Abstract Body (Do not enter title and authors here): Background: Dysregulated fibroblasts and macrophages have been implicated in vascular remodeling in pulmonary arterial hypertension (PAH). Macrophages are known to modulate fibroblast activity in fibrotic lung diseases. However, the spatial interactions between fibroblasts and macrophages within the lung tissue context of PAH remain largely unexplored.

Hypothesis: High-resolution spatial transcriptomics can reveal cell–cell interactions in PAH. We hypothesize that spatial interactions between macrophages and fibroblasts are increased in PAH lungs and may contribute to vascular remodeling.

Approach: Mice with Tie2Cre-mediated deletion of Egln1 (Egln1^Tie2Cre, or CKO) were used, as these animals exhibit spontaneous and severe pulmonary hypertension (PH) with pronounced vascular remodeling. Lung tissues from wild-type (WT) and CKO mice were analyzed using the high-resolution 10x Genomics Visium HD spatial transcriptomics platform. Whole-transcriptome data were collected at 2μm resolution, and adjacent squares were aggregated into 8μm regions to approximate single-cell dimensions. Cell identity was determined using the RCTD algorithm (v1.2.0) with integrated single-cell RNA-seq data from the WT and CKO mice. Machine learning techniques were employed to assign aggregated transcriptomic data to individual cells and calculate the spatial distances between fibroblasts and macrophages.

Results: Seventeen cell types and ten spatial regions were identified in WT and CKO lungs (Figure A), consistent with histological findings. CKO mice displayed prominent regions of fibrosis and inflammation. Specifically, regions corresponding to inflammation (Region 2), perivascular fibrosis (Region 5), and perivascular smooth muscle (Region 7) were expanded in CKO lungs, while capillary endothelial regions (Regions 4, 6, and 8) were markedly reduced. Notably, the spatial distance between fibroblasts and macrophages was significantly reduced in CKO lungs (Figure B).

Conclusion: High-resolution spatial transcriptomics (Visium HD) effectively delineates anatomical and molecular changes in PH lungs. The reduced spatial distance between fibroblasts and macrophages in PH mice suggests increased cellular interactions that may drive pulmonary vascular remodeling and contribute to PAH pathogenesis.

Keywords: Pulmonary Arterial Hypertension, Spatial transcriptomics, High resolution, Macrophages, Fibroblasts