SDF-1 expression in endothelial cells and regulation by VEGF.

HUVECs or human dermal microvascular endothelial cells were first starved of growth supplements by incubation in medium alone for 20 to 24 hours and then cultured in medium alone or in medium supplemented with VEGF. At the indicated time points, total RNA was extracted, and cell lysates or membrane extracts were prepared. (A) Semiquantitative RT-PCR analysis of SDF-1α and β expression in HUVECs cultured in medium alone, with VEGF (50 ng/mL), or with bFGF (25 ng/mL) for the indicated time periods. RNA preparations were tested by parallel RT-PCR amplification for G3PDH. (B) Western blot analysis of SDF-1 expression in HUVECs cultured in medium alone, with VEGF (50 ng/mL), or bFGF (25 ng/mL) and human dermal microvascular endothelial cells cultured with medium alone or VEGF (50 ng/mL) detected by affinity-purified rabbit antihuman SDF-1α antibodies. Loading accuracy was tested by membrane reprobing with antibodies to actin. (C) Flow cytometric analysis of surface and intracellular SDF-1 expression in HUVECs cultured for 24 hours in medium alone or with VEGF (20 or 50 ng/mL). Cells were stained with either a murine monoclonal antihuman–mouse SDF-1 antibody (IgG1, clone 79018.111) or isotype-matched control antibody (hybridoma clone 44716.14) followed by an FITC-labeled goat antimouse IgG antibody. (D) Western blot analysis of SDF-1 expression in membrane preparations of HUVECs cultured for 24 hours in medium alone or with VEGF (50 ng/mL) detected by rabbit anti–SDF-1 antibodies. Loading accuracy was verified by reprobing the membranes with anti-actin antibodies. Recombinant SDF-1 (5 ng) was run in parallel.