A role for toll-like receptor-4 in pulmonary angiogenesis following multiple exposures to swine barn air

Abstract

Swine barn air is a heterogeneous mixture of dust, bacteria and irritant chemicals including ammonia and hydrogen sulphide. Gram-negative bacteria are commonly found in swine barn air and significantly contribute to pulmonary disease in unprotected swine barn workers, through the endotoxin moiety, lipopolysaccharide (LPS). Toll-like Receptor-4 is the ligand for LPS. It is found on many cell types including monocytes, macrophages, neutrophils, endothelial cells, and to a lesser extent, epithelial cells. The severity and outcome of acute lung injury following barn air exposures depends upon the balance between epithelial and vascular endothelial repair mechanisms, including angiogenesis. Vascular Endothelial Growth Factor (VEGF) is an endothelial mitogen produced by mesenchymal and alveolar Type II epithelial cells and by activated bronchial airway epithelial cells. Research investigating the role of cytokines in angiogenesis has shown that close proximity of immune cells and endothelial cells modulates the production of various compounds that regulate vascular function. Given that LPS is the ligand for TLR4 there appeared to be a role for TLR4 in angiogenesis, particularly following endotoxin exposure. To determine whether this was occurring, we examined whether exposure to swine barn air alters vascular density in the lungs and the role of TLR4 using a murine model. Toll-like Receptor-4 wild-type (C3HeB/FeJ) and TLR4 mutant (C3H/HeJ) mice were obtained and exposed to swine barn air for 1-, 5-, or 20-days for 8 hours/day. Wild-type animals showed a 127% increase in vascular density after 20-days barn air exposure. Vascular Endothelial Growth Factor-A protein levels were decreased by 0.62-fold after one-day swine barn air exposure in wild-type animals, indicating that VEGF-A is being used as a pro-angiogenic mitogen. Transcription of VEGF-A mRNA was increased in wild-type animals after all swine barn air exposure periods. The receptor VEGFR-1 showed increased mRNA transcription over all time points. These effects were only observed in TLR4 wild-type animals, indicating that these effects are mediated by TLR4. Further, VEGF-A and VEGFR-1 appear to be involved in the manifestation of TLR4-induced angiogenesis in the lung.