acute lung injury ( ali ) is a devastating syndrome and an important cause of mortality in critically ill patients .
the syndrome is characterized by hypoxemia and respiratory failure due to exudation to alveolar spaces which impairs gas exchange .
the risk factors for developing this syndrome include pneumonia , gastric aspiration , sepsis , shock , and acute pancreatitis .
although acute pancreatitis represents one of the less common clinical disorders associated with acute respiratory distress syndrome ( ards ) , severe attacks of pancreatitis are frequently associated with acute lung injury and respiratory failure .
the underlying molecular mechanisms behind the development of lung injury are not fully understood , which may explain the lack of specific pharmacologic therapies .
transforming growth factor  ( tgf- ) is a multifunctional cytokine regulating inflammatory and fibrotic disorders .
it plays a critical role in embryonic development as well as in the resolution of tissue injury in multiple organs , including the lung .
tgf- exists in three isoforms and is a member of a large family of soluble proteins that modulate several cellular processes . of these isoforms , tgf-1 is generated in greatest abundance subsequent to tissue damage .
tgf- signaling is initiated via ligand - induced heteromeric complex formation of the tgf- type i and type ii serine / threonine kinases receptors . upon ligand
binding , the tgf- type ii receptor ( trii ) phosphorylates the type i receptor , which then propagates the signal through phosphorylation of the smad protein cascade .
smad proteins constitute three functional classes : the receptor - regulated smad ( r - smad ) , the comediator smad ( co - smad ) , and the inhibitory smad ( i - smad ) .
r - smads ( smad1 , 2 , 3 , 5 , and 8) are directly phosphorylated and activated by the type i receptor kinases and form heteromeric complexes with the co - smad , smad4 .
the activated smad complexes are translocated into the nucleus and , in conjunction with other nuclear cofactors , regulate the transcription of target genes .
the i - smad , smad6 and smad7 , negatively regulates tgf- signaling by competing with r - smads for receptor or co - smad interaction and by targeting the receptors for degradation .
tgf- has been most thoroughly evaluated for its crucial role in the development of pulmonary fibrosis and airway remodeling during the late phases of chronic lung injury [ 6 , 7 ] . however , the involvement and regulation of tgf- in acute lung injury are largely unknown .
murine models have demonstrated that the expression levels of several tgf--responsive genes involved in extracellular matrix modulation and fibrinolysis were dramatically increased in the early phase after induction of lung injury .
in addition , elevated levels of tgf-1 as well as tgf--inducible genes , such as procollagen type iii and 1 , have been demonstrated in the lungs of patients with ards [ 9 , 10 ] .
procollagen iii is one of the earliest predictors of the severity of ali [ 1114 ] .
tgf- has been shown to directly increase alveolar epithelial permeability by increasing the gaps between the endothelial cells [ 1518 ] .
increased epithelial permeability permits migration of neutrophils , which stimulates repair of the pulmonary epithelium .
we hypothesized that tgf- signaling might be active early in the lungs in ali and plays a significant part in the flooding of the alveolar spaces and lung injury .
the aim of the present study was to investigate the early activation of tgf- signaling in the lungs of a murine model of acute pancreatitis - associated ali .
antibodies against tgf-1 - 3 , trii , and alk5 ( tri ) were from santa cruz biotechnology inc .
( santa cruz , ca , usa ) . antibodies against smad2 , 3 , 4 , and 7 and p - smad2
810 -week- old male wild - type c57bl/6 mice were purchased from charles river , germany .
the mice were housed in appropriate facilities at lund university , under specific pathogen - free conditions and handled according to the institute guidelines with approval of the malmo - lund animal care ethics committee .
the animals were kept under 12/12  h light / dark regime in standard mesh cages with laboratory chow and drinking water ad libitum . 
 animal model .
acute pancreatitis was induced using the combined pancreatic duct and bile duct ( bpd ) ligation model as described previously .
the bpd ligation model is a highly acute model that elicits a pronounced pulmonary inflammatory response as early as 9  h after acute pancreatitis induction .
briefly , the mice were anesthetized and maintained with 24% isoflurane . under aseptic conditions ,
the bile duct , proximal to its entry into the pancreas , and the common bile - pancreatic duct , near its junction with the duodenum , were dissected and ligated ( bpd group ) .
the same procedure was applied to sham - operated control mice where the common bile - pancreatic duct and the bile duct were dissected , but not ligated , after which the abdomen was closed .
the mice recovered rapidly after surgery , and postoperative buprenorphine analgesia ( 0.05  mg / kg , s.c . ) was administered twice daily .
the animals ( n = 8 in each group ) were sacrificed by exsanguination through puncture of the abdominal aorta 9 and 24  h after pancreatitis - induced surgery .
lung biopsies were harvested , fixed in 4% paraformaldehyde for further immunohistochemical processing or snap - frozen in liquid nitrogen , and stored at 80c until western blot analyses .
endogenous peroxidase activity was quenched for 30  min in darkness by 1% h2o2 ( sigma aldrich , st . louis , mo , usa ) in methanol ( histolab , gothenburg , sweden ) . after blocking with avidin - biotin ( vector laboratories , burlingame , ca ) and normal goat serum ( 5% ) for 60  min , the sections were incubated with primary antibodies in tris - buffered saline ( tbs)/saponin buffer overnight .
biotinylated goat anti - rabbit igg secondary antibodies were added in 2% normal blocking serum .
slides were developed for 5  min in diaminobenzidine ( dab , vectastain , vector laboratories ) and counterstained with mayer 's hematoxylin ( histolab ) .
positively stained cells were quantified as average number of cells in 10 microscopic fields ( 20x magnification ) by two researchers blinded to the samples .
snap - frozen lung tissue was homogenized in liquid nitrogen using a mortar and pestle .
proteins were extracted in 1% tritonx-100 , 10  mm tris - hcl , 50  mm nacl , 5  mm edta , 30  mm sodium pyrophosphate , 50  mm naf , 0.1  mm na3vo4 , and complete protease inhibitor cocktail ( sigma ) .
lysates were dissolved in laemmli buffer , boiled and separated by sds - page ( 12% ) , and transferred to 0.2  m hybond - c extra nitrocellulose membrane ( amersham biosciences ) .
blots were blocked in 5% ( w / v ) milk in tris - buffered saline tween-20 ( tbst ) and incubated overnight at 4c with the primary antibodies ( 1  g / ml ) . following washing
, the membranes were incubated for 1  h with the secondary hrp - linked goat anti - rabbit antibody at 1  :  2000 .
immunoblotted proteins were visualized by supersignal west dura substrate ( pierce thermoscientific ) using li - cor odyssey fc imager ( li - cor biosciences ) and image studio vr 2.0 software .
statistical analyses were performed by two - tailed student 's t - test using prism software . a  p  value of < 0.05 was considered statistically significant .
to investigate the role of the tgf- system in the progression of ali due to acute pancreatitis , levels of tgf-1 , -2 , and -3 were examined in the lungs of ligated animals with acute pancreatitis compared to sham . in the lungs of the animals with ap , tgf-1 was associated predominantly with the bronchial epithelium and macrophages at both time points .
occasional fibroblast - like cells and type ii alveolar epithelial cells were also stained ( figure 1(b ) ) .
there was a 1.68-fold enhanced expression of tgf-1 in the lungs of this group compared to the sham - operated animals ( p < 0.05 ; figures 1(a ) , 1(b ) , 1(g ) , and 1(h ) ) .
these changes were more pronounced after 24  h as compared to 9  h ( p < 0.01 ; figures 1(g ) and 1(h ) ) .
staining for tgf-2 in airways of the animals with ap showed similar pattern to that for tgf-1 .
bronchial epithelial cells and smooth muscle cells were stained more intensely compared to other cell populations and fibroblast - like cells were stained more consistently compared to tgf-1 .
neutrophils were positive for tgf-2 as well as macrophages , sub - epithelial fibroblast - like cells which were stained moderately .
no obvious difference between the sham - operated and ligated animals at any of the time points ( figures 1(c ) and 1(d ) ) .
macrophages , fibroblast - like , and smooth muscle cells were also showing expression . in the ligated group
, macrophages were generally stained ; subepithelial fibroblast - like cells showed mixed but predominantly positive staining .
the bronchial epithelial cells showed a stronger staining tendency in the ligated group in both 9 ( figures 1(e ) and 1(f ) ) and 24  h ( data not shown ) .
protein extracts from the lungs of the sham - operated and ligated animals were analyzed by western blotting .
total tgf-1 protein levels were higher in the lung extracts of the ligated animals at 9 and 24  h compared to the lungs of the sham - operated group ( figures 1(g ) and 1(h ) ) .
there was not any difference for tgf-2 and 3 protein levels between the sham - operated animals and the acute pancreatitis group ( data not shown ) .
these results indicate that early modulation of tgf- ligands in the lungs of mice with acute pancreatitis mostly relates to induction of the tgf-1 isoform rather than tgf-2 and tgf-3 .
to assess modulation of two candidate receptors for tgf- signaling , the lung sections were stained for trii and alk5 ( tri ) .
both the acute pancreatitis ( figure 2(b ) ) and sham - operated groups ( figure 2(a ) ) demonstrated high levels of trii in bronchial epithelial and smooth muscle cells , while alveoli , fibroblasts , endothelial , and infiltrating cells showed moderate to weak staining ( figures 2(a ) and 2(b ) ) .
no obvious difference in the expression pattern of trii was observed between the acute pancreatitis group and sham controls at the time points studied .
bronchial epithelial and vascular endothelial cells were strongly positive for alk5 , while fibroblasts and alveoli were moderately positive for alk5 in the lungs of the pancreatitis - induced group ( figures 2(d ) and 2(f ) ) . a similar staining pattern was observed in the lungs of the sham - operated groups ( figures 2(c ) and 2(e ) ) .
interestingly , the bronchial epithelial cells demonstrated an enhanced nuclear accumulation of alk5 after 9  h that was further enhanced at 24  h after acute pancreatitis induction compared to sham controls .
in addition , acute pancreatitis following bpd - ligation was further associated with enhanced number of infiltrating cells that were more alk5-positive compared to sham ( figures 2(d ) and 2(f ) ) .
a significant overall increase in the number of alk5-positive cells per field was found in the pancreatitis - induced groups compared to sham at both 9 and 24  h ( figure 2(g ) , 53 versus 32 ; p < 0.001 and 45 versus 32 ; p < 0.05 ; resp . )
. the elevated alk5 levels in the lungs following acute pancreatitis induction were further confirmed by western blot of total protein extracts .
a pronounced increase in the total protein levels of alk5 was detected at both 9 and 24  h in the pancreatitis group compared to sham control ( figure 2(h ) ) .
these data indicate that the acute pancreatitis mediated regulation of tgf- responses at the receptor level predominantly involves induction of alk5 instead of trii .
smad2 was detected at moderate levels in bronchial epithelium , infiltrating cells , and fibroblasts , while low levels were observed in alveoli and vascular endothelial cells in both sham controls and 9  h after acute pancreatitis induction ( figures 3(a ) and 3(b ) ) .
high levels of smad3 were observed in bronchial epithelium , vascular endothelium , infiltrating , and fibroblast - like cells , while low levels were detected in alveolar cells in both sham controls and with acute pancreatitis induction ( figures 3(c ) and 3(d ) ) .
similarly , lungs from both sham controls and acute pancreatitis - associated ali showed moderate levels of smad4 in bronchial epithelial , vascular endothelial and infiltrating cells ( figures 3(e ) and 3(f ) ) .
a moderate smad4 induction was noted in the infiltrating cells in the ligated animals compared to sham controls ( figure 3(f ) ) .
however , neither immunostainings nor western blot ( figures 3(g ) and 3(h ) ) analyses showed a significant modulation of the smad24 protein levels in the lungs following 9 or 24  h acute pancreatitis induction compared to sham controls . to further study regulation of the tgf- signaling pathway ,
bronchial epithelial cells as well as vascular endothelial , smooth muscle , and inflammatory cells showed considerably higher levels of smad7 9  h after acute pancreatitis induction compared to sham controls ( figures 4(a ) and 4(b ) ) .
however , the smad7 induction appeared transient and with disease progression the levels of smad7 were reduced .
after 24  h the levels were not altered notably compared to control ( figures 4(c ) and 4(d ) ) .
analysis of the smad7 protein levels in lung extracts confirmed the immunohistochemistry findings , with 2.41-fold increased levels of smad7 ( p < 0.05 ) in the ligated group at 9  h , but not at 24  h , compared to sham controls ( figure 4(e ) ) . in order to study the level of active tgf- signaling during acute pancreatitis - associated acute lung injury , the activation and subcellular distribution of phosphorylated smad2 ( p - smad2 )
bronchial epithelial , vascular endothelial , infiltrating , and fibroblast - like cells in the sham - operated animals had a weak cytoplasmic expression of p - smad2 at both 9 and 24  h , with scattered cells showing a nuclear localization ( figures 5(a ) and 5(c ) ) .
in contrast , enhanced levels of cytoplasmic and importantly nuclear translocated p - smad2 were detected in the lungs , especially in vascular endothelial cells , 24  h after acute pancreatitis induction compared to sham control ( figures 5(c)5(f ) ) . despite differences at the cellular level
, the total protein level of p - smad2 was not altered between the sham - operated and ligated animals at any of the time points ( figure 5(g ) ) . a summary of the changes in expression of the tgf- signaling molecules is presented in table 1 .
tgf- has a well - established role in the fibrotic processes during chronic lung diseases . in the present study
, we show that tgf- signaling modulation starts as early as 9  h in the lungs of animals with acute pancreatitis - associated acute lung injury .
these changes included an early increase in the levels of tgf-1 and alk5 ( tri ) .
a parallel increase in the signaling regulator and general inhibitor of the tgf- signaling transduction , smad7 , was found at 9  h. later in the course of the disease at 24  h the levels of tgf1 and alk5 remained high , while levels of the inhibitory smad7 were reduced back to the level of the sham - operated animals .
these changes were associated with an enhanced nuclear translocation of phosphorylated smad2 indicating an active tgf- signaling .
this is the first report on the early modulation of tgf- signaling pathway in the acute lung injury due to acute pancreatitis .
tgf- signaling regulates various cellular processes , including cell proliferation , recognition , differentiation , apoptosis , and specification of developmental fate during embryogenesis as well as in mature tissues .
previous studies have indicated that tgf- not only participates in the late phase of acute lung injury , but also might be active early in acute lung injury and potentially could contribute to the development of pulmonary edema .
tgf- mrna level increased in murine intraparenchymal mononuclear cells and in alveolar macrophages within 1  h in a hemorrhage - induced acute lung injury model .
in addition to its classical role in fibrotic and extracellular matrix remodeling , tgf- signaling has a pivotal role in angiogenesis and vascular defects .
the in vitro effect of tgf- on the integrity of vascular endothelium started within 1 - 2  h and was maximal 8 - 9  h after exposure to tgf- .
tgf-1-induced lung endothelial cell barrier dysfunction is mediated by activation of rho and rho - kinase , which induce endothelial cell permeability .
this effect appears to result from endothelial cell contraction through the activation of a myosin light chain kinase - dependent signaling cascade .
our results showed an early increase ( 9  h ) in the protein level of tgf-1 in the lungs of animals with acute pancreatitis .
this can be a contributing factor to the lung injury due to tgf- effects on the vascular integrity .
we have previously shown a significant enrichment of inflammatory cells ( neutrophils and macrophages ) , recruited into the lung tissue of animals with acute pancreatitis at 9  h .
in addition to its effects on vascular permeability , tgf-1 has been shown to enhance monocyte migration .
tgf-1-driven monocyte motility was mediated via alk5-induced pi3  k and p38 signaling , independently of smad2/3 .
the early rise in both tgf-1 and alk5 observed herein may thus contribute to stimulate the monocyte enrichment in the lungs at 9  h. disruption of the vascular integrity is not the only way that tgf- signaling can play a role in the initiation and progression of acute lung injury .
tgf- activation plays a role in the development of acute lung injury through alteration of fibroproliferative responses , lung permeability , and inflammatory cell influx [ 18 , 2729 ] .
tgf- and tgf--inducible genes are able to modify lung permeability , epithelial ion transport , fibrinolysis , extracellular matrix , and surfactant homeostasis .
the integration of the changes in these molecular pathways by tgf- implicates it as a central mediator of acute lung injury .
mice treated with anti - tgf-antibodies showed decreased levels of pro - inflammatory cytokines compared to untreated one in a hemorrhage - induced acute lung injury model .
different human diseases have been associated with dysregulation of tgf- signaling , which shows the importance of a well - regulated signaling process .
smad7 is part of an autoinhibitory feedback control , which interacts with alk5 through various mechanisms .
smad7 resides in the nucleus in unstimulated cells and translocates to the plasma membrane following receptor activation , where it binds the receptors and inhibits further signaling .
smad7 has a central role in pathological processes by its antifibrotic and anti - inflammatory properties .
the anti - inflammatory activities of increased smad7 at early phase in our model can be interpreted as a protective mechanism in the lungs of the animals with acute pancreatitis . in this study , we showed increased levels of both tgf-1 and alk5 in the lungs as early as 9  h after the induction of acute pancreatitis .
however , increased tgf- signaling via the smad2 pathway was observed first after 24  h following acute pancreatitis induction . increased nuclear translocation of phosphorylated smad2 at 24  h can be due to the decreased smad7 levels in the lungs .
this time point was the peak of the inflammatory response associated with inflammatory cells recruitment to the lungs as well as pathological changes indicating the acute lung injury , in a previous report .
the later activation of the signaling pathway can be of interest in the acute lung injury , making the tgf- pathway a potential therapeutic target .
this is in accordance with previous report of attenuated lung injury by administration of a tgf- inhibitor 24  h after the injury .
in summary , the present study shows an early rise in tgf-1 and alk5 which may activate the tgf-/smad2 signaling or alternative pathways to contribute to ali .
acute lung injury is a critical syndrome due to a variety of precipitants , including acute pancreatitis .
improvements in outcome following ali over the past decade are in part due to improved strategies of mechanical ventilation and advanced support of other failing organs and not a specific treatment .
clinical and experimental studies indicate a role of tgf- in the early phases of ali , where tgf- contributes to the lung injury through epithelial and endothelial injuries .
our data showed an early rise in smad7 which can block the tgf-/smad2 signaling . with disease progression , the smad7 levels were reduced with a concomitant increase in nuclear translocation of phosphorylated smad2 , an indicator of active tgf-/smad2 signaling .
 acute lung injury is caused by many factors including acute pancreatitis . 
 there is no specific therapy directed at underlying pathophysiological mechanisms for acute lung injury . transforming growth factor- ( tgf- ) 
 is involved in the resolution of lung injury in later phases of the disease . 
 some evidence exists demonstrating that tgf- not only is involved in the late stages , but also contributes to lung injury early on in the progress of the disease . 
 acute pancreatitis was induced using ductal ligation in mice . 
 tgf-1 , 2 , and 3 , trii , alk-5 , smad2 , 3 , 4 , and 7 , and p - smad2 expression in the lungs were analyzed at 9 and 24  h. we demonstrate that tgf-1 levels in the lungs of mice with acute pancreatitis increase as early as 9  h after induction . 
 we observed an increased expression of alk-5 in acute pancreatitis at both 9 and 24  h. inhibitory smad7 expression was transiently increased at 9  h in acute pancreatitis , but reduced later at 24  h , with a concomitant increased nuclear translocation of phosphorylated smad2 . 
 our findings demonstrate activation of tgf- signaling in the lungs as early as 24  h after acute pancreatitis , suggesting that tgf- may represent a potential therapeutic candidate in acute pancreatitis - induced acute lung injury . 