Despite accumulating evidence that ICAM-1 mediates adhesion and extravasation of leukocytes to and through the endothelium (Carlos and Harlan, 1994) and endogenous NO modulates leukocyte adherence through the expression of adhesion molecules on leukocyte and ECs (Kubes et al., 1991; Tsao et al., 1994), the underlying signaling mechanisms of reduced NO-mediated increases in adhesiveness of microvessel remained poorly understood. NO reduction. The adhesion strength of EC ICAM-1 was assessed by atomic force microscopy (AFM) on live cells. Results showed that reduction of EC basal NO caused by the application of caveolin-1 scaffolding domain (AP-CAV) or NOS inhibitor, L-NMMA, for 30 min significantly increased phosphorylated ICAM-1 and its binding to mAb1A29 in the absence of altered ICAM-1 expression and its distribution at subcellular regions. The Src inhibitor, PP1, inhibited NO reduction-induced increases in ICAM-1 phosphorylation and adhesive binding. AFM detected significant increases in the binding force between AP-CAV-treated ECs and mAb1A29-coated probes. These results demonstrated that reduced EC basal NO lead to a rapid increase in ICAM-1 adhesive binding via Src-mediated phosphorylation without protein synthesis and translocation. This study suggests that a NO-dependent conformational change of constitutive EC membrane ICAM-1 might be the mechanism of rapid ICAM-1 dependent leukocyte adhesion observed observation at cellular and protein levels. We quantitatively measured real-time endothelial NO production in the absence and presence of AP-CAV, an endogenous eNOS inhibitor, and L-NMMA, and assessed the concomitant changes in sub-cellular quantity of ICAM-1, adhesive binding avidity of ICAM-1, as well as the signaling pathways involved in the activation status of ICAM-1. Open in a separate window Figure 1 Perfusion of rat mesenteric venules with AP-CAV induced basal NO-dependent ICAM-1 mediated leukocyte adhesion. Intact venules were perfused by AP-CAV for 30 min followed by resuming blood flow in the same vessel for 10 min. Leukocyte adhesion was quantified when each vessel was recannulated with BSA-Ringer solution (Xu et al., 2013). (A) Video images of a perfused venule under control conditions and after AP-CAV (10 M)-induced leukocyte adhesion, and the administration of a NO donor, sodium nitroprusside (SNP), in both perfusate (10 M) and superfusate (20 M) abolished AP-CAV-induced leukocyte adhesion. (B) AP-CAV induced dose-dependent increases in EC ICAM-1 binding to its blocking antibody mAb1A29. Confocal images of mAb1A29 (green) and vascular cell nuclei (red) immunofluorescence co-staining under control conditions, after AP-CAV perfusion, and after adding SNP to AP-CAV perfused vessels. (C) Perfusion of vessels with ICAM-1 inhibitory antibody, mAb1A29, significantly attenuated AP-CAV induced leukocyte adhesion (= 5 per group). * and ? indicate a significant increase and decrease from the control, respectively (modified from Xu et al., 2013 and used by original authors). Materials and methods Endothelial cell culture and treatments Primary human umbilical endothelial cells (HUVECs), endothelial growth media (EGM), and supplements were purchased from Lonza (CC-3122 and CC-4133, Walkersville, MD). HUVECs were seeded at a density of 2.5 x 103 cells per cm2 and cultured in EGM with supplements containing bovine brain extract, human epidermal growth factor, fetal bovine serum, hydrocortisone, ascorbic Acid, GA-1000 (Gentamicin, Amphotericin B), and heparin. The cell culture was performed in a humidified atmosphere of 5% CO2 at 37C. HUVECs were split routinely when they reached 90% confluence and used for experiments within 5 passages after purchasing. Cells were treated with AP-CAV, the fusion peptide of CAV scaffolding domain with AP, the Antennapedia internalization sequence from Drosophila Antennapedia homeodomain (synthesized by Tufts University), PP1, [(4-Amino-5-(methylphenyl)-7-(t-butyl)pyrazolo-(3,4-d)pyrimidine, Sigma], and/or N()-monomethyl-L-arginine acetate salt (L-NMMA, Sigma) for 30 min or 6 h at a concentration of 10 M. In some experiments, PP1 was added 30 min prior to AP-CAV peptide treatment. ECs cultured in petri dish were used for western blot, and microfluidic devices were used for NO measurements. The stock solutions of AP-CAV peptide, PP1, and L-NMMA were prepared in 100% DMSO, and the Talabostat final solution of each Talabostat agent was prepared by Talabostat 1:1,000 dilution of the stock with EC culture medium. NO measurements and fluorescence imaging in HUVECs cultured in microfluidic channels We recently demonstrated that ECs develop well-formed junctions when grown with continuous flow in the microchannels, and their intracellular calcium and nitric oxide responses to ATP were similar to Rabbit polyclonal to IL22 those observed in intact microvessels (Li et al., 2015). In this study, cultured EC-formed microvessel networks were used to measure changes in EC basal NO production rate before and after AP-CAV or L-NMMA was applied to the vessel lumen. The methods have been described in detail (Zhou and He, 2011a; Li et al., 2015; Xu et al., 2016). In brief, HUVECs were seeded into polydimethylsiloxane (PDMS) microfluidic microchannel network device and cultured under continuous perfusion with wall shear stress.