Figure 3

PAECs isolated from patients with pulmonary hypertension exhibit anoikis resistance as compared to commercial controls. (A) Some PAECs survive in suspension over 7 days and proliferate when reseeded on gelatin-coated plastic culture dishes, characteristic of anoikis resistance. Phase-contrast images of PAECs from a patient with hereditary pulmonary arterial hypertension (HPAH1) are shown, including cells at confluence (left-hand panel), 7 days following suspension in ultra-low attachment 6-well plates (middle panel), and 8 days after re-seeding the cells in suspension on a culture dish (right-hand panel). (B) The growth of anoikis-resistant cells is distinctive among patients with pulmonary hypertension. ~ 10–30% of HPAECs survived in suspension for 24 h, whereas ~ 5% of cells survived for 3- and 7-days (left-hand panel). The right-hand panel demonstrates percent confluency on day 4 (y-axis) of cells that have been in suspension for 1, 3 and 7 days (x-axis). Anoikis-resistant cells from one HPAH patient (HPAH1) exhibited rapid proliferation, whereas the growth capacity of anoikis-resistant cells from patients pulmonary veno-occlussive disease (PVOD), idiopathic PAH (IPAH1), IPAH2, and a second HPAH patient (HPAH2) was limited. (C) Representative phase-contrast images taken on the 14th day of incubation of the single-cell cloning wells from two different subjects are shown. The insert on the top demonstrates that the colony size in each well occupied less than 25% of the well. HPAH1 cells (left-hand panel), anoikis-resistant PAECs collected after 7 days in suspension from HPAH1 (HPAH1-ARD7, middle panel), and IPAH1 cells are shown. IPAH1 cells did not survive after 7 days in suspension. (D) Low density and single-cell proliferation are variable among PAECs isolated from patients with pulmonary hypertension. Confluency of serially diluted cells from six different cell types (Control, HPAH1, HPAH1-ARD7, PVOD, IPAH1, and IPAH2) is plotted after 10 days of culture, with densities ranging from 125K/well to 62 cells (left-hand panel). Anoikis-resistant cells are more replication-competent in single-cell cloning assays, 2 weeks after single-cell cloning (right-hand). (E) Lectin-induced agglutination discriminates endothelial cell phenotypes in vitro. Helix pomatia agglutinates PAECs, but not pulmonary microvascular endothelial cells. Helix pomatia agglutinated HPAECs from HPAH1, whereas Griffonia simplicifolia did not. Griffonia simplicifolia agglutinates pulmonary microvascular endothelial cells and endothelial progenitor cells. Here, Griffonia simplicifolia agglutinated anoikis resistant, highly proliferative cells from HPAH1-ARD7. Arrows indicate agglutinated cells. Data in panels (B) and (C) represent technical replicates for PH patients and n = 3 for commercial control. All phase-contrast images are taken with a 10 × objective, and the scale bar represents 100 μm. HPAH heritable pulmonary arterial hypertension. PVOD pulmonary vent-occlusive disease. IPAH idiopathic pulmonary arterial hypertension.