LAUSR

Introduction/Hypothesis: Sepsis-induced acute lung injury (ALI) in premature newborns is a risk factor for bronchopulmonary dysplasia (BPD). Angiopoietins are vascular growth factors that regulate endothelial homeostasis and inflammation. During sepsis and ALI, angiopoietin 2 (ANGPT2) is upregulated and pathologically antagonizes normal angiopoietin 1 (ANGPT1)mediated Tie 2 receptor phosphorylation and downstream signaling. Since limiting neonatal ALI may prevent BPD, we used our previously developed newborn mouse model of LPSinduced ALI and alveolar simplification (akin to BPD) to test whether exogenous ANGPT1 can antagonize ANGPT2 to prevent ALI. Methods: 6-day-old C57BL/6 mice (4-6 per group) were given ip LPS (2 mg/kg) ± 2 hr pretreatment with ip recombinant mouse ANGPT1 (rmAngpt1, 1 μg). Whole lungs were harvested after 24 hrs for mRNA and protein study. Apoptosis was seen by TUNEL stain. Separately, human pulmonary microvascular endothelial cell (HPMEC) cultures were stimulated with LPS ± recombinant human ANGPT1 (rhANGPT1) for 1 or 24 hrs to dissect effects on the angiopoietin-tie axis. Results: In mouse pups given ip LPS, rmAngpt1 pretreatment prevented lung NF-κB activation at 24 hours (P-p65:p65 fold change from control: 2.23±0.67 for LPS vs 1.04±0.11 rmAngpt1+LPS, p<0.05). rmAngpt attenuated lung gene expression of keratinocyte-derived cytokine, IL-1β, and the endothelial neutrophil attractant ICAM-1 by 25-35% (p<0.01), blunted the 4.6-fold rise in ICAM-1 protein by 27% (p<0.05), and protected against lung apoptosis (% apoptotic cells: 5.51±1.88 vs 1.59±0.29 [↓71%]; p<0.01). MMP-9, a marker of pathologic lung remodeling, had 10-fold higher lung gene expression post-LPS (p<0.0001), which was 58% lower with rmAngpt1+LPS (p<0.001). In HPMEC in vitro, trends in NF-κB activation, ICAM-1, and IL-8 paralleled in vivo data. Further, LPS-induced ANGPT2 (↑2.1-fold mRNA and ↑2.8-fold protein, p<0.05) was entirely suppressed by rhANGPT1. Tie 2 was dephosphorylated in endothelial cells 1 hour post-LPS alone though not with rhANGPT1+LPS. Conclusions: Therapeutic ANGPT1 attenuates lung apoptosis as well as key mediators of ALI and alveolar remodeling in an in vivo neonatal sepsis model. Restoring balance in the Angiopoietin-Tie axis during sepsis may potentially counter neonatal ALI and prevent BPD.