|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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The consequence of inactivation of the Lox gene, neonatal lethality, was highly surprising, considering that Lox has at least four additional isoenzymes, as discussed in previous sections, mice lacking elastin survive for up to four days (Li et al. 1998), and mice totally lacking type III collagen can survive for up six months (Liu et al. 1997b). Our results show that Lox has an essential function in the development of the embryonic cardiovascular system, and particulary in the maintenance of the structure and mechanical properties of the aortic wall. Deficiency in lysyl oxidase activity is likely to affect several aortic wall components, including its major components elastin and collagen types I and III (see Jacob et al. 2001, Silver et al. 2001 for review), but may also affect components found in minor amounts. The early development of the aorta, and the translation and secretion of the aortic wall extracellular matrix components, are likely to occur normally in Lox-/- embryos, as evidenced by the normal number of elastic lamellae and smooth muscle cell layers. Elastin exists in elastic fibers in two forms: as tropoelastin, a soluble precursor of elastin, and as a highly cross-linked elastin in the amorphous component (Cleary & Gibson 1996, Vrhovski & Weiss 1998). Assembly of tropoelastin into the amorphous component involves covalent cross-linking into a highly insoluble form (Cleary & Gibson 1996, Vrhovski & Weiss 1998). Lox has been localized immunologically to the mature elastic fibers (Kagan et al. 1986, Baccarani-Contri et al. 1989), and the formation of lysyl oxidase-catalysed cross-linking has been suggested to play a critical role in the nucleation of the elastin assembly (Brown-Augsburger et al. 1995). Our results, therefore, suggest that other lysyl oxidase isoezymes may also play a role in elastin cross-linking, since the amorphous and insoluble component of the elastic fibers exists in Lox-/- embryos, as seen in Figure 6. It is possible that the diminished amount of cross-links in Lox-/- embryos increases the susceptibility of elastin to degradation by matrix metalloproteinases (Tinker et al. 1990, Grange et al. 1997), which would lead to the fragmentation of the elastic fibers. As a result, the fragmented elastic fibers are probably unable to bear the hemodynamic stress, which leads to dilatation and rupture of the aortic wall. A similar destruction of the elastic fibers, as is seen in the aortic walls of the Lox-/- embryos, has been hypothesized to play a key role in the formation of an aneurysmal aortic dilatation, thus shifting the load produced by the blood pressure onto collagens, with consequent dilatation and eventually rupture of the aortic wall (MacSweeney et al. 1992, 1994). Disintegration of the intimal and medial elastic lamellaes, to which the endothelial cells and smooth muscle cells attach, also results in the degeneration of endothelial cells and disorganization of smooth muscle cells. In elastin null mice, smooth muscle cells proliferate and thereby stabilize the arterial structure, but finally obliterate the arterial lumen (Li et al. 1998). We did not observe any significant compensatory smooth muscle cell proliferation in the Lox-/- aortas, probably because normal amounts of elastin were produced, at least during the early stages. It has been suggested that elastin may control the proliferation of vascular smooth muscle cells (Li et al. 1998), but polymerization does not seem to be necessary for this regulatory function.
Figure 6. Structure of the aortic wall of the Lox+/+ and Lox-/- E18.5 embryos. Upper panels: Sections from the descending aorta at the site of the right crus of the diaphragm, stained with hematoxylin-eosin and illuminated in UV light. The lumen of the aorta is marked with an asterisk, and the single intimal elastic lamella is boxed. Lower panels: Electron micrographs from the site of a single elastic fiber from the boxed sections in upper panels: Amorphous elastin (aEL) in a continuous elastic fiber is seen in the Lox+/+ aortic wall (left). Despite the high degree of fragmentation of the elastic fiber, the remnants of the amorphous elastin (arrows) are still seen in the Lox-/- aortic wall (right).
To study the actual physiological consequences of structural abnormalities found in the Lox-/- aortic walls, we decided to use ultrasonographic studies. Increased pulsatility indices were measured in the umbilical artery, descending aorta, and intracranial arteries in Doppler ultrasonography of the E18.5 Lox-/- embryos. By comparison, although increased impedance of the umbilical artery and descending aorta is also found in human pregnancies with placental insufficiency, it is connected with a concomitant decrease in pulsatility indices in the cerebral circulation, as the fetus aims to maintain an adequate oxygen supply to the coronary and cerebral circulation (Reed 1997). Therefore, the universally increased arterial pulsatility indices observed in the Lox-/- embryos are likely to be due to abnormal elastic properties of the arterial walls, as discussed above, and not due to placental insufficiency.
The decreased mean velocities in the inflow and outflow regions suggest a lower cardiac output in Lox-/- than in Lox+/+ embryos. In order to maintain adequate blood supply and tissue perfusion, arterial blood pressure should be increased, as suggested in fetuses with intrauterine growth restriction and lower relative pulse amplitude in the descending aorta (Stale et al. 1991). The increase in systolic blood pressure in the Lox-/- embryos, in combination with aortic wall fragility, would then lead to the formation of aortic aneurysms and ruptures, which are otherwise rare in newborns. A decrease in the resilience of the arterial walls will lead to an increased afterload on the heart, as evidenced by the fact that the two Lox-/- embryos with the highest pulsatility indices in the descending aorta, and thus the greatest rise in the afterload, also had semilunar valve regurgitations. Furthermore, the increased pulsatility indices for veins in the ductus venosus imply an increase in systemic venous pressure, indicating congestive heart failure (Reed et al. 1997, Kiserud et al. 1999), which may contribute to the death of Lox-/- mice at the end of gestation or during delivery.
Reduced lysyl oxidase activity is found in animals with copper deficiency, in lathyrism, and in two X-linked, recessively inherited human disorders, Menkes syndrome and OHS, as discussed in Section 2.5. Lathyrism is caused by administration of β APN, a potent irreversible inhibitor of the LOX enzyme, while the low lysyl oxidase activity levels in Menkes disease, OHS, and copper-deficiency are secondary to abnormalities in copper metabolism. In all these conditions, all lysyl oxidase isoenzymes are thought to be affected, but their activities are not completely abolished. The present study provides the first evidence indicating that the lack of activity of the single isoenzyme Lox leads to major defects in the vascular elastic fibers. Mottled blotchy mice, a mouse model for OHS, die of aortic ruptures before six months of age, and the histological findings of early changes in their aortas (Brophy et al. 1988) are similar to those seen here in the Lox-/- embryonic aortas and closely resemble the pathological alterations in elastic fibers found in human aneurysms. In blotchy mice, the level of total lysyl oxidase activity in the aortic tissue is decreased by 59% (Moursi et al. 1995), but since these mice are born without aneurysms or other cardiovascular abnormalities, this level of activity must be sufficient during embryogenesis. Our data suggest that significantly reduced activity of the human LOX enzyme may also contribute to cardiovascular symptoms, and therefore alterations in LOX activity may play a critical role in human cardiovascular diseases.
All lysyl oxidase isoenzymes are likely to be able to use both elastin and collagens as their substrates, as has so far been shown for three of the five isoenzymes (Kagan 1986, Borel et al. 2001, Ito et al. 2001). Our data indicate, however, that the Lox functions were not compensated for by other isoenzymes, at least not to any significant extent. Thus, Lox clearly has a central role among the isoenzymes in the development of the cardiovascular system during embryogenesis, and the functions and significance of the other isoenzymes remain to be explored.
In conclusion, the pathophysiological sequence of events in the cardiovascular system of Lox-/- embryos suggests that deficient cross-linking of elastin and collagens causes a decline in the resilience and tensile strength of the arterial walls that leads to endothelial cell damage and disorganization of the smooth muscle cells, and causes severe abnormalities in the cardiovascular functions, finally contributing to perinatal death. In some of the cases, a diaphragmatic hernia may contribute to neonatal lethality. Our findings suggest that alterations in LOX activity may also play a critical role in human cardiovascular diseases. Therefore, this mouse model will be a useful tool for further studies.