Abstract Rationale Pulmonary hypertension (PH) is a progressive vascular disorder in which inflammation plays a pivotal role in the development of pulmonary vascular remodeling. The persistent accumulation of monocytes and macrophages within the pulmonary artery adventitia establishes a chronic proinflammatory niche that promotes disease progression. However, the mechanisms governing macrophage recruitment and their contribution to inflammatory signaling remain poorly defined. Precision-cut lung slices (PCLS) preserve native lung architecture and cellular composition, enabling ex vivo investigation of PH pathology. Yet, conventional PCLS systems lack immune cell infiltration, limiting their capacity to replicate immune-driven processes. To overcome this, we developed novel PCLS-immune co-culture models that integrate macrophage recruitment and activation to study immune-mediated vascular inflammation in PH. Methods PCLS from calves and/or humans with PH (PH-PCLS) and healthy controls (CO-PCLS) were used as ex vivo model systems. PCLS viability was confirmed using PrestoBlue and LIVE/DEAD fluorescence assays. CO-PCLS and PH-PCLS were subjected to hypoxia (1.5% O2), hypoxia plus interleukin-1β (IL-1β), or co-cultured with bone marrow-derived macrophages (BMDMs) isolated from CO and PH calves via transwell inserts (8 μm pores). Macrophage migration into PCLS was evaluated using CMFDA-labeled BMDMs (green) and Hoechst nuclear counterstain (blue) by Z-stack confocal microscopy. Expression level of pro-inflammatory genes were evaluated using RT-PCR. Transmission electron microscopy (TEM) was applied to assess extracellular vesicle (EV) localization and morphology, focusing on EV-mediated crosstalk among adventitial fibroblasts and macrophages. Results PH-PCLS demonstrated markedly elevated expression of key proinflammatory mediators, including IL6 and CCL2, compared with CO-PCLS, indicating persistent inflammatory activation in the hypertensive lung environment. However, CO-PCLS required combined stimulation with hypoxia, IL-1β, and BMDMs to induce a comparable proinflammatory gene signature to PH-PCLS, whereas hypoxia alone was insufficient. Confocal imaging confirmed increased macrophage infiltration into the PCLS under hypoxic conditions, aligning with observed transcriptional changes. Furthermore, TEM revealed abundant EVs within the adventitial compartment, closely associated with fibroblasts and macrophages, implicating EVs as mediators of immune-fibroblast communication driving vascular remodeling. Conclusion These novel PCLS-macrophage co-culture systems model macrophage-mediated inflammation in PH, provide an ex vivo platform to dissect immune-driven vascular remodeling and offer potential for developing immune cell-targeted therapeutic strategies in PH. This abstract is funded by: NHLBI P01HL014985; DOD/W81XWH1910259; DoD/W81XWH2010249
Zhang et al. (Fri,) studied this question.