Fig. 7: BAPN injection improves α-Klotho expression and cartilage integrity in aged mice.
A Principal component analysis (PCA) showing the separate clusters in ECM proteins from young and aged cartilage (n = 5/group). B Principal component 2 (PC2) in ECM protein expression is positively correlated with lamin A/C expression. C Heat map of the top 20 ECM proteins contributing to PC2, showing age-related ECM remodeling. Color indicates z score for each variable. Lox, an enzyme that induces collagen cross-linking, was in the top 20 proteins contributing to PC2 and was positively correlated with lamin A/C, supporting a connection between LOX-mediated increased collagen cross-linking and nuclear stiffness. D Schematic showing the experimental protocol for 4 weeks of daily BAPN injections in aged mice (n = 8 for saline, n = 9 for BAPN). E Schematic showing the atomic force microscopy measurement of murine medial tibial cartilage with 10 × 10 µm region of interest. F BAPN treatment significantly reduced Young’s modulus of medial tibial cartilage (n = 5/group). Young’s modulus data were transformed into Log MPa and normalized by Young’s modulus value in naive young group. G. Representative CellProfiler-generated nuclear images in murine medial tibial plateaus after 4-week injection of saline or BAPN. Nuclei were pseudocolored. White dashed lines indicate cartilage surface. Scale bar: 10 μm. BAPN injection in aged mice decreases nuclear eccentricity towards young level (n = 8 for saline, n = 9 for BAPN; 30–40 nuclei per individual mouse). H PCA showing the same cluster of young and aged+BAPN injection. I. BAPN injection in aged mice improves α-Klotho expression in articular cartilage quantified by immunofluorescence (n = 8 for saline, n = 9 for BAPN; 30–65 cells per individual sample). J BAPN injection in aged mice improves cartilage integrity (n = 8 for saline, n = 9 for BAPN). Representative histological sections stained with Safranin O/Fast Green are provided. Black arrows indicate loss of cartilage matrix. OARSI score (0–24 points; higher value indicates more severe cartilage degeneration) assessed by blinded assessor is provided. OARSI score in young cartilage is provided as a reference. K Beneficial effect of BAPN injection was blocked in Klotho+/− mice (n = 5 each for saline and BAPN). Statistical analyses were performed using linear regression analysis (B, C) or two-tailed Student t test (F, G, I–K). Data are presented as means ±95% confidence intervals. Portions of the figures were created with biorender.com. Source data are provided as a Source Data file.
