Figure 7.

Visualization of TM4SF1⁺ endothelial cells in situ, after isolation by FACS, and after coculture with fibroblasts. (A and B) Representative immunofluorescence images of distal pulmonary arteries from lung sections of (A) a 31-year-old male donor control subject versus a 30-year-old man with idiopathic PAH and (B) control, SuHx, and MCT rats stained for TM4SF1 (red), CD31 (green), SMA (white), and DAPI (blue). Scale bars: A, 50 μm; B, 20 μm. (C) Gating strategy for the isolation of TM4SF1⁺CD31⁺EPCAM⁻ (epithelial cell adhesion molecule) endothelial cells from rat lungs after gating for singlets and live cells. CD45 expression was also assessed. Percentages represent averages of three experiments. Full gating strategy is illustrated in Figure E9. (D) Representative confocal image of TM4SF1⁺CD31⁺ cells sorted from rat lungs and stained with antibodies to TM4SF1 (green) and CD31 (white) as well as Dil-acetylated low-density lipoprotein (Dil-Ac-LDL) (red) and DAPI (blue). Cells were pelleted after sorting, resuspended in a small volume of paraformaldehyde, and then left to dry on a coverslip before staining. Scale bars, 10 μm. (E) Representative confocal image of TM4SF1⁺CD31⁺ rat lung cells cocultured with human lung fibroblasts and stained after 6 hours with endothelial markers Dil-Ac-LDL (red), isolectin GS-IB4 (green), and DAPI (blue). Scale bars, 100 μm. SMA = smooth muscle actin.