Figure 1.
Ca2+ response in lung microvessels.
A–C Pseudocolor images show fluorescence emission in response to exciting Fura2-AM loaded microvessels at 340 nm (A) and 380 nm (B). The corresponding 340/380 fluorescence ratio was quantified on a pixel-by-pixel basis and displayed as a pseudocolored image (C). Scale bar = 20 µm. D. Tracings show temporal changes in F340/F380 in response to LPS infusions (100 µg/ml) into control (black tracing) and XeC-pretreated (gray tracing) lung microvessels. E, F Bar graphs show composite increase in the LPS-induced F340/F380 oscillation amplitude above baseline values for venules (E) and capillaries (F). Mean±SE. n = 4 vessels from 3 lungs per group. *-p<0.05 compared to XeC and XeC+LPS treatment groups.
Figure 2.
A–F Images show fluorescence emission intensity in response to exciting Fura2-AM loaded RLMVEC at 340 nm (A, D) and 380 nm (B, E) The corresponding 340/380 fluorescence ratio was quantified on a pixel-by-pixel basis and displayed as pseudocolored images (C, F). The images were obtained at baseline (A–C) and after OAG (100 µM) treatment (D-F). Scale bar = 20 µm. G. Bar graph shows increase in F340/F380 above baseline in RLMVEC treated with LPS (1 µg/ml) or OAG (100 µM), with and without XeC (10 µM) pretreatment (20 min). Mean±SE. n = 2−3 separate experiments and >50 RLMVECs per treatment group. *-p<0.05 compared to XeC+LPS treatment group.
Figure 3.
ICAM-1 expression in lung microvessels.
A–E Confocal images show immunofluorescence of ICAM-1 in lung microvessels following treatments as indicated. Treatment durations were as outlined in Methods. v-venule, c-capillary. Scale bar = 20 µm. F, G Bar graphs show ICAM-1 immunofluorescence intensity quantified along the vessel wall over the length of single microvessels for both venules (F) and capillaries (G). Mean±SE. n - number of vessels analyzed per group from a minimum of 2 lungs per group, except for XeC+LPS (3 lungs) and NBD (1 lung) groups. *-p<0.05 compared to HBS, XeC+LPS and NBD+LPS groups. BHQ = t-BHQ, NBD = IKK-NBD.
Figure 4.
Leukocyte retention in lung microvessels.
A–D Fluorescence images show retention of R6G-labeled leukocytes in lung microvessels following treatments, as indicated. Treatment durations and agent concentrations were as outlined in Methods. Scale bar = 20 µm. E, F Bar graphs show number of retained leukocytes per image frame in venules (E) and capillaries (F) following the indicated treatments. Mean±SE. n - number of images analyzed per group (3 lungs per group). *-p<0.05 compared to HBS, XeC+LPS and NBD+LPS groups. NBD = IKK-NBD.
Figure 5.
A–C Confocal immunofluorescence images show phosphorylation of the NF-κB p65 subunit in lung microvessels (green) and fluorescence of the nuclear marker Hoechst-33342 (blue) for the indicated treatments. Treatment durations and agent concentrations were as outlined in Methods. Scale bar = 20 µm. D, E Bar graphs show NF-κB p65 phosphorylation levels along the vessel wall over the length of single microvessels for both venules (D) and capillaries (E). Mean±SE. n - number of vessels analyzed per group. Each treatment repeated in 3 lungs each. *-p<0.05 compared to HBS and XeC+LPS groups.
Figure 6.
Nuclear localization of NF-kB.
Confocal images show immunofluorescence of NF-κB p65 subunit in lung microvessels (red, left column), fluorescence of the nuclear marker Hoechst-33342 (blue, column 2) and merge (column 3) for the indicated treatments. Pink color in the merged images indicates colocalization of NF-κB immunofluorescence and nuclear marker fluorescence. Treatment durations were as outlined in Methods. Nuclear localization of NF-κB immunofluorescence in the region marked by the rectangles (column 3 images) is shown magnified on far right column. Again, pink color indicates localization of NF-κB immunofluorescence (red) and nuclear marker fluorescence (blue). Treatment groups repeated in 3 lungs, except for NBD treatment group (n = 1). Note the higher colocalization in LPS-treated microvessels (B), compared to HBS-treated (A), and XeC- (C) and NBD-pretreated microvessels. NBD = IKK-NBD.