Characterization of ESL-1 deficient mice
Following RACE, amplified PCR fragments were sequenced and aligned with more than 300 base pairs of ESL-1 exon 1 sequence ().
Characterization of ESL-1 deficient mice
No ESL-1 null mice (ESL-1-/-) were present at 3 weeks of age while expected numbers of ESL-1+/- and ESL-1+/+ mice were present (), consistent with complete lethality due to ESL-1 deficiency. Timed breedings between ESL-1+/- mice were set up to determine at what stage lethality occurred. ESL-1+/- females were mated to ESL-1+/- males and pregnant females were then sacrificed at gestation day 10.5. DNA was analyzed from embryos with primers to amplify the gene trap and wild-type alleles. No ESL-1-/- embryos were identified (). These findings are consistent with a critical role of ESL-1 in early embryonic development.
To determine the effect of heterozyogous ESL-1 deficiency on ESL-1 expression, ESL-1 RNA levels were measured by quantitative RT-PCR from spleen RNA. RNA expression was significantly lower in ESL-1+/- mice compared to ESL-1+/+ mice (1.10 ± 0.09 versus 6.03 ± 2.72 relative expression, n=3 mice per group, p=0.03). ESL-1 protein levels, detected via Western blotting, were also lower in ESL-1+/- mice compared to ESL-1+/+ mice ().
To determine the effect of heterozygous ESL-1 deficiency on leukocyte-endothelial (L-E) interactions, intravital microscopy was performed on the cremaster venules of 8 week old male ESL-1+/- mice following challenge with locally injected tumor necrosis factor-alpha (TNF-α). ESL-1+/- mice demonstrated increased numbers of rolling leukocytes but decreased firmly attached leukocytes on endothelial cells compared to ESL-1+/+ littermates ().
To determine the effect of heterozygous ESL-1 deficiency on endothelial function, pressure myography10
was performed on mesenteric arteries from 18 week old mice which were fed 10 weeks of high-fat, high-sucrose diet. Endothelial dependent relaxation response to acetylcholine (10-4
mol/L) was impaired to the same extent in both ESL-1+/-
littermate mice (29.42±3.57 versus 24.46±6.21 %, respectively).
To assess the role of heterozygous ESL-1 deficiency on leukocyte extravasation, 10 weeks old ESL-1+/- and ESL-1+/+ mice were challenged with peritoneal thioglycollate injection. Compared to ESL-1+/+ mice, intraperitoneal leukocytes were reduced in ESL-1+/- mice (6.36±1.21×106 versus 2.71±0.61×106 cells/animal, n=4 mice per group, p=0.04).
Effect of reduced ESL-1 expression on atherosclerosis
To determine if heterozygous deficiency of ESL-1 would affect monocyte recruitment to atherosclerotic plaques, ApoE-/-, ESL-1+/- and ApoE-/-, ESL-1+/+ mice were compared. Following 9 weeks of a western diet, atherosclerosis was quantified by oil-red-o enface staining of the aorta and major branches. No difference in surface area staining was noted between the groups of mice (ApoE-/-, ESL-1+/- = 2.22±0.25 (n=8) versus ApoE-/-, ESL-1+/+ = 2.37±0.37% (n=14) lesion area, p=0.1). Similarly, lesion thickness quantified at the level of the aortic valve was not different between ApoE-/-, ESL-1+/- and ApoE-/-, ESL-1+/+ mice (2245.81±380.93 versus 1663.88±374.45 μm2, respectively, p=0.72). However, lesion analysis revealed greater collagen deposition () and fewer macrophages () in ApoE-/-, ESL-1+/- mice compared to ApoE-/-, ESL-1+/+ mice. Smooth muscle cell alpha actin staining of the aortic root was not different between ApoE-/-, ESL-1+/- and ApoE-/-, ESL-1+/+ mice (2.10 ± 0.56 versus 3.80 ± 1.66 %, respectively, p=0.1).
Reduced CD68 expression was also observed in liver tissue of ApoE-/-, ESL-1+/- compared to ApoE-/-, ESL-1+/+ mice (1.24±0.11 versus 1.69±0.11 respectively, n=4-5 per group, p=0.026), and this correlated with reduced macrophage content (7.55±0.96 cells per 400x field in ApoE-/-, ESL-1+/- compared to 13.58±1.37 cells per 400x field in ApoE-/-, ESL-1+/+ mice, n=6-7 per group, p< 0.0005).
No significant differences in LV chamber thickness were observed between ApoE-/-, ESL-1+/+ and ApoE-/-, ESL-1+/- mice (577.7±31.73 versus 582.1±26.85 μm, respectively, n=5 and 8 per group) or right ventricular chamber thickness (274.0±7.31 versus 313.7±24.1μm, respectively, n=5 and 8 per group). Myocardial collagen deposition was also not different between ApoE-/-, ESL-1+/+ and ApoE-/-, ESL-1+/- by Sirius Red staining.