P-selectin-deficient mice to study pathophysiology of sickle cell disease

Abstract

1. P-selectin–deficient SCD mice are protected from lung vaso-occlusion.

2. P-selectin–deficient SCD mice will be useful in assessing the benefits of anti–P-selectin therapy in diverse complications of SCD.

Figure 1.

Generation and characterization of SS-Selp^−/−mice. (A) Selp^−/− mice were bred to Townes SS mice to generate SS-Selp^−/− mice. Refer to supplemental Figure 1 for the breeding strategy. (B) Genomic PCR gel image showing the presence or absence of different alleles in C57BL/6 (WT) mice, Townes AS (AS) mice, Townes SS (SS) mice, Selp^−/− mice, and SS-Selp^−/− mice. Human WT β-globin (WT β^A), mouse β-globin, mouse α-globin, and mouse WT P-selectin alleles were absent in SS-Selp^−/− mice, but human β^S, human α-globin, and mouse mutant P-selectin (P-selectin^−/−) alleles were present in SS-Selp^−/− mice. (C) RT2qPCR analysis revealed a significant reduction in the mRNA levels of P-selectin in the aortas of SS-Selp^−/− mice compared with SS mice. Data are mean relative mRNA expression ± standard deviation (SD). (D) Western blot analysis of CD62P protein levels in platelets isolated from SS and SS-Selp^−/− mice. P-selectin (135 kDa) was expressed in platelets of SS mice but was absent in platelets of SS-Selp^−/− mice. Actin (37 kDa) was used as a loading control. Recombinant mouse P-selectin–Fc fusion protein (RP; 235 kDa; R&D Systems) was used as a positive control. Data in panels C-D are representative of 3 female SS mice and 3 female SS-Selp^−/− mice. Representative IHC images showing sinusoidal congestion by H&E staining (E) and fibrosis and collagen deposition by Sirius Red staining (F) in the liver sections of SS and SS-Selp^−/− mice. Original magnification ×40. (G) Percentage of sinusoidal congestion based on H&E staining. (H) Quantification of liver fibrosis as the percentage of the area positive for Sirius Red staining. Data in panels G-H are mean ± SE based on 3 male SS mice and 3 male SS-Selp^−/− mice. *P < .05.

Figure 2.

Genetic deletion of P-selectin attenuates pulmonary vaso-occlusion in SCD mice. (A) SS and SS-Selp^−/− mice were challenged IV with 0.1 µg/kg LPS, and qFILM was used 2 to 2.5 hours later to visualize the pulmonary microcirculation. (B) IV LPS (0.1 µg/kg) triggered occlusion of pulmonary arteriolar bottlenecks (large dotted ovals) in SS mice by large aggregates of neutrophils (red) and platelets (green). The same FOV is shown over 3 time points. A neutrophil (red; small dashed circles) flows toward the occlusion but cannot pass through it and is forced to flow toward another open vessel to the side of the occlusion. Supplemental Video 1 shows the complete time series for the FOV in panel B. (C) In contrast to SS mice, the majority of FOVs in SS-Selp^−/− mice were free of pulmonary vaso-occlusions. The same FOV is shown over 3 time points. A neutrophil (red; small dashed circles) is seen trafficking up the pulmonary arteriole that has no aggregates present. Supplemental Video 2 shows the complete time series for the FOV in panel C. The pulmonary microcirculation was labeled with FITC-Dextran and pseudo-colored purple. Neutrophils and platelets were labeled by IV administration of AF546-conjugated anti-Ly6G mAb and V450-conjugated anti-CD49b mAb, respectively. Neutrophils are shown in red, and platelets are pseudo-colored green. The arrows denote the direction of blood flow, and the asterisks (*) denote alveoli. Scale bars, 20 μm. The diameters of the vessels are 34 μm and 40 μm in panels B-C, respectively. (D-F) The neutrophil-platelet aggregates blocking pulmonary arterioles were quantified as described in Methods. After IV LPS administration, SS-Selp^−/− mice had a significantly decreased average number of pulmonary vaso-occlusions per FOV (D), percentage of FOVs with pulmonary vaso-occlusions (E), and large pulmonary vaso-occlusions (area >1000 μm²) (F) compared with SS mice. The average number of pulmonary vaso-occlusions per FOV and large pulmonary vaso-occlusions (area >1000 µm²) were compared between groups using the unpaired Student t test, and the percentage of FOVs with pulmonary vaso-occlusions were compared between groups using fourfold table with χ² analyses. SS mice: n = 1 male mice and 2 female mice, FOV = 45. SS-Selp^−/− mice: n = 1 male mice and 2 female mice, FOV = 46. Error bars are mean ± SE. *P < .05.