Figure 5

CTGF promotes HIF-1α-dependent VEGF expression and angiogenesis by inhibiting GPD1L expression and PHD2 hydroxylation activity by upregulation of miR-210. (a) Schematic 3′UTR representation of human GPD1L containing miR-210-binding site (upper panel). Cells were cotransfected with miR-210 mimic or inhibitor and wt-GPD1L-3′UTR or mt-GPD1L-3′UTR plasmid for 24 h followed by stimulation with CTGF, and relative luciferase activity was measured (lower panel). (b) OASFs were transfected with miR-210 mimic or inhibitor followed by CTGF stimulation, with GPD1L expression examined by RT-qPCR and western blot assay. (c) OASFs were transfected with miR-210 mimic or inhibitor followed by stimulation with CTGF, the expression of OH − HIF-1α (Pro564), PHD2, and HIF-1α in nucleus examined by western blot analysis. (d) OASFs were pretreated with HIF-1α inhibitor (10 μM) for 30 min followed by CTGF stimulation for 24 h, VEGF expression examined by RT-qPCR assay. (e and f) Medium was collected as CM, then applied to EPCs for 24 h. Cell capillary-like structure formation and migration in EPCs were examined by tube formation and Transwell assay. Results are expressed as mean±S.E.M. *P<0.05 compared with control; ^#P<0.05 compared with CTGF-treated group