Neuraminidase-1 is required for the normal assembly of elastic fibers

Abstract

The assembly of elastic fibers in tissues that undergo repeated cycles of extension and recoil, such as the lungs and blood vessels, is dependent on the proper interaction and alignment of tropoelastin with a microfibrillar scaffold. Here, we describe in vivo histopathological effects of neuraminidase-1 (Neu1) deficiency on elastin assembly in the lungs and aorta of mice. These mice exhibited a tight-skin phenotype very similar to the Tsk mouse. Normal septation of Neu1-null mice did not occur in neonatal mice, resulting in enlarged alveoli that were maintained in adults. The abnormal development of elastic fibers was remarkable under electron microscopy and confirmed by the overlapping distribution of elastin, fibrillin-1, fibrillin-2, and fibulin-5 (Fib-5) by the light microscopy immunostainings. Fib-5 fibers appeared diffuse and unorganized around the alveolar walls and the apex of developing secondary septal crests. Fibrillin-2 deposition was also abnormal in neonatal and adult lungs. Dispersion of myofibroblasts appeared abnormal in developing lungs of Neu1-null mice, with a random distribution of myofibroblast around the alveolar walls, rather than concentrating at sites of elastin synthesis. The elastic lamellae in the aorta of the Neu1-null mice were thinner and separated by hypertrophic smooth muscle cells that were surrounded by an excess of the sialic acid-containing moieties. The concentration of elastin, as measure by desmosine levels, was significantly reduced in the aorta of Neu1-null mice. Message levels for tropoelastin and Fib-5 were normal, suggesting the elastic fiber defects in Neu1-null mice result from impaired extracellular assembly.

Fig. 1.

Histology of neuraminidase-1 (Neu1)-null lungs. A and B: elastic fiber staining of 5-day control lungs (A) and Neu1-null littermate (B). Sections were stained with Hart's elastic stain and counterstained with tartrazine. Elastic fibers stain dark purple (arrows). C and D: control 7-day lung (C) compared with 7-day Neu1-null lung (D) illustrating the severe emphysema-like lesions in neonatal Neu1-null lungs. High magnification of 7-day control lungs (E) and Neu1-null lungs (F) depicting abnormal elastin deposition in the null lungs is shown (arrow). Bar: A and B = 22 μm; C and D = 44 μm; E and F = 11 μm.

Fig. 2.

Mean linear intercept (MLI) values as a function of age. Control lungs (solid line) and Neu1-null lungs (dashed line) are shown. Values represent the means from 2–5 mice at each time point.

Fig. 3.

Myofibroblast distribution in mouse lungs. Localization of myofibroblasts was determined by immunostaining for α-actin (red) in a 7-day control lung (A) and Neu1-null lung (B). Bar: A and B = 22 μm.

Fig. 4.

Fibulin-5 (Fib-5) immunostaining in mouse lung. Lung sections from 5-day-old mice showed a normal distribution of Fib-5 (red) in wild-type lungs, concentrating around the apex of developing secondary crest (A). Fib-5 immunostaining was less in the Neu1−/− lungs, and the fibers appeared disorganized (B). Control lungs at day 7 showed well-developed alveoli with prominent Fib-5 staining around the alveoli and heavy, organized fibers surrounded the blood vessels (C). Neu1-null lungs at 7 days were characterized by large alveoli and disordered Fib-5 around the alveolar walls (D). Fib-5 at 18 days was prominently displayed around the alveolar walls and blood vessels (E), whereas in Neu1−/− lungs, Fib-5 was sparse and disrupted, even in the small blood vessels (F). Bar = 22 μm.

Fig. 5.

Fibrillin-2 (FBN-2) immunostaining in mouse lung. Five-day-old wild-type lungs were uniformly and lightly stained for FBN-2 (red) around the alveolar walls (A). In 5-day Neu1-null lungs, the alveolar walls were much thicker, and very little if any staining for FBN-2 was evident (B). By 7 days of age, FBN-2 was visible as thin, well-organized fibers uniformly distributed around the alveolar walls of the control lung (C). FBN-2 was present in the alveolar walls of Neu1-null lungs but appeared diffuse and disorganized and not arranged in discrete fibers (D). At 18 days, a well-organized pattern of FBN-2-positive fibers was observed around the alveolar walls and around the endothelium of blood vessels of wild-type lungs (E). In Neu1-null lungs, the alveolar walls were thicker than normal, and although the FBN-2 staining was intense around the endothelium of blood vessels and epithelial lining of airways (F), it appeared diffuse and unorganized with no fibrillar structure (F). Bar = 22 μm.

Fig. 6.

Histochemical staining of control and Neu1-null aorta for elastin, mucins, and smooth muscle. Sections stained for elastin with Hart's stain: control aorta (A) stained for elastin showing 5–6 elastic lamellae and a Neu1-null aorta (B) showing the abnormal separation between the elastic lamellae. C: control aorta stained for sialomucins with Alcian blue (blue) with elastic lamellae appearing opaque. D: Neu1-null aorta stained for sialomucins indicating the large mass of positive material between elastic lamellae. E and F: control aorta (E) immunostained for smooth muscle with an antibody to α-smooth muscle actin (red) showing the thin area between elastic lamellae and Neu1-null aorta (F) showing the increased mass of smooth muscle between elastic lamellae, whereas some areas were devoid of smooth muscle (arrow). Bar = 22 μm.

Fig. 7.

Desmosine levels of aorta and lung of control and Neu1-null (KO) littermates. Desmosine levels at different ages in control aorta (A) are shown in white bars, and desmosine levels for Neu1-null aorta in black. *P < 0.01. Lung desmosine (B) for control mice are shown in white bars and Neu1-null in black. Numbers of animals in each group are shown at the bottom of each bar.

Fig. 8.

Electron microscopy of skin, lung, and aorta of control and Neu1-null littermates. In the skin of Neu1-null mice (top, right), the scarce elastic fibers look highly immature. They mostly consist of parallel-oriented microfibrils, lightly decorated with single patches, electron-dense elastin (arrows). These immature fibers differ from the well-developed counterparts containing the electron-dense core (arrows) made of cross-linked elastin embedding the microfibrillar scaffold (top, left). In contrast to well-developed elastic fibers (containing electron-dense elastin) seen in the lungs of control mice (middle, left), lungs of Neu1-null mice (middle, right) demonstrate scarce, thin, and fragmented elastic fibers deposited by the vacuolated fibroblasts or smooth muscle cells (SMCs) (middle, right). The aortic wall of the Neu1-deficient mice (bottom, right) show grossly abnormal organization of vacuolated SMCs coinciding with the presence of thinner and irregularly shaped elastic laminae that contained remarkably less electron-dense elastin than aortas of the age-matched control mice (bottom, left).

Fig. 9.

Serum α-1 antitrypsin levels of young and older mice. Percent inhibition of elastase with increasing concentrations of serum from control (solid line) and Neu1-null mice (dashed line). Each value represents the mean of 2 experiments of 6 mice each with the SE. OD, optical density.

Fig. 10.

Expression of tropoelastin (TE) and fibulin-5 (Fbln5) mRNA in control and Neu1-null mice. mRNA for TE is shown in A for tissues from control (white bars) and Neu1-null (black bars) mice. In B, mRNA levels for fibulin-5 are shown for control (white bars) and Neu1-null animals (black bars). Age of mice was 18, 31, and 65 days (D). Values represent mean and SE of 6 mice at 18 and 31 days and 2 mice at 31 days. WT, wild-type.