Blockade of EC-pericyte interactions in vivo leads to decreased basement membrane deposition and increased EC vessel width. (A) Quail CAM tissue from the controls, gefitinib/imatinib-treated quail embryos and α-PDGF-BB/HB-EGF–treated embryos, was isolated and immunostained for the basement membrane component fibronectin. Quantification of immunostaining intensity of extracellular basement membrane protein deposition displays a decrease in deposition under conditions of inhibited pericyte recruitment, most severely in conditions of combined PDGFR and EGFR inhibition. (B) Representative images of the fibronectin stains are shown, with arrows highlighting areas of decreased levels of extracellular basement membrane protein deposition. Overlays of QH1 staining (EC marker, green) versus fibronectin (red) are included for control versus α-PDGF-BB/HB-EGF treatments. (C) Measurements of EC tube width were done (from QH1 stains of EC tubes), demonstrating increased EC vessel width under conditions of inhibited pericyte recruitment to EC tubes. Furthermore, there was a decrease in the number of EC branch points in CAMs treated with these chemical inhibitors or blocking antibodies, further implicating direct vascular phenotypes. (D) Representative images of CAM tissue stained with the quail EC specific marker, QH1, are shown demonstrating the increased vessel width and decreased branch point phenotypes. Arrowheads indicate the “membrane-ruffled appearance” that was particularly observed in the gefitinib/imatinib condition and correlated with strongly reduced fibronectin deposition and increased vessel widths. n ≥ 5; P ≤ .01. *Significance from control conditions. +Significance from individual factor addition.