Figure 4. Pharmacologic inhibition using CIGS markedly blocks pericyte motility and recruitment as well as pericyte-induced capillary basement membrane deposition.

(A) EC-pericyte co-cultures were established under control conditions or with the CIGS drug combination added [20 μM CI-1020 (ET-1 receptor A antagonist), 1 μM imatinib (PDGFR inhibitor), 1 μM gefitinib (EGFR inhibitor), 10 μM SB 431542 (ALK5 inhibitor)]. Cultures were fixed at 72 hr and were immunostained with anti-CD31 antibodies to visualize ECs (red), while pericytes were GFP-labeled (green). Overlay images are shown in the upper and lower panels, while the middle panels show pericytes alone. Bar equals 200 μm. (B-D) EC-pericyte co-cultures were established with nuclear GFP-labeled pericytes and pericyte motility using time-lapse movie analysis was performed (B). Motility was examined in control cultures or cultures treated with CIGS from 0–72 hr (C) or separately, from 72–120 hr (D). Quantitation of pericyte motility under these conditions was performed. n≥8; Asterisks indicate significance relative to control at p<.05. (E) Control or CIGS treated co-cultures (120 hr) were immunostained for the indicated basement membrane components and imaged by confocal microscopy. Bar equals 200 μm.