Arpin-deficient mice are viable but show increased vascular permeability in the lung.
(A) Arpin mouse gene showing exon 3, which was deleted using the CRISP/Cas9 technology to generate the complete arpin-/- mouse model.
(B) Representative genotypification PCR of mice carrying the WT (arpin+/+), heterozygous (arpin+/-), and deleted (KO, arpin -/-) alleles.
(C) Representative Western blots for arpin in lysates of the indicated organ tissues from arpin+/+ and arpin-/- mice (n=3).
(D) Permeability assays in the lungs. 150 kDa FITC-dextran was injected into arpin+/+ and arpin-/- mice via the cannulated artery carotid. Animals were sacrificed, perfused, and lungs collected and homogenized in PBS. The homogenized tissue containing the leaked FITC-dextran was quantified using a fluorometer. (5 arpin+/+ and 8 arpin-/- mice were analyzed). Data are represented as mean ± SEM; *p<0.05; two-tailed student’s t-test with Welch’s correction.
(E) Representative Western blots for the indicated proteins in lysates of lung tissue from arpin+/+ and arpin-/- mice. The graph shows the quantification of the relative pixel intensity of the protein bands normalized to γ-tubulin as a loading control (n=3).
(F) Representative images of the hematoxylin and eosin staining of lung tissues of arpin+/+ (images 1 and 2) and arpin-/- (images 3-6) mice. Image 1 shows normal histology (10x objective). Image 2 shows normal structure of alveoli (*) and the blood vessels (arrowheads) without any pathology (40x objective, images 2-6). Image 3 shows some areas with alveolar volume reduced (*) in arpin-/- mice. Image 4 shows discrete interalveolar hemorrhages (arrow heads). Image 5 shows congestion and dilatation of the capillaries (arrow heads). Image 6 shows interalveolar edema (*) and microhemorrhage (arrowhead). The histological score of the lung tissue is shown in the graph below. A score of 0 indicates no inflammation; 1 low inflammation; 2 moderate inflammation; 3 high inflammation (9 images from arpin+/+ and 12 images from arpin-/- mice were analyzed). Data are represented as mean ± SEM; *p<0.05; two-tailed student’s t-test.
(G) Representative immunostaining for PECAM-1 and F-actin staining using phalloidin in cryosections from lungs of arpin+/+ and arpin-/- mice. Images in the bottom shown endothelial cell (EC) F-actin extracted using PECAM-1 as a templete of ECs using Imaris software (40x objective, scale bar = 50μm). Graph shows mean F-actin pixel intensity quantification in arpin-/- lungs normalized to the average of images from arpin+/+ lungs (20 images were analyzed from three mice in each group). Data are represented as mean ± SEM; **p<0.01; two-tailed student’s t-test.