Abstract: α-1 Antitrypsin (A1AT) is an abundant circulating serpin with a postulated function in the lung of potently inhibiting neutrophil-derived proteases. Emphysema attributable to A1AT deficiency led to the concept that a protease/anti-protease imbalance mediates cigarette smoke-induced emphysema. We hypothesized that A1AT has other pathobiological relevant functions in addition to elastase inhibition. We demonstrate a direct prosurvival effect of A1AT through inhibition of lung alveolar endothelial cell apoptosis. Primary pulmonary endothelial cells internalized human A1AT, which co-localized with and inhibited staurosporine-induced caspase-3 activation. In cell-free studies, native A1AT, but not conformers lacking an intact reactive center loop, inhibited the interaction of recombinant active caspase-3 with its specific substrate. Furthermore, overexpression of human A1AT via replication-deficient adeno-associated virus markedly attenuated alveolar wall destruction and oxidative stress caused by caspase-3 instillation in a mouse model of apoptosis-dependent emphysema. Our findings suggest that direct inhibition of active caspase-3 by A1AT may represent a novel anti-apoptotic mechanism relevant to disease processes characterized by excessive structural cell apoptosis, oxidative stress, and inflammation, such as pulmonary emphysema. α-1 Antitrypsin (A1AT) is an abundant circulating serpin with a postulated function in the lung of potently inhibiting neutrophil-derived proteases. Emphysema attributable to A1AT deficiency led to the concept that a protease/anti-protease imbalance mediates cigarette smoke-induced emphysema. We hypothesized that A1AT has other pathobiological relevant functions in addition to elastase inhibition. We demonstrate a direct prosurvival effect of A1AT through inhibition of lung alveolar endothelial cell apoptosis. Primary pulmonary endothelial cells internalized human A1AT, which co-localized with and inhibited staurosporine-induced caspase-3 activation. In cell-free studies, native A1AT, but not conformers lacking an intact reactive center loop, inhibited the interaction of recombinant active caspase-3 with its specific substrate. Furthermore, overexpression of human A1AT via replication-deficient adeno-associated virus markedly attenuated alveolar wall destruction and oxidative stress caused by caspase-3 instillation in a mouse model of apoptosis-dependent emphysema. Our findings suggest that direct inhibition of active caspase-3 by A1AT may represent a novel anti-apoptotic mechanism relevant to disease processes characterized by excessive structural cell apoptosis, oxidative stress, and inflammation, such as pulmonary emphysema. α-1 Antitrypsin (A1AT) is a prototypical serine protease inhibitor (serpin) with potent anti-neutrophil protease activities, such as against elastase and proteinase-3. The mechanism of protease inhibition by A1AT involves an exposed reactive loop, where the link of the P1-P1′ amino acids methionine and serine, respectively, is cleaved by the target protease, resulting in covalent binding between the serpin and its substrate, with irreversible trapping of the bound protease into the β-sheet A.1Lomas DA Parfrey H Alpha1-antitrypsin deficiency. 4: molecular pathophysiology.Thorax. 2004; 59: 529-535Crossref PubMed Scopus (120) Google Scholar Significant decreases in serum levels of human A1AT (hA1AT) have been associated with the development of emphysema,2Wiedemann HP Stoller JK Lung disease due to alpha 1-antitrypsin deficiency.Curr Opin Pulm Med. 1996; 2: 155-160Crossref PubMed Scopus (23) Google Scholar a chronic obstructive pulmonary disease characterized by permanent destruction of the small airways.3The definition of emphysema. Report of a National Heart, Lung, and Blood Institute, Division of Lung Diseases workshop.Am Rev Respir Dis. 1985; 132: 182-185PubMed Google Scholar A1AT deficiency is the most frequent form of genetically determined emphysema, usually the result of 342glutamic acid to lysine substitution (PiZ variant) or 264glutamic acid to valine (PiS variant).1Lomas DA Parfrey H Alpha1-antitrypsin deficiency. 4: molecular pathophysiology.Thorax. 2004; 59: 529-535Crossref PubMed Scopus (120) Google Scholar Approximately 60,000 Americans are predicated to be A1AT-deficient.4Stoller JK Aboussouan LS Alpha1-antitrypsin deficiency.Lancet. 2005; 365: 2225-2236Abstract Full Text Full Text PDF PubMed Scopus (503) Google Scholar In patients with A1AT deficiency, cigarette smoking triggers the disease decades earlier than in chronic obstructive pulmonary disease patients with normal serum A1AT levels (PiM variant). Because A1AT is an effective elastase inhibitor, emphysema in A1AT deficiency is believed to occur as a result of increased, unopposed destruction of the lung matrix by the neutrophil serine proteases elastase and proteinase-3 engaged by cigarette smoking.5Turino GM Senior RM Garg BD Keller S Levi MM Mandl I Serum elastase inhibitor deficiency and alpha 1-antitrypsin deficiency in patients with obstructive emphysema.Science. 1969; 165: 709-711Crossref PubMed Scopus (76) Google Scholar The decreased levels or activity of A1AT are attributed in part to excessive polymerization of the mutant protein6Carrell RW Lomas DA Alpha1-antitrypsin deficiency—a model for conformational diseases.N Engl J Med. 2002; 346: 45-53Crossref PubMed Scopus (368) Google Scholar and to post-translational changes induced by oxidative stress.7Taggart C Cervantes-Laurean D Kim G McElvaney NG Wehr N Moss J Levine RL Oxidation of either methionine 351 or methionine 358 in alpha 1-antitrypsin causes loss of anti-neutrophil elastase activity.J Biol Chem. 2000; 275: 27258-27265Abstract Full Text Full Text PDF PubMed Google Scholar, 8Carp H Miller F Hoidal JR Janoff A Potential mechanism of emphysema: alpha 1-proteinase inhibitor recovered from lungs of cigarette smokers contains oxidized methionine and has decreased elastase inhibitory capacity.Proc Natl Acad Sci USA. 1982; 79: 2041-2045Crossref PubMed Scopus (270) Google Scholar Polymerized serpin may get entangled in hepatocytes as it is synthesized and posttranslationally modified by glycosylation, contributing to low levels of circulating A1AT, less than 0.3 mg/ml for the PiZ patients (normal, 1 to 2.48 mg/ml).9Mahadeva R Chang WS Dafforn TR Oakley DJ Foreman RC Calvin J Wight DG Lomas DA Heteropolymerization of S, I, and Z alpha1-antitrypsin and liver cirrhosis.J Clin Invest. 1999; 103: 999-1006Crossref PubMed Scopus (169) Google Scholar The correlation between reduced serum levels of A1AT and pulmonary disease has been strengthened by the observations that mouse strain susceptibility to cigarette smoke-induced emphysema is inversely correlated with serum A1AT levels10Gardi C Cavarra E Calzoni P Marcolongo P de Santi M Martorana PA Lungarella G Neutrophil lysosomal dysfunctions in mutant C57 Bl/6J mice: interstrain variations in content of lysosomal elastase, cathepsin G and their inhibitors.Biochem J. 1994; 299: 237-245PubMed Google Scholar and that A1AT supplementation had a beneficial effect in cigarette smoke-induced emphysema in mice,11Churg A Wang RD Xie C Wright JL alpha-1-Antitrypsin ameliorates cigarette smoke-induced emphysema in the mouse.Am J Respir Crit Care Med. 2003; 168: 199-207Crossref PubMed Scopus (123) Google Scholar providing the rationale of A1AT augmentation in A1AT-deficient patients.4Stoller JK Aboussouan LS Alpha1-antitrypsin deficiency.Lancet. 2005; 365: 2225-2236Abstract Full Text Full Text PDF PubMed Scopus (503) Google Scholar As new functions of A1AT are uncovered, including a procellular survival effect in models of serum deprivation and ischemia-reperfusion injury,12Daemen MA Heemskerk VH van't Veer C Denecker G Wolfs TG Vandenabeele P Buurman WA Functional protection by acute phase proteins alpha(1)-acid glycoprotein and alpha(1)-antitrypsin against ischemia/reperfusion injury by preventing apoptosis and inflammation.Circulation. 2000; 102: 1420-1426Crossref PubMed Scopus (153) Google Scholar, 13Ikebe N Akaike T Miyamoto Y Hayashida K Yoshitake J Ogawa M Maeda H Protective effect of S-nitrosylated alpha(1)-protease inhibitor on hepatic ischemia-reperfusion injury.J Pharmacol Exp Ther. 2000; 295: 904-911PubMed Google Scholar, 14Ikari Y Mulvihill E Schwartz SM Alpha 1-proteinase inhibitor, alpha 1-antichymotrypsin, and alpha 2-macroglobulin are the antiapoptotic factors of vascular smooth muscle cells.J Biol Chem. 2001; 276: 11798-11803Crossref PubMed Scopus (76) Google Scholar and with the discovery of excessive alveolar cell (endothelial cell) apoptosis as an important mechanism of emphysema development,15Tuder RM Petrache I Elias JA Voelkel NF Henson PM Apoptosis and emphysema: the missing link.Am J Respir Cell Mol Biol. 2003; 28: 551-554Crossref PubMed Scopus (165) Google Scholar, 16Kasahara Y Tuder RM Taraseviciene-Stewart L Le Cras TD Abman S Hirth PK Waltenberger J Voelkel NF Inhibition of VEGF receptors causes lung cell apoptosis and emphysema.J Clin Invest. 2000; 106: 1311-1319Crossref PubMed Scopus (972) Google Scholar, 17Kasahara Y Tuder RM Cool CD Lynch DA Flores SC Voelkel NF Endothelial cell death and decreased expression of vascular endothelial growth factor and vascular endothelial growth factor receptor 2 in emphysema.Am J Respir Crit Care Med. 2001; 163: 737-744Crossref PubMed Scopus (613) Google Scholar, 18Petrache I Natarajan V Zhen L Medler TR Richter AT Cho C Hubbard WC Berdyshev EV Tuder RM Ceramide upregulation causes pulmonary cell apoptosis and emphysema-like disease in mice.Nat Med. 2005; 11: 491-498Crossref PubMed Scopus (431) Google Scholar, 19Zheng T Kang MJ Crothers K Zhu Z Liu W Lee CG Rabach LA Chapman HA Homer RJ Aldous D Desanctis G Underwood S Graupe M Flavell RA Schmidt JA Elias JA Role of cathepsin S-dependent epithelial cell apoptosis in IFN-{gamma}-induced alveolar remodeling and pulmonary emphysema.J Immunol. 2005; 174: 8106-8115Crossref PubMed Scopus (129) Google Scholar, 20Rangasamy T Cho CY Thimmulappa RK Zhen L Srisuma SS Kensler TW Yamamoto M Petrache I Tuder RM Biswal S Genetic ablation of Nrf2 enhances susceptibility to cigarette smoke-induced emphysema in mice.J Clin Invest. 2004; 114: 1248-1259Crossref PubMed Scopus (821) Google Scholar we hypothesized that A1AT has a direct anti-apoptotic effect on lung. To address the mechanisms underlying our recently described protective effects of hA1AT against a model of apoptosis-dependent emphysema induced by blockade of vascular endothelial growth factor,21Petrache I Fijalkowska I Zhen L Medler TR Brown E Cruz P Choe KH Taraseviciene-Stewart L Scerbavicius R Shapiro L Zhang B Song S Hicklin D Voelkel NF Flotte T Tuder RM A novel anti-apoptotic role for alpha-1 antitrypsin in the prevention of pulmonary emphysema.Am J Respir Crit Care Med. 2006; 173: 1222-1228Crossref PubMed Scopus (173) Google Scholar we investigated whether A1AT protects against apoptosis of structural parenchymal cells, in particular lung microvascular endothelial cells. We documented that A1AT and caspase-3 interact in cultured primary pulmonary endothelial cells and in cell-free systems and tested whether A1AT opposes the functional effects of capase-3 in the lung in vivo. Our results implicate an anti-apoptotic effect of A1AT in alveolar endothelial cells via direct caspase-3 inhibition. Primary antibodies are listed in Table 1. Recombinant active caspase-3, recombinant active caspase-8, and A1AT purified from human plasma were from Calbiochem (EMD Biosciences, San Diego, CA). His-tagged caspase-3 was from Chemicon, Temecula, CA. All other reagents were from Sigma-Aldrich, St. Louis, MO, unless otherwise specified.Table 1Primary Antibodies and Their SourcesPrimary antibodySourceDilutionApplicationFigureα-1 Antitrypsin goat polyclonalSanta Cruz Biotechnology, Inc., Santa Cruz, CA1:10,000IB endothelial cellsFigure 2, Figure 4α-1 Antitrypsin sheep polyclonalEnzyme Research Laboratories, South Bend, IN1:10,000IB endothelial cellsS2Active caspase-3 rabbit polyclonalCell Signaling Technology, Beverly, MA20 mg/mlIP endothelial cells2Caspase-3 rabbit monoclonalCell Signaling Technology1:2000 or 1:5000IB endothelial cells2, S220 mg/mlIP endothelial cells31:200IF endothelial cells3Active caspase-3 rabbit polyclonalAbcam, Cambridge, MA1:200IF endothelial cells3Caspase-3 rabbit polyclonalCell Signaling Technology1:200IF endothelial cells3Caspase-3 mouse monoclonalSanta Cruz Biotechnology1:1000IB endothelial cells2Lamin rabbit polyclonalSanta Cruz Biotechnology1:5000IB endothelial cellsNot shownActin mouse monoclonalSanta Cruz Biotechnology1:500IB endothelial cells2 Open table in a new tab Primary mouse lung microvascular endothelial cells were kindly provided by Dr. Patty Lee (Yale University, New Haven, CT), human lung microvascular cells were from Clonetics, San Diego, CA, and rat lung microvascular cells were a kind gift from Dr. Troy Stevens (University of Alabama, Birmingham, AL). Experiments were performed up to passage 18. Cells were maintained in complete culture medium consisting of Dulbecco's modified Eagle's medium (Life Technologies, Inc., Grand Island, NY), 20% fetal bovine serum, and penicillin/streptomycin (100 U/ml) at 37°C in 5% CO2 and 95% air. Experiments were performed at 80 to 100% confluence. Two hours before the addition of A1AT, the medium was replaced with serum-free medium, in which the cells were maintained for the duration of the experiments. Cells were pretreated with A1AT for a time ranging from 2 to 16 hours before the addition of staurosporine and harvested 2 hours later. A1AT pretreatment duration was 2 or 16 hours before UV (6 mJ/cm2) or tumor necrosis factor-α (20 ng/ml) exposure, respectively, and cells were harvested 16 hours later. The UV radiation was generated with an HL2000 Hybrylinker crosslinker with 8W short-wave UV (254 nm) tubes. Native or polymerized (by heat inactivation at 60°C for 2 hours) purified hA1AT was incubated with recombinant active caspase-3 (Calbiochem, La Jolla, CA) or recombinant caspase-8, and caspase activity was measured using a caspase-3 activity kit with either fluorescence (Promega, Madison, WI) or colorimetric readout (Calbiochem). We measured the interaction of A1AT with active caspase-3 in a functional assay of caspase-3 activity. We added active caspase-3 (4 U for most experiments; we also tested 5 U and 10 U) to wells containing buffer with increasing amounts of A1AT. We then added a fluorescently labeled tetrapeptide (Z-DEVD-R110, 25 μmol/L), which is a specific substrate for caspase-3. For these assays, human A1AT, salt-free lyophilized powder, was first resuspended in water and then incubated with recombinant caspase-3 in caspase-3 buffer (50 mmol/L HEPES, pH 7.4, 0.1% Chaps, 0.1 mmol/L ethylenediaminetetraacetic acid, and 1 mmol/L dithiothreitol) at room temperature for 15 minutes before the addition of the caspase-3 substrate. Fluorescence was measured at room temperature for 30 to 90 minutes. We calculated the slope of OD (fluorescence signal) versus time and the ratios of fluorescent signal in the presence and absence of A1AT in the reaction. As control, we used similar volumes of water or phosphate-buffered saline (PBS) as the reactions containing A1AT. The elastase inhibitory activity of A1AT was assessed in parallel with that of caspase activity, using aliquots from the same batch of A1AT, to ensure that the dilutions of A1AT tested in the caspase-3 assays had preserved their serpin activity. The elastase activity assays were performed by A1AT incubation with purified active porcine pancreatic elastase (2 U) followed by the addition of fluorescently labeled substrate (DQ-elastin, 25 μg/ml) in a fluorescent elastase activity assay using the Enzchek elastase assay kit (Molecular Probes, Eugene, OR). The reaction was allowed to occur at room temperature, and measurements of fluorescence were performed every 5 to 10 minutes for up to 2 to 3 hours. Conformers of A1AT were generated following established methods22Janciauskiene S Eriksson S Callea F Mallya M Zhou A Seyama K Hata S Lomas DA Differential detection of PAS-positive inclusions formed by the Z, Siiyama, and Mmalton variants of alpha1-antitrypsin.Hepatology. 2004; 40: 1203-1210Crossref PubMed Scopus (53) Google Scholar and tested for loss of activity against elastase before caspase-3 inhibitory assays. The conformers' concentrations are given as native A1AT that was input into their generation. The proteinases used to generate cleaved conformers22Janciauskiene S Eriksson S Callea F Mallya M Zhou A Seyama K Hata S Lomas DA Differential detection of PAS-positive inclusions formed by the Z, Siiyama, and Mmalton variants of alpha1-antitrypsin.Hepatology. 2004; 40: 1203-1210Crossref PubMed Scopus (53) Google Scholar were not removed from the modified A1AT solution, but the mixture was allowed to incubate for >3 hours, which we predict might have led to the inactivation of any remaining active proteinase. The remaining proteinase did not have any significant influence on the results, because the negative control used identically treated mixtures (minus the A1AT) as part of the caspase-3 activity assays. Conformer activity was compared with these controls to exclude nonspecific effect of residual active proteinase. Dose-response inhibitory activities of A1AT against both caspase and elastase were checked in duplicate for a minimum of three independent experiments. Caspase-3 activity was measured in cell lysates or cell-free mixtures using a fluorescence-based assay (Promega) as reported.18Petrache I Natarajan V Zhen L Medler TR Richter AT Cho C Hubbard WC Berdyshev EV Tuder RM Ceramide upregulation causes pulmonary cell apoptosis and emphysema-like disease in mice.Nat Med. 2005; 11: 491-498Crossref PubMed Scopus (431) Google Scholar Caspase-8 activity was measured with a specific rhodamine-labeled caspase-8 substrate (Z-IETD-R110; Molecular Probes). The amount of intracytoplasmic cytochrome c was measured with a cytochrome c ELISA kit from Calbiochem, following the manufacturer's protocol, using a plate reader (Molecular Dynamics, Sunnyvale, CA) set at 450 nm. Endothelial cell lysates were loaded in equal amounts (10 μg protein, unless otherwise noted) determined by Bradford assay (Pierce Biotechnology, Rockford, IL). Proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) (Novex, San Diego, CA), or native PAGE, followed by immunoblotting as previously described.23Petrache I Verin AD Crow MT Birukova A Liu F Garcia JG Differential effect of MLC kinase in TNF-alpha-induced endothelial cell apoptosis and barrier dysfunction.Am J Physiol. 2001; 280: L1168-L1178Google Scholar The chemiluminescent signals were quantified by densitometry (ImageQuant; Amersham) and normalized by actin. His-specific antibody was from Zymed (South San Francisco, CA). Mouse lung endothelial cells were exposed to serum-depleted medium, or serum-depleted medium with hA1AT (0.05 mg/ml) 2 to 24 hours before treatment with staurosporine (600 nmol/L, 2 hours). For immunofluorescence, cells were grown on coverslips, and hA1AT was added to live cells after being labeled with Dylight (procedure described below). Coverslips were fixed with 3% formaldehyde and visualized with the fluorescence or confocal microscope. Co-staining was performed in fixed cells that were then permeabilized with 0.25% Triton X and stained with antibodies specific for procaspase-3 or active caspase-3 (Cell Signaling, Beverly, MA), or isotype control antibody, as described.24Petrache I Birukov K Zaiman AL Crow MT Deng H Wadgaonkar R Romer LH Garcia JG Caspase-dependent cleavage of myosin light chain kinase (MLCK) is involved in TNF-alpha-mediated bovine pulmonary endothelial cell apoptosis.FASEB J. 2003; 17: 407-416Crossref PubMed Scopus (85) Google Scholar For co-immunoprecipitation assays of caspase-3 and A1AT, mouse lung endothelial cells were cultivated in Dulbecco's modified Eagle's medium and then exposed to serum-depleted medium or serum-depleted medium supplemented with hA1AT (0.05 mg/ml) 24 hours before treatment with staurosporine (600 nmol/L, 2 hours). Cell lysates were collected in RIPA buffer, diluted in PBS, and precleared with protein A-Sepharose. In control experiments, mouse IgG was also used for preclearance. The supernatant was incubated with polyclonal rabbit anti-caspase-3 antibody at 4°C, 16 hours, followed by protein A-Sepharose (2 hours). Monoclonal rabbit anti-caspase-3 and mouse monoclonal anti-caspase-3 antibody were also tested (Table 1). After centrifugation pellets were suspended in sample buffer with reducing agent, boiled for SDS-PAGE, and Western-immunoblotted with caspase-3 and A1AT antibodies (Table 1). Rabbit serum and mouse IgG were used as controls for Western blotting and no cross-reactivity was observed. When detection of immunoprecipitated protein was performed with the same antibody source used as that used for immunoprecipitation (ie, rabbit monoclonal anti-caspase-3), IgG bands were observed at 45 kd as expected and clearly distinct from the expected molecular weight of caspase-3 or A1AT (data not shown). To test the specificity of the primary anti-A1AT antibody, the presence of A1AT in the precipitate was verified with goat antibody anti-A1AT (G-17) and with the same antibody blocked with 20-fold molar excess of G-17 peptide (Santa Cruz Biotechnology, Santa Cruz, CA). hA1AT (5 mg/ml stock) was labeled with DyLight 547 NHS ester (Pierce) following the manufacturer's protocol and used at a final concentration of 0.05 mg/ml. The dye itself or dye excess eluted from Bio Spin column was used as controls. A nuclear/cytosol fractionation kit (BioVision) was used and the subcellular fractions were tested for the presence of hA1AT by Western blotting (Table 1). Isothermal calorimetry measured the heat released by interacting liquid solutions using VP-ITC Microcal (noise level, 1 ncal/second). Caspase-3 peptide (RGTELDCGIETD, corresponding to amino acids 164 to 175; lyophilized, resuspended in water) or purified elastase (lyophilized and then resuspended in water) was injected into a thermally controlled cell containing hA1AT (lyophilized and then resuspended in water). Injection amount and waiting time were adjusted according to the relaxation time of reactants and the response-function of the isothermal calorimetry. The free energy ΔG0, enthalpy ΔH0, and entropy ΔS0 change on binding were related through the thermodynamic equation ΔG0 = − RT ln K = ΔH0 − T ΔS0. The biotin label transfer assay was performed according to the manufacturer's (Pierce) instructions. A1AT was labeled with a tri-functional cross-linking reagent sufo-SBED that contains sulfonated N-hydroxysuccinimide active ester, photo-activable aryl azide, and biotin, used for identification. After separation of an unbound label on the column (Pierce), labeled hA1AT was incubated for 60 minutes with human recombinant caspase-3 at a 5:1 ratio, in the presence of 20% albumin. After UV exposure for 15 minutes to complete cross-linking, the mixture was then reduced with dithiothreitol, yielding biotinylated caspase-3. The transfer of biotin label from A1AT onto caspase-3 as a result of transient interaction was tested by Western immunoblotting using Neutravidin-horseradish peroxidase, rabbit monoclonal antibody against caspase-3, and sheep polyclonal antibody against A1AT. Animal studies were approved by the Animal Care and Use Committee and Animal Care of The Johns Hopkins University School of Medicine. Male C57Bl/6 mice (3 months old, 25 g) were from The Jackson Laboratory, Bar Harbor, ME. The experiment was performed in the same shipment lot, n = 4 to 6 mice/group. All experiments were performed with a recombinant adeno-associated virus vector construct, rAAV-CB-AAT. The detailed structure of this construct has previously been reported.25Song S Embury J Laipis PJ Berns KI Crawford JM Flotte TR Stable therapeutic serum levels of human alpha-1 antitrypsin (AAT) after portal vein injection of recombinant adeno-associated virus (rAAV) vectors.Gene Ther. 2001; 8: 1299-1306Crossref PubMed Scopus (140) Google Scholar In brief, it consists of adeno-associated virus (AAV) serotype 2 inverted terminal repeats flanking an expression cassette that drives hA1AT expression from a hybrid cytomegalovirus enhancer/β-actin promoter. This DNA cassette was pseudotyped into AAV serotype 5 capsids, using a published co-transfection method with purification on an iodixanol gradient.26Zolotukhin S Potter M Zolotukhin I Sakai Y Loiler S Fraites Jr, TJ Chiodo VA Phillipsberg T Muzyczka N Hauswirth WW Flotte TR Byrne BJ Snyder RO Production and purification of serotype 1, 2, and 5 recombinant adeno-associated viral vectors.Methods. 2002; 28: 158-167Crossref PubMed Scopus (471) Google Scholar, 27Sirninger J Muller C Braag S Tang Q Yue H Detrisac C Ferkol T Guggino WB Flotte TR Functional characterization of a recombinant adeno-associated virus 5-pseudotyped cystic fibrosis transmembrane conductance regulator vector.Hum Gene Ther. 2004; 15: 832-841PubMed Google Scholar All vector preparations were titrated using a DNA dot-blot hybridization method,26Zolotukhin S Potter M Zolotukhin I Sakai Y Loiler S Fraites Jr, TJ Chiodo VA Phillipsberg T Muzyczka N Hauswirth WW Flotte TR Byrne BJ Snyder RO Production and purification of serotype 1, 2, and 5 recombinant adeno-associated viral vectors.Methods. 2002; 28: 158-167Crossref PubMed Scopus (471) Google Scholar and doses were based on vector genome equivalents. Mice received 9.6 × 1010 particles of UF11 622 or 9.6 × 1010 particles of A1AT-AAV (pTR2-CB-AAT) intramuscularly, diluted in sterile 0.9% NaCl. The intratracheal administration of active caspase-3 was performed in mice by injection of 50 μl of PBS solutions containing either active caspase-3 (0.8 μg; MBL) + Chariot (1 μl; Active Motif, Carlsbad, CA) or Chariot alone (1 μl).28Aoshiba K Yokohori N Nagai A Alveolar wall apoptosis causes lung destruction and emphysematous changes.Am J Respir Cell Mol Biol. 2003; 28: 555-562Crossref PubMed Scopus (319) Google Scholar The preparation of trachea, the intratracheal delivery, and lung processing were performed as previously described.29Tuder RM Zhen L Cho CY Taraseviciene-Stewart L Kasahara Y Salvemini D Voelkel NF Flores SC Oxidative stress and apoptosis interact and cause emphysema due to vascular endothelial growth factor receptor blockade.Am J Respir Cell Mol Biol. 2003; 29: 88-97Crossref PubMed Scopus (347) Google Scholar The left lung was inflated and fixed, followed by paraffin-embedding, hematoxylin and eosin staining, and standardized morphometry, performed on coded slides as described.29Tuder RM Zhen L Cho CY Taraseviciene-Stewart L Kasahara Y Salvemini D Voelkel NF Flores SC Oxidative stress and apoptosis interact and cause emphysema due to vascular endothelial growth factor receptor blockade.Am J Respir Cell Mol Biol. 2003; 29: 88-97Crossref PubMed Scopus (347) Google Scholar The hA1AT protein was oxidized by incubation with either hydrogen peroxide or cigarette smoke extract, the latter obtained as previously described.18Petrache I Natarajan V Zhen L Medler TR Richter AT Cho C Hubbard WC Berdyshev EV Tuder RM Ceramide upregulation causes pulmonary cell apoptosis and emphysema-like disease in mice.Nat Med. 2005; 11: 491-498Crossref PubMed Scopus (431) Google Scholar, 30Tuder RM Wood K Taraseviciene L Flores SC Voekel NF Cigarette smoke extract decreases the expression of vascular endothelial growth factor by cultured cells and triggers apoptosis of pulmonary endothelial cells.Chest. 2000; 117: 241S-242SCrossref PubMed Scopus (44) Google Scholar The catalase activity was measured in the lung tissue lysates with a catalase assay kit (Cayman Chemical, Ann Arbor, MI), following the manufacturer's instructions, using bovine liver catalase as a positive control. The statistical analysis was performed with SPSS for windows software package (SPSS Inc.). The differences between groups were compared using unpaired Student's t-test or one-way analysis of variance with Student-Newman-Keuls post hoc test. All of the data are expressed as mean ± SEM. Statistical difference was accepted at P < 0.05. hA1AT supplementation before the addition of staurosporine, a broad protein kinase inhibitor that induces endothelial cell apoptosis, significantly inhibited staurosporine-induced apoptosis of primary mouse and human (not shown) lung endothelial cells in culture, as measured by caspase-3 activity, cytochrome c release, and assessment of 4,6-diamidino-2-phenylindole (DAPI)-stained nuclei (Figure 1, a–c, respectively). Pretreatment with A1AT also inhibited UV (6 mJ/cm2) or tumor necrosis factor-α (20 ng/ml with cycloheximide 100 μg/ml)-induced apoptosis, as measured by cytochrome c release and capase-3 activity, respectively (Supplementary Figure 1 at http://ajp.amjpathol.org). To evaluate whether the prosurvival effect of A1AT, a 52-kd circulating protein, was exerted at the plasma membrane or intracellular membrane, endothelial cells were incubated with fluorescently labeled hA1AT at similar concentrations found to inhibit their apoptosis (50 μg/ml). Labeled hA1AT permeated primary lung endothelial cells, localizing intracellularly but not in the nucleus or on plasma membrane fractions, as demonstrated by cell fractionation (Figure 2a), fluorescence microscopy (Figure 2, b–d), and confocal microscopy (Figure 2e). In contrast, polymeric hA1AT, obtained by heating the native hA1AT in PBS at 60°C for 2 hours and verified by gel electrophoresis, was not internalized by endothelial cells at 2 hours and 4 hours but rather remained attached to the plasma membrane (not shown). The finding of a granular and homogeneous pattern of labeled hA1AT expression suggested both a cytoplasmic and endosoma