Background-The lysyl oxidases are extracellular copper enzymes that initiate the crosslinking of collagens and elastin, 5 human isoenzymes having been characterized so far. The crosslinks formed provide the tensile strength and elastic properties for various extracellular matrices, including vascular walls. We studied the role of the first described isoenzyme Lox by inactivating its gene in mice. Methods and Results-Murine Lox gene was disrupted by routine methods. Lox Ϫ/Ϫ mice died at the end of gestation or as neonates, necropsy of the live-born pups revealing large aortic aneurysms. In light microscopy, hazy and unruffled elastic lamellae in the Lox Ϫ/Ϫ aortas were observed, and electron microscopy of the aortic walls of the Lox Ϫ/Ϫ fetuses showed highly fragmented elastic fibers and discontinuity in the smooth muscle cell layers in Lox Ϫ/Ϫ fetuses. The wall of the aorta in the Lox Ϫ/Ϫ fetuses was significantly thicker, and the diameter of the aortic lumen was significantly smaller than that in the Lox ϩ/ϩ aortas. In Lox Ϫ/Ϫ fetuses, Doppler ultrasonography revealed increased impedance in the umbilical artery, descending aorta, and intracranial artery blood velocity waveforms, decreased mean velocities across cardiac inflow and outflow regions, and increased pulsatility in ductus venosus blood velocity waveforms. Conclusions-Lox

Lysyl oxidases, a family comprising LOX and four LOX-like enzymes, catalyze crosslinking of elastin and collagens. Mouse Lox was recently shown to be crucial for development of the cardiovascular system because null mice died perinatally of aortic aneurysms and cardiovascular dysfunction. We show here that Lox is also essential for development of the respiratory system and the integrity of elastic and collagen fibers in the lungs and skin. The lungs of E18.5 Lox(-/-) embryos showed impaired development of the distal and proximal airways. Elastic fibers in E18.5 Lox(-/-) lungs were markedly less intensely stained and more disperse than in the wild type, especially in the mesenchyme surrounding the distal airways, bronchioles, bronchi, and trachea, and were fragmented in pulmonary arterial walls. The organization of individual collagen fibers into tight bundles was likewise abnormal. Similar elastic and collagen fiber abnormalities were seen in the skin. Lysyl oxidase activity in cultured Lox(-/-) skin fibroblasts and aortic smooth muscle cells was reduced by approximately 80%, indicating that Lox is the main isoenzyme in these cells. LOX abnormalities may thus be critical for the pathogenesis of several common diseases, including pulmonary, skin, and cardiovascular disorders.

Hypoxia-inducible factor (HIF) has a pivotal role in oxygen homeostasis and cardioprotection mediated by ischemic preconditioning. Its stability is regulated by HIF prolyl 4-hydroxylases (HIF-P4Hs), the inhibition of which is regarded as a promising strategy for treating diseases such as anemia and ischemia. We generated a viable Hif-p4h-2 hypomorph mouse line (Hifp4h-2 gt/gt ) that expresses decreased amounts of wild-type Hifp4h-2 mRNA: 8% in the heart; 15% in the skeletal muscle; 34 -47% in the kidney, spleen, lung, and bladder; 60% in the brain; and 85% in the liver. These mice have no polycythemia and show no signs of the dilated cardiomyopathy or hyperactive angiogenesis observed in mice with broad spectrum conditional Hif-p4h-2 inactivation. We focused here on the effects of chronic Hif-p4h-2 deficiency in the heart. Hif-1 and Hif-2 were stabilized, and the mRNA levels of glucose transporter-1, several enzymes of glycolysis, pyruvate dehydrogenase kinase 1, angiopoietin-2, and adrenomedullin were increased in the Hifp4h-2 gt/gt hearts. When isolated Hif-p4h-2 gt/gt hearts were subjected to ischemia-reperfusion, the recovery of mechanical function and coronary flow rate was significantly better than in wild type, while cumulative release of lactate dehydrogenase reflecting the infarct size was reduced. The preischemic amount of lactate was increased, and the ischemic versus preischemic

Lysyl oxidase (LOX)3 catalyzes the oxidation of specific lysine residues within extracellular elastin and collagen thus generating residues of ␣-aminoadipic-␦-semialdehyde within these proteins (1). These peptidyl aldehydes can then undergo condensation with vicinal ␣-aminoadipic-␦-semialdehyde or unmodified lysine residues to form inter-and intrapeptide covalent cross-linkages that stabilize these fibrous proteins. In addition to the non-ionic aldehyde product, the LOX-catalyzed reaction acting on protonated lysine produces stoichiometric amounts of hydrogen peroxide and ammonium, as shown in Reaction 1.