Heterogeneous distribution of Weibel-Palade bodies (WPbs) in pulmonary vascular endothelium. Representative transmission electron micrographs of cross sections of extra-alveolar vessels in human (A and B), rat (C–E), and mouse (F) lung. A: low-resolution micrograph; arrow-directed area is exhibited in higher resolution in B. WPbs are indicated with arrowheads. C: low-resolution micrograph taken at the junction (Jct) where an extra-alveolar vessel (EAV) connects to an alveolar capillary (Cap); higher resolution of the boxed area is shown in D and highlighted in E. Note that the endothelial cell (EC) exhibiting WPbs (arrowheads) is at the extra-alveolar side of the junction. F: higher resolution micrograph demonstrating a cluster of WPbs (arrowheads).

Heterogeneous distribution of WPbs in pulmonary vascular endothelium. Micrographs of a cross section of an alveolar capillary in human lung. A: low-resolution micrograph; higher resolution of the boxed areas are shown in B and C; no WPbs were detected, while detailed cell ultrastructure clearly was revealed. N, nucleus; BM, basement membrane. *Endothelial caveolae: note that their ∼70-nm outer diameter is consistent with that previously described (42).

Heterogeneous distribution of WPbs in pulmonary vascular endothelium. Summarized data from a total of 188 measurements (human 47, rat 51, and mouse 90) of the diameter of the vessels where the WPbs were or were not detected, i.e., +WPb or −WPb, respectively, obtained from human, rat, and mouse lungs (n = 5, 4, and 5, respectively). Symbols, scatter plot; gray lines, median with range. Note the abrupt transitions of the vessel calibers from the vessels possessing WPbs to vessels possessing no WPbs in all 3 species examined.

von Willebrand factor (vWF) is expressed but not colocalized with P-selectin in alveolar capillary endothelium. A–C: immunohistochemistry for vWF in human and mouse lung sections. A: human alveolar capillary (∧) and extra-alveolar vessel (*) endothelium regularly displayed considerable immunoreactivity to vWF. B and C: mouse pulmonary endothelium displayed heterogeneous immunoreactivity to vWF: weak and irregular in capillary endothelium (∧) and prominent in extra-alveolar vessel endothelium (*). Images acquired at 21°C on a Nikon Eclipse E600W upright optical microscope with a ×20, 0.50 numerical aperture objective (for A) and a ×60, 0.85 numerical aperture objective (for B and C) (replicated 3 times). D and E: double immunofluorescence labeling of vWF and P-selectin in mouse lung sections. Representative projected images of all parts of Z-stacks combining both fluorescence and differential interference contrast mode taken at 0.2-μm intervals from the areas of an alveolus (D) and an extra-alveolar vessel (E). Immunofluorescence was performed with an anti-vWF antibody followed by a DyLight 488-conjugated secondary antibody (green) and an anti-P-selectin antibody followed by a biotinylated secondary antibody with cyanine 3-conjugated streptavidin (red) and nuclear fluorescence staining with DAPI (blue). Colocalization of vWF and P-selectin is evidenced by the yellow color. Images acquired at 21°C on a Nikon A1 confocal laser microscope with a ×60, 1.20 numerical aperture water immersion objective (replicated 3 times).

Subcellular localization of P-selectin is WPb independent in alveolar capillary endothelium. Representative transmission electron micrographs of mouse lung cryostat sections immunolabeled for P-selectin and visualized with Qdot 655. A: low-resolution micrograph of a cross section of an extra-alveolar vessel; arrow-directed area is shown in higher resolution in B. Quantum dots (arrowheads in B) are seen within the confinement of a tangentially sectioned WPb. C: low-resolution micrograph of a cross section of an alveolar capillary; arrow-directed area is shown in higher resolution in D. Quantum dots are seen in clusters in the endothelial cytosol (circle and arrowhead in D) and reveal no association with any intracellular structures. Some quantum dots are seen at the endothelial surface (ellipse in D).

Both alveolar capillary and extra-alveolar vessel endothelium exhibit regulated P-selectin surface expression. Representative micrographs of immunohistochemistry for surface P-selectin in tissue sections from mouse lungs that had been perfused with regular perfusate (4%BSA/EBSS) either alone (A) or with thrombin (5 U/ml; B and C) for 10 min. A: in control lung, extra-alveolar endothelium exhibited continuous and intense immunoreactivity to P-selectin (arrows); alveolar septal endothelium exhibited only sporadic immunoreactivity to P-selectin (arrowheads). B: in thrombin-stimulated lung, extra-alveolar endothelium exhibited the same immunoreactivity to P-selectin (arrows) as in A; alveolar septal endothelium exhibited extensive focal immunoreactivity to P-selectin (note the dark brown staining in boxed area, also denoted by arrowheads in C). C: enlarged boxed area in B. Images acquired at 21°C on a Nikon Eclipse E600W upright optical microscope with a ×40, 0.75 numerical aperture objective (replicated 3 times each). D–F: immunofluorescence imaging of endothelial surface P-selectin in extra-alveolar vessels. Representative merged FITC-fluorescence images of a serial optical sections taken at 0.5-μm intervals in ∼200-μm viable slices of the mouse lung. Lungs were perfused with an FITC-conjugated P-selectin antibody alone (D), thrombin (5 U/ml) followed by an FITC-conjugated isotype IgG (E), or thrombin followed by the FITC-conjugated P-selectin antibody (F). Only minimal fluorescent signal was present in D and E. Note the appearance of substantial FITC fluorescence on the luminal surface of an extra-alveolar vessel in F. Margins for the extra-alveolar vessel (denoted by EAV) are depicted by blue dotted lines in D and E. Images acquired at 21°C on a PerkinElmer UltraView RS confocal microscope with a ×60, 1.20 numerical aperture water immersion objective (replicated 3 times each).

Pharmacologic T-type channel blockade or genetic α_1G knockout mitigates thrombin-stimulated P-selectin surface expression in alveolar capillary endothelium. A–C, immunohistochemistry for surface P-selectin in tissue sections from wild-type (α_1G^+/+) lungs stimulated with thrombin alone (A) or together with mibefradil (B) or from α_1G-deficient (α_1G^−/−) lungs stimulated with thrombin (C). Thrombin-stimulated P-selectin surface expression in alveolar septal endothelium in wild-type lungs (note the dark brown staining in A) but not in mibefradil-administered or α_1G-deficient lungs (B and C). Replicated 3 times each. D–F: immunofluorescence imaging of surface P-selectin in extra-alveolar endothelium from lungs stimulated with thrombin alone (D) or together with mibefradil (E), or from α_1G-deficient lungs stimulated with thrombin (F). Thrombin-stimulated P-selectin surface expression in extra-alveolar endothelium was not altered by mibefradil or α_1G knockout. Replicated 3 times each.

Pulmonary microvessel endothelium possesses a functional T-type Ca²⁺ channel: depolarization-induced endothelial cytosolic Ca²⁺ ([Ca²⁺]_i) response in pulmonary microvessels. A: fura 2-loaded endothelial cells of lung microvessels were imaged in situ by real-time fluorescence microscopy. Representative images of the 340-nm/380-nm ratio pseudocolor coded for [Ca²⁺]_i at baseline (left) and after high K⁺ concentration (40 mmol/l) perfusion in control lungs (top) and lungs pretreated with mibefradil (10 μmol/l; bottom). White lines, vessel margins; n = 5 each (number of animals studied). B: group data of endothelial [Ca²⁺]_i in situ. [Ca²⁺]_i was determined at 5-min intervals at baseline as well as during high K⁺ concentration perfusion in control lungs (○) and lungs pretreated with the T-type Ca²⁺ channel blocker mibefradil (●). Data are means ± SE. *P < 0.05 vs. control, Mann-Whitney U-test.

Pharmacologic T-type channel blockade or genetic α_1G knockout abolishes depolarization-induced P-selectin surface expression in alveolar capillary endothelium. Immunohistochemistry for surface P-selectin in tissue sections from wild-type (α_1G^+/+) lungs perfused with high K⁺ (40 mmol/l) alone (A) or together with mibefradil (10 μmol/l; B) or from α_1G-deficient (α_1G^−/−) lungs perfused with high K⁺ (C). Depolarizing endothelial membrane evoked P-selectin surface expression in alveolar septal endothelium in wild-type lungs (note the dark brown staining in A) but not in mibefradil-administered or α_1G-deficient lungs (B and C) (replicated 3 times each).

Morphometric assessment of alveolar endothelium P-selectin expression using a point-counting strategy, yielding a measure of the P-selectin volume fraction. P-selectin volume fraction increased significantly in wild-type (α_1G^+/+) lungs stimulated with thrombin or challenged with high K⁺. Such increase in P-selectin volume fraction was not observed in wild-type lungs with mibefradil treatment (α_1G^+/+/Mib) or in lungs deficient for α_1G (α_1G^−/−). Data are means ± SE. ^#P < 0.001, compared with all other groups. *P < 0.001, compared with the group of α_1G^+/+ with thrombin or with high K⁺, by one-way ANOVA with Student-Newman-Keuls post hoc test.