Abstract Body (Do not enter title and authors here): Background: Pulmonary capillary endothelial cells (ECs) are essential for lung function, maintaining vascular integrity, angiogenesis, and gas/nutrient exchange. Recent advances in single-cell RNA sequencing (scRNA-seq) have revealed significant heterogeneity among general capillary ECs (gCaps), providing new insights into pulmonary vascular homeostasis and disease. This study examines the role of gCap subtypes in pulmonary hypertension (PH).

Methods: scRNA Seq was performed on Control, Sugen Hypoxia (SuHx) PH rat model and EC’s derived from Control and PH patients.

Results: Five gCap subpopulations (gCapA-E) were identified, all expressing the Apelin receptor and marked by unique genes: Clic4 (gCapA), Flot1 (gCapB), Fabp4 (gCapC), Scn7a (gCapD), and Lingo2 (gCapE). Of these, the gCapC cells showed strong angiogenic potential with enriched angiogenesis and tip cell markers. Depletion of gCapCs via FACS impaired angiogenesis in-vitro and in a 3D vessel-on-a-chip model, confirming gCapCs as regulators of angiogenesis. gCapB cells (Flot1+) localized at the interface of arterial and capillary ECs and displayed a progenitor-like phenotype. RNA velocity and PAGA analysis suggested gCapB cells give rise to gCapD, gCapA, and arterial ECs. gCapB cells showed high mitotic activity, G2M/S-phase signatures, increased Fos/Jun signaling, and stemness markers CDKn1a and CDKn1c. Longitudinal profiling of 2, 6 and 12 wk-old rats revealed gCapB cell expansion and loss of Foxm1+ progenitors, suggesting a developmental shift. Furthermore, gCapB cell depletion impaired EC proliferation. Seahorse analysis revealed high oxidative metabolism in gCapB and gCapC, supporting roles in repair and angiogenesis.
In the SuHx PH model, global capillary loss was highly pronounced for Aerocytes (aCaps). This was coupled with gCapB activation and proliferative (Mki67+) gCaps expansion and decreased maturation and angiogenic sprouting of gCapCs. We also observed the activation of EC plasticity toward mesenchymal and hematopoietic transition. Complementing these findings, scRNA-seq analysis of human PH lungs revealed a global depletion of Aerocytes with upregulation of Mki67 in gCaps, underscoring the translational relevance of cellular changes in the Su/Hx model.

Conclusions: Aerocytes (gas exchange regulators), gCapCs (angiogenesis regulators) and gCapBs (repair regulators) are disrupted in PH. Their dysfunction highlights new therapeutic targets for vascular repair in PH.