Journal: Blood Advances

Article Title: Nbeal2 knockout mice are not protected against hypoxia-induced pulmonary vascular remodeling and pulmonary hypertension

doi: 10.1182/bloodadvances.2024013880

Figure Lengend Snippet: Platelet activation increased in WT and Nbeal2 −/− hypoxic mice. (A) Representative western blot of platelet lysates for PF4. (B) Quantification of western blot demonstrates platelet PF4 is reduced in Nbeal2 −/− mice compared with WT mice at baseline. (C) Platelet PF4 levels are reduced in Nbeal2 −/− mice compared with WT mice at baseline. Platelet PF4 is decreased in hypoxic WT mice at days 3 and 21 but not Nbeal2 −/− mice. (D) Plasma PF4 was decreased at baseline in Nbeal2 −/− compared with WT mice. Plasma PF4 increased in hypoxic WT and Nbeal2 −/− at day 3. Hypoxia-induced increase in plasma PF4 was attenuated in Nbeal2 −/− mice. (E) The percentage of P-selectin expressing platelets increased in hypoxic WT and Nbeal2 −/− mice at day 3. Hypoxia-induced increase in P-selectin expressing platelets was attenuated in Nbeal2 −/− compared with WT mice. (F) The percentage of activation of αIIBβ3 (JONA) expressing platelets increased in hypoxic WT and Nbeal2 −/− mice at day 3. The hypoxia-induced increase in the percentage of platelets expressing activated αIIBβ3 (JONA) was attenuated in Nbeal2 −/− compared with WT mice. (G-I) PLAs are increased in hypoxic WT mice at day 3 but not Nbeal2 −/− mice. PMAs and PNAs are similar between WT and Nbeal2 −/− mice at baseline and unchanged in hypoxia. 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 −/− . NMX, normoxia.

Article Snippet: Membranes were incubated with rabbit anti-mouse PF4 primary antibody (PF4; Abclonal, A3694; 1:2500) overnight at 4°C followed by donkey anti-rabbit horseradish peroxidase (HRP)–conjugated secondary antibody (Santa Cruz, 2313; 1:10 000).

Techniques: Activation Assay, Western Blot, Clinical Proteomics, Expressing

Journal: Blood Advances

Article Title: Nbeal2 knockout mice are not protected against hypoxia-induced pulmonary vascular remodeling and pulmonary hypertension

doi: 10.1182/bloodadvances.2024013880

Figure Lengend Snippet: 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. (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.

Article Snippet: Membranes were incubated with rabbit anti-mouse PF4 primary antibody (PF4; Abclonal, A3694; 1:2500) overnight at 4°C followed by donkey anti-rabbit horseradish peroxidase (HRP)–conjugated secondary antibody (Santa Cruz, 2313; 1:10 000).

Techniques: Staining, Imaging, Software, Microscopy, Expressing

Journal: Blood Advances

Article Title: Nbeal2 knockout mice are not protected against hypoxia-induced pulmonary vascular remodeling and pulmonary hypertension

doi: 10.1182/bloodadvances.2024013880

Figure Lengend Snippet: Lung IMs were increased at baseline and hypoxia-induced increase in lung IMs was prevented in Nbeal2 −/− mice. (A) Lung IMs were increased in Nbeal2 −/− mice at baseline compared with WT mice. (A-D) At day 3, total IMs and IM1, IM2, and IM3 subsets increased in WT but not Nbeal2 −/− mice. At day 21, total lung IMs and IM1s were increased in Nbeal2 −/− compared with WT mice. (E) Lung sections were stained with anti-CD31, anti-α-SMA, anti-CD68, anti-PF4, anti-CD41, and anti-CD45, and DAPI. Whole-slide scans were obtained using the Vectra Polaris spatial imaging scope (×40 objective, 0.5-micron resolution). Representative images of pulmonary vessels were captured. Representative image of IMs in WT and Nbeal2 −/− mice (original magnification ×40; scale bars, 50 μm). 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.

Article Snippet: Membranes were incubated with rabbit anti-mouse PF4 primary antibody (PF4; Abclonal, A3694; 1:2500) overnight at 4°C followed by donkey anti-rabbit horseradish peroxidase (HRP)–conjugated secondary antibody (Santa Cruz, 2313; 1:10 000).

Techniques: Staining, Imaging