ApoA-I prevents the binding of fluid-phase Bio-VWF to VWF fibers in synthetic microvessels. The synthetic microvessels containing HUVECs were activated with 200 ng/mL of PMA for 40 minutes at 37°C and then washed with phosphate-buffered saline (PBS) containing 6% BSA for 5 minutes. ApoA-I (500 μg/mL in SFM) or SFM was perfused over the activated microvessels for 5 minutes followed by perfusion of Bio-VWF (10 μg/mL in SFM). At the end of the experiments, the microvessels were washed with PBS containing 6% BSA for 5 minutes before staining for VWF and ApoA-I. Vessels perfused with either buffer or ApoA-I were both stained with antibody to ApoA-I. There was no signal for ApoA-I in the vessels perfused with buffer (image not shown). Secreted VWF in the microvessels self-associated to form transluminal fibers. (A) In the absence of ApoA-I, Bio-VWF (red) perfused through the activated microvessel bound to a transluminal VWF fiber, accumulating on its upstream side. (B) Colocalization of Bio-VWF (yellow) with the transluminal VWF fiber (green). (C) Absence of Bio-VWF localization in a microvessel when ApoA-I was perfused through the vessel before Bio-VWF. (D) VWF fibers (green) secreted by the activated microvessels. (E) ApoA-I (white) localization in a microvessel after perfusion. (F) Colocalization of ApoA-I (white) with VWF fibers (green). Nuclei of HUVECs are in blue. Scale bars, 50 μm (A-F). Arrows indicate direction of flow.