusion-positive non-small cell lung cancer with FGFR1- and FGF2-high mRNA expression at baseline. The combination of FGFR and targeted TKIs enhanced cell growth inhibition and apoptosis induction in basal FGFR1- and FGF2-high protein expressing cells with ALK-rearranged and epidermal growth factor receptor (EGFR)-mutated NSCLC, human epidermal growth factor receptor 2 (HER2)-amplified breast cancer, or v-raf murine sarcoma viral oncogene homolog B1 (BRAF)-mutated melanoma by preventing compensatory extracellular signal-regulated kinase (ERK) reactivation. These results suggest that a targeted TKI-induced DTP state results from an oncogenic switch from activated oncogenic driver signaling to the FGFR1 pathway in basal FGFR1- and FGF2-high expressing cancers and initial dual blockade of FGFR and driver oncogenes based on FGFR1 and FGF2 expression levels at baseline is a potent treatment strategy to prevent acquired drug resistance to targeted TKIs through DTP cells regardless of types of driver oncogenes. Show less

The interleukin-6 (IL-6)/IL-12 family of cytokines plays critical roles in the induction and regulation of innate and adaptive immune responses. Among the various cytokines, only this family has the unique characteristic of being composed of two distinct subunits, α- and β-subunits, which form a heterodimer with subunits that occur in other cytokines as well. Recently, we found a novel intracellular role for one of the α-subunits, Epstein-Barr virus-induced gene 3 (EBI3), in promoting the proper folding of target proteins and augmenting its expression at the protein level by binding to its target protein and a well-characterized lectin chaperone, calnexin, presumably through enhancing chaperone activity. Because calnexin is ubiquitously and constitutively expressed but EBI3 expression is inducible, these results could open an avenue to establish a new paradigm in which EBI3 plays an important role in further increasing the expression of target molecules at the protein level in collaboration with calnexin under inflammatory conditions. This theory well accounts for the heterodimer formation of EBI3 with p28, and probably with p35 and p19 to produce IL-27, IL-35, and IL-39, respectively. In line with this concept, another β-subunit, p40, plays a critical role in the assembly-induced proper folding of p35 and p19 to produce IL-12 and IL-23, respectively. Thus, chaperone-like activities in proper folding and maturation, which allow the secretion of biologically active heterodimeric cytokines, have recently been highlighted. This review summarizes the current understanding of chaperone-like activities of EBI3 to form heterodimers and other associations together with their possible biological implications. Show less

Epstein-Barr virus-induced gene 3 (EBI3) is a subunit common to IL-27, IL-35, and IL-39. Here, we explore an intracellular role of EBI3 that is independent of its function in cytokines. EBI3-deficient naive CD4+ T cells had reduced IFN-γ production and failed to induce T cell-dependent colitis in mice. Similarly reduced IFN-γ production was observed in vitro in EBI3-deficient CD4+ T cells differentiated under pathogenic Th17 polarizing conditions with IL-23. This is because the induction of expression of one of the IL-23 receptor (IL-23R) subunits, IL-23Rα, but not another IL-23R subunit, IL-12Rβ1, was selectively decreased at the protein level, but not the mRNA level. EBI3 augmented IL-23Rα expression via binding to the chaperone molecule calnexin and to IL-23Rα in a peptide-dependent manner, but not a glycan-dependent manner. Indeed, EBI3 failed to augment IL-23Rα expression in the absence of endogenous calnexin. Moreover, EBI3 poorly augmented the expression of G149R, an IL-23Rα variant that protects against the development of human colitis, because binding of EBI3 to the variant was reduced. Taken together with the result that EBI3 expression is inducible in T cells, the present results suggest that EBI3 plays a critical role in augmenting IL-23Rα protein expression via calnexin under inflammatory conditions. Show less

The exchange of genetically engineered mouse strains between research facilities requires transporting fresh mouse sperm under refrigerated temperatures. Although sperm generally maintains fertility for 48 h at cold temperatures, in vitro fertilization rates of C57BL/6 mouse sperm are low after 48-h cold storage. Furthermore, 48 h is often not sufficient for the specimens to reach their destinations. To increase the availability of this technology, we aimed to extend the cold storage period while maintaining sperm fertility. In this study, we determined the optimal medium for sperm preservation and evaluated the effect of reduced glutathione in the fertilization medium on sperm fertility after cold storage. We found that higher fertility levels were maintained after 72-h cold storage in the preservation medium Lifor compared with storage in paraffin oil, M2 medium, or CPS-1 medium. In addition, 1.0 mM glutathione enhanced sperm fertility. After transporting sperm from Asahikawa Medical University to our laboratory, embryos were efficiently produced from the cold-stored sperm. After transfer, these embryos developed normally into live pups. Finally, we tested the transport system using genetically engineered mouse strains and obtained similar high fertilization rates with all specimens. In summary, we demonstrated that cold storage of sperm in Lifor maintains fertility, and glutathione supplementation increased the in vitro fertilization rates of sperm after up to 96 h of cold storage. This improved protocol provides a simple alternative to transporting live animals or cryopreserved samples for the exchange of genetically engineered mouse strains among research facilities. Show less

We previously studied fatty acid metabolism in the liver of nonalcoholic fatty liver disease (NAFLD) and reported the activation of the LXRalpha-SREBP-1c pathway in hepatocytes. LXRalpha regulates cholesterol metabolism as well as fatty acid metabolism, and its agonistic ligands are oxysterols. Moreover, there is some evidence that excess cholesterol intake is involved in the onset of NAFLD. Therefore, in this study, we examined the expression of cholesterol metabolism-associated genes in the NAFLD liver by real-time PCR. Expression of LXRalpha and ACAT1 was up-regulated in NAFLD and this was more noticeable in non-obese rather than in obese patients. Although the expression of the LDL receptor, which acts on cholesterol uptake, and of SREBP-2, a positive key regulator of cholesterol, was suppressed, the expression of enzymes that promote cholesterol synthesis was uniformly increased in NAFLD. Gene expression of apoB100 and microsomal triglyceride transfer protein, which are associated with VLDL secretion, and ABCG5, which is involved in cholesterol excretion, was significantly elevated in NAFLD. Because cholesterol accumulates in hepatocytes in NAFLD liver, cholesterol uptake and synthesis should be physiologically down-regulated. However, cholesterol synthesis was activated in NAFLD liver, meaning that cholesterol metabolism is dysregulated in NAFLD. Overproduction of cholesterol may lead to an increased level of oxysterols, activation of LXRalpha and SREBP-1c, and enhanced fatty acid synthesis. Show less