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• W2043157186 abstract "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 ∼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. 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 ∼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. Lysyl oxidases are extracellular copper-dependent enzymes that catalyze the formation of lysine and hydroxylysine-derived cross-links in collagens and lysine-derived cross-links in elastin. These cross-links are essential for the tensile strength of collagens and the rubber-like properties of elastin, both abundant extracellular matrix proteins that are necessary for the structural integrity and function of connective tissues.1Kielty CM Grant ME The collagen family: structure, assembly, and organization in the extracellular matrix.in: Royce PM Steinmann B Connective Tissue and Its Heritable Disorders. Molecular, Genetic, and Medical Aspects. Wiley-Liss, Inc., New York2002: 159-221Crossref Google Scholar, 2Kagan HM Li W Lysyl oxidase: properties, specificity, and biological roles inside and outside of the cell.J Cell Biochem. 2003; 88: 660-672Crossref PubMed Scopus (689) Google Scholar, 3Myllyharju J Kivirikko KI Collagens, modifying enzymes and their mutations in humans, flies and worms.Trends Genet. 2004; 20: 33-43Abstract Full Text Full Text PDF PubMed Scopus (862) Google Scholar In addition to the first characterized lysyl oxidase, LOX,4Trackman PC Pratt AM Wolanski A Tang SS Offner GD Troxler RF Kagan HM Cloning of rat aorta lysyl oxidase cDNA: complete codons and predicted amino acid sequence.Biochemistry. 1990; 29: 4863-4870Crossref PubMed Scopus (107) Google Scholar, 5Hämäläinen ER Jones TA Sheer D Taskinen K Pihlajaniemi T Kivirikko KI Molecular cloning of human lysyl oxidase and assignment of the gene to chromosome 5q23.3-31.2.Genomics. 1991; 11: 508-516Crossref PubMed Scopus (117) Google Scholar four LOX-like proteins are currently known: LOXL1, LOXL2, LOXL3, and LOXL4.6Kenyon K Modi WS Contente S Friedman RM A novel human cDNA with a predicted protein similar to lysyl oxidase maps to chromosome 15q24–q25.J Biol Chem. 1993; 268: 18435-18437Abstract Full Text PDF PubMed Google Scholar, 7Kim Y Boyd CD Csiszar K A new gene with sequence and structural similarity to the gene encoding human lysyl oxidase.J Biol Chem. 1995; 270: 7176-7182Crossref PubMed Scopus (104) Google Scholar, 8Saito H Papaconstantinou J Sato H Goldstein S Regulation of a novel gene encoding a lysyl oxidase-related protein in cellular adhesion and senescence.J Biol Chem. 1997; 272: 8157-8160Crossref PubMed Scopus (96) Google Scholar, 9Jourdan-Le Saux C Tronecker H Bogic L Bryant-Greenwood GD Boyd CD Csiszar K The LOXL2 gene encodes a new lysyl oxidase-like protein and is expressed at high levels in reproductive tissues.J Biol Chem. 1999; 274: 12939-12944Crossref PubMed Scopus (83) Google Scholar, 10Huang Y Dai J Tang R Zhao W Zhou Z Wang W Ying K Xie Y Mao Y Cloning and characterization of a human lysyl oxidase-like 3 gene (hLOXL3).Matrix Biol. 2001; 20: 153-157Crossref PubMed Scopus (29) Google Scholar, 11Jourdan-Le Saux C Tomsche A Ujfalusi A Jia L Csiszar K Central nervous system, uterus, heart, and leukocyte expression of the LOXL3 gene, encoding a novel lysyl oxidase-like protein.Genomics. 2001; 74: 211-218Crossref PubMed Scopus (58) Google Scholar, 12Mäki JM Kivirikko KI Cloning and characterization of a fourth human lysyl oxidase isoenzyme.Biochem J. 2001; 355: 381-387Crossref PubMed Scopus (76) Google Scholar, 13Asuncion L Fogelgren B Fong KS Fong SF Kim Y Csiszar K A novel human lysyl oxidase-like gene (LOXL4) on chromosome 10q24 has an altered scavenger receptor cysteine rich domain.Matrix Biol. 2001; 20: 487-491Crossref PubMed Scopus (67) Google Scholar, 14Mäki JM Tikkanen H Kivirikko KI Cloning and characterization of a fifth human lysyl oxidase isoenzyme: the third member of the lysyl oxidase-related subfamily with four scavenger receptor cysteine-rich domains.Matrix Biol. 2001; 20: 493-496Crossref PubMed Scopus (82) Google Scholar All four are likely to catalyze cross-link formation in collagens and elastin, as has been shown so far for LOXL1 and LOXL4,15Borel A Eichenberger D Farjanel J Kessler E Gleyzal C Hulmes DJ Sommer P Font B Lysyl oxidase-like protein from bovine aorta. Isolation and maturation to an active form by bone morphogenetic protein-1.J Biol Chem. 2001; 276: 48944-48949Crossref PubMed Scopus (118) Google Scholar, 16Jung ST Kim MS Seo JY Kim HC Kim Y Purification of enzymatically active human lysyl oxidase and lysyl oxidase-like protein from Escherichia coli inclusion bodies.Protein Expr Purif. 2003; 31: 240-246Crossref PubMed Scopus (43) Google Scholar, 17Kim MS Kim SS Jung ST Park JY Yoo HW Ko J Csiszar K Choi SY Kim Y Expression and purification of enzymatically active forms of the human lysyl oxidase-like protein 4.J Biol Chem. 2003; 278: 52071-52074Crossref PubMed Scopus (47) Google Scholar but the specific functions of these isoenzymes are as yet unknown. Expression of LOX is markedly increased in fibrotic tissues, including models of dermal, lung, liver, and arterial fibrosis.18Smith-Mungo LI Kagan HM Lysyl oxidase: properties, regulation and multiple functions in biology.Matrix Biol. 1998; 16: 387-398Crossref PubMed Scopus (441) Google Scholar Although no human disease caused by a primary LOX deficiency has been identified so far, at least three vertebrate conditions are associated with reduced lysyl oxidase activity. Administration of β-amino-propionitrile (β-APN), a lysyl oxidase inhibitor, leads to abnormal cross-linking of collagens and elastin and results in lathyrism, a disease characterized by kyphoscoliosis, bone deformities, weakening of the epiphyseal plates, tendons, ligament attachments, skin and cartilage, dislocation of joints, loss of teeth, hernias, and vascular rupture.19Steinmann B Royce PM Superti-Furga A The Ehlers-Danlos Syndrome.in: Royce PM Steinmann B Connective Tissue and Its Heritable Disorders. Molecular, Genetic, and Medical Aspects. Wiley-Liss Inc., New York2002: 431-523Crossref Google Scholar A deficiency in copper, a lysyl oxidase cofactor, leads to manifestations closely resembling those of lathyrism.19Steinmann B Royce PM Superti-Furga A The Ehlers-Danlos Syndrome.in: Royce PM Steinmann B Connective Tissue and Its Heritable Disorders. Molecular, Genetic, and Medical Aspects. Wiley-Liss Inc., New York2002: 431-523Crossref Google Scholar Lysyl oxidase activity is also reduced in two X-linked recessively inherited disorders, Menkes disease and its milder form, occipital horn syndrome, both caused by mutations in a copper transporter gene and characterized by neurological and connective tissue symptoms.20Horn N Tümer Z Menkes disease and occipital horn syndrome.in: Royce PM Steinmann B Connective Tissue and Its Heritable Disorders. Molecular, Genetic, and Medical Aspects. Wiley-Liss Inc., New York2002: 651-685Crossref Google Scholar LOX has also been reported to have novel biological roles as a tumor suppressor, affecting cell adhesion and growth control and having intracellular and intranuclear functions.2Kagan HM Li W Lysyl oxidase: properties, specificity, and biological roles inside and outside of the cell.J Cell Biochem. 2003; 88: 660-672Crossref PubMed Scopus (689) Google Scholar, 18Smith-Mungo LI Kagan HM Lysyl oxidase: properties, regulation and multiple functions in biology.Matrix Biol. 1998; 16: 387-398Crossref PubMed Scopus (441) Google Scholar, 21Csiszar K Lysyl oxidases: a novel multifunctional amine oxidase family.Prog Nucleic Acid Res Mol Biol. 2001; 70: 1-32Crossref PubMed Google Scholar Inactivation of the mouse Lox gene has recently been shown to lead to perinatal death caused by aortic aneurysms, cardiovascular dysfunction, and diaphragmatic rupture.22Mäki JM Räsanen J Tikkanen H Sormunen R Mäkikallio K Kivirikko KI Soininen R Inactivation of the lysyl oxidase gene Lox leads to aortic aneurysms, cardiovascular dysfunction, and perinatal death in mice.Circulation. 2002; 106: 2503-2509Crossref PubMed Scopus (397) Google Scholar, 23Hornstra IK Birge S Starcher B Bailey AJ Mecham RP Shapiro SD Lysyl oxidase is required for vascular and diaphragmatic development in mice.J Biol Chem. 2003; 278: 14387-14393Crossref PubMed Scopus (258) Google Scholar The wall of the aorta in Lox-null (Lox−/−) embryos was significantly thicker and the aortic lumen markedly smaller than in wild-type (WT) embryos, and the elastic fibers in the smooth muscle cell layers of their aortic walls were highly fragmented and discontinuous.22Mäki JM Räsanen J Tikkanen H Sormunen R Mäkikallio K Kivirikko KI Soininen R Inactivation of the lysyl oxidase gene Lox leads to aortic aneurysms, cardiovascular dysfunction, and perinatal death in mice.Circulation. 2002; 106: 2503-2509Crossref PubMed Scopus (397) Google Scholar, 23Hornstra IK Birge S Starcher B Bailey AJ Mecham RP Shapiro SD Lysyl oxidase is required for vascular and diaphragmatic development in mice.J Biol Chem. 2003; 278: 14387-14393Crossref PubMed Scopus (258) Google Scholar These results suggested that Lox has an essential role in the development and function of the cardiovascular system. Elastin cross-links and immature collagen cross-links were decreased in amount in the aorta and lungs of the Lox−/− embryos by ∼60% and 40%, respectively.23Hornstra IK Birge S Starcher B Bailey AJ Mecham RP Shapiro SD Lysyl oxidase is required for vascular and diaphragmatic development in mice.J Biol Chem. 2003; 278: 14387-14393Crossref PubMed Scopus (258) Google Scholar Some of the Loxl proteins thus appear to participate in the cross-linking of collagens and elastin, but they cannot fully compensate for the absence of Lox activity. Inactivation of the mouse Loxl1 gene is not lethal, but Loxl1−/− mice show disturbed regeneration of elastic fibers in the postpartum intrauterine tract and also develop pelvic prolapse, enlarged air spaces in the lung, laxity of the skin, and vascular abnormalities that coincide with accumulation of the elastin precursor tropoelastin.24Liu X Zhao Y Gao J Pawlyk B Starcher B Spencer JA Yanagisawa H Zuo J Li T Elastic fiber homeostasis requires lysyl oxidase-like 1 protein.Nat Genet. 2004; 36: 178-182Crossref PubMed Scopus (510) Google Scholar In contrast to Lox, Loxl1 was localized to sites of elastogenesis and interacted with fibulin-5, which is known to bind tropoelastin.24Liu X Zhao Y Gao J Pawlyk B Starcher B Spencer JA Yanagisawa H Zuo J Li T Elastic fiber homeostasis requires lysyl oxidase-like 1 protein.Nat Genet. 2004; 36: 178-182Crossref PubMed Scopus (510) Google Scholar The phenotypes of the Lox−/− and Loxl1−/− knockout mice suggest that members of the Lox family are likely to show functional differences in vivo. In the current study we examined the Lox−/− mouse line further. The distal and proximal airways of the lungs in embryos were found to be enlarged and their numbers were found to be decreased, leading to a condition resembling adult emphysema. Histochemistry and electron microscopy of the lungs demonstrated generalized elastinopathy and structurally abnormal collagen bundles, which were also seen in the skin. The amount of lysyl oxidase activity was reduced by ∼80% in cultured Lox−/− skin fibroblasts and aortic smooth muscle cells, the Loxl proteins evidently being responsible for the remaining 20% of the activity. This is the first demonstration at the level of lysyl oxidase activity that the Loxl proteins also contribute to the total activity in vivo, although Lox is responsible for the majority of it, at least in the cell types studied. Thus, in addition to proper development of the cardiovascular system,22Mäki JM Räsanen J Tikkanen H Sormunen R Mäkikallio K Kivirikko KI Soininen R Inactivation of the lysyl oxidase gene Lox leads to aortic aneurysms, cardiovascular dysfunction, and perinatal death in mice.Circulation. 2002; 106: 2503-2509Crossref PubMed Scopus (397) Google Scholar Lox activity is essential for normal development of the respiratory system and the skin. Tissue samples and whole embryos were fixed overnight in 10% buffered formalin or in Bouin's solution or Carnoy's solution for 2 to 12 hours, depending on the fixation method used, and embedded in paraffin. The lung samples used in the morphometric analysis were fixed under a 50-mbar vacuum. Sections were stained with hematoxylin and eosin (H&E), Masson's trichrome, and Hart's modified elastin stain. The WT and Lox−/− samples were processed and sectioned in parallel and analyzed without knowledge of the genotype. Paraffin sections (5 μm) from the WT and Lox−/− animals were used for immunohistochemical analyses. Antibodies against tropoelastin domains 7 to 26 (Elastin Products Co. Inc., Owensville, MO), type I collagen (Rockland, Gilbertsville, PA), type III collagen (Rockland), type IV collagen (Chemicon Int. Inc., Temecula, CA), and smooth muscle α-actin (α-SMA) (Sigma, St. Louis, MO) were used at a dilution of 1:50 to 1:200 in phosphate-buffered saline (PBS). Samples stained with the collagen antibodies were pretreated with 0.4% pepsin in 0.01 mol/L HCl, pH 2.0, at 37°C for 5 minutes. Before application of the primary antibodies, the sections were treated with 0.3% H2O2 in methanol to suppress endogenous peroxidase activity. The primary antibody binding was visualized using the Histomouse SP (AEC) kit (Zymed Laboratories, Inc., San Francisco, CA) according to the manufacturer's instructions. Sections were counterstained with hematoxylin. Digital images with appropriate magnification were captured from H&E-stained paraffin sections of the left lobe of the lung at E15.5 and E18.5, E15.5 being the earliest time point analyzed morphometrically. Immunostaining with α-SMA antibody was used to identify the pulmonary arteries. Eight to ten randomly selected paraffin sections from different parts of the left lobe of the lung of at least six embryos per genotype were analyzed. The numbers of distal airways, bronchioles, and arteries per mm2 were counted, and also the numbers of epithelial cells in the bronchioles, and the thicknesses of the bronchial epithelium and pulmonary arterial walls were measured. In the latter case, the thicknesses of arteries within the typical 70- to 130-μm circumference range at E18.5 were measured using 8 to 10 randomly selected sections from three individual embryos per genotype. Statistical analyses were performed using the F- and t-tests. Lung and skin biopsies from E18.5 embryos were fixed in a mixture of 1% glutaraldehyde and 4% formaldehyde in 0.1 mol/L phosphate buffer, postfixed in 1% osmium tetroxide, dehydrated in acetone, and embedded in Epon EMBed 812. For elastin contrasting, specimens treated with osmium tetroxide were immersed in 2% aqueous tannic acid for 1 hour and further processed as described above. Thin sections were cut with a Reichert Ultracut ultramicrotome and examined in a Philips CM100 transmission electron microscope (FEI Co., Eindhoven, The Netherlands). Images were captured with a charge-coupled device camera equipped with TCL-EM-Menu version 3 from Tietz Video and Image Processing Systems GmbH (Gaunting, Germany). Samples for immunoelectron microscopy were fixed in 4% paraformaldehyde in 0.1 mol/L phosphate buffer with 2.5% sucrose for 2 hours at room temperature, after immersion in 2.3 mol/L sucrose, and frozen in liquid nitrogen. Thin cryosections were cut with a Leica Ultracut UCT microtome. Sections for immunolabeling were first incubated in 0.05 mol/L glycine in PBS, followed by incubation in 5% bovine serum albumin (BSA) with 0.1% cold water fish skin gelatin (Aurion, Wageningen, The Netherlands) in PBS. The sections were then incubated with a polyclonal antibody against tropoelastin (Elastin Products Co. Inc.) for 60 minutes, followed by a protein-A gold complex. The antibodies and the gold conjugate were diluted in 0.1% BSA-C (Aurion) in PBS and all of the washing steps were performed with 0.1% BSA-C in PBS. The controls were prepared by performing the labeling procedure without the primary antibody. The sections were embedded in methylcellulose and examined as described above. The aortas were aseptically removed from E18.5 WT, Lox+/− and Lox−/− embryos and placed in an AmnioMAX C-100 complete medium (Invitrogen Corp., Grand Island, NY). Fat tissue surrounding the aorta and the adventitia were grossly stripped away. The aorta was positioned longitudinally and scraped to remove the intima and the cleaned aorta was cut into pieces, which were placed into individual wells of a plastic six-well culture dish containing AmnioMAX C-100 medium supplemented with penicillin/streptomycin. The cultures of smooth muscle cells that formed around the tissue were trypsinized (0.05% trypsin, 0.53 mmol/L ethylenediamine tetraacetic acid), passaged, expanded, and genotyped. The cells were stained with α-SMA (Sigma) and analyzed for the classic hill-and-valley morphology to confirm their smooth muscle cell status. Skin fibroblasts were isolated from E18.5 embryos by placing 1- to 2-mm skin biopsies into a cell culture plate with Dulbecco's modified Eagle's medium (Invitrogen Corp.) containing 20% fetal bovine serum (v/v), 1% nonessential amino acids, and 100 U/ml penicillin and streptomycin. Cultures of the fibroblasts that formed around the tissue were trypsinized (0.05% trypsin, 0.53 mmol/L ethylenediamine tetraacetic acid), passaged, expanded, and genotyped. Lysyl oxidase activity was measured in the conditioned medium of the aortic smooth muscle cell and skin fibroblast cultures using a tritiated recombinant human tropoelastin as a substrate.25Bedell-Hogan D Trackman P Abrams W Rosenbloom J Kagan H Oxidation, cross-linking, and insolubilization of recombinant tropoelastin by purified lysyl oxidase.J Biol Chem. 1993; 268: 10345-10350Abstract Full Text PDF PubMed Google Scholar The cell culture medium was changed to a serum-free Dulbecco's modified Eagle's medium (Life Technologies Inc., Grand Island, NY) containing 0.5% bovine serum albumin, 1% nonessential amino acids, and 100 U/ml penicillin and streptomycin at 70% confluency, and the cells were incubated for 16 hours. The 0.8-ml reaction mixtures contained 600 μl of the conditioned media, 0.1 mol/L borate, 0.15 mol/L NaCl, pH 8.0, and 300,000 cpm of tritiated tropoelastin in the presence or absence of 50 μg/ml β-APN (Sigma). The reactions were incubated for 3 hours at 37°C, followed by distillation under a vacuum. The radioactivity in 0.5-ml aliquots of the distillates was determined by liquid scintillation spectrometry. Protein concentrations in the cell layers were analyzed by the method of Bradford26Bradford MM A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding.Anal Biochem. 1976; 72: 248-254Crossref PubMed Scopus (211983) Google Scholar to normalize the enzyme activities between samples. Aortic smooth muscle cells and skin fibroblasts isolated from WT and Lox−/− E18.5 embryos were seeded onto glass coverslips and grown for 9 to 12 days after confluency to ensure extracellular matrix production, fixed for 8 minutes in 2% paraformaldehyde at room temperature, and incubated in 2% BSA/PBS (pH 7.2) for 30 minutes. The samples were incubated with a polyclonal anti-tropoelastin antibody (Elastin Products Company Inc.) at a 1:100 dilution for 1 hour, washed with PBS, and incubated with a tetramethylrhodamine B isothiocyanate-conjugated goat anti-rabbit secondary antibody (DAKO A/S, Glostrup, Denmark) diluted according to the manufacturer's instructions for 1 hour at room temperature. After extensive washing with PBS, the coverslips were mounted onto microscope slides with Immu-Mount (Thermo Shandon, Pittsburgh, PA) and analyzed by fluorescence microscopy. H&E staining of the E18.5 Lox−/− embryonic lungs showed enlarged distal and proximal airways (Figure 1, a and b). The Lox−/− distal airways (ie, acinar tubule derivatives, including alveolar ducts, sacs, and primitive alveolar structures) and bronchioles were dilated and the walls of the distal airways were thickened, but no destruction of the walls was observed. These abnormalities were even more severe at P0, the distal airways of Lox−/− lungs being extremely dilated with further thickening of their walls, and numerous atelectatic areas being observed (Figure 1, c and d). The number of proximal airways, ie, developing bronchioles, in the E15.5 Lox−/− lungs (122 ± 25, n = 8) was ∼22% lower (P < 0.05) than in the WT siblings (156 ± 44, n = 12), indicating defective branching of the airways. No differences in the gross morphology of the lungs were apparent between the null and WT embryos until E17.5, when a marked decrease in the staining of elastic fibers was observed in the Lox−/− lungs (see below). The weight of the E18.5 Lox−/− lungs, ie, 1 day before birth, was reduced by ∼17% (P < 0.01) relative to the WT, with concomitant decreases of 23% (P < 0.05) and 21% (P < 0.001), respectively, in the numbers of bronchioles and distal airways (Table 1). The epithelial cells in the Lox−/− bronchioles were disorganized (data not shown) and reduced in number by 8% (P < 0.01), the thickness of the epithelium also being significantly reduced (P < 0.001) (Table 1). The number of pulmonary arteries was reduced by ∼11%, but this was not statistically significant (Table 1).Table 1Morphometric Analysis of WT and Lox−/− Lungs at E18.5WT−/−Body weight (g)1.246 ± 0.1571.161 ± 0.149NS(n = 14)(n = 18)Lung weight/body weight (g)0.0391 ± 0.0060.0324 ± 0.006†P < 0.01;(n = 14)(n = 18)Number of bronchioles/mm28.66 ± 1.976.67 ± 0.77*P < 0.05;(n = 6)(n = 6)Number of distal airways/mm2229.37 ± 8.00180.31 ± 27.10‡P < 0.001.(n = 6)(n = 6)Number of arteries/mm215.93 ± 0.9614.25 ± 2.48NS(n = 6)(n = 6)Thickness of the bronchial epithelium (μm)13.08 ± 1.1511.75 ± 0.75‡P < 0.001.(n = 33§n refers to the number of bronchioles or arterioles calculated from randomly selected sections of three embryos. Otherwise n refers to the number of embryos included in the analysis)(n = 25§n refers to the number of bronchioles or arterioles calculated from randomly selected sections of three embryos. Otherwise n refers to the number of embryos included in the analysis)Number of epithelial cells in bronchioles (cells/mm)166.42 ± 17.94153.52 ± 16.26†P < 0.01;(n = 32§n refers to the number of bronchioles or arterioles calculated from randomly selected sections of three embryos. Otherwise n refers to the number of embryos included in the analysis)(n = 25§n refers to the number of bronchioles or arterioles calculated from randomly selected sections of three embryos. Otherwise n refers to the number of embryos included in the analysis)Thickness of the pulmonary arterial walls versus diameter (%)29.85 ± 4.9331.44 ± 4.94NS(n = 37§n refers to the number of bronchioles or arterioles calculated from randomly selected sections of three embryos. Otherwise n refers to the number of embryos included in the analysis)(n = 49§n refers to the number of bronchioles or arterioles calculated from randomly selected sections of three embryos. Otherwise n refers to the number of embryos included in the analysis)* P < 0.05;† P < 0.01;‡ P < 0.001.§ n refers to the number of bronchioles or arterioles calculated from randomly selected sections of three embryos. Otherwise n refers to the number of embryos included in the analysis Open table in a new tab Histochemical elastin staining showed less prominent staining of the elastic fibers in the E18.5 Lox−/− lung sections than in the WT (Figure 2, a and b), and staining was virtually absent in the Lox−/− lung mesenchyme surrounding the distal airways and less prominent in the bronchioles (Figure 2, a and b), bronchi, and trachea (data not shown). Individual elastic fibers in the Lox−/− lung sections appeared thinner than in the WT, and were irregular with a hazy appearance. The intensity of elastin staining in the Lox−/− pulmonary arterial walls was not as markedly reduced relative to the WT as in the lung mesenchyme, but the elastic fibers seemed to be partially destructed (Figure 2, a and b). The staining of elastic fibers was decreased to an even greater extent in the lungs of Lox−/− neonates than in the E18.5 Lox−/− lungs (data not shown). Immunohistochemical analysis with a tropoelastin antibody likewise showed reduced staining of elastic fibers in the E18.5 Lox−/− lungs (Figure 2; c to f). Furthermore, the elastic lamellae around the Lox−/− pulmonary arteries were highly fragmented (Figure 2, c and d). No such fragmentation was observed in the Lox−/− bronchiolar lamina propria, but the staining intensity of elastin was slightly decreased relative to the WT (Figure 2, e and f). Electron microscopy of the E18.5 Lox−/− lungs showed a dramatic decrease in the staining of individual elastic fibers, with very little if any amorphous material, the individual fibers being highly dispersed (data not shown). Staining of elastic fibers was essentially absent from the Lox−/− walls of the distal airways, but was prominent in the WT walls (Figure 2, g and h). Immunohistochemical analysis with type I and IV collagen antibodies showed prominent staining in both the E18.5 Lox−/− and WT lungs (Figure 3; a to d). Likewise, no obvious differences were observed in the immunostaining of type III collagen (data not shown). However, electron microscopy analysis showed clear differences in the organization of collagen fibers between the Lox−/− and the WT E18.5 lungs (Figure 3, e and f). The individual collagen fibers in the Lox−/− lungs were dispersed, short, and did not form tight bundles as observed in the WT lungs (Figure 3, e and f). As described previously, diaphragmatic hernias are occasionally found in E18.5 Lox−/− embryos and more frequently at P0.22Mäki JM Räsanen J Tikkanen H Sormunen R Mäkikallio K Kivirikko KI Soininen R Inactivation of the lysyl oxidase gene Lox leads to aortic aneurysms, cardiovascular dysfunction, and perinatal death in mice.Circulation. 2002; 106: 2503-2509Crossref PubMed Scopus (397) Google Scholar, 23Hornstra IK Birge S Starcher B Bailey AJ Mecham RP Shapiro SD Lysyl oxidase is required for vascular and diaphragmatic development in mice.J Biol Chem. 2003; 278: 14387-14393Crossref PubMed Scopus (258) Google Scholar The diaphragmatic rupture occurs in the E18.5 Lox−/− embryos at the site of the collagen-rich diaphragmatic central tendon, allows abdominal contents to enter the thoracic cavity and therefore disturbs the respiratory movements (Figure 4). Electron microscopy (data not shown) showed clear differences in the organization of collagen fibers in the diaphragmatic muscle of the E18.5 Lox−/− embryos, similar to those shown in the lung and skin, and the surfaces of individual fibers contained undefined loosely associated material, which was also seen in other tissues studied (see below). Because diaphragmatic herniation has been shown to have a negative effect on the development of the lungs and branching of the alveobronchial tree in early embryos,27Kitagawa M Hislop A Boyden EA Reid L Lung hypoplasia in congenital diaphragmatic hernia. A quantitative study of airway, artery, and alveolar development.Br J Surg. 1971; 58: 342-346Crossref PubMed Scopus (314) Google Scholar, 28Inselman LS Mellins RB Growth and development of the lung.Pediatrics. 1981; 98: 1-13Abstract Full Text PDF Scopus (115) Google Scholar, 29Hasleton PS Embryology and development of the lung. Spenser's Pathology of the Lung. McGraw-Hill, Ne" @default.
• W2043157186 created "2016-06-24" @default.
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• W2043157186 date "2005-10-01" @default.
• W2043157186 modified "2023-10-17" @default.
• W2043157186 title "Lysyl Oxidase Is Essential for Normal Development and Function of the Respiratory System and for the Integrity of Elastic and Collagen Fibers in Various Tissues" @default.
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