Effect of sodium lactate (NaLA) and lactic acid (LA) on Hedgehog signaling gene expression, migration, and invasion in LM8 osteosarcoma cells

Dupuytren's contracture is a superficial fibrotic disease of the hands that causes flexion contractures of the affected fingers. Diabetes mellitus (DM) is a risk factor for Dupuytren's contracture. However, the exact underlying mechanisms by which DM is involved in its development and progression remain unknown. This study investigated the involvement of glycometabolic disorders in the pathogenesis of Dupuytren's contracture. RNA sequencing revealed that S100A4 expression was significantly increased in Dupuytren's contracture-derived fibroblasts under high-glucose conditions compared with low-glucose conditions, and this finding was confirmed by immunoblotting and enzyme-linked immunosorbent assay. S100A4 expression in Dupuytren's contracture tissues was significantly higher in patients with diabetes than in those without. S100A4 was expressed in several cell types, including fibroblasts, myofibroblasts, and macrophages. However, the expression of its receptor, Toll-like receptor 4 (TLR4), was predominantly detected in CD68-expressing macrophages. Furthermore, recombinant S100A4 treatment significantly increased transforming growth factor-beta 1 (TGF-β1) expression, which is a central mediator of fibrosis, in macrophages. Pharmacological inhibition of TLR4 suppresses TGF-β1 upregulation via S100A4. Thus, the S100A4-TLR4-TGF-β axis could be a potential therapeutic target for Dupuytren's contracture in diabetic patients.

Full membrane images supporting Figs. 6A, 6G, and Supplementary Fig.6C

Vismodegib suppresses lung metastasis of murine osteosarcoma in vivo. A, Representative tumor images from vehicle (DMSO)- and vismodegib-treated mice subcutaneously implanted with LM8 cells. Scale bars, 10 mm. B, Tumor growth curves based on volume measurements at the indicated time points (n = 8 biological replicates per group). C, Bouin’s solution–fixed lung tissues from vehicle- and vismodegib-treated mice. Scale bars, 10 mm. D, Representative hematoxylin and eosin–stained lung sections from the vehicle- and vismodegib-treated groups. The top row at low magnification (scale bars, 1,000 μm) and the bottom at high magnification (scale bars, 50 μm). E, Quantification of metastatic area relative to total lung area in vehicle- and vismodegib-treated groups (n = 8 biological replicates per group). Data are presented as the mean ± SEM or median (IQR). P values were calculated using the Mann–Whitney U test (B and E). *, P < 0.05 vs. vehicle.

Abstract Osteosarcoma is the most common malignant bone tumor in children and adolescents, with a high propensity for lung metastasis, leading to poor prognosis. Lactate dehydrogenase A (LDHA), a key glycolytic enzyme, has been implicated in the progression and metastasis of various cancers. In this study, we demonstrated that Ldha knockdown reduced cell migration and invasion in murine osteosarcoma cells and decreased lung metastasis. RNA sequencing analysis revealed that Ldha knockdown was associated with the downregulation of Hedgehog signaling. Consistently, pharmacologic inhibition of Hedgehog signaling using vismodegib suppressed cell migration, invasion, and lung metastases in murine osteosarcoma cells. In human osteosarcoma cells, LDHA silencing downregulated Hedgehog signaling, and inhibition of Hedgehog signaling suppressed cell migration and invasion. These findings highlight the involvement of the Ldha–Hedgehog signaling axis in osteosarcoma lung metastases, providing insights into its molecular mechanisms and potential therapeutic targets. Significance: Ldha knockdown downregulates Hedgehog signaling and inhibits osteosarcoma lung metastasis, presenting a potential therapeutic target for osteosarcoma.

Images of tumors and lung metastasis of all mice implanted with LM8-shCTL or shLdha cells

Expression levels of epithelial–mesenchymal transition-related genes in LM8-shCTL, shLdha1, and shLdha2 cells

Primer sequences used in this study

Ldha knockdown increases the generation of reactive oxygen species

Vismodegib suppresses the proliferation, migration, and invasion of murine LM8 osteosarcoma cells in vitro. A, Wound healing assay to evaluate the migration capacity of vismodegib-treated LM8 cells. Scale bars, 200 μm. B, Quantification of wound healing rate from (A; n = 3; independent experiments). C, Transwell migration assay (without Matrigel) assessing migration capacity in vismodegib-treated LM8 cells. Scale bars, 50 μm. D, Quantification of migration ability from (C), showing the number of migrated cells after 24 hours (n = 3; independent experiments). E, Transwell invasion assay (with Matrigel) evaluating invasion capacity in vismodegib-treated LM8 cells. Scale bars, 50 μm. F, Quantification of invasion ability from (E), showing the number of invaded cells after 48 hours (n = 3; independent experiments). Data are presented as the mean ± SEM. P values were calculated using one-way ANOVA test (B, D, and F). *, P < 0.05; **, P < 0.01; ***, P < 0.001; and ****, P < 0.0001 vs. 0 μmol/L.

Expression of Hedgehog signaling genes in the clinical samples and cells of human osteosarcoma

LDHA knockdown leads to the downregulation of Hedgehog signaling. A, Volcano plot illustrating false discovery rate and log2 fold change in gene expression between LM8-shCTL and shLdha1 cells. B, Volcano plot displaying false discovery rate and log2 fold change in gene expression between LM8-shCTL and shLdha2 cells. C, Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis of significantly upregulated and downregulated genes from (A; LM8-shLdha1 vs. LM8-shCTL). The top 10 significantly enriched terms are displayed. D, KEGG pathway analysis of significantly upregulated and downregulated genes from (B; LM8-shLdha2 vs. LM8-shCTL). The top 10 significantly enriched terms are displayed. E, mRNA expression levels of Hedgehog signaling–related genes (n = 3; independent experiments). The expression level of shCTL was set to 1. F, Protein expression of Hedgehog signaling–related proteins in LM8-shCTL, LM8-shLdha1, and LM8-shLdha2 cells. Data are presented as the mean ± SEM. P values were calculated using one-way ANOVA test (E and F). *, P < 0.05; **, P < 0.01; ***, P < 0.001 vs. shCTL; ****, P < 0.0001.

Background Duloxetine is a potent analgesic that reduces pain associated with knee osteoarthritis. This study aimed to evaluate the analgesic effects of regular postoperative duloxetine after high tibial osteotomy (HTO) and its influence on knee function, pain severity, and daily life. Methods This open-label randomised controlled trial was designed as a single-centre, parallel-group study. Patients undergoing HTO were randomly allocated to the control and duloxetine groups. Patients allocated to the duloxetine group were administered oral duloxetine for the first 12 weeks postoperatively and were followed up for 6 months following surgery. The primary outcome was pain assessed by the visual analogue scale (VAS), and secondary outcomes included evaluations using the Brief Pain Inventory, the Knee Society Score, and the 2011 Knee Society Score. Results A total of 38 patients in the duloxetine group and 37 patients in the control group were analyzed. The duloxetine group demonstrated significantly lower pain VAS scores at rest 6 months postoperatively compared with the control group (0.5 ± 1.0 vs 1.3 ± 2.0, respectively; p = 0.029). There were no significant differences between the groups in the other scores. However, patients in the duloxetine group showed significantly lower consumption of acetaminophen in the third postoperative week (0.0 ± 0.0 vs 0.3 ± 0.8 tablets/day, respectively). Conclusions Duloxetine demonstrated a modest analgesic benefit following HTO, with reduced pain at rest at 6 months and decreased rescue medication use during the third postoperative week. Routine use after HTO may therefore not be warranted in unselected patients. Due to the open-label design, performance and detection bias cannot be excluded. Trial registration The study protocol was registered in the UMIN clinical trial registration before starting recruitments: UMIN000040026 (registration date: June 1, 2020) https://center6.umin.ac.jp/cgi-open-bin/ctr_e/ctr_view.cgi?recptno=R000045630 .

Effects of vismodegib and purmorphamine on LM8 and LM8-shLdha osteosarcoma cells

Objectives:: Life space (LS) is an important indicator of functional recovery after total hip arthroplasty (THA). In this study, we aimed to investigate the association between restoration of LS to preoperative levels 6 months after THA and perioperative improvements in walking performance, adjusting for demographic and clinical factors. Methods:: This retrospective cohort study included 104 patients with hip osteoarthritis undergoing primary unilateral THA between January 2019 and April 2023. Participants were classified into restoration and non-restoration groups based on whether their LS returned to preoperative levels at 6 months postoperatively. Logistic regression analysis was conducted with LS restoration as the dependent variable, perioperative changes in gait speed as the main explanatory variable, and preoperative gait speed and preoperative LS score as covariates. Model performance was assessed using the area under the receiver operating characteristic (ROC) curve. Results:: LS repair was associated with improved gait speed during the perioperative period [odds ratio (OR) 18.3, 95% confidence interval (CI) 1.44-233.0], high preoperative gait speed (OR 9.4, 95% CI 2.28-69.0), and low preoperative LS score (OR 0.967, 95% CI 0.94-0.99). The predictive model demonstrated good discriminatory ability (area under the ROC curve 0.791; 95% CI 0.699-0.883). Conclusions:: Perioperative gait speed improvement and high preoperative walking ability are major factors associated with LS restoration after THA. Conversely, patients with low preoperative LS may have a higher potential for restoration. These findings emphasize the importance of targeted interventions aimed at improving ability to promote community mobility and independence after THA.

Abstract Osteocytes are long-lived cells that play a central role in bone homeostasis, yet age-related changes in their functional states remain poorly understood, particularly because skeletal aging involves multiple processes beyond cellular senescence. We generated an osteocyte-specific Mepe Cre mouse line and combined osteocyte ablation in young and middle-aged mice with skeletal phenotyping, single-cell transcriptomics, and senolytic treatment. Mepe Cre -driven recombination was largely confined to osteocytes, with minimal off-target activity. Osteocyte ablation increased bone mass at both ages, indicating that osteocytes constrain bone accrual as part of their role in skeletal homeostasis. However, the accompanying remodeling changes differed with age: enhanced osteoblast activity predominated in young mice, whereas reduced osteoclast-mediated bone resorption predominated in middle-aged mice. Single-cell transcriptomics revealed distinct osteocyte subpopulations whose relative abundance shifted with age, from a predominantly matrix-enriched state in young mice to an expanded aging-transitional state in middle-aged mice. Although this state showed partial enrichment of senescence-associated transcriptional signatures, senolytic treatment failed to recapitulate the increase in bone mass induced by osteocyte ablation. Osteocyte therefore regulate bone mass through age-dependent mechanisms that coincide with shifts in osteocyte-state composition. These changes emerge by middle age and may contribute to early remodeling imbalance before overt cellular senescence during skeletal aging. Graphical Abstract Graphical summary of the findings of this study. AA, amino acids; NA, nucleic acid; UA, uric acid; TCA, tricarboxylic acid.