Expression of platelet-type 12-LOX in endothelial cells and its role in cell proliferation.
(A) RT-PCR detection of 12-LOX expression in endothelial cells. Total RNA was isolated and processed for double-round RT-PCR using primers designed on the basis of human platelet-type 12-LOX sequence as described in “Materials and Methods.” Control, no RNA present in PCR or RT reaction mixtures as controls for the quality of PCR; RT(−), no reverse transcriptase present in RT reaction mixtures as controls for the possible contamination of DNA in RNA samples; RT(+), reverse transcription present. (B) RT-PCR detection of platelet-type 12-LOX expression in RV-ECT cells. The primer combinations for 1st, 2nd, and 3rd are described in “Materials and methods.” The target sizes of the final PCR product from 1st, 2nd, and 3rd primer combinations are 111 bp, 62 bp, and 88 bp, respectively. (C) Immunoblot analysis of 12-LOX expression in endothelial cells. The blot was probed with a rabbit polyclonal antibody to human platelet-type 12-LOX. (D) Inhibition of VEGF-stimulated 12-LOX activity by BHPP. Cell treatment and measurement of 12(S)-HETE are detailed in “Materials and methods.” Columns, average levels of 12(S)-HETE per 1 × 106 cells (n = 3); bars, SE. a,P < .01 when compared with the unstimulated control; b,P < .05 when compared to the VEGF-stimulated cells. (E) Involvement of endothelial 12-LOX in bFGF- or VEGF-stimulated cell proliferation. HUVEC cells were plated in 96-well culture plates. Cell proliferation was stimulated with 10 ng/mL bFGF (open triangle) or 10 ng/mL VEGF (filled circle) in EBM-2 with 2% FBS. Cells with no bFGF or VEGF stimulation were used as controls (open circle). 48 hours after treatment with graded levels of BHPP, cell numbers were measured by an MTS method as described in “Materials and methods.” Data point, mean from quadruplicate determination; bars, SE from quadruplicate of treatment.
