(A) Representative phase-contrast and immunofluorescence images of cultured BM-derived ECs from young and aged mice. Scale bars: 200 μm (phase-contrast) and 50 μm (immunofluorescence). (B) Representative flow plots of cultured ECs stained for VECAD⁺CD31⁺ demonstrating highly purified EC populations. (C–E) AFM analysis of elasticity in cultured young and aged ECs showing an increase in aged EC stiffness. (C) Representative reconstructed images of EC monolayers. (D) Box plots of the median stiffness in cultured young and aged ECs (n = 3 biological replicates). (E) Normalized relative EC stiffness (n = 3 biological replicates). (F and G) Proliferation status of cultured young and aged ECs. (F) Representative histograms of Edu incorporation following cell-cycle synchronization. (G) Quantification of Edu incorporation demonstrating an early inhibition of cell-cycle entry into the S phase in aged ECs that was resolved by 24 hours (n = 3 biological replicates). (H) Quantification of SA β-gal activity in young and aged ECs (n = 3 biological replicates). (I and J) In vitro scratch wound-healing assay showing a functional delay in cell migration in aged ECs. (I) Representative phase-contrast images (dashed lines demarcate the initial scratch wound). Scale bar: 400 μm. (J) Quantification of EC wound healing (n = 3 biological replicates). (K) Normalized gene expression in cultured young and aged ECs (n = 3 biological replicates). *P < 0.05, **P < 0.01, and ***P < 0.001. Significance was determined using an unpaired, 2-tailed Student’s t test, with error bars representing the sample mean ± SEM. A nonparametric, 1-sided Wilcoxon rank-sum test was used to compare median endothelial stiffness in D. Data are presented as box plots, with whiskers representing an IQR of ± 1.5. Relative endothelial stiffness in E was normalized to young ECs and presented as a 95% CI.