Nestin overexpression promotes endothelial proliferation.

(A) Co-immunofluorescence staining of PCNA and Nestin reveals multiple Nestin+ PCNA+ cells (arrow) in the intima of a concentric lesion from a PAH patient. In contrast, no Nestin+ PCNA+ cells were detected in the intima of a pulmonary artery from a control subject. Scale bars: 50 μm (overview), 25 μm (detail images). For PAH, the image on the left shows an overview of the blood vessel, whereas the images in the middle demonstrate orthogonal views of z-stacks from the area indicate by a dotted box. The image on the right is an intensity projection of the whole z-stack. (B) Transient Nestin overexpression in rat lung ECs 72h after adenoviral transduction (qRT-PCR). β2-microglobulin (B2m) was used as housekeeping gene. n = 6 per group. (C-D) Increased BrdU incorporation over 4 h following Nestin overexpression in rat lung ECs (72h after adenovirus inoculation). (C) Representative histograms and (D) quantification. n = 6 per group. (E) Transient Nestin overexpression (qRT-PCR) in human control PAECs 72h after adenovirus transduction (qRT-PCR). TATA-binding protein (TBP) was used as housekeeping gene. (n = 4 per group). (F) qRT-PCR of MKI67 (Ki-67, a proliferation marker) and CXCL12 in hPAECs 72h after AdNES or AdDL70 treatment. n = 4 per group. All data shown as mean+SEM. *PPG) Representative Western blots demonstrating the transgenic Nestin protein expression and elevated CXCL12 protein expression in PAECs 72h after adenoviral transfection with AdNES vs. AdDL70. β-actin was used as loading control.