Fig. 2. Pericyte-derived heme-binding protein 1 (Hebp1) induces angiogenesis under high glucose (HG) conditions. (A-C) Tube formation assays (A), aortic ring assays (B), and migration assays (C) of cells treated with phosphate-buffered saline (PBS) or Hebp1 (200 ng/mL) under normal glucose (NG) and HG conditions. ×40 magnification. (D) Number of migrated endothelial cells was quantified by using ImageJ software (n=4). ***p<0.001 vs. HG group. (E) Representative western blots for Hebp1 in mouse corpus cavernosum pericytes (MCPs) infected with shCon or shHebp1 lentivirus at three doses (1 µL, 1×104 TU; 5 µL, 5×104 TU; 10 µL, 1×105 TU/mL culture medium). (F) Tube formation assay of mouse corpus cavernosum endothelial cells (MCECs) exposed to MCP culture media at indicated conditions. ×40 magnification. (G) The number of master junctions (n=4, left) and intensity of area of microvessels sprouting from aortic rings were quantified by use of ImageJ software (n=4, right). ***p<0.001 vs. HG group. (H) Normalized band intensity values were quantified by use of ImageJ software (n=4). **p<0.01 vs. shCon group. (I) Number of master junctions was quantified by use of ImageJ software (n=4). ***p<0.001 vs. complement medium group, **p<0.01 vs. MCPs-shCon cultured medium group. The relative ratio of the NG, shCon, or complement medium group was arbitrarily set to 1. shCon, small hairpin RNA scramble control; shHebp1, shRNA heme-binding protein 1.
