|Lysyl oxidases: Cloning and characterization of the fourth and the fifth human lysyl oxidase isoenzymes, and the consequences of a targeted inactivation of the first described lysyl oxidase isoenzyme in mice|
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ES cells carrying an inactivated Lox gene were generated by a two-step targeting method comprising homologous recombination followed by cre recombination (Figure 1A-D in III). The targeted ES cells were used to generate chimeras, of which one chimeric male transmitted the mutation to his offspring when mated with wild-type females. Deletion of the first exon, including the transcription start sites, led to complete inactivation of the Lox gene, as seen in the Northern blots (Figure 1E in III). Heterozygous Lox+/- mice, which are viable and fertile, were crossbred, and the offspring were genotyped at two weeks of age. The numbers of Lox+/+ and Lox+/- pups (n = 111) were in perfect balance, comprising 32.4% and 67.6%, respectively, but no Lox-/- pups were obtained.
To study the time of death of the Lox-/- mice, pregnant Lox+/- females were sacrificed 18.5 days after breeding with Lox+/- males, and the embryos were genotyped. Here, a normal Mendelian ratio of all three genotypes was obtained (26.7% Lox+/+, 45% Lox+/-, 28.3% Lox-/-, n = 127). Pregnant females were then carefully observed during the assumed day of delivery, and in this way four live-born Lox-/- pups were obtained for analysis and three were found dead. In addition, the remnants of three Lox-/- pups were recovered, which were used for DNA analysis. The gross appearance of the four newborn Lox-/- pups was normal, but they had cyanotic skin and their condition was poor. They were breathing, but were sluggish and did not suck milk (Figure 2A in III). Autopsy revealed large aneurysms (Figure 2B in III) in the aortas of three of the four. A narrowed lumen was observed in paraffin sections of the abdominal aorta of all four pups, and a large aneurysmal dilatation, of a diameter approximately 3 times larger than in the aortas of their wild-type littermates, in sections from two pups (Figure 2C and D in III). The three neonatal Lox-/- pups that had died immediately after birth had a congenital diaphragmatic hernia, and two of them also had a large hemorrhage in the upper chest region.
Since preliminary analyses indicated defects in blood vessels, aortas from embryos at different stages were taken for detailed analysis. Alterations in the structure of the aortic wall were already seen at E14.5, which was the earliest time point analyzed, but were most obvious in the E18.5 embryos. Light microscopy revealed hazy and unruffled elastin lamellae in the Lox-/- samples, whereas the lamellae in the wild-type aorta were well defined and ruffled. The wall of the aorta in the Lox-/- embryos was significantly thicker (P<0.005; see Table 1 in III) and the diameter of the aortic lumen was significantly smaller (P<0.001; see Table 1 in III) than in the Lox+/+ aortas. Most of the endothelial cells on the internal elastic lamellae in the Lox-/- aortic wall were rounded in appearance, whereas those in the Lox+/+ aorta were flat (Figure 3A and B in III). No diaphragmatic hernias were found in the E16.5-18.5 Lox-/- embryos, even though these were seen in three out of the seven Lox-/- neonates.
Electron microscopy of the Lox-/- aortas indicated that the elastic laminae were fragmented, having only remnants of their elastic fibers, and the smooth muscle cell layer was disorganized. The endothelial cells showed degenerative changes such as vacuolization, blebbing, and even detachment from the elastic lamina (Figure 3C and D in III). No major differences in the collagen fibers of the aortic adventitia were seen between the Lox-/- and Lox+/+ embryos by electron microscopy.
Since the Lox+/- adult mice seemed to be fertile and have a normal life-span, we continued to compare the data from Lox-/- and Lox+/+ embryos only. No difference in fetal heart rate was found between the two groups, but the pulsatility indices for the descending aorta, umbilical artery, and intracranial arteries were significantly higher, and the mean outflow and inflow velocities were significantly lower in the Lox-/- embryos (Table 2 in III). In addition, embryos with the highest pulsatility indices in the descending aorta had semilunar valve regurgitation. The pulsatility indices for veins in the ductus venosus were found to be significantly higher in the Lox-/- embryos (Table 2 in III), and were diagnosed as a sign of congestive heart failure. In addition, embryos with the highest pulsatility indices in the descending aorta had semilunar valve regurgitation (see Figure 4B-C in III).