Angiotensin II increases PlGF mRNA expression (A, B) and protein production (C, D) in EA.Hy 926 endothelial cells and HUASMCs. Growth-arrested EA.Hy 926 cells and HUASMCs were stimulated with Angiotensin II (10-6 mol/L) for different durations. (A) Quantitative RT-PCR (Q-PCR) results. Total RNA was isolated from cells. After reverse transcription, they were subjected to quantitative PCR analysis to determine PlGF mRNA level. Graph is representative of relative PlGF mRNA levels in the various conditions. Experiments were performed five times with the similar results (n = 5 in each group). * indicates P < 0.05 vs control EA.Hy 926 cells or HUASMCs. (B) Induction of PlGF mRNA expression is Ang II concentration-dependent. * indicates P < 0.05 vs control EA.Hy 926 cells or HUASMCs. (C) Release of PlGF protein measured by ELISA. Cells were incubated with Ang II (10-6 mol/L) for 8, 24 or 48 h, and PlGF protein released into cell culture media was measured by ELISA (n = 3 in each group). * indicates P < 0.05 vs control EA.Hy 926 cells or HUASMCs. (D) Expression of PlGF protein detected by immunofluorescence assay. Immunofluorescence detecting of PlGF expression in untreated control cells, and cells stimulated with Ang II (10-6 mol/L) for 24 hours (original magnification of 200 ×). Mean optical density (MOD) of PlGF staining was measured on the images by using the ImageJ software. The figure shows one of three similar experiments. * P < 0.05 vs control EA.Hy 926 cells or HUASMCs.