Figure 2. nAChR mediated endothelial cell proliferation and survival.
To assess the role of the homomeric nAChRs in EC proliferation and survival, siRNA methodology was employed. HMVEC were transfected with siRNA against the α7 or α9 nAChR subunits or scrambled siRNA or exposed to transfection vehicle without siRNA, for 72 hours. (A) Gene silencing of the specific nAChR was validated by RT-PCR. Total RNA was isolated and subjected to RT-PCR. The data represent 3 different experiments carried out in triplicate. The values are expressed as relative fold change of each condition vs. control without siRNA with error bars showing SEM (p<0.05).
(B) Gene silencing of the specific nAChR was validated by Western immunoblots for α7 or α9 nAChR and β-Actin expression.
(C) Effect of siRNA on EC proliferation. The proliferation assay utilized BrdU incorporation. Nicotine stimulated HMVEC proliferation, an effect that was abrogated by siRNA against α7 nAChR. By contrast, siRNA against α9 nAChR increased basal EC proliferation. Results are shown as fold change (mean±SEM of 3 different experiments each carried out in triplicate) compared to the nonstimulated control cells. *p<0.001 vehicle versus nicotine treated cells. # p<0.05, vs. non-transfected cells exposed to same stimulus.
(D) Effect of siRNA on EC survival. Cells were first transfected with siRNA as described in (A). Subsequently, serum free conditions were used to induce apoptosis for 24 hours, then either vehicle or nicotine(10−10M) was added to the medium and the cells were further cultured for another 24 hours. The cell survival assay is described in Materials and methods. *p<0.05 vehicle versus nicotine treated cells. # p<0.05, vs. non-transfected cells exposed to same stimulus.