Inhibition of endothelial-cell adhesion, survival, and proliferation by Sema3A and reversal by VEGF165. (A) Representative images depicting Sema3A inhibition of endothelial-cell attachment to tissue culture wells. HUVECs were incubated (16 hours) in medium (MEDIUM199, 1% FBS) only, with Sema3A, VEGF165, VEGF121, with Sema3A plus VEGF165 or VEGF121. Cells were visualized under an Olympus IX51 phase-contrast microscope equipped with a 4 ×/0.13 PhL objective lens and a 10 × eyepiece (Olympus Optical, Melville, NY), and were photographed with a Retiga 1300 digital camera (Qimaging, Burnaby, BC, Canada). Images obtained via QCapture software (Qimaging) were imported into Adobe Photoshop 6.0 software (Adobe Systems, San Jose, CA) for processing. Original magnification, × 40. (B) Correlation between endothelial-cell attachment and viability. Endothelial cells were incubated (16 hours) in medium alone, with Sema3A, VEGF165, VEGF121, or with Sema3A plus VEGF165 or VEGF121. Cell viability was measured by absorbance at 450 nm after addition of Cell Counting Kit-8. The adherent cells were counted by NIH image analysis after removal of nonadherent cells. The results reflect the mean (± SD) of triplicate cultures (representative experiment of 3 performed). (C) Sema3A inhibits endothelial-cell proliferation. Cells were cultured for 3 days in medium alone, Sema3A, VEGF165, VEGF121, or with Sema3A plus VEGF165 or VEGF121. 3H thymidine uptake was measured during the final 20 hours of incubation. The results reflect the mean cpm of triplicate cultures (representative experiment of 3 performed).
