Figure 3.
Hypoxia-induced increase in lung PF4 and accumulation of lung platelets was delayed in Nbeal2−/− mice. (A) Lung PF4 increased in hypoxic WT mice at days 3 and 14. Lung PF4 increased in hypoxic Nbeal2−/− mice at day 14. Hypoxia-induced increase in lung PF4 was decreased at day 3 and increased at day 14 in Nbeal2−/− compared with WT mice. (B) Platelets accumulated in the distal lung of hypoxic WT mice at day 3, which resolved by day 21. Accumulation of distal lung platelets was not observed in hypoxic Nbeal2−/− mice. (C) Whole-slide scans were obtained using the Leica Aperio VERSA brightfield scope (×40 objective, 0.5-micron resolution). Representative images of CD41 staining in the distal lung (original magnification ×20; scale bars, 100 μm. As previously described, CD41+ pixels were quantified in whole-lung sections using a random, nonbiased approach using ImageScope, version 12.4.3.500 (Leica Biosystems Imaging, Inc, Deer Park, IL) pixel quantification software.1 (D) Lung sections were stained with anti-PF4, anti-CD41, anti-CD45, and DAPI. Whole-slide scans were obtained using the 3I Marianas Confocal Microscope (×100 objective, 0.1-micron resolution). Representative images of intracellular and extracellular PF4 were captured. Original magnification ×100; scale bars, 50 μm. White arrows indicate extracellular PF4 (magenta), and yellow arrows indicate intracellular PF4 within platelets (yellow). (E) Lung sections were probed with a PF4 mRNA primer and anti-CD41, anti-CD45, and DAPI antibodies. Whole-slide scans were obtained using (×40 objective, 0.5-micron resolution). Representative images of pulmonary vessels and the distal lung were captured. Representative images of peak Pf4 mRNA expression within megakaryocytes and leukocytes in the pulmonary vessels and distal lung of WT (day 3) and Nbeal2−/− (day 14) vs their baseline, as well as other unidentified cells (original magnification ×40; scale bars, 50 μm). Yellow arrows indicate PF4 mRNA (magenta) within megakaryocytes (yellow), and blue arrows indicate Pf4 mRNA within leukocytes (cyan). Statistics: P ≤.05 by 2-way ANOVA with Tukey post hoc analysis. Comparisons: (∗) WT compared with WT baseline, (#) Nbeal2−/− compared with Nbeal2−/− baseline, (^) WT compared with Nbeal2−/−. HPX, hypoxia; NMX, normoxia.

Hypoxia-induced increase in lung PF4 and accumulation of lung platelets was delayed in Nbeal2−/− mice. (A) Lung PF4 increased in hypoxic WT mice at days 3 and 14. Lung PF4 increased in hypoxic Nbeal2−/− mice at day 14. Hypoxia-induced increase in lung PF4 was decreased at day 3 and increased at day 14 in Nbeal2−/− compared with WT mice. (B) Platelets accumulated in the distal lung of hypoxic WT mice at day 3, which resolved by day 21. Accumulation of distal lung platelets was not observed in hypoxic Nbeal2−/− mice. (C) Whole-slide scans were obtained using the Leica Aperio VERSA brightfield scope (×40 objective, 0.5-micron resolution). Representative images of CD41 staining in the distal lung (original magnification ×20; scale bars, 100 μm. As previously described, CD41+ pixels were quantified in whole-lung sections using a random, nonbiased approach using ImageScope, version 12.4.3.500 (Leica Biosystems Imaging, Inc, Deer Park, IL) pixel quantification software.1 (D) Lung sections were stained with anti-PF4, anti-CD41, anti-CD45, and DAPI. Whole-slide scans were obtained using the 3I Marianas Confocal Microscope (×100 objective, 0.1-micron resolution). Representative images of intracellular and extracellular PF4 were captured. Original magnification ×100; scale bars, 50 μm. White arrows indicate extracellular PF4 (magenta), and yellow arrows indicate intracellular PF4 within platelets (yellow). (E) Lung sections were probed with a PF4 mRNA primer and anti-CD41, anti-CD45, and DAPI antibodies. Whole-slide scans were obtained using (×40 objective, 0.5-micron resolution). Representative images of pulmonary vessels and the distal lung were captured. Representative images of peak Pf4 mRNA expression within megakaryocytes and leukocytes in the pulmonary vessels and distal lung of WT (day 3) and Nbeal2−/− (day 14) vs their baseline, as well as other unidentified cells (original magnification ×40; scale bars, 50 μm). Yellow arrows indicate PF4 mRNA (magenta) within megakaryocytes (yellow), and blue arrows indicate Pf4 mRNA within leukocytes (cyan). Statistics: P ≤.05 by 2-way ANOVA with Tukey post hoc analysis. Comparisons: (∗) WT compared with WT baseline, (#) Nbeal2−/− compared with Nbeal2−/− baseline, (^) WT compared with Nbeal2−/−. HPX, hypoxia; NMX, normoxia.

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