Figure 2
SDF-1α induces expression of active form of E-selectin ligands in EC.
(A) Increased expression of E-selectin ligand, CD44, in luminal EC of ischemic hindlimb wounds injected with γmSDF-1α compared to PBS injected non-wounded hindlimb in NOD mice (n = 8 mice/group). (A) right: Co-expression (yellow) of CD44 (green) and CD31 (red) in vessels was detected by immunostaining. Representative images are shown (images of isotype-matched non-specific control Ab are not shown). Left: Quantification of CD44 expression in vessels. Data are presented as mean ± SD of ratio of CD44:CD31 signals from 5 random selected sections of high power field (LPF, X 20) of each wound sample. CD31 signal was established as “1” in each section and relative amount of CD44 signal was normalized accordingly. (B) Immunoblotting analysis of SDF-1α-induced expression of three E-selectin ligands in HMVEC at various time points (γhSDF-1α: 100 ng/ml). β-actin is used as loading control. Experiments were repeated three times and similar results were obtained. (C) Binding of FITC-conjugated HECA452 to HMVEC stimulated with γhSDF-1α or BSA. Fluorescent signals were quantified (top). Representative fluorescent images were exhibited (bottom). (D) Binding of FITC-conjugated HECA452 to human EPC stimulated with γhSDF-1α or BSA (100 ng/ml). Fluorescent signals were quantified (top). Representative fluorescent images were exhibited (bottom). Data are analyzed by 2-tailed Student’s t-test and presented as mean ± SEM of fluorescent signals based on triplicate wells in each condition and totally three independent experiments in (C,D).
