Figure 6. Decreased PTEN and αSMA expression in coronary arteries exposed to CF-LVADs.

Coronary arteries from explanted hearts of non–left ventricular assist device (non-LVAD) or continuous-flow LVAD (CF-LVAD) patients were immunofluorescently stained for α-smooth muscle actin (αSMA) (A) and PTEN (C). (A and C) Representative confocal images of stained vessels showing the arterial media (M). Scale bars: 100 μm. (B) Relative area of αSMA expression for the arterial media was measured by ImageJ and averaged from 4 or 5 confocal images (original magnification, ×63) per vessel. (D) Relative area of PTEN staining was determined by ImageJ as described in B. Non-LVAD: N = 13 individual vessels from 7 independent hearts; CF-LVAD: N = 12 individual vessels from 9 independent hearts. Horizontal lines show the mean ± SD. Significant P values are shown, as determined by 2-tailed Student’s t test. (E) Left: Relative area of DAPI expression in the media per vessel was measured by ImageJ and averaged from 4 or 5 confocal images (original magnification, ×63) of media per vessel. Non-LVAD: N = 13 individual vessels; CF-LVAD: N = 12 individual vessels. Middle and Right: The mean gray value within cell boundary ROI for single-cell analysis of medial smooth muscle cells for αSMA (middle) and PTEN (right) levels was determined using ImageJ, as described in Figure 3. Nonatherosclerotic hyperplasia (NAH): N = 180 individual cells from 8 vessels and 5 independent hearts; LVAD: N = 217 individual cells from 7 vessels and 6 independent hearts. ^#P ≤ 0.0001 by Mann-Whitney 2-tailed t test. Plotted data include the median gray value (horizontal bar), interquartile range (box boundary), and minimum to maximum range of data values (vertical bar).