Altered stability of pulmonary surfactant in SP-C-deficient mice - PubMed (original) (raw)

Altered stability of pulmonary surfactant in SP-C-deficient mice

S W Glasser et al. Proc Natl Acad Sci U S A. 2001.

Abstract

The surfactant protein C (SP-C) gene encodes an extremely hydrophobic, 4-kDa peptide produced by alveolar epithelial cells in the lung. To discern the role of SP-C in lung function, SP-C-deficient (-/-) mice were produced. The SP-C (-/-) mice were viable at birth and grew normally to adulthood without apparent pulmonary abnormalities. SP-C mRNA was not detected in the lungs of SP-C (-/-) mice, nor was mature SP-C protein detected by Western blot of alveolar lavage from SP-C (-/-) mice. The levels of the other surfactant proteins (A, B, D) in alveolar lavage were comparable to those in wild-type mice. Surfactant pool sizes, surfactant synthesis, and lung morphology were similar in SP-C (-/-) and SP-C (+/+) mice. Lamellar bodies were present in SP-C (-/-) type II cells, and tubular myelin was present in the alveolar lumen. Lung mechanics studies demonstrated abnormalities in lung hysteresivity (a term used to reflect the mechanical coupling between energy dissipative forces and tissue-elastic properties) at low, positive-end, expiratory pressures. The stability of captive bubbles with surfactant from the SP-C (-/-) mice was decreased significantly, indicating that SP-C plays a role in the stabilization of surfactant at low lung volumes, a condition that may accompany respiratory distress syndrome in infants and adults.

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Figures

Figure 1

SP-C targeting construct and Southern blot identification of targeted ES cells and mice. (A) A restriction map of the SP-C gene is shown above the targeting vector. Exons are numbered 1–6. The bottom line diagrams the targeted SP-C gene. Precise homologous recombination between lines 1 and 2 results in exon 2 interrupted by the neomycin resistance (pGKneo) gene and loss of the nonhomologous herpes simplex virus thymidine kinase (HSV-TK) gene (bottom line). (B) Southern blot analysis of _Bsu_36I genomic DNA from ES cells. _Bsu_36I-digested DNA was probed with a 5′ Sph-Pst fragment adjacent to the targeting DNA, which detects 3.7-kb upstream and 5.5-kb downstream endogenous bands (+/+, lane 1). DNA from a targeted ES cell colony (+/−) shows the 5.5-kb allele and the targeted allele that are increased by the size of the inserted pGKneo cassette to 7.2 kb. (C) Southern blot analysis of genomic DNA from SP-C gene-targeted mice. The 5′ probe was used for blots of _Bgl_II-digested DNA. A single, 5.7-kb _Bgl_II band is detected in wild-type mice by the 5′ probe. Two bands are detected in mice heterozygous for the targeted site (+/−). Homozygous SP-C (−/−) mice produce only the 7.4-kb, larger upper band, indicating that both SP-C alleles are interrupted.

Figure 2

Northern blot analysis of RNA from the lungs of SP-C (+/+), (+/−), and (−/−) mice. Total RNA was separated by gel electrophoresis, and blots were probed with an SP-C cDNA probe (Left) or SP-B cDNA probe (Right). An SP-C band of ≈900 bp was detected in (+/+) mice. Reduced SP-C hybridization was detected in (+/−) lung RNA. No full-length SP-C mRNA was detected in the (−/−) lung RNA, whereas a smaller minor band was detected that lacks the SP-C coding sequences of exon 2. The alternate splicing of the disrupted gene is diagrammed below the blot (B).

Figure 3

Surfactant protein levels in alveolar lavage of SP-C-targeted mice. Alveolar lavage containing the pulmonary surfactant was collected from SP-C (+/+), (+/−), and (−/−) mice. Samples were normalized to saturated phosphatidylcholine and resolved by SDS/PAGE followed by immunoblot analysis with a polyclonal antibody specific to each surfactant protein. (A) Levels of mature SP-C were assessed with an antibody specific to recombinant SP-C peptide. SP-C (+/+) lavage samples were run in lanes 1–3, SP-C (+/−) samples were run in lanes 4–6, and SP-C (−/−) samples were run in lanes 7–9. The amount of mature SP-C peptide is reduced in the SP-C (+/−) mice by approximately half and is entirely absent in the SP-C (−/−) lanes. (B) Abundance of the other three surfactant proteins was evaluated in lung lavage from SP-C (+/+) and SP-C (−/−) mice. Immunoblots of lavage samples are shown for SP-A (Top), SP-B (Middle), and SP-D (Bottom). Lanes 1–4 are from SP-C (+/+) mice and lanes 5–8 are from SP-C (−/−) mice.

Figure 4

Ultrastructure of type II epithelial cells in SP-C-deficient mice. Lung tissue from adult mice was prepared for electron microscopy. Intact lamellar bodies (intracellular form of surfactant) were detected in SP-C (+/+) type II cells (A) and in SP-C (−/−) type II cells (B), ×10,400. Extracellular tubular myelin forms of surfactant were detected in SP-C (+/+) mice (C) and SP-C (−/−) mice (D), ×26,000.

Figure 5

Lung mechanics. Hysteresivity, which is the viscoelasticity of the lung tissue, was calculated as Gtissue/Htissue at PEEP of 0, 1, 2, and 4 cm H2O. Hysteresivity was significantly lower for SP-C (−/−) mice than SP-C (+/+) mice overall (ANOVA,P < 0.05) and for each value of PEEP (P values shown in figure). Data shown are group mean and SD.

Figure 6

Surfactant function. Three pools of large aggregate fractions (three mice/pool) of surfactant from SP-C (+/+) and SP-C (−/−) mice were used to assess equilibrium surface tension, and minimal surface tension was achieved on a captive-bubble surfactometer. The equilibrium surface tension and minimum surface tension were measured by using a standard bubble size (A). Surface tension measurements with a reduced bubble volume (B) demonstrated bubble-surface film instability with surfactant preparation from SP-C (−/−) mice whereas bubble stability was maintained with surfactant preparations from SP-C (+/+) mice.

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References

1. Jobe A H. N Engl J Med. 1993;328:861–868. - PubMed
1. Creuwels L A J, vanGolde L M G, Haagsman H P. Lung. 1997;175:1–39. - PMC - PubMed
1. Mason R J, Greene K, Voelker D R. Am J Physiol. 1998;275:L1–L13. - PubMed
1. Ikegami M, Whitsett J A, Jobe A, Ross G, Fisher J, Korfhagen T. Am J Physiol. 2000;279:L468–L476. - PubMed
1. Weaver T E. Biochim Biophys Acta. 1998;1408:173–179. - PubMed

Altered stability of pulmonary surfactant in SP-C-deficient mice - PubMed (original) (raw)