TGF-beta inhibition to promote lung growth in mechanically ventilated newborn mice: a novel strategy to prevent ventilator-induced lung injury
Final Report Abstract
Prolonged mechanical ventilation with O2-rich gas (MV-O2) leads to failed formation of alveoli and pulmonary micro-vessels, resulting in lung growth arrest as seen in neonatal chronic lung disease (CLD). Reports of premature infants with evolving CLD and of newborn mice exposed to MV-O2 show that increased TGFβ and reduced VEGF signalling are linked to lung cell apoptosis. Studies of the Bland laboratory demonstrated that TGFβ blockade and inhibition of elastase activity using the serineelastase inhibitor elafin prevented those changes, thereby enabling lung growth. Other studies showed that MV-induced lung injury is linked to aberrant epidermal growth factor (EGF) receptor signalling. The overall goal of the research fellowship was to identify mechanisms by which TGFβ blockade or elafin treatment protects against MV-induced lung injury. This project had four specific hypotheses: (1) TGFβ inhibition preserves pro-angiogenic signalling and formation of micro-vessels in lungs of newborn mice during MV-O2; (2) cyclic stretch triggers TGFβ signalling in cultured human pulmonary micro-vascular endothelial cells (HPMVEC), which in turn adversely affects endothelial cell homeostasis; (3) MV-O2 disrupts EGFR signalling in lungs of newborn mice; and (4) elafin preserves EGFR activation during prolonged MV-O2, suppresses apoptosis and promotes lung growth. (1) We showed that prolonged MV-O2 increases phosphorylation of Smad2, reduces expression of VEGFR-2 and increases tropoelastin in lungs of newborn mice. Treatment with a neutralizing antibody to TGFβ (TGFβ-nAb), however, attenuated both the decrease in VEGF-R2 and the increase in tropoelastin that occurred during MV-O2 of IgG-treated (control) pups. Protein abundance and phosphorylation of VEGFR-2 were greater in mechanically ventilated pups that had been treated with TGFβ-nAb than in those treated with IgG. The preserved alveolar formation shown previously by the Bland laboratory was accompanied by prevention of the 30% decrease in micro-vessels after TGFβ inhibition during MV-O2. (2) We linked cyclic stretch to increased TGFβ activity in cultured HPMVEC by showing that cyclic stretch for up to 24h induces pSmad2. Moreover, exposure of cultured HPMVEC to active TGFβ1 showed increased apoptosis and reduced migration and tube formation. Gene expression and protein abundance of VEGFR-2 were markedly reduced by TGFβ1, demonstrating a pivotal role of TGFβ in regulating VEGF signalling. (3) MV-O2 increased lung elastase activity, inhibited EGFR activation and downstream Akt signalling, and resulted in increased apoptosis in lungs of newborn mice. Since total EGFR and EGFR ligands, such as amphiregulin, were increased in mechanically ventilated lungs, reduced EGFR-pAkt was not the result of down-regulated EGFR or lack of ligand. (4) Treatment with elafin prevented these changes. Moreover, exposure of mouse lung epithelial cells (MLE-12) to elafin activated EGFR and suppressed apoptosis associated with EGFR inhibition. Major findings: TGFβ inhibition during MV-O2 blocks the adverse effects of TGFβ signalling on VEGFR-2 and promotes micro-vascular formation in lungs of newborn mice. Elafin promotes lung growth during MV-O2 in two ways: first, it does so by inhibiting elastase activity; and second by preserving EGFR signalling. This could account for elafin’s benefit in suppressing apoptosis and lung growth in mechanically ventilated lungs. These findings identify novel mechanisms by which lung growth could be enhanced during prolonged MV, while forging new approaches for developing novel strategies to treat or prevent CLD.
Publications
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Elafin inhibits elastase activity, preserves epithelial growth factor (EGF) signaling and prevents apoptosis in lungs of mechanically ventilated newborn mice. Pediatric Academy Societies’, 2014, Vancouver; 3665.7
Alejandre Alcazar MA, Kaschwich M, Preuss S, Mujahid S, Ertsey R, Rabinovitch M, Bland RD
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Lung matrix and vascular remodeling in mechanically ventilated elastin haplo-insufficient (Eln+/-) newborn mice. Am J Physiol Lung Cell Mol Physiol. 2014 Dec 24
Hilgendorff A, Parai K, Ertsey R, Navarro EF, Jain N, Carandang F, Peterson J, Mokres LM, Milla C, Preuss S, Alejandre Alcazar MA, Khan S, Masumi J, Ferreira-Tojais N, Mujahid S, Starcher BC, Rabinovitch M, Bland RD
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Tgfβ blockade preserves vascular endothelial growth factor (VEGF) signaling and enables alveolar formation in mechanically ventilated newborn mice. American Thoracic Society International Conference; Am J Respir Crit Care Med 189;2014:A2323
Alejandre Alcazar MA, Parai K, Hilgendorff A, Preuss S, Kaschwich M, Ertsey R, Mokres L, Navarro EF, Rabinovitch M, Bland RD
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The relationship between serial [(18)F]PBR06 PET imaging of microglial activation and motor function following stroke in mice. Mol Imaging Biol. 2014 Dec;16(6):821-9
Lartey FM, Ahn GO, Ali R, Rosenblum S, Miao Z, Arksey N, Shen B, Colomer MV, Rafat M, Liu H, Alejandre Alcazar MA, Chen JW, Palmer T, Chin FT, Guzman R, Loo BW Jr, Graves E
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Elafin preserves a novel EGF-Receptor-Krueppel-like factor 4 axis and protects against lung cell apoptosis in lungs of mechanically ventilated newborn mice. American Thoracic Society International Conference, 2015
Alejandre Alcazar MA, Kaschwich M, Ertsey R, Preuss S, Mujahid, S, Masumi J, Khan S, Mokres L, Rabinovitch M, Bland RD
