Inhibition of VEGF receptors causes lung cell apoptosis and emphysema

Abstract

Pulmonary emphysema, a significant global health problem, is characterized by a loss of alveolar structures. Because VEGF is a trophic factor required for the survival of endothelial cells and is abundantly expressed in the lung, we hypothesized that chronic blockade of VEGF receptors could induce alveolar cell apoptosis and emphysema. Chronic treatment of rats with the VEGF receptor blocker SU5416 led to enlargement of the air spaces, indicative of emphysema. The VEGF receptor inhibitor SU5416 induced alveolar septal cell apoptosis but did not inhibit lung cell proliferation. Viewed by angiography, SU5416-treated rat lungs showed a pruning of the pulmonary arterial tree, although we observed no lung infiltration by inflammatory cells or fibrosis. SU5416 treatment led to a decrease in lung expression of VEGF receptor 2 (VEGFR-2), phosphorylated VEGFR-2, and Akt-1 in the complex with VEGFR-2. Treatment with the caspase inhibitor Z-Asp-CH(2)-DCB prevented SU5416-induced septal cell apoptosis and emphysema development. These findings suggest that VEGF receptor signaling is required for maintenance of the alveolar structures and, further, that alveolar septal cell apoptosis contributes to the pathogenesis of emphysema.

Figure 1

Histology of rat lungs. (a) Section of lung from a control rat showing normal alveolar structure. (b and c) Sections of lungs from an SU5416-treated rat showing enlarged airspaces. (d) Section of lung from a rat treated with SU5416 plus Z-Asp-CH₂-DCB (caspase inhibitor) showing normal alveolar structure. Hematoxylin-and-eosin staining was used. Scale bars, 100 μm. (e) Emphysema in SU5416-treated lungs assessed by mean linear intercept. There were significantly greater mean linear intercept values in the SU5416-treated rat lungs (n = 6) when compared with the lungs from control rats (n = 6). Treatment with Z-Asp-CH₂-DCB (n = 6) prevented the increase in mean linear intercept in SU5416-treated rats. ^AP < 0.05 (one-factor ANOVA, Scheffe F-test).

Figure 2

(a) Barium-gelatin angiograms of the left lungs of three SU5416-treated rats (below) compared with three control rat lungs (above). Barium filling was reduced in SU5416-treated rat lungs compared with control rat lungs. (b) Peripheral arterial density quantification of control and SU5416-treated lungs (^AP < 0.05).

Figure 3

(a) Caspase 3–like activity in lungs during the SU5416 treatment. SU5416 treatment led to progressive increase of caspase 3–like activity in the rat lungs. (b) Comparison of caspase 3–like activity in lungs after 3 weeks of treatment. Caspase 3–like activity in SU5416-treated rat lungs (n = 6) was significantly higher than that of control rat lungs (n = 6), whereas lungs from rats treated with SU5416 + Z-Asp-CH₂-DCB (n = 6) showed no increase in apoptotic activity. ^AP < 0.05 (one-factor ANOVA, Fisher’s protected least significant difference test).

Figure 4

Immunohistochemistry for active caspase 3 counterstained with methyl green. (a) Control rat lung, showing specific staining for caspase 3 in the alveolar septa (arrow). (b) SU5416-treated rat lung, showing an increase of positive cells in the alveolar septa (arrow). (c) SU5416 plus Z-Asp-CH₂-DCB–treated rat lung. (d) SU5416-treated rat lung. Positive cells (arrow) are observed in the endothelial cell layer of the pulmonary artery. Original magnifications, a–d: ×600.

Figure 5

Immunohistochemistry for PCNA counterstained with hematoxylin, showing positive staining (brown) for proliferating cells in the intra-alveolar space and alveolar septa. (a) Control rat lungs. (b) SU5416-treated rat lungs. Original magnifications, a and b: ×400.

Figure 6

Lung sections stained by the TUNEL technique, counterstained with methyl green. (a) Control rat lungs, showing specific staining for DNA strand breaks in the intra-alveolar cells (arrows). (b) SU5416-treated rat lungs show abundant staining of cells in the alveolar septa (arrows). Original magnifications, a and b: ×400.

Figure 7

In situ ligation of labeled DNA fragments. (a) Control rat lung. (b) SU5416-treated rat lung. Positive reactions (arrows) were seen in alveolar septa. Original magnifications, a and b: ×600.

Figure 8

(a) Detection of internucleosomal ladders using LM-PCR ladder assay. Internucleosomal ladders were observed in lung samples obtained from SU5416-treated rats. M, 200-bp DNA size markers; P, positive control calf thymus DNA; H, no DNA in H₂O. Control samples consist of vehicle-treated lungs. (b) Densitometric units of internucleosomal DNA ladders in control (n = 2), and SU5416-treated lungs for 3 (n = 3), 7 (n = 3), and 21 days (n = 3). Results are shown as mean ± SE and are significant at P < 0.01 by the single-factor ANOVA.

Figure 9

(a) Expression of VEGFR-2 in lungs of rats treated with vehicle (control); SU5416 for 3, 7, or 21 days; or SU5416 + Z-Asp-CH₂-DCB for 21 days. Each data point represents the average of expression in two lungs per experimental group. The bands were quantitated by densitometry, followed by normalization for loading and determination of protein integrity using β-actin expression. (b) Assessment of phosphorylated (active) VEGFR-2 in lungs of rats treated with vehicle (control); SU5416 for 3, 7, or 21 days; or SU5416 + Z-Asp-CH₂-DCB for 21 days, by immunoprecipitation with anti–VEGFR-2 antibody and Western blot for VEGFR-2 and its phosphorylated form. Each data point represents the average of expression in two lungs per experimental group, and the data are expressed as the ratio of phosphorylated VEGFR-2 to nonphosphorylated VEGFR-2. (c) Assessment of Akt-1 in the complex with VEGFR-2 performed by immunoprecipitation with anti–VEGFR-2 antibody, and Western blot for VEGFR-2 and Akt-1. Data are expressed as the ratio of Akt-1 to VEGFR-2. (d) Assessment of PI3 kinase in the complex with VEGFR-2 performed by immunoprecipitation with anti-VEGFR-2 antibody, and Western blot for VEGFR-2 and PI3 kinase. Data are expressed as the ratio of PI3 kinase to VEGFR-2.