Figure 5.

Distending pressure transfection achieves siRNA delivery and MARCKS silencing in human saphenous vein. A) Left panel: triple staining of untransfected vein segments using confocal microscopy shows overlapping green and red autofluorescence of the elastic fibers with no blue Cy5 fluorescence. Right panel: vein segments transfected with Cy5 siRNA demonstrate discrete areas of blue fluorescence within all layers, independent of the green/red autofluorescence. Micrographs (×100) correspond to one representative experiment of 3 performed. B) Quantitative analysis of Cy5 fluorescence in confocal micrographs reveals significantly greater siRNA delivery to the endothelium (E), as compared to the media (M) or adventitia (A). n = 5 to 6 vein segments per condition. C) Left panel: distending pressure transfection with MARCKS siRNA reduced MARCKS mRNA levels at day 2, as measured by qRT-PCR. Data represent averages of 5 independent experiments. Right panel: Western blot analysis of whole-vein extracts recovered 5 days after transfection showed a reduction in MARCKS protein in vein segments transfected with MARCKS siRNA (MKS) vs. control siRNA (Con). Western blots correspond to 1 representative experiment of 10 performed. *P < 0.05.