B17-09 Recruited Macrophages Disrupt Saccular Stage Lung Microvascular Development

Abstract Background Normal lung microvascular development is critical for distal airspace formation in the saccular stage lung. Inflammation disrupts critical morphogenic cues required for microvascular formation, leading to alveolar simplification and bronchopulmonary dysplasia (BPD). We previously developed a transgenic mouse model to study the effects of mesenchyme-driven inflammation in the saccular stage lung. In this model, activated human IKKB was expressed in TBx4-lung enhancer-positive cells in a doxycycline (Dox) inducible manner (IKKβTbx4). Lungs of saccular stage IKKβTbx4mice demonstrated macrophage-predominant inflammatory cell recruitment and developed a BPD lung phenotype characterized by abnormal vascular organization and reduced distal airspaces. In this study, we examined the effects of recruited macrophages on the formation of the saccular stage pulmonary microvasculature. Methods We activated the transgene in neonatal IKKβTbx4 mice during the saccular stage (postnatal [PN] day 0-5) by placing lactating dams on Dox (2g/L). A small molecule CCR2 antagonist (or vehicle DMSO) was administered intraperitoneally (IP) daily to neonatal IKKβTbx4 mice for macrophage depletion experiments. We evaluated lungs at PN5 and 2 months. Lung parenchymal cell populations were identified and quantified using immunofluorescent staining and flow cytometry. Results Transgene activation led to a significant reduction in total endothelial cell number (ERG+ nuclei [per high-powered field], control: 354±50, IKKβTbx4: 208 ±10; P < 0.05), distal lung microvascular area (as determined by Endomucin staining), and expression of angiogenic markers in saccular stage IKKβTbx4 mice. These data were corroborated by flow cytometry, which demonstrated a significant reduction in endothelial cell numbers (CD31+ viable cells [per lung], control: 8.3x105± 271,872, IKKβTbx4: 4.6x105±114,095; P < 0.05), including both Cap1 (CD31+/Kit+) and Cap2 (CD31+/Car4+) populations. Microvasculature-associated lung pericytes (CD31-/CD146+/PDGFRβ+) were also reduced in IKKβTbx4 lungs. At 2 months of age, IKKβTbx4 mice demonstrated a simplified lung microvascular network along with muscularization of the pulmonary arteries and anatomic right ventricular hypertrophy (Fulton’s Index [RV/(LV+Septum) mass], control: 0.23±0.04; IKKβTbx4: 0.35±.04; P < 0.05), indicative of chronic pulmonary hypertension. Administration of a small molecule CCR2 antagonist to saccular stage IKKβTbx4 mice restored expression of critical angiogenic factors, rescued organization of the lung microvascular network, and preserved normal lung development. Conclusions Our results highlight a critical role for recruited CCR2-positive macrophages in inhibiting normal lung microvascular organization during the saccular stage, thus setting the stage for BPD and its associated long-term pulmonary vascular complications. This abstract is funded by: HL094296, HD087023, HL105334, HL157373