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Figure 4
Establishment and functional analysis of the RAMP2O/E line. (A) Plasmid vector used to overexpress RAMP2 (see Methods). (B) Western blot analysis of the membrane protein fraction from RAMP2O/E cells showing expression of the transfected gene. (C–E) Capillary formation by EAhy926 cells on Matrigel. RAMP2O/E cells or control ECs were cultured in 24-well culture plates coated with Matrigel in medium containing 10^–7 M AM, and capillary formation was monitored microscopically. (C) Capillary area relative to day-1 cell surface area. RAMP2O/E cells exhibited greater angiogenesis than control. n = 8 per group. (D and E) Representative photomicrographs of RAMP2O/E and control cells. (F) In vitro vascular permeability assay (see Methods). The permeability of the monolayer, assessed using a fluorescence microplate reader, is expressed relative to control at 5 min. RAMP2O/E cells showed significantly lower permeability than control ECs. n = 10 per group. *P < 0.05, **P < 0.01 vs. control. (G–J) Immunostaining of ZO-1. ECs were cultured until confluent on chamber slides in the presence of 10^-7 M AM. Two hours after treatment with 0.5 mM H₂O₂, the cells were immunostained using anti–ZO-1 antibody and Hoechst 33342. (K) Comparison of the tight junctions illustrated by the immunostaining in G–J. Tight junctions were better preserved after H₂O₂ treatment in RAMP2O/E cells than control ECs. **P < 0.01 vs. H₂O₂-treated control; comparison in 4 microscopic fields each from 3 independent experiments. (L) Quantitative real-time PCR analysis of gene expression in ECs cultured on Matrigel. Values are relative to control ECs treated with 10^–7 M AM. RAMP2O/E cells showed stronger expression of VEGF, eNOS, and CDN5 than control cells; this effect was blocked by LY294002 (10^-6 M) or a PKA inhibitor (10^-6 M). n = 6 per group. ^##P < 0.01 and ^#P < 0.05 vs. AM-treated control. **P < 0.01 and *P < 0.05 vs. AM-treated RAMP2O/E. Scale bars: 50 μm (D and E); 10 μm (G–J).