Immunohistochemistry.

<p>Lung endothelial cells are labeled with anti-ERG antibody (green) and proliferating cells are marked with anti-Ki67 antibody (red). Double-stained Ki67-positive endothelial cells appear yellow (arrows). Representative sections of control and VEGF-treated lungs harvested on POD 2 (A) and 4 (B) are shown at 20X magnification. Topical VEGF treatment significantly increases endothelial proliferation on both POD 2 and 4 (C).</p

Morphometric analyses.

<p>Lungs of VEGF-treated mice demonstrate increased parenchymal volume (A) and a trend toward higher alveolar volume (B). Septal surface area (C) was higher in the VEGF group but there was no difference in mean septal thickness (D). VEGF-treated mice had increased total alveolar count (E). Data are expressed as mean ± SE.</p

Lung tissue protein expression analyses.

<p>ELISA reveals increased VEGF levels in VEGF-treated lungs (A) on POD 4. However, quantitative polymerase chain reactions (qPCR) show no difference in mRNA transcript levels of VEGF, VEGFR1, or VEGFR2 between the two groups (B). Immunoblot demonstrates an increase in the levels of P-VEGFR2, VEGFR2, P-EGFR, EGFR, and heparin-binding EGF-like growth factor (HB-EGF) with VEGF treatment (C-D). Data are expressed as mean ± SE.</p

Lung volume and functional assessments.

<p>Nasal instillation resulted in less systemic absorption of VEGF compared to intraperitoneal injection as measured by enzyme-linked immunosorbent assay (ELISA) (A). VEGF-treated mice display higher post-euthanasia lung volume on post-operative day (POD) 4 and 10 (B). There is a trend toward increased total lung capacity (C) and pulmonary compliance (D) with VEGF treatment on POD 4. Although not reaching statistical significance, mice in the VEGF group performed better on post-treadmill rest time (E), walking distance (F), basic movement count (G), and fine movement count (H) than the control mice on POD 10. Data are expressed as mean ± standard error of the mean (SE).</p

Co-culture assays of human bronchial epithelial cells (HBEC) and human lung microvascular endothelial cells (HMVEC-L).

<p>VEGF₁₆₅ at 10 ng/mL activated HMVEC-L (A). HMVEC-L treated with VEGF₁₆₅ show upregulation of proteases such as cathepsin B and V, matrix metalloprotease (MMP)-7, CD10, and kallikrein 13 (B) as well as a more than 6-fold increase in HB-EGF concentration in the conditioned medium (C). HBEC treated directly with VEGF show no increase in proliferation (D). However, in a co-culture model with HMVEC-L (E), proliferation increases when HBEC is co-cultured with VEGF-treated HMVEC-L (F). When an HB-EGF neutralizing antibody is added to co-cultured HBEC (G), proliferation decreases at higher concentrations of the antibody (H). Activation of EGFR on co-cultured HBEC is confirmed to decrease in the presence of an HB-EGF neutralizing antibody (I-J).</p