Leukocyte transmigration through endothelial cells. (A-D) Endoglin overexpressing endothelial cells. (A) Flow cytometry analysis of endoglin transfectants. The tetracycline-inducible bovine endothelial cell line GM7372-EL, engineered to express human endoglin were incubated or not with Dox for 24 hours. Flow cytometry analysis was carried out on the Dox inducible green fluorescence protein (GFP) used here as a marker of human endoglin expression. (B-D) Transmigration assays. Transwell experiments were performed using confluent monolayers of bovine endothelial cell lines GM7372-EL and GM7372-M. To test the migration rate, Nalm6 (B), THP-1 (C), or U937 (D) cells were seeded on top of the transwell and 100 ng/mL CXCL12 was added to the bottom of chambers, unless otherwise stated. Soluble endoglin (Sol-Eng) was added to U937 cells, as indicated (D). After incubation for 2 hours at 37°C, transmigrated cells were counted by flow cytometry. There is a significant increased transmigration through GM7372-EL versus GM7372-M monolayers. Each bar represents the mean value of 3 assays each performed in triplicates. **P < .005; *P < .01. (E-I) Primary cultures of endothelial cells. (E) Blood outgrowth endothelial cells (BOECs). Confluent monolayers from healthy subjects (Control) or 3 different HHT patients (Nos. 1, 2, and 3) were subjected to transmigration assays using THP-1 cells as in previous figures. Endothelial cells from HHT No. 1 correspond to an HHT1 patient with a pathogenic mutation (R171X) in the endoglin gene and endothelial cells from HHT no. 2 and no. 3 show decreased expression of endoglin protein.²⁸  There is a significant decreased transmigration through HHT versus control monolayers of BOECs. (F-I) Transmigration through human umbilical vein endothelial cells (HUVECs). Confluent monolayers of HUVECs were tested for the migration rate of Nalm6 (F), U937 (G), T lymphocytes (H), and B lymphocytes (I) in the presence or absence of 100 ng/mL CXCL12 at the bottom of chambers. Soluble endoglin (Sol-Eng) at 1 μg/mL or the human endoglin-derived RGDK peptide were added, as indicated. (J) Transmigration of murine SP2 cells through mouse MS1 endothelial cells was carried out as above, but in the presence of mouse soluble endoglin (mSol-Eng) at 1 μg/mL or mouse endoglin-derived TDD peptide, as indicated. After incubation for 2 hours at 37°C, transmigrated cells were counted by flow cytometry. In the presence of CXCL12 there is a marked increased transmigration of human Nalm6 and U937 cells as well as T and B lymphocytes through HUVECs that is inhibited in the presence of soluble endoglin. A similar behavior was observed with murine SP2 cells migrating through MS1 mouse endothelial cells. Each bar represents the mean value of 3 experiments, each performed in triplicates. **P < .005; *P < .01.