Hiroyuki Tomita

The endothelial glycocalyx (eGCX) plays a crucial role in mechanotransduction, maintaining vascular barrier integrity, and facilitating leukocyte–endothelial crosstalk. However, accurately quantifying it is challenging due to fragmented methodologies. As the eGCX is a highly hydrated, labile layer that readily collapses upon chemical fixation and dehydration, reported thickness values span orders of magnitude depending on the specific measurement principle employed. Consequently, relying on single-metric claims risks both false-positive and false-negative conclusions. Compounding this challenge, eGCX composition, morphology, and injury responses vary significantly across organs and vascular beds. This spatial heterogeneity is a factor that standard static in vitro cultures fundamentally fail to recapitulate. To overcome these limitations, we present a cross-platform evaluation framework organized around three orthogonal axes: structure (coverage, penetrability, and component localization), function (permeability and perfusion), and turnover (shedding versus synthesis). We delineate how each readout corresponds to a distinct physical sub-compartment, critically appraising methods that include confocal lectin staining, rapid-freeze electron microscopy (EM), atomic force microscopy (AFM), transendothelial resistance, intravital exclusion-zone imaging, perfused boundary region analysis, and circulating shedding biomarkers. For each platform, including in vitro, in vivo, and clinical settings, we propose minimal yet robust endpoint packages that integrate assay intent, organ context, and hemodynamic covariates. This approach shifts the field toward convergent, translatable inference for eGCX-targeted therapeutics. Beyond cataloguing techniques, the central contribution of this framework is the explicit mapping of each commonly used readout to the specific physical sub-compartment it interrogates, coupled with organ- and vascular-compartment-aware prioritization, so that convergent agreement among orthogonal structural, functional, and turnover measurements—rather than any single value—becomes the basis for falsifiable, translatable inference.

LDHA–Hedgehog signaling axis regulates the migration and invasion of human osteosarcoma cells. A, Protein expression of GLI1 in human osteosarcoma cell lines. B, IHC staining of human osteosarcoma specimens using an anti-GLI1 antibody. Representative cases with negative (left; case #16) and positive (right; case #14) GLI1 staining. Scale bars, 50 μm. C, Cell proliferation assay of SaOS-2-shCTL or shLDHA cells, assessed using CCK-8 (n = 8; independent experiments). The absorbance at 0 hour was set to 1. D, Transwell migration assay (without Matrigel) to assess migration capacity in SaOS-2-shCTL or shLDHA cells. Graph quantifying migration ability, showing the number of migrated cells after 24 hours (n = 3; independent experiments). Scale bars, 50 μm. E, Transwell invasion assay (without Matrigel) to assess migration capacity in SaOS-2-shCTL or shLDHA cells. Graph quantifying invasion ability, showing the number of invaded cells after 48 hours (n = 3; independent experiments). Scale bars, 50 μm. F, mRNA expression levels of LDHA mRNA and Hedgehog signaling–related genes in SaOS-2 cells with or without LDHA knockdown (n = 3; independent experiments). G, Protein expression levels of LDHA and Hedgehog signaling components in SaOS-2 cells with or without LDHA knockdown. Quantification of protein expression normalized to β-actin and represented in the bar graph (n = 3; independent experiments). H, Transwell migration assay (without Matrigel) evaluating the migration capacity of vismodegib-treated SaOS-2 cells. Graph quantifying migration ability, showing the number of migrated cells after 24 hours (n = 3; independent experiments). Scale bars, 50 μm. I, Transwell invasion assay (with Matrigel) assessing the invasion capacity of vismodegib-treated SaOS-2 cells. Graph quantifying invasion ability, showing the number of invaded cells after 48 hours (n = 3; independent experiments). Scale bars, 50 μm. Data are presented as the mean ± SEM. P values were calculated using an unpaired two-tailed Student t test (C–I). *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001 vs. shCTL or 0 μmol/L.

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

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

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.

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.

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