Figure 4.

The PMV-induced calcium oscillations in VSMCs and wound closure are abolished by the TRPV4 antagonist GSK219 but not by Nife. (A) The color images represent the time-lapse FRET images of the changes in cytoplasmic calcium upon PMV treatment of VSMCs pretreated with GSK219 (middle), Nife (bottom) and the DMSO control (top). The hot and cold colors represent high and low FRET ratios, indicating high and low levels of cytoplasmic calcium change, respectively. Scale bar: 30 μm. (B) The time courses represent the normalized FRET/ECFP ratio averaged over the cell body in VSMCs pretreated with GSK219 (n = 19, blue), Nife (n = 8, red) and DMSO (n = 12, orange) after PMV treatment, and all shadowed areas indicate the S.E.M. Comparation of the max normalized FRET/ECFP ratio (C) and frequency of cytoplasmic calcium oscillations (D) among VSMCs pretreated with GSK219 (n = 19, blue), Nife (n = 8, red) and DMSO control (n = 12, orange) after PMV treatment. (E) The migration of VSMCs pretreated with GSK219 (middle), Nife (bottom) and DMSO control (top) after PMV treatment. Scale bar: 200 μm. (F) The histogram shows the fold change in the level of GSK219- and Nife-pretreated VSMC migration relative to the that of DMSO control-pretreated VSMC migration. The values are shown as the mean ± S.E.M. for each condition (n = 6). * P < 0.05, ** P < 0.01. (G) The protein level of TRPV4 was increased after PMV treatment for 24 h. *** P < 0.001 vs. DMEM control (n = 5). (H) Immunofluorescence staining of TRPV4 (red) and nuclei (blue) of VSMCs after treatment with PMVs (top panel) or the DMEM control (bottom panel) for 5 min. Arrows indicate typical regions of dense and punctate TRPV4 staining. Scale bar: 40 μm. (I) TRPV4 inhibitor GSK219 attenuated neointimal hyperplasia in injured carotid arteries on Day 28 after the surgery. Scale bar: 50 μm.