Cigarette smoke regulates VEGFR2-mediated survival signaling in rat lungs

Abstract

Background: Vascular endothelial growth factor (VEGF) and VEGF receptor 2 (VEGFR2)-mediated survival signaling is critical to endothelial cell survival, maintenance of the vasculature and alveolar structure and regeneration of lung tissue. Reduced VEGF and VEGFR2 expression in emphysematous lungs has been linked to increased endothelial cell death and vascular regression. Previously, we have shown that CS down-regulated the VEGFR2 and its downstream signaling in mouse lungs. However, the VEGFR2-mediated survival signaling in response to oxidants/cigarette smoke (CS) is not known. We hypothesized that CS exposure leads to disruption of VEGFR2-mediated endothelial survival signaling in rat lungs.

Methods: Adult male Sprague-Dawley rats were exposed CS for 3 days, 8 weeks and 6 months to investigate the effect of CS on VEGFR2-mediated survival signaling by measuring the Akt/PI3-kinase/eNOS downstream signaling in rat lungs.

Results and discussion: We show that CS disrupts VEGFR2/PI3-kinase association leading to decreased Akt and eNOS phosphorylation. This may further alter the phosphorylation of the pro-apoptotic protein Bad and increase the Bad/Bcl-xl association. However, this was not associated with a significant lung cell death as evidenced by active caspase-3 levels. These data suggest that although CS altered the VEGFR2-mediated survival signaling in the rat lungs, but it was not sufficient to cause lung cell death.

Conclusion: The rat lungs exposed to CS in acute, sub-chronic and chronic levels may be representative of smokers where survival signaling is altered but was not associated with lung cell death whereas emphysema is known to be associated with lung cell apoptosis.

Figure 1

VEGFR2-PI-3K association in rat lungs exposed to CS. (A) A representative immunoblot picture of immunoprecipitated VEGFR2 probed for the p85 catalytic subunit of PI-3K after 3 days, 8 weeks and 6 months of CS exposure in rat lungs. The interaction of the p85 subunit of PI-3K was unaltered after 3 days and 8 weeks of CS exposure but was significantly increased after 6 months of CS exposure compared to sham-exposed animals (n = 6). (B) Histograms represent the Mean ± SE of percentage of VEGFR2/PI-3K association. ** p < 0.01 compared to sham-exposed animals.

Figure 2

Effect of CS on Akt phosphoryaltion. (A) A representative immunoblot picture of Akt phosphorylation after 3 days, 8 weeks and 6 months of CS exposure in rat lungs. Akt phosphorylation was significantly reduced both at 8 weeks and 6 months, but not after 3 days of CS exposure, compared to sham-exposed animals (n = 6). (B) Histograms represent the Mean ± SE of percentage of Akt phosphorylation. *** p < 0.001 compared to sham-exposed animals.

Figure 3

Effect of CS on Bad phosphorylation. (A) A representative immunoblot picture of Bad phosphorylation (Ser136) after 3 days, 8 weeks and 6 months of CS exposure in rat lung. Bad phosphorylation (Ser136) was unaltered after 3 days and 8 weeks but significantly decreased after 6 months of CS exposure compared to sham-exposed animals (n = 6). (B) Histograms represent the Mean ± SE of percentage of phosphorylated Bad levels. ** p < 0.01 compared sham-exposed animals.

Figure 4

Bad-Bcl-xl interaction in CS exposed rat lungs. (A) A representative immunoblot picture of immunoprecipitated Bcl-xl probed for Bad after 3 days, 8 weeks and 6 months of CS exposed rat lungs. Bad interaction with Bcl-xl was significantly increased at 3 days, 8 weeks and 6 months CS exposure compared to sham-exposed animals (n = 6). (B) Histograms represent the Mean ± SE of percentage of Bad interaction with Bcl-xl. ** p < 0.01 compared to sham-exposed animals.

Figure 5

Bcl-2 mRNA levels in CS exposed rat lung. (A) A representative RT-PCR picture of Bcl-2 mRNA levels after 3 days, 8 weeks and 6 months of CS exposure. Bcl-2 mRNA expression remains unaltered at all time point compared to sham-exposed animals (n = 6). (B) Histograms represent the Mean ± SE of percentage of Bcl-2 mRNA expression levels.

Figure 6

Phosphorylated and total eNOS levels in CS exposed rat lung. (A) A representative immunoblot picture of phosphorylated and total eNOS after 3 days and 8 weeks of CS exposure in rat lungs. Phophorylated and total eNOS levels were significantly reduced in 8 weeks, but not after 3 days of CS exposure compared to sham-exposed animals (n = 6). (B) Histograms represent the Mean ± SE of percentage of eNOS phosphorylation. *** p < 0.001 compared to sham-exposed animals.

Figure 7

Effect of CS smoke on active caspase 3 activation in rat lungs. (A) Representative pictures show the IHC staining of active caspase 3 at 3 days, 8 weeks and 6 months CS smoke-exposed rat lungs. Arrows represent cells positively stained for active caspase 3. (B) Graph representing positive cell counts in rat lungs. There was no difference in the number of positively stained cells between sham-exposed and CS-exposed animals after either 3 days, 8 weeks or 6 months of CS exposure in rat lungs (n = 6).

Figure 8

Hypothesized mechanism of CS-impaired VEGFR2-mediated survival signaling. CS decreases VEGFR2 levels thereby alters the survival signaling via PI-3K. Downregulation of VEGFR2-PI-3 K-Akt pathways will lead to reduction of eNOS and NO bioavailability as well as reduced survival signaling via increasing the Bad-Bcl-xL interaction.