- Email: [email protected]
Acute Lung Injury Does Not Impair Adenoviral-Mediated Gene Transfer to the Alveolar Epithelium
pathogenesis of IPF has been intensely studied and the candidate genes playing the key roles have been revealed, and (3) single gene delivery or single gene knockout has been reported to be effective for reducing fibrosis in the lungs of an experimental IPF model. We have examined the effect of lung regeneration by gene delivery of hepatocyte growth factor to bleomycin-induced fibrosis of mouse lung. We also revealed antifibrotic effect of decorin, a small leucine-rich proteoglycan, known as an intrinsic negative regulator of transforming growth factor-, a key inducer of pulmonary fibrosis, using an adenoviral vector expressing human decorin, driven by cytomegalovirus promotor (AdCMV.DC). With intratracheal administration of AdCMV.DC to C57BL/6 mice with bleomycin-induced lung injury, the relative content of hydroxyproline was reduced but lung inflammation was prolonged. No reduction of hydroxyproline content by IV administration of AdCMV.DC suggested that adenoviral-mediated decorin treatment is effective only by direct administration to the injured lung, a finding which is different from the results of hepatocyte growth factor. We overviewed our gene delivery trials to pulmonary fibrosis, from the points of anti-inflammation, antifibrosis, and regeneration.
Acute Lung Injury Does Not Impair Adenoviral-Mediated Gene Transfer to the Alveolar Epithelium* Vidas Dumasius; Michael Mendez, MD; Go¨ khan M. Mutlu, MD; and Phillip Factor, DO, FCCP
(CHEST 2002; 121:33S–34S) Abbreviations: ad-gal ⫽ first-generation human type 5 recombinant adenovirus that expresses an e. coli lac z gene; adnull ⫽ first generatoin human type 5 recombinant adenovirus that expresses no cDNA; ALI ⫽ acute lung injury
have shown that overexpression of genes such S astudiessuperoxide dismutase, catalase, Na,K-adenosine 1–5
triphosphatase, and interleukin-10 in the distal lung can protect from acute injury. All of these prior studies transduced the lungs prior to the onset of injury. In order for gene transfer to be clinically useful for acute lung injury (ALI), it will be necessary to transduce the alveolar epithelium after the onset of injury. However, the pathobiology of ALI includes alveoli filled with fluid, fibrin, inflammatory cells, and cytokines, all of which can impair
*From Pulmonary and Critical Care Medicine (Drs. Dumasius and Mendez), Evanston Hospital, Evanston; and Northwestern University Medical School (Drs. Mutlu and Factor), Chicago, IL. Supported by the American Heart Association, Evanston Northwestern Healthcare Research Institute, grants HL-48129, HL66211, and HL-66185. Correspondence to: Phillip Factor, DO, FCCP, Pulmonary and Critical Care Medicine, Evanston Northwestern Healthcare, 2650 Ridge Rd, Evanston, IL 60201; e-mail: [email protected]
alveolar gene transfer.6 These biomechanical processes represent formidable hurdles that have limited the development of gene therapy for ALIs. In the current study, we tested whether recombinant adenovectors could affect gene transfer to the alveolar epithelium of rats following the establishment of a severe lung injury1,7 induced by exposure to hyperoxia. Acute hyperoxia (⬎ 95% normobaric oxygen) produces a lung injury characterized by a homogeneous alveolitis, polymorphic cellular infiltrate, pulmonary edema, increased alveolar epithelial and endothelial permeability, and a high death rate (lethal dose for 50% of test animals, 72 h).1,7 To test the hypothesis that gene transfer could be achieved in the setting of ALI, we exposed adult, male SpragueDawley rats to ⬎ 95% oxygen for 60 h, 64 h, or 68 h. The lungs of these animals were then infected with 4 ⫻ 109 plaque forming units of E1a-/E3- recombinant adenoviruses that express an Escherichia coli lac Z gene under the control of a human immediate-early cytomegalovirus promoter-enhancer (ad-gal) or no complementary DNA (adnull). Adenovectors were delivered endotracheally to lightly sedated, orally intubated, spontaneously breathing animals using a 50% surfactant (Survanta; Abbott Laboratories; Columbus, OH) vehicle. We have previously shown that this vectordelivery strategy produces widespread gene transfer to the alveolar epithelium of normal rats.1,8 All of the animals exposed to 64 h or 68 h of hyperoxia (n ⫽ 10 per group) and 7 of 16 rats (44%) exposed for 60 h died during virus delivery. The surviving six ad-gal rats and three adnull rats were compared to four uninfected hyperoxic control rats, and to four uninfected rats, four ad-gal rats, and four adnullinfected, room-air, control rats. All of the hyperoxic control rats had large bilateral pleural effusions, histologic signs of injury, and increased total lung water (wet/dry weight ratios [⫾ SD]: hyperoxia, 5.47 ⫾ 0.73; room air, 3.64 ⫾ 0.28; p ⫽ 0.012) consistent with the presence of pulmonary edema. To assess the extent and distribution of gene transfer in this model, adnull-infected and ad-gal–infected lungs were stained with X-gal (pH 8.0 in Tris-buffered saline solution) 72 h postinfection. X-gal staining of hyperoxic lungs infected with ad-gal revealed diffuse transgene expression that was indistinguishable from similarly infected room air ad-gal control rats in all segments of the lung. No -galactosidase activity was noted in any of the uninfected or adnull-infected control rats. Gene transfer was quantitated by enumerating the number of alveolar cells with perinuclear blue color in 10 high-power microscopic fields randomly selected from longitudinal sections of left lungs. Further quantitation was pursued using a spectrophotometric -galactosidase activity assay (ONPG [O-nidrophenyl--D-galactopyranoside] hydrolysis per microgram of protein) of homogenates produced from the right lung. The number of cells per high-power field in the hyperoxic lungs was slightly greater than room-air lungs infected with ad-gal, whereas galactosidase activity was greater in the room-air lungs. No evidence of -galactosidase activity was noted in any of the uninfected room air lungs, hyperoxic control lungs, or CHEST / 121 / 3 / MARCH, 2002 SUPPLEMENT
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adnull lungs. Histologic analysis demonstrated significant lung injury and transgene expression within areas of lung injury. A growing body of data indicates that the transfer of protective genes to the alveolar epithelium can, in experimental models, ameliorate ALI. Previously, the development of therapeutic gene transfer strategies for ALI was limited by concerns that the associated pathophysiologic processes would preclude efficient transduction of the alveolar epithelium. The results of this study indicate otherwise and show that adenovectors are capable of gene transfer to severely injured, edematous lungs with an efficiency that is not different from uninjured, room-air, control lungs. These results provide support for the development of gene therapies for ALIs.
References 1 Factor P, Dumasius V, Saldias F, et al. Adenovirus-mediated transfer of an Na⫹/K⫹-ATPase 1 subunit gene improves alveolar fluid clearance and survival in hyperoxic rats. Hum Gene Ther 2000; 11:2231–2242 2 Danel C, Erzurum SC, Prayssac P, et al. Gene therapy for oxidant injury-related diseases: adenovirus-mediated transfer of superoxide dismutase and catalase cDNAs protects against hyperoxia but not against ischemia-reperfusion lung injury. Hum Gene Ther 1998; 9:1487–1496 3 Epperly MW, Bray JA, Krager S, et al. Intratracheal injection of adenovirus containing the human MnSOD transgene protects athymic nude mice from irradiation-induced organizing alveolitis. Int J Radiat Oncol Biol Phys 1999; 43:169 – 181 4 Morrison DF, Foss DL, Murtaugh MP. Interleukin-10 gene therapy-mediated amelioration of bacterial pneumonia. Infect Immun 2000; 68:4752– 4758 5 Stern M, Ulrich K, Robinson C, et al. Pretreatment with cationic lipid-mediated transfer of the Na⫹K⫹-ATPase pump in a mouse model in vivo augments resolution of high permeability pulmonary oedema. Gene Ther 2000; 7:960 – 966 6 Otake K, Ennist DL, Harrod K, et al. Nonspecific inflammation inhibits adenovirus-mediated pulmonary gene transfer and expression independent of specific acquired immune responses. Hum Gene Ther 1998; 9:2207–2222 7 Crapo J, Barry B, Foscue H, et al. Structural and biochemical changes in rat lungs occurring during exposure to lethal and adaptive doses of oxygen. Am Rev Respir Dis 1980; 122:123– 143 8 Factor P, Saldias F, Ridge K, et al. Augmentation of lung liquid clearance via adenoviral-mediated gene transfer of the Na,K-ATPase 1 subunit. J Clin Invest 1998; 102: 1142–1150
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Adenovirally Mediated Expression of Urokinase Receptor Binding Site on Integrin ␣-Chain Blocks Adhesion and Migration of Human Lung Fibroblasts* Sha Zhu, PhD; Candece L. Gladson, MD; Jerry Stewart; Kimberly E. White; Harold A. Chapman, Jr., MD; and Mitchell A. Olman, MA, MD
(CHEST 2002; 121:34S–35S) Abbreviations: mAb ⫽ monoclonal antibody; u-PAR ⫽ urokinase plasminogen activator receptor
and migration of fibroblasts to the vitronecA ttachment tin- and fibronectin-rich alveolar matrices are critical
processes in wound repair after acute lung injury. This study was undertaken to determine the mechanism by which human lung fibroblasts attach and migrate toward provisional alveolar matrix proteins, and to develop mechanism-based, novel therapeutic agents designed to specifically inhibit these functions. We have previously demonstrated urokinase plasminogenactivator receptor (u-PAR) and a functional fibrinolytic system on these cells. Analysis of surface integrins reveals expression of the vitronectin receptors ␣v3, ␣v5, ␣v1, and ␣81. Fibroblast attachment to vitronectin was inhibited by 50 g/mL of anti– uPAR IgG (70%), monoclonal antibody (mAb) anti-1 integrin (80%), and mAb anti-␣v5 integrin (30%), but not by mAb to ␣v3, ␣3, and ␣5 integrins in the presence of 1 mM Ca⫹⫹. These data suggest that u-PAR–integrin interactions play an important role in fibroblast attachment to provisional alveolar matrix proteins. Furthermore, a peptide homologous to the u-PAR binding site on integrin ␣-chains also blocked cell attachment to vitronectin in a concentration-dependent manner (70% at 100 M), while the control scrambled peptide had no effect. These data suggest that u-PAR interacts with ␣v1 or ␣81 in mediating fibroblast attachment to vitronectin. A replication-deficient adenovirus encoding for the u-PAR binding region of the integrin ␣-chain was generated, and adenoviral infection of human lung fibroblasts was optimized. Adenovirally infected fibroblasts (multiplicity of infection ⫽ 500, 48 h) had reduced migration toward *From the Departments of Medicine (Dr. Zhu and Ms. White) and Pathology (Drs. Gladson, Olman, and Mr. Stewart), Division of Pulmonary and Critical Care Medicine, University of Alabama, Birmingham AL; and the Cardiovascular Research Institute (Dr. Chapman), University of California at San Francisco, San Francisco, CA. Supported by the Veterans Affairs Merit Review Board, and National Heart, Lung, and Blood Institute grant HL-58655 (Dr. Olman). Correspondence to: Mitchell A. Olman, MD, Department of Medicine, Division of Pulmonary and Critical Care Medicine, University of Alabama at Birmingham Medical Center, 1900 University Blvd, 215 THT, Birmingham, AL 35294; e-mail: [email protected].
Thomas L. Petty 44th Annual Aspen Lung Conference: Pulmonary Genetics, Genomics, Gene Therapy
Acute Lung Injury Does Not Impair Adenoviral-Mediated Gene Transfer to the Alveolar Epithelium* Vidas Dumasius; Michael Mendez, MD; Go¨ khan M. Mutlu, MD; and Phillip Factor, DO, FCCP
(CHEST 2002; 121:33S–34S) Abbreviations: ad-gal ⫽ first-generation human type 5 recombinant adenovirus that expresses an e. coli lac z gene; adnull ⫽ first generatoin human type 5 recombinant adenovirus that expresses no cDNA; ALI ⫽ acute lung injury
have shown that overexpression of genes such S astudiessuperoxide dismutase, catalase, Na,K-adenosine 1–5
triphosphatase, and interleukin-10 in the distal lung can protect from acute injury. All of these prior studies transduced the lungs prior to the onset of injury. In order for gene transfer to be clinically useful for acute lung injury (ALI), it will be necessary to transduce the alveolar epithelium after the onset of injury. However, the pathobiology of ALI includes alveoli filled with fluid, fibrin, inflammatory cells, and cytokines, all of which can impair
*From Pulmonary and Critical Care Medicine (Drs. Dumasius and Mendez), Evanston Hospital, Evanston; and Northwestern University Medical School (Drs. Mutlu and Factor), Chicago, IL. Supported by the American Heart Association, Evanston Northwestern Healthcare Research Institute, grants HL-48129, HL66211, and HL-66185. Correspondence to: Phillip Factor, DO, FCCP, Pulmonary and Critical Care Medicine, Evanston Northwestern Healthcare, 2650 Ridge Rd, Evanston, IL 60201; e-mail: [email protected]
alveolar gene transfer.6 These biomechanical processes represent formidable hurdles that have limited the development of gene therapy for ALIs. In the current study, we tested whether recombinant adenovectors could affect gene transfer to the alveolar epithelium of rats following the establishment of a severe lung injury1,7 induced by exposure to hyperoxia. Acute hyperoxia (⬎ 95% normobaric oxygen) produces a lung injury characterized by a homogeneous alveolitis, polymorphic cellular infiltrate, pulmonary edema, increased alveolar epithelial and endothelial permeability, and a high death rate (lethal dose for 50% of test animals, 72 h).1,7 To test the hypothesis that gene transfer could be achieved in the setting of ALI, we exposed adult, male SpragueDawley rats to ⬎ 95% oxygen for 60 h, 64 h, or 68 h. The lungs of these animals were then infected with 4 ⫻ 109 plaque forming units of E1a-/E3- recombinant adenoviruses that express an Escherichia coli lac Z gene under the control of a human immediate-early cytomegalovirus promoter-enhancer (ad-gal) or no complementary DNA (adnull). Adenovectors were delivered endotracheally to lightly sedated, orally intubated, spontaneously breathing animals using a 50% surfactant (Survanta; Abbott Laboratories; Columbus, OH) vehicle. We have previously shown that this vectordelivery strategy produces widespread gene transfer to the alveolar epithelium of normal rats.1,8 All of the animals exposed to 64 h or 68 h of hyperoxia (n ⫽ 10 per group) and 7 of 16 rats (44%) exposed for 60 h died during virus delivery. The surviving six ad-gal rats and three adnull rats were compared to four uninfected hyperoxic control rats, and to four uninfected rats, four ad-gal rats, and four adnullinfected, room-air, control rats. All of the hyperoxic control rats had large bilateral pleural effusions, histologic signs of injury, and increased total lung water (wet/dry weight ratios [⫾ SD]: hyperoxia, 5.47 ⫾ 0.73; room air, 3.64 ⫾ 0.28; p ⫽ 0.012) consistent with the presence of pulmonary edema. To assess the extent and distribution of gene transfer in this model, adnull-infected and ad-gal–infected lungs were stained with X-gal (pH 8.0 in Tris-buffered saline solution) 72 h postinfection. X-gal staining of hyperoxic lungs infected with ad-gal revealed diffuse transgene expression that was indistinguishable from similarly infected room air ad-gal control rats in all segments of the lung. No -galactosidase activity was noted in any of the uninfected or adnull-infected control rats. Gene transfer was quantitated by enumerating the number of alveolar cells with perinuclear blue color in 10 high-power microscopic fields randomly selected from longitudinal sections of left lungs. Further quantitation was pursued using a spectrophotometric -galactosidase activity assay (ONPG [O-nidrophenyl--D-galactopyranoside] hydrolysis per microgram of protein) of homogenates produced from the right lung. The number of cells per high-power field in the hyperoxic lungs was slightly greater than room-air lungs infected with ad-gal, whereas galactosidase activity was greater in the room-air lungs. No evidence of -galactosidase activity was noted in any of the uninfected room air lungs, hyperoxic control lungs, or CHEST / 121 / 3 / MARCH, 2002 SUPPLEMENT
33S
adnull lungs. Histologic analysis demonstrated significant lung injury and transgene expression within areas of lung injury. A growing body of data indicates that the transfer of protective genes to the alveolar epithelium can, in experimental models, ameliorate ALI. Previously, the development of therapeutic gene transfer strategies for ALI was limited by concerns that the associated pathophysiologic processes would preclude efficient transduction of the alveolar epithelium. The results of this study indicate otherwise and show that adenovectors are capable of gene transfer to severely injured, edematous lungs with an efficiency that is not different from uninjured, room-air, control lungs. These results provide support for the development of gene therapies for ALIs.
References 1 Factor P, Dumasius V, Saldias F, et al. Adenovirus-mediated transfer of an Na⫹/K⫹-ATPase 1 subunit gene improves alveolar fluid clearance and survival in hyperoxic rats. Hum Gene Ther 2000; 11:2231–2242 2 Danel C, Erzurum SC, Prayssac P, et al. Gene therapy for oxidant injury-related diseases: adenovirus-mediated transfer of superoxide dismutase and catalase cDNAs protects against hyperoxia but not against ischemia-reperfusion lung injury. Hum Gene Ther 1998; 9:1487–1496 3 Epperly MW, Bray JA, Krager S, et al. Intratracheal injection of adenovirus containing the human MnSOD transgene protects athymic nude mice from irradiation-induced organizing alveolitis. Int J Radiat Oncol Biol Phys 1999; 43:169 – 181 4 Morrison DF, Foss DL, Murtaugh MP. Interleukin-10 gene therapy-mediated amelioration of bacterial pneumonia. Infect Immun 2000; 68:4752– 4758 5 Stern M, Ulrich K, Robinson C, et al. Pretreatment with cationic lipid-mediated transfer of the Na⫹K⫹-ATPase pump in a mouse model in vivo augments resolution of high permeability pulmonary oedema. Gene Ther 2000; 7:960 – 966 6 Otake K, Ennist DL, Harrod K, et al. Nonspecific inflammation inhibits adenovirus-mediated pulmonary gene transfer and expression independent of specific acquired immune responses. Hum Gene Ther 1998; 9:2207–2222 7 Crapo J, Barry B, Foscue H, et al. Structural and biochemical changes in rat lungs occurring during exposure to lethal and adaptive doses of oxygen. Am Rev Respir Dis 1980; 122:123– 143 8 Factor P, Saldias F, Ridge K, et al. Augmentation of lung liquid clearance via adenoviral-mediated gene transfer of the Na,K-ATPase 1 subunit. J Clin Invest 1998; 102: 1142–1150
34S
Adenovirally Mediated Expression of Urokinase Receptor Binding Site on Integrin ␣-Chain Blocks Adhesion and Migration of Human Lung Fibroblasts* Sha Zhu, PhD; Candece L. Gladson, MD; Jerry Stewart; Kimberly E. White; Harold A. Chapman, Jr., MD; and Mitchell A. Olman, MA, MD
(CHEST 2002; 121:34S–35S) Abbreviations: mAb ⫽ monoclonal antibody; u-PAR ⫽ urokinase plasminogen activator receptor
and migration of fibroblasts to the vitronecA ttachment tin- and fibronectin-rich alveolar matrices are critical
processes in wound repair after acute lung injury. This study was undertaken to determine the mechanism by which human lung fibroblasts attach and migrate toward provisional alveolar matrix proteins, and to develop mechanism-based, novel therapeutic agents designed to specifically inhibit these functions. We have previously demonstrated urokinase plasminogenactivator receptor (u-PAR) and a functional fibrinolytic system on these cells. Analysis of surface integrins reveals expression of the vitronectin receptors ␣v3, ␣v5, ␣v1, and ␣81. Fibroblast attachment to vitronectin was inhibited by 50 g/mL of anti– uPAR IgG (70%), monoclonal antibody (mAb) anti-1 integrin (80%), and mAb anti-␣v5 integrin (30%), but not by mAb to ␣v3, ␣3, and ␣5 integrins in the presence of 1 mM Ca⫹⫹. These data suggest that u-PAR–integrin interactions play an important role in fibroblast attachment to provisional alveolar matrix proteins. Furthermore, a peptide homologous to the u-PAR binding site on integrin ␣-chains also blocked cell attachment to vitronectin in a concentration-dependent manner (70% at 100 M), while the control scrambled peptide had no effect. These data suggest that u-PAR interacts with ␣v1 or ␣81 in mediating fibroblast attachment to vitronectin. A replication-deficient adenovirus encoding for the u-PAR binding region of the integrin ␣-chain was generated, and adenoviral infection of human lung fibroblasts was optimized. Adenovirally infected fibroblasts (multiplicity of infection ⫽ 500, 48 h) had reduced migration toward *From the Departments of Medicine (Dr. Zhu and Ms. White) and Pathology (Drs. Gladson, Olman, and Mr. Stewart), Division of Pulmonary and Critical Care Medicine, University of Alabama, Birmingham AL; and the Cardiovascular Research Institute (Dr. Chapman), University of California at San Francisco, San Francisco, CA. Supported by the Veterans Affairs Merit Review Board, and National Heart, Lung, and Blood Institute grant HL-58655 (Dr. Olman). Correspondence to: Mitchell A. Olman, MD, Department of Medicine, Division of Pulmonary and Critical Care Medicine, University of Alabama at Birmingham Medical Center, 1900 University Blvd, 215 THT, Birmingham, AL 35294; e-mail: [email protected].
Thomas L. Petty 44th Annual Aspen Lung Conference: Pulmonary Genetics, Genomics, Gene Therapy