Selective induction and functional requirement of integrins with affinity for basement membrane matrices during EC-pericyte tube coassembly. (A) PCR analysis of selected α integrin chains was performed to examine expression in both the bovine pericytes and ECs (HUVECs) when cultured alone or in coculture. G3PDH is shown as a loading control. (B) Five-day images from time lapse movies are shown, highlighting the differences in vessel width between control EC-only cultures and EC-pericyte cocultures. Arrows show the differences in width. (C) Average vessel widths were measured from images obtained from time lapse movies for quantitation. (D) Integrin-blocking antibodies were added to the culture media of EC alone versus EC-pericyte cocultures either from the beginning of the experiment or starting at day 3 or 5 at 20 μg/mL. Analysis of total vessel area is shown, demonstrating a requirement for α₂β₁ using EC-only cultures. Analysis of average vessel width is reported showing increases in EC tube width with blockade of α₅β₁, α₃β₁, and α₆β₁ only when pericytes are present, and more significantly increased tube widths when antibodies to α₁β₁, α₃β₁, and α₆β₁ are mixed (bottom panels). Statistical significance, P < .01. Species specificity of the antibodies can be found in supplemental Table 2.