Functions of Type II Pneumocyte-Derived Vascular Endothelial Growth Factor in Alveolar Structure, Acute Inflammation, and Vascular Permeability
2010; Elsevier BV; Volume: 176; Issue: 4 Linguagem: Inglês
10.2353/ajpath.2010.090209
ISSN1525-2191
AutoresMarco Mura, Matthew Binnie, Bing Han, Chengjin Li, Cristiano Feijó Andrade, Atsushi Shiozaki, Yu Zhang, Napoleone Ferrara, David Hwang, Thomas K. Waddell, Shaf Keshavjee, Mingyao Liu,
Tópico(s)Respiratory Support and Mechanisms
ResumoVascular endothelial growth factor-A (VEGF) is a potent regulator of vascular permeability, inflammatory response, and cell survival in the lung. To explore the functions of VEGF produced locally in type II pneumocytes, we generated mice with a conditional deletion of VEGF-A using Cre recombinase driven by the human surfactant protein C (SPC) promoter. In 7- to 10-week-old VEGF-knockout (SPC-VEGF-KO) mice, lung histology and physiology were essentially normal, except for higher dynamic lung compliance and lower pulmonary vascular permeability. Emphysema was seen in 28- to 32-week-old animals. To investigate the role of type II pneumocyte-derived VEGF in acute lung injury, we challenged 7- to 10-week-old SPC-VEGF-KO mice and their wild-type littermates with intestinal ischemia-reperfusion. Bronchoalveolar lavage fluid total cell count, pulmonary permeability, and lung injury score were significantly attenuated, and total lung VEGF levels were significantly lower in SPC-VEGF-KO mice compared with wild-type controls. In SPC-VEGF-KO mice, activated caspase 3-positive type II epithelial cells were increased after intestinal ischemia-reperfusion, even though there was no significant difference in the total number of cells positive for terminal deoxynucleotidyl transferase dUTP nick-end labeling. We conclude that VEGF in type II cells helps protect alveolar epithelial cells from caspase-dependent apoptosis. However, VEGF produced from type II cells may contribute to increased vascular permeability during acute lung injury. Vascular endothelial growth factor-A (VEGF) is a potent regulator of vascular permeability, inflammatory response, and cell survival in the lung. To explore the functions of VEGF produced locally in type II pneumocytes, we generated mice with a conditional deletion of VEGF-A using Cre recombinase driven by the human surfactant protein C (SPC) promoter. In 7- to 10-week-old VEGF-knockout (SPC-VEGF-KO) mice, lung histology and physiology were essentially normal, except for higher dynamic lung compliance and lower pulmonary vascular permeability. Emphysema was seen in 28- to 32-week-old animals. To investigate the role of type II pneumocyte-derived VEGF in acute lung injury, we challenged 7- to 10-week-old SPC-VEGF-KO mice and their wild-type littermates with intestinal ischemia-reperfusion. Bronchoalveolar lavage fluid total cell count, pulmonary permeability, and lung injury score were significantly attenuated, and total lung VEGF levels were significantly lower in SPC-VEGF-KO mice compared with wild-type controls. In SPC-VEGF-KO mice, activated caspase 3-positive type II epithelial cells were increased after intestinal ischemia-reperfusion, even though there was no significant difference in the total number of cells positive for terminal deoxynucleotidyl transferase dUTP nick-end labeling. We conclude that VEGF in type II cells helps protect alveolar epithelial cells from caspase-dependent apoptosis. However, VEGF produced from type II cells may contribute to increased vascular permeability during acute lung injury. Acute lung injury (ALI) and its severe form, acute respiratory distress syndrome (ARDS), are characterized by increased capillary permeability, leading to interstitial and alveolar edema, an influx of circulating inflammatory cells, and formation of hyaline membranes. However, the molecular mechanisms of ALI/ARDS are still largely unknown. Vascular endothelial growth factor-A (VEGF) is a potent regulator of vascular permeability, a key player in angiogenesis, and a survival factor for endothelial cells.1Ferrara N Gerber HP LeCouter J The biology of VEGF and its receptors.Nat Med. 2003; 9: 669-676Crossref PubMed Scopus (7822) Google Scholar The biological properties of VEGF are mainly mediated by VEGF receptor 2, also called Flk-1 (Fetal liver kinase 1), which is expressed on endothelial and epithelial cells.2Gille H Kowalski J Li B LeCouter J Moffat B Zioncheck TF Pelletier N Ferrara N Analysis of biological effects and signaling properties of Flt-1 (VEGFR-1) and KDR (VEGFR-2). A reassessment using novel receptor-specific vascular endothelial growth factor mutants.J Biol Chem. 2001; 276: 3222-3230Crossref PubMed Scopus (547) Google Scholar, 3Brown KR England KM Goss KL Snyder JM Acarregui MJ VEGF induces airway epithelial cell proliferation in human fetal lung in vitro.Am J Physiol Lung Cell Mol Physiol. 2001; 281: L1001-L1010PubMed Google Scholar VEGF also induces monocyte activation and migration, which has been found to be mediated through VEGF receptor 1, also called Flt-1 (FMS-like tyrosine kinase 1).4Barleon B Hauser S Schollmann C Weindel K Marme D Yayon A Weich HA Differential expression of the two VEGF receptors flt and KDR in placenta and vascular endothelial cells.J Cell Biochem. 1994; 54: 56-66Crossref PubMed Scopus (141) Google Scholar VEGF is considered to be both a vascular permeability factor and a pro-inflammatory cytokine. VEGF increases endothelial permeability via specific signal transduction pathways.5Becker PM Verin AD Booth MA Liu F Birukova A Garcia JG Differential regulation of diverse physiological responses to VEGF in pulmonary endothelial cells.Am J Physiol Lung Cell Mol Physiol. 2001; 281: L1500-L1511PubMed Google Scholar, 6Godzich M Hodnett M Frank JA Su G Pespeni M Angel A Howard MB Matthay MA Pittet JF Activation of the stress protein response prevents the development of pulmonary edema by inhibiting VEGF cell signaling in a model of lung ischemia-reperfusion injury in rats.FASEB J. 2006; 20: 1519-1521Crossref PubMed Scopus (40) Google Scholar Blocking VEGF has been shown to reduce tissue inflammation and damage in vivo. For example, VEGF-induced brain edema has some features that resemble stroke,7Paul R Zhang ZG Eliceiri BP Jiang Q Boccia AD Zhang RL Chopp M Cheresh DA Src deficiency or blockade of Src activity in mice provides cerebral protection following stroke.Nat Med. 2001; 7: 222-227Crossref PubMed Scopus (305) Google Scholar and VEGF antagonism reduced edema formation and tissue damage in the brain after ischemia-reperfusion in mice.8van Bruggen N Thibodeaux H Palmer JT Lee WP Fu L Cairns B Tumas D Gerlai R Williams SP van Lookeren Campagne M Ferrara N VEGF antagonism reduces edema formation and tissue damage after ischemia/reperfusion injury in the mouse brain.J Clin Invest. 1999; 104: 1613-1620Crossref PubMed Scopus (390) Google Scholar However, the role of VEGF in ALI/ARDS is controversial.9Mura M dos Santos CC Stewart D Liu M Vascular endothelial growth factor and related molecules in acute lung injury.J Appl Physiol. 2004; 97: 1605-1617Crossref PubMed Scopus (156) Google Scholar Intrapulmonary overexpression of VEGF with an adenoviral vector resulted in high-permeability edema in murine lungs.10Kaner RJ Ladetto JV Singh R Fukuda N Matthay MA Crystal RG Lung overexpression of the vascular endothelial growth factor gene induces pulmonary edema.Am J Respir Cell Mol Biol. 2000; 22: 657-664Crossref PubMed Scopus (246) Google Scholar Using an inducible transgenic mouse model, it was shown that VEGF overexpression in airway epithelium also induces pulmonary edema.11Lee CG Link H Baluk P Homer RJ Chapoval S Bhandari V Kang MJ Cohn L Kim YK McDonald DM Elias JA Vascular endothelial growth factor (VEGF) induces remodeling and enhances TH2-mediated sensitization and inflammation in the lung.Nat Med. 2004; 10: 1095-1103Crossref PubMed Scopus (495) Google Scholar Transfection of sFlt-1 gene attenuated bleomycin-induced pneumopathy in mice.12Hamada N Kuwano K Yamada M Hagimoto N Hiasa K Egashira K Nakashima N Maeyama T Yoshimi M Nakanishi Y Anti-vascular endothelial growth factor gene therapy attenuates lung injury and fibrosis in mice.J Immunol. 2005; 175: 1224-1231PubMed Google Scholar Similarly, pretreatment of rats with adenovirus encoding soluble Flk-1 prevented ischemia-reperfusion-induced lung injury.6Godzich M Hodnett M Frank JA Su G Pespeni M Angel A Howard MB Matthay MA Pittet JF Activation of the stress protein response prevents the development of pulmonary edema by inhibiting VEGF cell signaling in a model of lung ischemia-reperfusion injury in rats.FASEB J. 2006; 20: 1519-1521Crossref PubMed Scopus (40) Google Scholar These results all suggest that VEGF signaling through its receptors may promote pulmonary permeability and inflammation. However, intriguingly, ischemia-reperfusion does not affect total VEGF protein levels in the lung.6Godzich M Hodnett M Frank JA Su G Pespeni M Angel A Howard MB Matthay MA Pittet JF Activation of the stress protein response prevents the development of pulmonary edema by inhibiting VEGF cell signaling in a model of lung ischemia-reperfusion injury in rats.FASEB J. 2006; 20: 1519-1521Crossref PubMed Scopus (40) Google Scholar In addition, VEGF levels in the alveolar compartment of patients with ARDS are indistinguishable from those in patients with hydrostatic pulmonary edema.13Ware LB Kaner RJ Crystal RG Schane R Trivedi NN McAuley D Matthay MA VEGF levels in the alveolar compartment do not distinguish between ARDS and hydrostatic pulmonary oedema.Eur Respir J. 2005; 26: 101-105Crossref PubMed Scopus (47) Google Scholar Lung epithelial lining fluid from ARDS patients actually contained lower levels of VEGF than that from at-risk subjects, and increased levels at Day 4 were associated with better recovery.14Thickett DR Armstrong L Millar AB A role for vascular endothelial growth factor in acute and resolving lung injury.Am J Respir Crit Care Med. 2002; 166: 1332-1337Crossref PubMed Scopus (149) Google Scholar Consistent with this finding, VEGF blockade has been shown to result in increased markers of oxidative stress, alveolar cell apoptosis and alveolar enlargement,15Tuder 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 (342) Google Scholar, 16Tang K Rossiter HB Wagner PD Breen EC Lung-targeted VEGF inactivation leads to an emphysema phenotype in mice.J Appl Physiol. 2004; 97: 1559-1566; discussion 1549Crossref PubMed Scopus (190) Google Scholar suggesting a protective role for VEGF in the epithelial barrier. These interesting observations also suggest that the expression and function of VEGF may be regulated locally at sites of lung injury, without causing dramatic changes in overall lung VEGF levels. VEGF is highly compartmentalized in the lung17Kaner RJ Crystal RG Compartmentalization of vascular endothelial growth factor to the epithelial surface of the human lung.Mol Med. 2001; 7: 240-246PubMed Google Scholar and is produced by multiple sources, such as epithelial and mesenchymal cells, myofibroblasts, and macrophages.1Ferrara N Gerber HP LeCouter J The biology of VEGF and its receptors.Nat Med. 2003; 9: 669-676Crossref PubMed Scopus (7822) Google Scholar, 18Corne J Chupp G Lee CG Homer RJ Zhu Z Chen Q Ma B Du Y Roux F McArdle J Waxman AB Elias JA IL-13 stimulates vascular endothelial cell growth factor and protects against hyperoxic acute lung injury.J Clin Invest. 2000; 106: 783-791Crossref PubMed Scopus (138) Google Scholar However, type II alveolar epithelial cells are considered to be the main source of VEGF in the lung parenchyma,17Kaner RJ Crystal RG Compartmentalization of vascular endothelial growth factor to the epithelial surface of the human lung.Mol Med. 2001; 7: 240-246PubMed Google Scholar although the physiological role of VEGF from this source remains unclear. VEGF released by alveolar epithelial cells may act as an autocrine trophic factor for epithelial cells,19Roberts JR Perkins GD Fujisawa T Pettigrew KA Gao F Ahmed A Thickett DR Vascular endothelial growth factor promotes physical wound repair and is anti-apoptotic in primary distal lung epithelial and A549 cells.Crit Care Med. 2007; 35: 2164-2170Crossref PubMed Scopus (51) Google Scholar and modulate functions of the adjacent vascular endothelium in a paracrine fashion.20Shifren JL Doldi N Ferrara N Mesiano S Jaffe RB In the human fetus, vascular endothelial growth factor is expressed in epithelial cells and myocytes, but not vascular endothelium: implications for mode of action.J Clin Endocrinol Metab. 1994; 79: 316-322Crossref PubMed Scopus (171) Google Scholar To investigate the roles of locally produced VEGF in the lung, we generated transgenic mice in which the expression of VEGF was selectively deleted in distal lung epithelial cells using a Cre recombinase system driven by the surfactant protein C (SPC)-promoter. While young transgenic mice showed a normal phenotype, older animals spontaneously developed typical features of initial emphysema. The severity of ALI, pulmonary permeability and inflammation, and the expression of cell death and stress markers were compared between transgenic young mice and their wild-type littermates after intestinal ischemia-reperfusion, a model of extrapulmonary ARDS.21Souza DG Soares AC Pinho V Torloni H Reis LF Teixeira MM Dias AA Increased mortality and inflammation in tumor necrosis factor-stimulated gene-14 transgenic mice after ischemia and reperfusion injury.Am J Pathol. 2002; 160: 1755-1765Abstract Full Text Full Text PDF PubMed Scopus (171) Google Scholar This study was approved by the Animal Use and Care Committee of the University Health Network. All animals received humane care in compliance with the Principles of Laboratory Animal Care formulated by the National Society for Medical Research and the Guide for the Care and Use of Laboratory Animals prepared by the Institute of Laboratory Animal Resources and published by the National Institutes of Health (NIH publication 0-309-05337-3, revised 1996), and the Guide for the Care and Use of Experimental Animals formulated by the Canadian Council on Animal Care. SPC-Cre transgenic mice were generated by cloning a cDNA encoding Cre recombinase and a nuclear localization signal downstream of the 3.7-kb human SPC promoter sequence, thus driving Cre expression in distal lung epithelium. This promoter confers gene expression throughout the lung epithelium during gestation and in a subset of type II pneumocytes postnatally. Transgene positive founders were identified by Southern blotting for Cre and tested for Cre activity by mating them to Z/EG reporter mice (ICR background), in which Cre-mediated excision of a stop codon leads to cell-specific expression of enhanced jellyfish green fluorescent protein (GFP).22Novak A Guo C Yang W Nagy A Lobe CG Z/EG, a double reporter mouse line that expresses enhanced green fluorescent protein upon Cre-mediated excision.Genesis. 2000; 28: 147-155Crossref PubMed Scopus (722) Google Scholar, 23Chalfie M Tu Y Euskirchen G Ward WW Prasher DC Green fluorescent protein as a marker for gene expression.Science. 1994; 263: 802-805Crossref PubMed Scopus (5487) Google Scholar To confirm the specificity of gene targeting, dual transgenic SPC-Cre Z/EG offspring from one founder line were tested for GFP/prosurfactant protein C colocalization with immunofluorescent staining. The same SPC-Cre recombinase line (ICR background) was bred to floxed VEGF mice (ICR background) which have loxP sites inserted around the third exon (Figure 1A). Site-specific recombination between the two loxP sites of the VEGF gene results in a null VEGF allele. To generate homozygous floxed VEGF-Cre recombinase mice, mice carrying both a SPC-Cre transgene and one floxed VEGF allele were bred to homozygous floxed VEGF mice. The presence of the floxed VEGF gene was detected by PCR.24Eremina V Sood M Haigh J Nagy A Lajoie G Ferrara N Gerber HP Kikkawa Y Miner JH Quaggin SE Glomerular-specific alterations of VEGF-A expression lead to distinct congenital and acquired renal diseases.J Clin Invest. 2003; 111: 707-716Crossref PubMed Scopus (1076) Google Scholar Only homozygous knock-out animals (ie, Spc-Cre+/0 VEGFlox/lox) (SPC-VEGF KO) and wild-type littermates were used. Recombination of the floxed allele deletes all VEGF isoforms (VEGF120, 164, and 188). Genomic DNA was isolated from mouse tails and used for genotypic analysis. The presence of the floxed VEGF gene was detected by PCR using the oligonucleotide primers muVEGF 419.F (5′-CCTGGCCCTCAAGTACACCTT-3′) and muVEGF 567.R (5′-TCCGTACGACGCATTTCTAG-3′) (Sigma-Genosys, The Woodlands, TX), which generates a 140-bp fragment of the VEGF allele in the presence of the loxP-1 site and a 100-bp fragment in the wild-type allele. To test for specific Cre recombinase expression in distal lung epithelium, colocalization of Cre-dependent GFP and the type II pneumocyte marker, surfactant protein C precursor protein (proSPC), was confirmed by double immunofluorescent staining. Lungs were chopped into fragments with a blade, fixed with 4% buffered formalin, processed, and embedded in paraffin; 5 μm sections were deparaffinized and rehydrated. Sections were boiled in 0.01 mol/L NaCitrate [pH 6.0] for 10 minutes and then cooled to room temperature. Sections were blocked with 5% goat serum in PBS for 1 hour at room temperature, incubated with primary antibody overnight at 4°C, washed in PBS, then stained with secondary antibody for 1 hour at room temperature, washed in PBS, and mounted in Vectashield medium with 4,6-diamidino-2-phenylindole (Vector labs, Burlingame CA). The antibodies used were rabbit anti-mouse proSPC (AB3786 1:500; Millipore, Billerica MA) and chicken anti-GFP (ab13970 1:1000; Abcam, Cambridge, MA) fluorescein isothiocyanate-conjugated anti-chicken IgY (1:500) and Cy3-conjugated anti-rabbit IgG (1:500; Jackson Immunoresearch Laboratories, West Grove, PA) in the dark for 1 hour. After washing, slides were mounted with GVA mounting solution (Invitrogen, Carlsbad, CA). Thirty randomly chosen fields from two animals at ×200 magnification were analyzed. The average number of GFP-positive cells was quantified as a percentage of total proSPC-positive cells counted. To determine the specificity of staining, the primary antibody was omitted from the staining protocol as a negative control. We also used lung tissue slide from wild-type animals, to show the lack of anti-GFP staining. Quantitative real-time reverse transcriptase-PCR analysis of the RNA expression of VEGF was performed on RNA isolated from frozen lung tissues as previously described.25dos Santos CC Han B Andrade CF Bai X Uhlig S Hubmayr R Tsang M Lodyga M Keshavjee S Slutsky AS Liu M DNA microarray analysis of gene expression in alveolar epithelial cells in response to TNFalpha, LPS, and cyclic stretch.Physiol Genomics. 2004; 19: 331-342Crossref PubMed Scopus (131) Google Scholar Total RNA extraction was performed with the RNeasy kit (Qiagen, Mississauga, Ontario, Canada). Results are expressed as the ratio of VEGF and the housekeeping gene B2M expression levels. Primers were designed using the Primer Express 1.5 software (Applied Biosystems) and purchased from ACGT (Toronto, Ontario, Canada). For morphometric studies, the mean linear intercept was used as a measure of interalveolar wall distance, as described.26Yao H Edirisinghe I Yang SR Rajendrasozhan S Kode A Caito S Adenuga D Rahman I Genetic ablation of NADPH oxidase enhances susceptibility to cigarette smoke-induced lung inflammation and emphysema in mice.Am J Pathol. 2008; 172: 1222-1237Abstract Full Text Full Text PDF PubMed Scopus (88) Google Scholar The mean linear intercept was calculated for each sample based on 20 random fields observed at a magnification of ×400 using a cross-line. Fields containing airways and blood vessels were excluded. The total number of alveolar intercepts encountered in each field was counted and the mean value for each animal (20 fields) was calculated. A lower mean linear intercept count represents more pronounced airspace enlargement. Seven- to ten-week-old male mice (weight 24.2 ± 4.9 g in wild-type group, 26.7 ± 5.3 g in SPC-VEGF KO group) were anesthetized with 5% isoflurane, intubated with a cannula (1.0 mm, Harvard Apparatus Canada, St. Laurent, Canada), and connected to a volume-controlled constant flow ventilator (Inspira Advanced Safety Ventilator, Harvard Apparatus). After intubation, anesthesia was continuously maintained with isoflurane and body temperature was maintained at 37°C by an immersion thermostat throughout baseline observation or IIR surgical procedure. The animals were ventilated with a tidal volume of 6 ml/kg, inspiratory oxygen fraction 1.0, inspiratory/expiratory ratio 1:2, and a frequency of 140 breaths/min. Dynamic lung compliance was measured with an esophageal catheter. After 10 minutes of observation, either at baseline level or after 30 minutes of IIR, animals were sacrificed by exsanguination. Arterial blood gas analysis was performed in four 7- to 10-week-old wild-type animals, three 7- to 10-weeks-old SPC-VEGF KO animals, and four SPC-VEGF KO 28- to 32-week-old animals. HSE-USB acquisition system and Pulmodyn software (H. Sachs Elektronik, March-Hugstetten, Germany) were used for all pulmonary function measurements. The IIR procedure was performed by occluding the superior mesenteric artery below the celiac trunk with an arterial microclamp for 30 minutes, as previously described27Mura M Andrade CF Han B Seth R Zhang Y Bai XH Waddell TK Hwang D Keshavjee S Liu M Intestinal ischemia-reperfusion-induced acute lung injury and oncotic cell death in multiple organs.Shock. 2007; 28: 227-238Crossref PubMed Scopus (85) Google Scholar, 28Mura M Han B Andrade CF Seth R Hwang D Waddell TK Keshavjee S Liu M The early responses of VEGF and its receptors during acute lung injury: implication of VEGF in alveolar epithelial cell survival.Crit Care. 2006; 10: R130Crossref PubMed Scopus (55) Google Scholar; the reperfusion period was extended to 24 hours without mechanical ventilation or supplemental oxygen. Intestinal ischemia was confirmed by paleness of the jejunum and ileum. After 30 minutes the clamp was removed, 0.5 ml of sterile saline at 37°C was injected into the peritoneal cavity and the skin was sutured. Anesthesia was terminated and animals were observed until a spontaneous breath appeared. Mechanical ventilation was then stopped and animals were let wean until a regular pattern of breathing was achieved. After 24 hours, animals were resubmitted to anesthesia and laparotomy and sacrificed by exsanguination. For sham-operated animals, the same procedures were performed but the mesenteric artery was not clamped. Separate subgroups of lungs were 1) fixed for histological evaluation and immunohistochemistry studies; 2) snap-frozen in liquid nitrogen for protein or RNA analysis; 3) used for bronchoalveolar lavage (BAL) or Evans blue dye assay. Blood samples were collected after 24 hours of reperfusion by puncture of the aorta and centrifuged (4000 × g, 10 minutes). Plasma samples were stored at −80°C. ALI was investigated by evaluating BAL cell counts, pulmonary vascular permeability, lung histology, and fibrin deposition with procedures previously described.27Mura M Andrade CF Han B Seth R Zhang Y Bai XH Waddell TK Hwang D Keshavjee S Liu M Intestinal ischemia-reperfusion-induced acute lung injury and oncotic cell death in multiple organs.Shock. 2007; 28: 227-238Crossref PubMed Scopus (85) Google Scholar, 28Mura M Han B Andrade CF Seth R Hwang D Waddell TK Keshavjee S Liu M The early responses of VEGF and its receptors during acute lung injury: implication of VEGF in alveolar epithelial cell survival.Crit Care. 2006; 10: R130Crossref PubMed Scopus (55) Google Scholar Evans blue dye permeability assay was performed with the method described by Kaner et al.10Kaner RJ Ladetto JV Singh R Fukuda N Matthay MA Crystal RG Lung overexpression of the vascular endothelial growth factor gene induces pulmonary edema.Am J Respir Cell Mol Biol. 2000; 22: 657-664Crossref PubMed Scopus (246) Google Scholar To assess fibrin deposition, fibrinogen immunohistochemistry was performed.29Swaisgood CM French EL Noga C Simon RH Ploplis VA The development of bleomycin-induced pulmonary fibrosis in mice deficient for components of the fibrinolytic system.Am J Pathol. 2000; 157: 177-187Abstract Full Text Full Text PDF PubMed Scopus (152) Google Scholar The degree of lung injury was determined using the grading system developed by Ginsberg et al.30Ginsberg HS Horswood RL Chanock RM Prince GA Role of early genes in pathogenesis of adenovirus pneumonia.Proc Natl Acad Sci USA. 1990; 87: 6191-6195Crossref PubMed Scopus (70) Google Scholar The lung injury score comprises the assessment of four parameters: 1) alveolar hemorrhage, 2) vascular congestion, 3) infiltration or aggregation of neutrophils in the airspace or vessel wall, and 4) fibrin deposition in alveoli. The severity of each parameter was scored as 0, absent; 1, mild; 2, moderate; and 3, severe. The combined score of all four parameters was taken for each animal.30Ginsberg HS Horswood RL Chanock RM Prince GA Role of early genes in pathogenesis of adenovirus pneumonia.Proc Natl Acad Sci USA. 1990; 87: 6191-6195Crossref PubMed Scopus (70) Google Scholar Ten randomly chosen fields from each animal with approximately the same number of alveoli were analyzed by a pulmonary pathologist (D.H.), who was blinded to the group allocation. VEGF gene expression was assessed by in situ hybridization using a probe kindly provided by Dr. Andras Nagy (University of Toronto, Canada). Lung tissues were prepared as previously described.31Han B Mura M Andrade CF Okutani D Lodyga M dos Santos CC Keshavjee S Matthay M Liu M TNFalpha-induced long pentraxin PTX3 expression in human lung epithelial cells via JNK.J Immunol. 2005; 175: 8303-8311PubMed Google ScholarIn situ hybridization was conducted by a staff member of the core facility in our institute. For quantitative analysis, 10 optical fields of alveolar area from each animal (3 mice/group), not including major airways or vessels, were randomly chosen at ×1000 magnification. The number of VEGF-positive cells as well as the total cell number of cell nuclei in the chosen fields were counted in a blinded fashion. The percentage of positive-stained cells for each optical field was quantified as a percentage of total cells counted. VEGF levels in lung homogenates, BAL fluid and plasma were determined using an ELISA kit (R&D Systems, Minneapolis, MN)28Mura M Han B Andrade CF Seth R Hwang D Waddell TK Keshavjee S Liu M The early responses of VEGF and its receptors during acute lung injury: implication of VEGF in alveolar epithelial cell survival.Crit Care. 2006; 10: R130Crossref PubMed Scopus (55) Google Scholar that recognizes VEGF isoforms with either 120 or 164 amino acids. The lower detection limit was 31 pg/ml. ELISA values in lung homogenates were standardized to total protein concentration. Terminal transferase dUTP nick end labeling (TUNEL) staining, and TUNEL/activated caspase 3 double staining have been described.27Mura M Andrade CF Han B Seth R Zhang Y Bai XH Waddell TK Hwang D Keshavjee S Liu M Intestinal ischemia-reperfusion-induced acute lung injury and oncotic cell death in multiple organs.Shock. 2007; 28: 227-238Crossref PubMed Scopus (85) Google Scholar Activated caspase 3/Surfactant Protein B (SPB) double immunofluorescent staining was also performed. SPB was selected as a marker of Type II cells because SPC is known to be down-regulated in animal models of ALI.32Abadie Y Bregeon F Papazian L Lange F Chailley-Heu B Thomas P Duvaldestin P Adnot S Maitre B Delclaux C Decreased VEGF concentration in lung tissue and vascular injury during ARDS.Eur Respir J. 2005; 25: 139-146Crossref PubMed Scopus (83) Google Scholar Clara cells are also SPB-positive, and therefore airways were excluded from the semiquantitative analysis. The staining was performed on 4 μm lung slides. After deparaffinization and dehydration, antigen retrieval was performed with 10 μg/ml proteinase K in 10 mmol/L Tris/HCl [pH 7.4 to 8] for 15 minutes. Slides were incubated with a rabbit anti-SPB polyclonal antibody (1:50 in PBS-1% bovine serum albumin[BSA]) for 6 hours, washed with PBS, and then incubated with a Texas Red mouse anti-rabbit IgG (1:100 in PBS-1% BSA; Jackson Immunoresearch) for 1 hour. Slides were next incubated with a goat polyclonal antibody against the activated (cleaved) form of caspase 3 (1:50 in PBS-1% BSA) (Santa Cruz) overnight at 4°C, washed with PBS and then incubated with a fluorescein isothiocyanate-conjugated donkey anti-goat IgG (1:500 in PBS-1% BSA, Biotium, Hayward, CA) at room temperature for 1 hour. The nuclei were counterstained with Hoechst 33258 (1:5000, 10 minutes). All slides were subjected to identical exposure times. Nonimmune serum instead of primary antibody for caspase 3 was used for negative controls. Spleen sections (secondary follicles) were used as positive control.33Krajewski S Zapata JM Krajewska M VanArsdale T Shabaik A Gascoyne RD Reed JC Immunohistochemical analysis of in vivo patterns of TRAF-3 expression, a member of the TNF receptor-associated factor family.J Immunol. 1997; 159: 5841-5852PubMed Google Scholar The average number of activated caspase 3-positive cells was quantified as a percentage of total Hoescht-positive cells counted; the average number of activated caspase 3-positive epithelial cells was also quantified as a percentage of total SPB-positive cells counted. The detailed protocols published previously were followed.28Mura M Han B Andrade CF Seth R Hwang D Waddell TK Keshavjee S Liu M The early responses of VEGF and its receptors during acute lung injury: implication of VEGF in alveolar epithelial cell survival.Crit Care. 2006; 10: R130Crossref PubMed Scopus (55) Google Scholar The primary antibodies used were anti-VEGF (1:200) (Santa Cruz Biotechnology, Santa Cruz, CA) and chicken anti-fibrinogen (1:400, GenWay Biotech San Diego, CA). Slides were then washed and incubated with biotinylated secondary antibody (1:600
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