Pulmonary function tests of mutant mice with reduced bioavailable VEGF indicate a restrictive lung disease.

<p>Plethysmography was performed at 12 weeks on anesthetized mutant mice and littermate controls. (A) Pressure volume curves of mutant mice were shifted downward compared to control mice. Data are expressed as mean ± SEM. (B, C) Mutant mice also demonstrated a 30% decrease in hysteresis and compliance. (D, E) They also showed a 1.5-fold increase in static elastance and a 2-fold increase in resistance, all indicative of a restrictive lung disease. All differences were statistically significant. Green bars represent mean values, **P<0.01, ***P<0.001, ****P<0.0001 compared to controls.</p

Summary of effects of VEGF sequestration leading to restrictive lung disease phenotype.

<p>Summary of effects of VEGF sequestration leading to restrictive lung disease phenotype.</p

Mesenchymal VEGF sequestration does not affect pulmonary angiogenesis but affects acinar air space morphogenesis.

<p>Western blot analyses of (A) total VEGFR-2 and phosphorylated VEGFR-2, and (B) endothelial markers PECAM-1 and VE-cad. No significant difference in expression between control and mutant mice was noted on either analysis. (C-D) Representative H&E images of peripheral lung sections from control and mutant mice demonstrate larger acinar air spaces in mutants. This was confirmed by mean linear intercept analysis. Images at 20x magnification, scale bars represent 100 μm.</p

Reduced VEGF disrupts the parenchyma around airways and vasculature.

<p>(A, B) Representative images of immunofluorescent staining for α smooth muscle actin (αSMA) reveals fewer putative myofibroblasts surrounding the airways in mutant mice (B) compared to controls (A). (C) Quantification by Western blot confirms a statistically significant decrease in the relative abundance of αSMA in mutants. (D, E) Representative images of Sirius red stain for Type 1 collagen demonstrates decreased peri-vascular staining in mutants (E) compared to controls (D). (F) Mutant mice express significantly less collagen around pulmonary veins based on optical density quantification. <i>White and black arrows</i> point to magnified areas shown in insets. Scale bars represent 50 μm. Data are expressed as mean ± SD, *P< 0.05, **P< 0.01 versus control.</p

VEGF sequestration decreases PI3K-Akt and MAPK-ERK pathway signaling.

<p>Western blot data for (A) PI3K, (B) phosphorylated and total Akt, and (C) phosphorylated and total ERK1/2 demonstrate decreased activation of these signaling pathways in mutants. (D) Western blot data also show decreased relative production of HIF-1α in mutant mice. (E) Mutant mice also express decreased relative levels of activated EGFR, which could further decrease surfactant production through its synergistic effect on VEGF expression. Data are expressed as mean ± SD, *P<0.05 vs control.</p