Yoko Itakura

Phenotypic switching between contractile (differentiated state) and proliferative (dedifferentiated state) vascular smooth muscle cells (VSMCs) is a hallmark of vascular remodeling that contributes to atherosclerotic diseases. Gangliosides, a group of glycosphingolipids, have been detected in atherosclerotic lesions and are suspected to contribute to the disease process. However, the underlying mechanism, specifically with respect to their role in VSMC phenotype switching, is not clear. In this study, we sought to reveal the endogenous expression of gangliosides and their functional significance in VSMCs during atherosclerosis. We found that switching from the contractile to proliferative phenotype was accompanied by upregulation of a- and b-series gangliosides, which in turn, were regulated by polycomb repressor complex 2 (PRC2). Downregulation of ganglioside expression using an siRNA targeting ST3GAL5, which is required for the synthesis of a- and b-series gangliosides, attenuated the proliferation and migration of dedifferentiated VSMCs. Therefore, we concluded that the increased expression of a- and b-series gangliosides via PRC2 activity during dedifferentiation is involved in the proliferation and migration of VSMCs. Gangliosides may be an effective target in VSMCs for atherosclerosis prevention and treatment.

The newly established mouse cortical-bone-derived stem cells (mCBSCs) are unique stem cells compared to mouse mesenchymal stem cells (mMSCs). The mCBSC-treated hearts after myocardial infarction have been reported to have greater improvement in myocardial structure and functions. In this study, we examined the stemness features, cell surface glycan profiles, and paracrine functions of mCBSCs compared with mMSCs. The stemness analysis revealed that the self-renewing capacity of mCBSCs was greater than mMSCs; however, the differentiation capacity of mCBSCs was limited to the chondrogenic lineage among three types of cells (adipocyte, osteoblast, chondrocyte). The cell surface glycan profiles by lectin array analysis revealed that α2-6sialic acid is expressed at very low levels on the cell surface of mCBSCs compared with that on mMSCs. In contrast, the lactosamine (Galβ1-4GlcNAc) structure, poly lactosamine- or poly N-acetylglucosamine structure, and α2-3sialic acid on both N- and O-glycans were more highly expressed in mCBSCs. Moreover, we found that mCBSCs secrete a greater amount of TGF-β1 compared to mMSCs, and that the TGF-β1 contributed to the self-migration of mCBSCs and activation of fibroblasts. Together, these results suggest that unique characteristics in mCBSCs compared to mMSCs may lead to advanced utility of mCBSCs for cardiac and noncardiac repair.

Purpose: Fibroblast growth factor receptor 4 (FGFR4), one of the four tyrosine kinase receptors for FGFs, is involved in various cellular processes. Activation of the FGF19/FGFR4 signaling is strongly associated with cancer development and progression. BLU9931 is an irreversible and highly selective small-molecule inhibitor of FGFR4. In this study, we aimed to elucidate the role of FGF19/FGFR4 signaling using BLU9931 in human PDAC. Experimental procedures: The expression of FGFR4 in eight human PDAC cell lines were examined using real-time PCR, immunoblotting, and FACS analysis. The effects of BLU9931 on FGFR4-positive PDAC cells were examined using growth assay, cell cycle assay, immunoblotting of downstream targets of FGF19/FGFR4 signaling, real-time PCR analysis of stemness markers, and sphere forming assays. Motility was evaluated using migration and invasion assays. Matrix metalloproteinase activity was evaluated using gelatin zymography. Induction of senescence was evaluated using senescence-associated β-galactosidase activity and real-time PCR analysis of senescence-associated, phenotype-related secretory genes. The synergistic effects of senolytic drugs on BLU9931-treated cells were evaluated using ATP assay. Expression and clinicopathological features of FGFR4 in PDAC patients were examined using immunohistochemical analysis of tissue microarrays. Results: In human PDAC cases, FGFR4 expression was positively correlated with larger primary tumors and more advanced disease stage. Among the eight PDAC cell lines, FGFR4 was expressed at the highest level in PK-1 cell line, in which the single-nucleotide polymorphism G388R in FGFR4 was detected. Inhibition of signal transduction through the ERK, AKT, and STAT3 pathways by BLU9931 reduced proliferation of FGF19/FGFR4 signaling-activated PDAC cells. In contrast, BLU9931 did not alter stemness features, including stemness marker expression, anticancer drug resistance, and sphere-forming ability of PK-1 cells. However, BLU9931 inhibited cell invasion, in part, by downregulating membrane-type matrix metalloproteinase-1 (MT1-MMP) expression in FGF19/FGFR4 signaling-activated PDAC cells. Furthermore, downregulation of SIRT1 and SIRT6 by BLU9931 contributed to senescence induction, priming these cells for quercetin-induced death, a process termed senolysis. Conclusions: We demonstrated that inhibition of signal transduction pathways through the ERK, AKT, and STAT3 pathways by BLU9931 inhibited PDAC cell proliferation and invasion, in part by downregulating MT1-MMP expression in autocrine/paracrine FGF19/FGFR4 signaling-positive PDAC cells. Furthermore, downregulation of SIRT1 and SIRT6 by BLU9931 may have contributed to senolysis in these cells. Thus, we propose that BLU9931 may be a promising drug for the treatment of FGFR4-positive PDAC. Citation Format: Norihiko Sasaki, Fujiya Gomi, Hisashi Yoshimura, Masami Yamamoto, Yoko Matsuda, Masaki Michishita, Hitoshi Hatakeyama, Yoichi Kawano, Yoko Itakura, Masashi Toyoda, Murray Korc, Toshiyuki Ishiwata. FGFR4 inhibitor BLU9931 induces cellular senescence in pancreatic ductal adenocarcinoma cell lines promoting sensitivity to senolytic therapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2449.

Insulin in physiological concentrations is important to maintain vascular function. Moreover, vascular insulin resistance contributes to vascular impairment. In the elderly, other factors including hypertension, dyslipidemia, and chronic inflammation amplify senescence of vascular endothelial and smooth muscle cells. In turn, senescence increases the risk for vascular-related diseases such as arteriosclerosis, diabetes, and Alzheimer’s disease. Recently, it was found that GM1 ganglioside, one of the glycolipids localized on the cell membrane, mediates vascular insulin resistance by promoting senescence and/or inflammatory stimulation. First, it was shown that increased GM1 levels associated with aging/senescence contribute to insulin resistance in human aortic endothelial cells (HAECs). Second, the expression levels of gangliosides were monitored in HAECs treated with different concentrations of tumor necrosis factor-alpha (TNFα) for different time intervals to mimic in vivo acute or chronic inflammatory conditions. Third, the levels of insulin signaling-related molecules were monitored in HAECs after TNFα treatment with or without inhibitors of ganglioside synthesis. In this review, we summarize the molecular mechanisms of insulin resistance in aged/senescent and TNFα-stimulated endothelial cells mediated by gangliosides and highlight the possible roles of gangliosides in vascular insulin resistance-related diseases.

Glycans are associated with and serve as biomarkers for various biological functions. We previously reported that cell surface sialylated glycoproteins of dermal fibroblasts decreased with cellular senescence and human aging. There is little information on the changes in glycoprotein expression and subcellular localization during the aging process. Here, we examined intracellular glycan profiles of fibroblasts undergoing cellular senescence and those derived from aging human subjects using lectin microarray analysis. We found a sequential change of the intracellular glycan profiles was little during cellular senescence. The intracellular glycans of cells derived from aged fetus and from elderly subjects showed similar localized patterns while repeating unsteady changes. The ratio of α2-3/2-6sialylated intracellular glycoproteins in total cell extracts increased, except for a part of α2-3sialylated O-glycans. These findings are in contrast to those for membrane glycoprotein, which decreased with aging. Interestingly, the ratio of increasing sialylated glycoproteins in the fetus-derived cells showing cellular senescence was similar to that in cells derived from the elderly. Thus, intracellular glycans may maintain cellular functions such as ubiquitin/proteasome-mediated degradation and/or autophagy during aging by contributing to the accumulation of intracellular glycosylated proteins. Our findings provide novel mechanistic insight into the molecular changes that occur during aging.

Chitin-binding lectins form the hevein family in plants, which are defined by the presence of single or multiple structurally conserved GlcNAc (N-acetylglucosamine)-binding domains. Although they have been used as probes for chito-oligosaccharides, their detailed specificities remain to be investigated. In this study, we analyzed six chitin-binding lectins, DSA, LEL, PWM, STL, UDA, and WGA, by quantitative frontal affinity chromatography. Some novel features were evident: WGA showed almost comparable affinity for pyridylaminated chitotriose and chitotetraose, while LEL and UDA showed much weaker affinity, and DSA, PWM, and STL had no substantial affinity for the former. WGA showed selective affinity for hybrid-type N-glycans harboring a bisecting GlcNAc residue. UDA showed extensive binding to high-mannose type N-glycans, with affinity increasing with the number of Man residues. DSA showed the highest affinity for highly branched N-glycans consisting of type II LacNAc (N-acetyllactosamine). Further, multivalent features of these lectins were investigated by using glycoconjugate and lectin microarrays. The lectins showed substantial binding to immobilized LacNAc as well as chito-oligosaccharides, although the extents to which they bound varied among them. WGA showed strong binding to heavily sialylated glycoproteins. The above observations will help interpret lectin-glycoprotein interactions in histochemical studies and glyco-biomarker investigations.

Fibroblasts are key players in maintaining skin homeostasis and in orchestrating physiological tissue repair and skin regeneration. Dysfunctions in fibroblasts that occur with aging and the senescent process lead to the delayed healing observed in elderly people. The molecular mechanisms leading to fibroblast dysfunction during aging and the senescent process have not yet been clarified. Previously, changes in patterns of glycosylation were observed in fibroblasts in aging and the senescent process, but the effect of these changes on the function of fibroblasts has not been well documented. Here, we investigated whether changes in glycosylation during the process to senescence may have functional effects on fibroblasts. The changes in cell surface glycans on skin fibroblasts during the process to senescence were examined in early-passage (EP) and late-passage (LP) skin fibroblasts by fluorescence-activated cell sorting analysis using lectins. The contributors to the changes in cell surface glycans were examined by real-time polymerase chain reaction or Western blot analysis. The effects of changes in glycosylation on proliferation, migration, induction of cellular senescence, and myofibroblast differentiation induced by transforming growth factor (TGF)-β1 stimulation were examined in EP fibroblasts. The changes in glycosylation were performed by GalNAc-α-O-benzyl or sialidase treatment. A decrease in sialylation of glycoproteins and an increase in sialidase NEU1 were observed in LP fibroblasts. The reduction of sialylation did not have any effect on proliferation, migration, or induction of cellular senescence. On the other hand, myofibroblast differentiation was inhibited by the reduction of sialylation, indicating that sialylation is important for myofibroblast differentiation. The localization of CD44 in lipid rafts, which is required for myofibroblast differentiation, was inhibited by the reduction of sialylation. Furthermore, reduced myofibroblast differentiation in LP fibroblasts was restored by a sialidase inhibitor. Desialylation of CD44 with increased sialidase during the process to senescence reduced the localization of CD44 in lipid rafts after TGF-β1 stimulation, leading to the inhibition of myofibroblast differentiation. Thus, regulation of sialylation may be an attractive strategy for the prevention and regenerative therapy of age-related skin diseases, cosmetic skin alterations, and chronic wounds caused by delayed healing in elderly people.

Glycans play essential roles in biological functions such as differentiation and cancer. Recently, glycans have been considered as biomarkers for physiological aging. However, details regarding the specific glycans involved are limited. Here, we investigated cellular senescence- and human aging-dependent glycan changes in human diploid fibroblasts derived from differently aged skin donors using a lectin microarray. We found that α2-6sialylated glycans in particular differed between elderly- and fetus-derived cells at early passage. However, both cell types exhibited sequentially decreasing α2-3sialylated O-glycan structures during the cellular senescence process and showed similar overall glycan profiles. We observed a senescence-associated decrease in sialylation and increase in galactose exposure. Therefore, glycan profiling using lectin microarrays might be useful for the characterization of biomarkers of aging.

Background: Cell-based regeneration therapies have great potential for application in new areas in clinical medicine, although some obstacles still remain to be overcome for a wide range of clinical applications. One major impediment is the difficulty in large-scale production of cells of interest with reproducibility. Current protocols of cell therapy require a time-consuming and laborious manual process. To solve this problem, we focused on the robotics of an automated and high-throughput cell culture system. Automated robotic cultivation of stem or progenitor cells in clinical trials has not been reported till date. The system AutoCulture® used in this study can automatically replace the culture medium, centrifuge cells, split cells, and take photographs for morphological assessment. We examined the feasibility of this system in a clinical setting. Results: We observed similar characteristics by both the culture methods in terms of the growth rate, gene expression profile, cell surface profile by fluorescence-activated cell sorting, surface glycan profile, and genomic DNA stability. These results indicate that AutoCulture® is a feasible method for the cultivation of human cells for regenerative medicine. Conclusions: An automated cell-processing machine will play important roles in cell therapy and have widespread use from application in multicenter trials to provision of off-the-shelf cell products.