Adenoviral transfer of HSP-70 into pulmonary epithelium ameliorates experimental acute respiratory distress syndrome

Abstract

The acute respiratory distress syndrome (ARDS) provokes three pathologic processes: unchecked inflammation, interstitial/alveolar protein accumulation, and destruction of pulmonary epithelial cells. The highly conserved heat shock protein HSP-70 can limit all three responses but is not appropriately expressed in the lungs after cecal ligation and double puncture (2CLP), a clinically relevant model of ARDS. We hypothesize that restoring expression of HSP-70 using adenovirus-mediated gene therapy will limit pulmonary pathology following 2CLP. We administered a vector containing the porcine HSP-70 cDNA driven by a CMV promoter (AdHSP) into the lungs of rats subjected to 2CLP or sham operation. Administration of AdHSP after either sham operation or 2CLP increased HSP-70 protein expression in lung tissue, as determined by immunohistochemistry and Western blot hybridization. Administration of AdHSP significantly attenuated interstitial and alveolar edema and protein exudation and dramatically decreased neutrophil accumulation, relative to a control adenovirus. CLP-associated mortality at 48 hours was reduced by half. Modulation of HSP-70 production reduces pathologic changes and may improve outcome in experimental ARDS.

Figure 1

Representative HSP-70–immunostained lung sections obtained following administration of AdGFP or AdHSP. C48 indicates 2CLP with virus administration; tissue was fixed 48 hours afterward. Primary antibody was polyclonal goat anti-rat HSP-70; secondary antibody was rabbit anti-goat IgG conjugated to horseradish peroxidase. Detection was performed with immunoperoxidase/avidin/biotin and metal-enhanced 3,3-diamino-benzidine. Arrows indicate HSP-70 staining, primarily in type II pulmonary epithelial cells.

Figure 2

Immunoreactive HSP-70 levels in lung homogenate. (a) Representative immunoblot. Homogenate was obtained 48 hours after virus administration and operative intervention. Ten micrograms of homogenate were loaded per lane. Primary antibody was polyclonal goat anti-rat HSP-70; secondary antibody was rabbit anti-goat IgG. Detection was with enhanced chemiluminescence. Lane 1, purified HSP-70 (positive control); lane 2, unoperated control without AdHSP; lane 3, unoperated control with AdHSP, lane 4, sham operation without AdHSP; lane 5, sham operation with AdHSP; lane 6, 2CLP without AdHSP; lane 7, 2CLP with AdHSP; lane 8, 2CLP with AdGFP. (b) Graphic representation of densitometric quantification of HSP-70 immunoblots. n = 3 for each intervention. *Significantly different (P < 0.05) from value without virus administration. Values are arbitrary densitometric units, expressed as mean ± SD. (c) Graphic representation of densitometric quantification of AdHSP and AdGFP expression over time following sham operation. Values are mean ± SD of densitometric measurements made on three immunoblots. *Significantly different (P < 0.05) from all other measurements. White bars, no virus; black bars, AdHSP, gray bars, AdGFP.

Figure 3

Sections from lungs harvested 48 hours after virus administration with or without 2CLP, stained with hematoxylin and eosin. Magnifications are indicated at top of figure. No op, no operation.