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. 2017 Apr 24;18(1):70.
doi: 10.1186/s12931-017-0549-2.

Exogenous Gene Transfer of Rab 3 8 Small GTPase Ameliorates Aberrant Lung Surfactant Homeostasis in Ruby Rats

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Free PMC article

Exogenous Gene Transfer of Rab 3 8 Small GTPase Ameliorates Aberrant Lung Surfactant Homeostasis in Ruby Rats

Kazuhiro Osanai et al. Respir Res. .
Free PMC article

Abstract

Background: Rab38 small GTPase regulates intracellular transport in melanocytes and alveolar type II epithelial cells. Ruby rats carrying Rab38 and other gene mutations exhibit oculocutaneous albinism, bleeding diathesis, and hence, are a rat model of human Hermansky-Pudlak syndrome (HPS). We previously showed that Long Evans Cinnamon (LEC) rats, one strain of the Ruby rats, developed aberrant lung surfactant homeostasis with remarkably enlarged lamellar bodies in alveolar type II cells.

Methods: A replication-deficient recombinant adenovirus expressing rat Rab38 (Ad-Rab38) was constructed. Alveolar type II cells were isolated from the LEC rats and tested for lung surfactant phosphatidylcholine secretion. The rats were also examined whether exogenous expression of Ad- Rab38 could rescue the altered lung surfactant homeostasis in the lungs.

Results: Isolated type II cells infected with Ad-Rab38 exhibited improved secretion patterns of [3H]phosphatidylcholine, i.e. increased basal hyposecretion and decreased agonist-induced hypersecretion. Endobronchial administration of Ad-Rab38 improved the morphology of type II cells and lamellar bodies, reducing their sizes close to those of wild-type rats. The increased amounts of phosphatidylcholine and surfactant protein B in the lamellar body fractions were decreased in the Ad-Rab38 infected lungs.

Conclusions: These results provide strong evidence that the aberrant lung surfactant homeostasis in the LEC rats is caused by Rab38 deficit, and suggest that endobronchial delivery of the responsive transgene could be an effective method to ameliorate the abnormal lung phenotype in the animal model of HPS.

Keywords: Adenovector; Gene transfer; Hermansky-Pudlak syndrome; Lung surfactant; Rab38 GTPase; Ruby rats.

Figures

Fig. 1
Fig. 1
Construction of recombinant adenovirus. A multi-cloning site (MCS) of a shuttle plasmid pDC315 was digested with BamH1 and Sal1 restriction enzymes. cDNA (lacZ or Rab38) was digested with the two enzymes and inserted into the shuttle plasmid. The recombinant shuttle plasmid (lacZ or Rab38) and the adenovirus genomic plasmid were simultaneously added to HEK293 cells. Recombinant viral plaques appeared within 2–3 weeks. See the text for detail
Fig. 2
Fig. 2
Expression of Rab38 protein in LEC type II cells infected with Ad-Rab38. (a) Western blot shows expression of Rab38 protein by Ad-Rab38 infection from day 1 through day 21. (b) The β-galactosidase (lacZ) assay shows ~100% of transduction efficiency of rat type II cells by Ad-lacZ infection at MOI = 5. Bar = 20 μm. (c) Immunofluorescence staining of exogenously expressed Rab38 in LEC type II cells. Adherent cells were infected with Ad-lacZ (ad) or Ad-Rab38 (el). Objective lens magnification, a − h: ×40, i − l:×100. Bar = 25 μm. The exogenous Rab38 appeared to at least partially co-localize with granularly distributed SP-B in the cells (l, arrow heads)
Fig. 3
Fig. 3
[3H]phosphatidylcholine secretion from cultured type II cells isolated from SD or LEC rats. The cells were infected with Ad-lacZ or Ad-Rab38 and radiolabeled with [3H]choline. Secretion (%) was calculated as [3H]PC activity in 100 × supernatant/(supernatant + cell). a Basal secretion (%) was without an agonist. b Agonist-induced secretion value was normalized by basal secretion value in each experiment group, i.e. fold of the basal secretion value. *P < 0.05, **P < 0.01. n = 6 of separate experiments performed in duplicate dishes
Fig. 4
Fig. 4
Transduction efficiency by a single endobronchial administration of Ad-lacZ as evaluated by ex vivo endobronchial staining with lacZ assays. a Ad-lacZ was delivered into the left lungs by a single endobronchial administration. Fourteen days later, the lungs were stained for 4 h with intratracheal administration of 0.5 mg/ml X-gal (13). Left: Control, Right: Ad-lacZ. b, c The lung tissues were sectioned into 5-μm slices, and counter-stained with nuclear fast red (Kernechtrot). Magnification × 200, Bar = 40 μm. b: Control, c: Ad-lacZ
Fig. 5
Fig. 5
Phosphatidylcholine levels in Ad-Rab38-infected LEC rat lungs. Ad-lacZ or Ad-Rab38 recombinant adenovector was delivered into the left lungs by an endobronchial administration at 14 days prior to sacrifice. The left lungs were lavaged, homogenized, and lamellar body (LB) fractions were isolated. After lipid extraction, phosphatidylcholine levels were analyzed by two-dimensional thin layer chromatography. *P < 0.05, **P < 0.01 (n = 6 rats). Note the different magnitude of a vertical scale
Fig. 6
Fig. 6
Surfactant protein B is decreased in the lamellar body fraction in Ad-Rab38-infected LEC rat lungs, whereas surfactant protein A is not altered. Fixed amounts of the lamellar body fractions (5 μg protein) were loaded onto SDS-PAGE and transferred to a nitrocellulose membrane. The same membrane was incubated twice with two different primary antibodies after being stripped of previous antibody complexes. a A representative result from three independent experiments, each with similar results. Densitometry of the Western blots was performed for SP-A (b) and SP-B (c). **P < 0.01, n = 3 rats
Fig. 7
Fig. 7
Electron microscopic appearance of type II cells and lamellar bodies in Ad-Rab38-infected LEC rat lungs. a Ultra-thin sections (60 nm-thick) prepared from the left lungs were examined using a transmission electron microscope, and more than 25 fields were randomly photographed at a magnification of × 6,000. Star (*) indicates lamellar body. Bar: 6.6 μm. b Using ~25 electron microscopic photographs per experimental group, the areas of cells and lamellar bodies were quantified by an area-calculating software, and the numbers of lamellar bodies per single cell were counted. *P < 0.05, **P < 0.01, ***P < 0.001

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