Figure 1.
A CD32+ subpopulation of human liver ECs (ECs and HLEC) expresses FVIII. A data set for scRNA-seq of human liver cells was analyzed and a subpopulation of ECs was purified and studied. (A) Human liver cells contained 3 subpopulations of vascular ECs. (B) Differentially expressed genes identify subpopulations of ECs, including CD32 (FCGR2B), which marks endothelial cluster 11. Differentially expressed genes were plotted for all-liver cell clusters; the size of the circle indicates the percentage of cells in each population expressing each gene, and the intensity of the color indicates the level of expression. (C) scRNA-Seq demonstrates that CD32+ HLECs express higher levels of F8 and CD36 but lower levels of VWF. The expression of selected genes in each endothelial cluster and hepatocytes is plotted. (D) Expression of CD32 in CD32+ HLEC. HLEC were sorted by CD32+ expression, and CD32 mRNA expression was measured by quantitative reverse transcription polymerase chain reaction (qRT-PCR) (N = 3, mean ± standard deviation [SD], ∗∗P < .005). (E) Localization of CD32 in CD32+ HLEC. CD32+ HLEC were isolated, hybridized with antibody to CD32, and imaged by confocal microscope (green = CD32, blue = DNA). Representative images are shown using the left 20× objective and the right 63× immersion objective (scale bar, 50 μM). (F) CD32+ HLEC express F8. CD32+ mRNA was purified from HLEC and HUVEC, and F8 mRNA expression was measured by qRT-PCR (N = 3, mean ± SD, ∗∗∗P = .0001). (G) CD32+ HLEC release FVIII into the media. Media were collected from CD32+ HLEC at 2, 4, 6, 8, and 24 hours. Top: FVIII antigen was measured by enzyme-linked immunosorbent assay (ELISA) (N = 3, mean ± SD). Bottom: FVIII activity was measured using a chromogenic FVIII activity assay (N = 6, mean ± SD). (H) FVIII was localized in a punctate pattern in CD32+ HLEC. CD32+ HLEC were hybridized with antibody to FVIII and imaged using a confocal microscope. Representative images are shown using the 20× objective and the 63X immersion objective (scale bar, 100 μM and 50 μM, respectively). (I) FVIII and COPII are colocalized in CD32+ HLEC. CD32+ HLEC were hybridized with antibodies to FVIII and COPII and imaged using confocal microscope using the 20× objective and the 63X immersion objective (scale bar, 100 μM and 50 μM, respectively). The Pearson correlation coefficient = 0.62 ± 0.08. (J) CD32+ HLEC express minimal levels of VWF and release minimal levels of VWF into the media. mRNA was isolated from CD32+ HLEC and HUVEC and analyzed by qRT-PCR (top) (N = 6, mean ± SD, ∗∗∗∗P < .0001). Ten thousand HUVEC and HLEC CD32+ cells were seeded in p96-collagen coated plates, cultured for 3 days, the media was replaced, cells were treated with media or 10 μM histamine for 1 hour, and the concentrations of VWF released into the media were measured by ELISA (bottom). (N = 8, mean ± SD, ∗∗∗∗P < .0001).