Introduction|NNP The|DT concept|NN of|IN lung|NN fibroblasts|NNS as|IN effector|NN cells|NNS in|IN the|DT pathogenesis|NNS of|IN idiopathic|JJ pulmonary|JJ fibrosis|NNS (|( IPF|NNP )|) has|VBZ recently|RB evolved|VBN [|NN 1|CD 2|CD ]|NN .|. 
Lung|NNP fibroblasts|NNS respond|VBP ,|, in|IN vitro|NN ,|, to|TO inflammatory|JJ cytokines|NNS by|IN producing|VBG growth|NN factors|NNS and|CC collagen|NN ,|, resulting|VBG in|IN fibroblast|NN proliferation|NN and|CC extracellular|NN matrix|NN deposition|NN [|NN 2|CD 3|CD 4|CD ]|NN .|. 
In|IN addition|NN ,|, activated|VBN lung|NN fibroblasts|NNS have|VBP been|VBN shown|VBN to|TO produce|VB large|JJ amounts|NNS of|IN inflammatory|JJ cytokines|NNS and|CC chemokines|NNS ,|, in|IN vitro|NN ,|, and|CC hence|RB ,|, these|DT cells|NNS may|MD also|RB have|VB a|DT role|NN as|IN effector|NN -|- inflammatory|JJ cells|NNS [|NN 1|CD 2|CD ]|NN .|. 
This|DT capacity|NN to|TO produce|VB both|DT inflammatory|JJ and|CC fibrotic|JJ factors|NNS could|MD mean|VB that|IN phenotypically|RB altered|VBN lung|NN fibroblasts|NNS act|VBP simultaneously|RB as|IN effector|NN and|CC target|NN cells|NNS ,|, via|IN paracrine|NN and|CC autocrine|NN mechanisms|NNS ,|, perpetuating|VBG the|DT fibrotic|JJ process|NN [|NN 2|CD ]|NN .|. 
Prostanoids|NNP are|VBP important|JJ regulators|NNS of|IN fibroblast|NN function|NN [|NN 5|CD 6|CD 7|CD 8|CD 9|CD ]|NN .|. 
Prostaglandin|NNP (|( PG|NNP )|) E|NN 2|CD is|VBZ thought|VBN to|TO have|VB antifibrotic|JJ properties|NNS in|IN vitro|NN ,|, but|CC also|RB can|MD have|VB proinflammatory|NN effects|NNS both|DT in|IN vivo|NN and|CC in|IN vitro|NN [|NN 10|CD 11|CD 12|CD ]|NN .|. 
Thromboxane|NNP (|( TX|NNP )|) A|DT 2|CD increases|NNS proliferation|NN ,|, and|CC DNA|NNP and|CC RNA|NNP synthesis|NN in|IN several|JJ cell|NN types|NNS ,|, including|VBG fibroblasts|NNS and|CC smooth|JJ muscle|NN like|IN glomerular|NN mesangial|NN cells|NNS [|NN 13|CD 14|CD 15|CD 16|CD ]|NN .|. 
Conversely|RB ,|, prostacyclin|NN (|( PGI|NNP 2|CD )|) decreases|NNS smooth|JJ muscle|NN cell|NN proliferation|NN and|CC collagen|NN synthesis|NN [|NN 17|CD 18|CD ]|NN .|. 
Many|JJ cell|NN types|NNS ,|, including|VBG lung|NN fibroblasts|NNS ,|, contain|VB cyclooxygenase|NN (|( COX|NNP )|) ,|, a|DT proximal|NN enzyme|NN in|IN prostanoid|NN production|NN ,|, and|CC can|MD generate|VB prostanoids|NNS [|NN 19|CD ]|NN .|. 
It|PRP has|VBZ been|VBN previously|RB reported|VBN that|IN IPF|NNP lung|NN fibroblasts|NNS have|VBP decreased|VBN COX-2|NNP expression|NN compared|VBN to|TO normal|JJ lung|NN fibroblasts|NNS and|CC ,|, hence|RB ,|, have|VBP decreased|VBN PGE|NNP 2|CD production|NN [|NN 12|CD 20|CD 21|CD ]|NN .|. 
Because|IN of|IN these|DT findings|NNS and|CC the|DT fact|NN that|IN PGs|NNP are|VBP important|JJ fibroblast|NN regulators|NNS ,|, we|PRP sought|VBD to|TO investigate|VB whether|IN abnormalities|NNS in|IN COX-2|NNP expression|NN could|MD be|VB associated|VBN with|IN an|DT altered|VBN balance|NN between|IN profibrotic|JJ and|CC antifibrotic|JJ PGs|NNP .|. 
We|PRP hypothesized|VBN that|IN fibroblasts|NNS from|IN the|DT lungs|NNS of|IN patients|NNS with|IN IPF|NNP (|( HF-IPF|NNP )|) have|VBP an|DT altered|VBN PG|NNP balance|NN compared|VBN to|TO normal|JJ lung|NN fibroblasts|NNS (|( HF-NL|NNP )|) .|. 
This|DT phenotypical|JJ abnormality|NN could|MD be|VB an|DT important|JJ factor|NN in|IN the|DT pathogenesis|NNS of|IN IPF|NNP .|. 

Materials|NNS and|CC methods|NNS Primary|JJ lung|NN fibroblasts|NNS 
Fibroblasts|NNP from|IN the|DT lungs|NNS of|IN seven|CD patients|NNS (|( 6|CD males|NNS )|) with|IN IPF|NNP (|( HF-IPF|NNP )|) were|VBD harvested|VBN :|: a|DT )|) from|IN excised|VBD lung|NN at|IN the|DT time|NN of|IN lung|NN transplantation|NN ;|: b|SYM )|) during|IN an|DT autopsy|NN performed|VBN within|IN 4|CD hours|NNS from|IN death|NN ;|: or|CC c|SYM )|) during|IN open|NN or|CC transbronchial|NN lung|NN biopsies|NNS at|IN the|DT time|NN of|IN diagnosis|NN .|. 
Of|IN the|DT seven|CD patients|NNS ,|, five|CD subjects|NNS had|VBD advanced|VBN lung|NN fibrosis|NNS and|CC were|VBD receiving|VBG prednisone|NN ±|NN immunosuppressive|JJ agents|NNS ;|: 2|CD patients|NNS were|VBD at|IN an|DT earlier|JJR stage|NN of|IN their|PRP$ disease|NN and|CC were|VBD not|RB receiving|VBG immunosuppressive|JJ drugs|NNS .|. 
The|DT mean|JJ age|NN of|IN the|DT patients|NNS was|VBD 59|CD (|( range|NN 43|CD -|- 71|CD )|) ]|NN .|. 
HF-NL|NNP were|VBD cultured|JJ from|IN five|CD human|JJ lungs|NNS that|WDT arrived|VBD at|IN our|PRP$ transplant|NN center|NN with|IN the|DT intention|NN of|IN being|VBG used|VBN for|IN transplantation|NN ,|, but|CC for|IN various|JJ reasons|NNS could|MD not|RB be|VB transplanted|VBN ;|: these|DT were|VBD macroscopically|RB and|CC microscopically|RB normal|JJ .|. 
The|DT cells|NNS were|VBD harvested|VBN and|CC cultured|JJ as|IN per|IN the|DT protocol|NN described|VBN by|IN Kumar|NNP et|CC al.|NN [|NN 22|CD ]|NN .|. 
Briefly|NNP ,|, lung|NN tissue|NN sections|NNS were|VBD finely|RB cut|VB with|IN sterile|JJ scissors|NNS and|CC incubated|JJ with|IN serum|NN free|JJ DMEM|NNP containing|VBG trypsin|NN ,|, DNAse|NNP and|CC collagenase|NN for|IN 30|CD min.|NN 
The|DT procedure|NN was|VBD repeated|VBN twice|RB ,|, and|CC the|DT supernatants|NNS were|VBD pooled|VBN and|CC cultured|JJ in|IN one|CD 100|CD mm|NN plate|NN and|CC incubated|JJ at|IN 37|CD °|NN C|NNP in|IN a|DT 5|CD %|NN CO|NNP 2|CD humidified|JJ atmosphere|NN .|. 
Culture|NNP medium|NN (|( DMEM|NNP with|IN 5|CD %|NN fetal|JJ bovine|JJ serum|NN [|NN FBS|NNP ]|NN and|CC penicillin|NN /|NN streptomycin|NN )|) was|VBD replaced|VBN three|CD times|NNS per|IN week|NN and|CC fibroblasts|NNS were|VBD passed|VBN (|( 1|CD :|: 2|CD split|NN )|) at|IN the|DT time|NN they|PRP became|VBD confluent|NN .|. 
On|IN passage|NN 4|CD the|DT cells|NNS were|VBD resuspended|JJ in|IN 1|CD ml|NN of|IN DMEM|NNP with|IN 20|CD %|NN FBS|NNP and|CC DMSO|NNP and|CC frozen|VBN at|IN -|: 70|CD °|NN C.|NN 
For|IN each|DT experiment|NN described|VBD below|IN the|DT cells|NNS were|VBD thawed|JJ ,|, cultured|JJ and|CC passed|VBN at|IN least|JJS once|RB .|. 
All|DT the|DT experiments|NNS were|VBD conducted|VBN with|IN cells|NNS at|IN passages|NNS 6|CD to|TO 8|CD .|. 

Inducible|NNP cyclooxygenase|NN (COX)-2|NNP expression|NN and|CC eicosanoid|NN production|NN COX-2|NNP activity|NN was|VBD determined|VBN by|IN measuring|VBG PGE|NNP 2|CD ,|, 6-keto-PGF|NN 1|CD α|NN (|( stable|JJ PGI|NNP 2|CD metabolite|NN )|) ,|, TXB|NNP 2|CD (|( stable|JJ TXA|NNP 2|CD metabolite|NN )|) ,|, and|CC PGF|NNP 2|CD α|NN production|NN in|IN stimulated|VBN fibroblasts|NNS .|. 
HF-IPF|NNP (|( n|NN =|SYM 7|CD )|) and|CC HF-NL|NNP (|( n|NN =|SYM 5|CD )|) were|VBD brought|VBN to|TO >|NN 90|CD %|NN confluency|NN in|IN 100|CD mm|NN plates|NNS and|CC then|RB placed|VBN on|IN serum|NN free|JJ DMEM|NNP for|IN 24|CD hours|NNS to|TO render|VB them|PRP quiescent|JJ .|. 
Fibroblasts|NNP were|VBD then|RB incubated|JJ in|IN DMEM|NNP with|IN 5|CD %|NN FBS|NNP alone|RB or|CC in|IN the|DT same|JJ medium|NN with|IN IL-1β|NNP (|( 2.5|CD ng|NN /|NN ml|NN )|) for|IN 24|CD hours|NNS .|. 
At|IN the|DT end|NN of|IN the|DT incubation|NN period|NN the|DT supernatant|NN was|VBD aspirated|JJ and|CC fresh|JJ media|NNS containing|VBG 30|CD μM|NN of|IN arachidonic|JJ acid|NN was|VBD added|VBN to|TO the|DT plates|NNS .|. 
After|IN 30|CD min|NN of|IN incubation|NN the|DT supernatant|NN was|VBD collected|VBN and|CC saved|VBN at|IN -|: 70|CD °|NN C|NNP for|IN later|RB eicosanoid|NN analysis|NN .|. 
The|DT cells|NNS were|VBD then|RB resuspended|JJ and|CC divided|VBN into|IN two|CD aliquots|NNS ,|, which|WDT were|VBD used|VBN for|IN RNA|NNP and|CC protein|NN extractions|NNS ,|, respectively|RB .|. 
The|DT above|IN experiments|NNS were|VBD repeated|VBN in|IN HF-IPF|NNP (|( n|NN =|SYM 2|CD )|) and|CC HF-NL|NNP (|( n|NN =|SYM 2|CD )|) using|VBG serum|NN free|JJ media|NNS conditions|NNS .|. 
Prostanoids|NNP were|VBD measured|VBN by|IN modified|VBN stable|JJ isotope|NN dilution|NN assays|NNS that|WDT used|VBD gas|NN chromatography|NN -|- negative|JJ ion|NN -|- chemical|JJ ionization|NN mass|NN spectrometry|NN as|RB previously|RB described|VBD [|NN 23|CD ]|NN .|. 
Briefly|NNP ,|, deuterium|NN -|- labeled|JJ internal|JJ standards|NNS of|IN PGE|NNP 2|CD ,|, PGF|NNP 2|CD α|NN ,|, TXB|NNP 2|CD ,|, and|CC 6-keto-PGF|NN 1|CD α|NN were|VBD added|VBN to|TO the|DT supernatants|NNS with|IN isopropyl|NN alcohol|NN .|. 
Isopropyl|NNP alcohol|NN was|VBD removed|VBN by|IN evaporation|NN under|IN nitrogen|NN .|. 
After|IN acidification|NN to|TO pH|NN 3.5|CD ,|, the|DT samples|NNS were|VBD extracted|VBN on|IN preconditioned|JJ C-18|NNP PrepSep|NNP columns|NNS (|( Fisher|NNP Scientific|NNP ,|, Fair|NNP Lawn|NNP ,|, NJ|NNP )|) ,|, and|CC eluted|JJ with|IN ethyl|NN acetate|NN .|. 
The|DT extract|NN was|VBD then|RB converted|VBN to|TO a|DT pentafluorobenzyl|NN ester|NN by|IN treatment|NN with|IN a|DT mixture|NN of|IN 12.5|CD %|NN pentafluorobenzyl|NN bromide|NN in|IN acetonitrile|NN and|CC disopropylethylamine|NN at|IN room|NN temperature|NN for|IN 30|CD min.|NN 
After|IN evaporation|NN of|IN reagents|NNS ,|, the|DT residue|NN was|VBD subjected|VBN to|TO TLC|NNP plates|NNS ,|, using|VBG the|DT solvent|JJ system|NN chloroform|NN /|NN ethanol|NN (|( 93|CD :|: 7|CD ,|, vol|NN /|NN vol|NN )|) for|IN PGF|NNP 2|CD α|NN and|CC TXB|NNP 2|CD ,|, and|CC ethyl|NN acetate|NN /|NN methanol|NN (|( 93|CD :|: 2|CD ,|, vol|NN /|NN vol|NN )|) for|IN 6-keto-PGF|NN 1|CD α|NN and|CC PGE|NNP 2|CD .|. 
Then|RB PGF|NNP 2|CD α|NN was|VBD converted|VBN to|TO trimethylsilyl|NN ether|NN derivative|JJ by|IN treatment|NN with|IN N,O-bis|NNP (|( trimethylsilyl|NN )|) trifluoroacetamide|NN and|CC dimethylformamide|NN .|. 
The|DT methoxime|NN derivative|NN of|IN TXB|NNP 2|CD ,|, PGE|NNP 2|CD and|CC 6-keto-PGF|NN 1|CD α|NN was|VBD made|VBN by|IN treatment|NN with|IN 2|CD %|NN methoxamine|NN hydrochloride|NN in|IN pyridine|NN at|IN 70|CD °|NN C|NNP for|IN 60|CD min|NN ,|, followed|VBN by|IN evaporation|NN of|IN the|DT pyridine|NN ,|, addition|NN of|IN water|NN ,|, and|CC extraction|NN with|IN ethyl|NN acetate|NN .|. 
Derivatization|NNP was|VBD completed|VBN by|IN formation|NN of|IN the|DT trimethylsilyl|NN derivatives|NNS by|IN treatment|NN with|IN N,O-bis|NNP (|( trimethylsilyl|NN )|) trifluoroacetamide|NN and|CC pyridine|NN .|. 
Eicosanoids|NNP were|VBD quantified|VBN by|IN measuring|VBG the|DT ratio|NN of|IN the|DT intensity|NN of|IN ions|NNS m|NN /|NN z|SYM 569/573|CD for|IN PGF|NNP 2|CD α|NN ,|, m|NN /|NN z|SYM 614/618|CD for|IN TXB|NNP 2|CD and|CC 6-keto-PGF|NN 1|CD α|NN ,|, and|CC m|NN /|NN z|SYM 524/528|CD for|IN PGE|NNP 2|CD .|. 
An|DT analytical|JJ blank|NN for|IN each|DT of|IN these|DT products|NNS was|VBD determined|VBN by|IN measuring|VBG the|DT amount|NN of|IN nondeuterated|JJ material|NN ,|, detected|VBD after|IN extracting|VBG and|CC analyzing|VBG 2|CD ml|NN of|IN saline|NN to|TO which|WDT the|DT deuterium-labeled|JJ internal|JJ standards|NNS had|VBD been|VBN added|VBN .|. 

Western|JJ analysis|NN After|IN washing|VBG with|IN PBS|NNP at|IN pH|NN 7.4|CD ,|, pellets|NNS were|VBD lyzed|JJ in|IN solubilization|NN buffer|NN containing|VBG 50|CD mM|NN TRIS|NNP at|IN pH|NN 8|CD ,|, 1|CD %|NN Tween|NNP 20|CD ,|, 10|CD mM|NN phenylmethylsulphonyl|NN fluoride|NN ,|, diethyldithiocarbamic|JJ acid|NN ,|, leupeptin|NN and|CC pepstatin|NN A|DT (|( all|DT from|IN Sigma|NNP Chemical|NNP )|) ,|, sonicated|JJ ,|, boiled|VBD with|IN gel|NN loading|NN buffer|NN (|( 62.5|CD mM|NN TRIS-HCl|NNP ,|, at|IN pH|NN 6.8|CD ,|, 10|CD %|NN glycerol|NN ,|, 2|CD %|NN SDS|NNP ,|, 5|CD %|NN β-mercaptoethanol|JJ ,|, and|CC bromophenol|NN blue|JJ )|) ,|, and|CC centrifuged|JJ at|IN 15,000|CD x|SYM g|SYM for|IN 10|CD min.|NN 
Equal|NNP amounts|NNS of|IN protein|NN (|( 70|CD to|TO 100|CD μg|NN )|) were|VBD separated|VBN by|IN electrophoresis|NNS .|. 
SDS-PAGE|NNP was|VBD performed|VBN using|VBG a|DT 7.5|CD %|NN separating|VBG gel|NN with|IN a|DT 4|CD %|NN stacking|VBG gel|NN .|. 
The|DT resolved|VBN proteins|NNS were|VBD transferred|VBN electrophoretically|RB to|TO nitrocellulose|NN membranes|NNS (|( Hybond-|NNP ECL|NNP ,|, Amersham|NNP Corp|NNP .|. )|) .|. 
After|IN transfer|NN ,|, the|DT filters|NNS were|VBD incubated|JJ overnight|JJ at|IN 4|CD °|NN C|NNP in|IN a|DT blocking|VBG solution|NN (|( 20|CD mM|NN TRIS|NNP base|NN ,|, 137|CD mM|NN sodium|NN chloride|NN at|IN pH|NN 7.6|CD ,|, 5|CD %|NN powdered|JJ milk|NN ,|, 3|CD %|NN BSA|NNP )|) ,|, and|CC incubated|JJ with|IN primary|JJ polyclonal|NN rabbit|NN antibodies|NNS against|IN COX-2|NNP at|IN a|DT dilution|NN 1|CD :|: 1000|CD (|( Cayman|NNP Chemical|NNP ,|, Ann|NNP Arbor|NNP ,|, MI|NNP )|) ,|, for|IN 1|CD hour|NN at|IN room|NN temperature|NN .|. 
The|DT filters|NNS were|VBD washed|VBN (|( TBS|NNP -|- 0.1|CD %|NN Tween|NNP 20|CD at|IN pH|NN 7.6|CD )|) and|CC incubated|JJ with|IN horseradish|NN peroxidase|NN linked|VBD secondary|JJ antibodies|NNS at|IN a|DT dilution|NN 1|CD :|: 4000|CD (|( Amersham|NNP )|) .|. 
After|IN washing|VBG ,|, the|DT membranes|NNS were|VBD incubated|JJ with|IN luminol|NN based|VBD chemiluminescence|NN reagent|NN (|( DuPont|NNP NEN|NNP Research|NNP Products|NNPS ,|, Boston|NNP ,|, MA|NNP )|) .|. 

Northern|JJ analysis|NN Cell|NNP pellets|NNS were|VBD lyzed|JJ and|CC RNA|NNP extracted|VBD using|VBG the|DT RNeasy|NNP method|NN ®|NN (|( Qiagen|NNP )|) ,|, following|VBG the|DT manufacturer|NN 's|POS instructions|NNS .|. 
RNA|NNP was|VBD quantified|VBN by|IN determining|VBG light|JJ absorbance|NN at|IN 260|CD nm|NN and|CC then|RB fractioned|JJ by|IN electrophoresis|NNS (|( 10|CD μg|NN per|IN lane|NN )|) on|IN a|DT 1|CD %|NN agarose|NN MOPS|NNP /|NN formaldehyde|NN gel|NN .|. 
The|DT RNA|NNP was|VBD denatured|JJ prior|RB to|TO loading|NN by|IN incubating|VBG the|DT RNA|NNP at|IN 65|CD °|NN C|NNP for|IN 10|CD min|NN in|IN a|DT loading|NN buffer|NN comprising|VBG 1|CD X|NNP MOPS|NNP ,|, 50|CD %|NN formamide|NN ,|, 6.5|CD %|NN formaldehyde|NN ,|, 5|CD %|NN glycerol|NN ,|, 0.1|CD mM|NN EDTA|NNP ,|, 0.025|CD %|NN bromophenol|NN blue|JJ ,|, 0.025|CD %|NN xylene|NN cyanol|NN .|. 
The|DT RNA|NNP was|VBD transferred|VBN by|IN gravity|NN -|- assisted|VBN capillary|JJ method|NN with|IN 6|CD X|NNP SSC|NNP to|TO nylon|NN hybridization|NN membrane|NN ,|, and|CC then|RB fixed|VBN to|TO the|DT membrane|NN by|IN UV|NNP crosslinking|VBG (|( Stratalinker|NNP 1200|CD μj|NN /|NN cm|NN 2|CD )|) .|. 
Prehybridization|NNP and|CC hybridization|NN were|VBD performed|VBN at|IN 42|CD °|NN C|NNP and|CC using|VBG Quick|NNP Hyb|NNP ®|NN (|( Stratagene|NNP )|) as|IN hybridization|NN solution|NN .|. 
The|DT COX-2|NNP probe|NN was|VBD random|JJ primed|VBN following|VBG the|DT directions|NNS of|IN the|DT manufacturer|NN (|( Megaprime|NNP ®|NN ,|, Amersham|NNP /|NN Pharmacia|NNP )|) .|. 
The|DT membrane|NN was|VBD then|RB washed|VBN at|IN a|DT final|JJ stringency|NN of|IN 0.2|CD X|NNP SSC|NNP ,|, 0.1|CD %|NN SDS|NNP ,|, at|IN 60|CD °|NN C|NNP for|IN 30|CD min.|NN 
The|DT membrane|NN was|VBD wrapped|VBN in|IN plastic|NN wrap|NN and|CC exposed|VBN to|TO Kodak|NNP XR|NNP film|NN at|IN -|: 70|CD °|NN C|NNP with|IN intensifier|NN screen|NN overnight|JJ .|. 

Statistical|NNP methods|NNS All|DT results|NNS are|VBP presented|VBN as|IN medians|NNS with|IN their|PRP$ range|NN .|. 
Comparisons|NNP between|IN HF-IPF|NNP and|CC HF-NL|NNP were|VBD done|VBN using|VBG the|DT Mann|NNP -|- Whitney|NNP test|NN .|. 
A|DT P|NN value|NN of|IN <|NN 0.05|CD 
was|VBD considered|VBN significant|JJ .|. 

Results|NNS Baseline|NNP and|CC stimulated|VBN COX-2|NNP activity|NN in|IN HF-IPF|NNP and|CC HF-NL|NNP Unstimulated|NNP eicosanoid|NN production|NN was|VBD similar|JJ in|IN both|DT HF-IPF|NNP and|CC HF-NL|NNP (|( Fig.|NN 1|CD ,|, a-d|JJ )|) .|. 
When|WRB fibroblasts|NNS were|VBD stimulated|VBN with|IN IL-1β|NNP there|EX was|VBD a|DT significant|JJ and|CC similar|JJ upregulation|NN of|IN PGE|NNP 2|CD production|NN in|IN both|DT HF-IPF|NNP and|CC HF-NL|NNP (|( 28.35|CD [|NN range|NN :|: 9.09|CD -|: 89.09|CD ]|NN versus|CC 17.12|CD [|NN 8.58|CD -|: 29.33|CD ]|NN ng|NN /|NN 10|CD 6|CD cells|NNS /|NN 30|CD min|NN ,|, respectively|RB ;|: P|NN =|SYM 0.25|CD ;|: [|NN Fig.|NN 1|CD a|DT ]|NN )|) .|. 
IL-1β-stimulated|NNP production|NN of|IN TXB|NNP 2|CD (|( stable|JJ metabolite|NN of|IN the|DT active|JJ TXA|NNP 
2|CD )|) ,|, PGF|NNP 2|CD α|NN ,|, and|CC 6-keto-PGF|NN 1|CD α|NN (|( stable|JJ metabolite|NN of|IN PGI|NNP 2|CD )|) increased|VBN modestly|RB in|IN every|DT case|NN ,|, except|IN TXB|NNP 2|CD production|NN by|IN HF-NL|NNP ,|, which|WDT decreased|VBD (|( 0.75|CD [|NN 0.15|CD -|: 2.58|CD ]|NN ng|NN /|NN 10|CD 6|CD cells|NNS /|NN 30|CD min|NN at|IN baseline|NN versus|CC 0.61|CD [|NN 0.21|CD -|: 1.64|CD ]|NN ng|NN /|NN 10|CD 6|CD cells|NNS /|NN 30|CD min|NN with|IN IL-1β|NNP stimulation|NN )|) (|( Fig.|NN 1|CD b|SYM )|) .|. 
Results|NNS of|IN PGE|NNP 2|CD production|NN were|VBD similar|JJ when|WRB experiments|NNS were|VBD conducted|VBN in|IN serum|NN free|JJ media|NNS conditions|NNS (|( results|NNS not|RB shown|VBN )|) .|. 
IL-1β|NNP stimulated|VBN TXB|NNP 2|CD production|NN was|VBD significantly|RB greater|JJR in|IN HF-IPF|NNP (|( 1.92|CD [|NN 1.27|CD -|: 2.57|CD ]|NN ng|NN /|NN 10|CD 6|CD cells|NNS /|NN 30|CD min|NN )|) than|IN in|IN HF-NL|NNP (|( 0.61|CD [|NN 0.21|CD -|: 1.64|CD ]|NN ng|NN /|NN 10|CD 6|CD cells|NNS /|NN 30|CD min|NN ;|: P|NN =|SYM 0.007|CD )|) (|( Fig.|NN 1|CD b|SYM )|) ;|: baseline|NN TXB|NNP 2|CD production|NN was|VBD not|RB significantly|RB different|JJ between|IN the|DT two|CD cell|NN groups|NNS (|( 1.73|CD [|NN 0.77|CD -|: 2.53|CD ]|NN versus|CC 0.75|CD [|NN 0.15|CD -|: 2.58|CD ]|NN ng|NN /|NN 10|CD 6|CD cells|NNS /|NN 30|CD min|NN ,|, in|IN HF-IPF|NNP and|CC HF-NL|NNP ,|, respectively|RB ;|: P|NN =|SYM 0.17|CD [|NN Fig.|NN 1|CD b|SYM ]|NN )|) .|. 
Because|IN PGI|NNP 2|CD and|CC TXA|NNP 2|CD have|VBP opposing|VBG effects|NNS in|IN vivo|NN ,|, we|PRP calculated|VBD the|DT ratio|NN of|IN their|PRP$ metabolites|NNS (|( 6-keto-PGF|NN 1|CD α|NN :|: TXB|NNP 2|CD )|) and|CC found|VBD a|DT significantly|RB lower|JJR ratio|NN in|IN HF-IPF|NNP at|IN baseline|NN (|( 0.08|CD [|NN 0.04|CD -|: 0.52|CD ]|NN versus|CC 0.12|CD [|NN 0.11|CD -|: 0.89|CD ]|NN in|IN HF-IPF|NNP and|CC HF-NL|NNP ,|, respectively|RB ;|: P|NN =|SYM 0.028|CD )|) and|CC a|DT similar|JJ trend|NN under|IN stimulated|VBN conditions|NNS (|( 0.24|CD [|NN 0.05|CD -|: 1.53|CD ]|NN versus|CC 1.08|CD [|NN 0.51|CD -|: 3.79|CD ]|NN in|IN HF-IPF|NNP and|CC HF-NL|NNP ,|, respectively|RB ;|: P|NN =|SYM 0.09|CD [|NN Fig.|NN 2|CD ]|NN )|) .|. 

Baseline|NNP and|CC stimulated|VBN COX-2|NNP expression|NN Western|JJ blot|NN in|IN unstimulated|JJ fibroblasts|NNS showed|VBD no|DT detectable|JJ COX-2|NNP protein|NN in|IN either|DT group|NN of|IN cells|NNS ,|, while|IN IL-1β|NNP significantly|RB induced|VBD COX-2|NNP to|TO a|DT similar|JJ degree|NN in|IN IPF|NNP and|CC normal|JJ lung|NN fibroblasts|NNS (|( Fig.|NN 3|CD )|) .|. 
Northern|NNP blot|NN showed|VBD minimal|JJ COX-2|NNP mRNA|NN in|IN unstimulated|JJ cells|NNS and|CC significant|JJ upregulation|NN of|IN COX-2|NNP mRNA|NN expression|NN when|WRB stimulated|VBN with|IN IL-1β|NNP in|IN both|DT HF-IPF|NNP and|CC HF-NL|NNP (|( Fig.|NN 
4|CD )|) .|. 

Discussion|NNP Several|JJ factors|NNS modulate|VBP fibroblast|NN proliferation|NN and|CC collagen|NN production|NN ,|, including|VBG mitogenic|JJ cytokines|NNS (|( e.g.|NN ,|, transforming|VBG growth|NN factor|NN β|NN [|NN TGFβ|NNP ]|NN ,|, platelet|NN -|- derived|VBN growth|NN factor|NN [|NN PDGF|NNP ]|NN ,|, basic|JJ fibroblast|NN growth|NN factor|NN [|NN bFGF|NN ]|NN )|) ,|, eicosanoids|NNS (|( i.e.|NN ,|, PGE|NNP 2|CD ,|, TXB|NNP 2|CD ,|, and|CC PGI|NNP 2|CD )|) ,|, and|CC antifibrogenic|JJ cytokines|NNS (|( e.g.|NN IFN-γ|NNP )|) [|NN 1|CD 2|CD 3|CD ]|NN .|. 
It|PRP is|VBZ very|RB likely|JJ that|IN a|DT complex|JJ interaction|NN among|IN these|DT factors|NNS exists|VBZ in|IN the|DT tissue|NN repair|NN process|NN ,|, and|CC it|PRP is|VBZ possible|JJ that|IN pathologic|JJ fibrosis|NNS ,|, as|IN in|IN IPF|NNP ,|, results|VBZ from|IN phenotypical|JJ alterations|NNS in|IN fibroblasts|NNS that|WDT affect|VBP the|DT "|'' normal|JJ "|'' interaction|NN of|IN these|DT factors|NNS .|. 
Our|PRP$ results|NNS show|VBP that|DT stimulation|NN of|IN primary|JJ cultures|NNS of|IN human|JJ lung|NN fibroblasts|NNS with|IN the|DT proximal|NN cytokine|NN IL-1β|NNP upregulates|NNS COX-2|NNP protein|NN and|CC mRNA|NN expression|NN to|TO a|DT similar|JJ degree|NN in|IN normal|JJ and|CC IPF|NNP fibroblasts|NNS .|. 
TXA|NNP 2|CD production|NN tended|VBD to|TO be|VB greater|JJR in|IN IPF|NNP than|IN in|IN normal|JJ fibroblasts|NNS at|IN baseline|NN ;|: when|WRB stimulated|VBN with|IN IL-1β|NNP this|DT difference|NN became|VBD statistically|RB significant|JJ .|. 
The|DT ratio|NN of|IN PGI|NNP 2|CD to|TO TXA|NNP 2|CD metabolites|NNS was|VBD lower|JJR in|IN IPF|NNP fibroblasts|NNS at|IN baseline|NN and|CC with|IN IL-1β|NNP stimulation|NN .|. 
The|DT above|IN results|NNS suggest|VBP that|IN a|DT decreased|VBN PGI|NNP 2|CD :|: TXA|NNP 2|CD ratio|NN could|MD be|VB a|DT phenotypic|JJ alteration|NN present|JJ in|IN IPF|NNP fibroblasts|NNS ,|, resulting|VBG in|IN a|DT loss|NN of|IN their|PRP$ capacity|NN to|TO autoregulate|NN proliferation|NN and|CC extracellular|NN matrix|NN production|NN .|. 
The|DT effects|NNS of|IN PGs|NNP on|IN cell|NN proliferation|NN and|CC collagen|NN production|NN have|VBP been|VBN widely|RB studied|VBN in|IN different|JJ cell|NN types|NNS [|NN 13|CD 14|CD 15|CD 16|CD 17|CD 26|CD ]|NN .|. 
TXA|NNP 2|CD has|VBZ been|VBN studied|VBN extensively|RB because|IN of|IN its|PRP$ apparent|JJ role|NN in|IN atherosclerosis|NNS ,|, due|JJ to|TO its|PRP$ prothrombotic|JJ and|CC mitogenic|JJ activities|NNS on|IN vascular|NN smooth|JJ muscle|NN cells|NNS [|NN 15|CD 16|CD ]|NN .|. 
These|DT mitogenic|JJ effects|NNS are|VBP potentiated|JJ by|IN growth|NN factors|NNS [|NN 15|CD 16|CD 27|CD 28|CD ]|NN .|. 
In|IN vascular|NN smooth|JJ muscle|NN cells|NNS TXA|NNP 2|CD stimulates|NNS synthesis|NN of|IN bFGF|NN and|CC increases|VBZ the|DT expression|NN of|IN the|DT proto|JJ -|- oncogenes|NNS c-fos|JJ ,|, c-myc|JJ ,|, and|CC egr-1|NN ,|, which|WDT are|VBP associated|VBN with|IN entry|NN into|IN the|DT cell|NN growth|NN cycle|NN [|NN 15|CD ]|NN .|. 
In|IN addition|NN ,|, TXA|NNP 2|CD increases|NNS proliferation|NN of|IN fibroblasts|NNS [|NN 13|CD ]|NN and|CC smooth|JJ muscle|NN -|- like|IN glomerular|NN mesangial|NN cells|NNS [|NN 14|CD ]|NN .|. 
On|IN the|DT other|JJ hand|NN ,|, PGI|NNP 2|CD decreases|NNS vascular|NN smooth|JJ muscle|NN cell|NN proliferation|NN and|CC collagen|NN and|CC glycosaminoglycane|NN synthesis|NN ,|, via|IN activation|NN of|IN adenylyl|NN cyclase|NN and|CC subsequent|JJ production|NN of|IN cAMP|NN [|NN 17|CD ]|NN .|. 
Betaprost|NNP ,|, an|DT analog|NN of|IN PGI|NNP 2|CD ,|, decreases|NNS procollagen|NN I|PRP and|CC III|NNP mRNA|NN expression|NN in|IN cardiac|JJ fibroblasts|NNS [|NN 18|CD ]|NN .|. 
These|DT effects|NNS may|MD counteract|VB the|DT profibrotic|JJ effects|NNS seen|VBN with|IN TXA|NNP 2|CD and|CC it|PRP is|VBZ possible|JJ that|IN an|DT alteration|NN of|IN a|DT "|'' normal|JJ "|'' physiologic|JJ balance|NN between|IN PGI|NNP 2|CD and|CC TXA|NNP 2|CD could|MD increase|VB tendency|NN towards|IN fibrogenesis|NNS .|. 
It|PRP is|VBZ important|JJ to|TO mention|VB that|IN our|PRP$ experiments|NNS were|VBD conducted|VBN at|IN similar|JJ passage|NN levels|NNS (|( passage|NN 6|CD to|TO 8|CD )|) in|IN both|DT groups|NNS ,|, since|IN senescence|NN of|IN fibroblasts|NNS is|VBZ associated|VBN with|IN a|DT shift|NN from|IN the|DT biosynthesis|NNS of|IN PGI|NNP 2|CD to|TO TXA|NNP 2|CD [|NN 24|CD 25|CD ]|NN .|. 
It|PRP is|VBZ possible|JJ that|IN the|DT difference|NN seen|VBN in|IN our|PRP$ study|NN between|IN HF-IPF|NNP and|CC HF-NL|NNP could|MD result|VB from|IN comparing|VBG fibroblasts|NNS of|IN different|JJ ages|NNS .|. 
HF-IPF|NNP might|MD have|VB been|VBN harvested|VBN from|IN fibrotic|JJ lesions|NNS where|WRB fibroblasts|NNS had|VBD previously|RB undergone|VBN a|DT greater|JJR number|NN of|IN cell|NN divisions|NNS than|IN HF-NL|NNP ,|, obtained|VBN from|IN nonfibrotic|JJ lungs|NNS .|. 
Although|IN this|DT is|VBZ a|DT possibility|NN ,|, the|DT age|NN -|- related|VBN shift|NN in|IN PG|NNP production|NN has|VBZ only|RB been|VBN shown|VBN at|IN very|RB high|JJ cell|NN passages|NNS and|CC has|VBZ not|RB been|VBN documented|VBN in|IN vivo|NN .|. 
We|PRP also|RB found|VBD that|IN both|DT HF-IPF|NNP and|CC HF-NL|NNP had|VBD similar|JJ PGE|NNP 
2|CD production|NN at|IN baseline|NN ,|, and|CC a|DT similar|JJ increase|NN when|WRB stimulated|VBN with|IN IL-1β|NNP .|. 
PGE|NNP 2|CD can|MD decrease|VB fibroblast|NN proliferation|NN and|CC collagen|NN synthesis|NN ,|, and|CC increase|NN collagen|NN degradation|NN [|NN 5|CD 6|CD 7|CD 8|CD ]|NN .|. 
Recent|JJ reports|NNS suggesting|VBG decreased|VBN COX-2|NNP expression|NN and|CC PGE|NNP 2|CD production|NN in|IN IPF|NNP fibroblasts|NNS have|VBP received|VBN significant|JJ attention|NN [|NN 12|CD 20|CD 21|CD ]|NN .|. 
In|IN our|PRP$ study|NN we|PRP found|VBD that|IN both|DT COX-2|NNP protein|NN expression|NN and|CC PGE|NNP 2|CD production|NN were|VBD upregulated|JJ to|TO a|DT similar|JJ degree|NN in|IN IPF|NNP and|CC normal|JJ lung|NN fibroblasts|NNS .|. 
We|PRP believe|VBP that|IN differences|NNS in|IN methodology|NN and|CC patient|NN selection|NN may|MD explain|VB the|DT discrepancies|NNS with|IN other|JJ studies|NNS .|. 
Vancheri|NNP and|CC collaborators|NNS [|NN 20|CD ]|NN found|VBD that|IN TNF-α-stimulated|NNP fibrotic|JJ lung|NN fibroblasts|NNS had|VBD decreased|VBN COX-2|NNP expression|NN and|CC PGE|NNP 2|CD production|NN ,|, but|CC they|PRP further|RBR showed|VBD that|IN these|DT findings|NNS were|VBD a|DT result|NN of|IN decreased|VBD expression|NN of|IN TNF-α|NNP receptors|NNS .|. 
The|DT latter|JJ finding|NN would|MD argue|VB against|IN a|DT primary|JJ defect|NN in|IN COX-2|NNP expression|NN ,|, since|IN no|DT other|JJ stimulus|NN ,|, other|JJ than|IN TNF-α|NNP ,|, was|VBD tested|VBN .|. 
In|IN another|DT study|NN ,|, Keerthisingam|NNP et|CC al.|NN [|NN 21|CD ]|NN reported|VBD that|IN fibrotic|JJ lung|NN fibroblasts|NNS had|VBD decreased|VBN COX-2|NNP expression|NN and|CC PGE|NNP 2|CD production|NN in|IN response|NN to|TO TGFβ|NNP stimulation|NN .|. 
This|DT study|NN differed|VBD from|IN ours|PRP in|IN that|IN a|DT different|JJ stimulus|NN was|VBD used|VBN .|. 
Of|IN significance|NN is|VBZ the|DT fact|NN that|IN the|DT COX-2|NNP gene|NN is|VBZ known|VBN to|TO be|VB NF-κB|NNP dependent|JJ ,|, and|CC IL-1β|NNP ,|, but|CC not|RB TGFβ|NNP ,|, is|VBZ a|DT potent|JJ inducer|NN of|IN NF-κB|NNP activation|NN .|. 
Hence|RB ,|, the|DT pathway|NN involved|VBN in|IN the|DT induction|NN of|IN the|DT COX-2|NNP gene|NN by|IN IL-1β|NNP and|CC TGFβ|NNP may|MD be|VB different|JJ .|. 
Furthermore|RB ,|, a|DT significant|JJ proportion|NN of|IN the|DT fibroblasts|NNS used|VBN in|IN the|DT study|NN by|IN Keerthisingam|NNP et|CC al.|NN [|NN 21|CD ]|NN were|VBD obtained|VBN from|IN patients|NNS with|IN systemic|JJ sclerosis|NN ,|, which|WDT makes|VBZ their|PRP$ fibroblast|NN population|NN more|RBR heterogeneous|JJ .|. 
Wilborn|NNP et|CC al.|NN [|NN 12|CD ]|NN also|RB reported|VBD a|DT decreased|VBN production|NN of|IN PGE|NNP 2|CD by|IN IL-1β-stimulated|NNP IPF|NNP fibroblasts|NNS ,|, due|JJ to|TO decreased|VBD COX-2|NNP expression|NN [|NN 12|CD ]|NN .|. 
There|EX is|VBZ a|DT possibility|NN that|DT patient|NN selection|NN may|MD have|VB differed|VBD between|IN the|DT two|CD studies|NNS .|. 
However|RB ,|, we|PRP feel|VBP certain|JJ that|IN the|DT diagnostic|JJ accuracy|NN of|IN our|PRP$ patient|NN population|NN was|VBD high|JJ ,|, due|JJ to|TO the|DT fact|NN that|IN 5|CD out|IN of|IN a|DT total|NN of|IN 7|CD IPF|NNP subjects|NNS included|VBN in|IN our|PRP$ study|NN underwent|VBD lung|NN transplantation|NN with|IN confirmatory|NN pathology|NN results|NNS consistent|JJ with|IN IPF|NNP .|. 
The|DT other|JJ 2|CD subjects|NNS had|VBD biopsy|NN -|- proven|VBN IPF|NNP .|. 
In|IN addition|NN ,|, our|PRP$ results|NNS were|VBD similar|JJ when|WRB comparing|VBG lung|NN fibroblasts|NNS obtained|VBN from|IN 5|CD subjects|NNS with|IN advanced|JJ stage|NN IPF|NNP with|IN those|DT of|IN 2|CD subjects|NNS at|IN an|DT earlier|JJR stage|NN of|IN their|PRP$ disease|NN ,|, who|WP had|VBD received|VBN no|DT therapy|NN .|. 
Although|IN the|DT reasons|NNS for|IN our|PRP$ different|JJ results|NNS are|VBP unclear|JJ ,|, the|DT fact|NN that|IN we|PRP found|VBD similar|JJ COX-2|NNP expression|NN and|CC PGE|NNP 2|CD production|NN in|IN normal|JJ and|CC IPF|NNP lung|NN fibroblasts|NNS suggests|VBZ that|DT loss|NN of|IN COX-2|NNP expression|NN is|VBZ not|RB a|DT universal|JJ characteristic|NN of|IN fibroblasts|NNS cultured|JJ from|IN the|DT lungs|NNS of|IN subjects|NNS with|IN IPF|NNP .|. 

Conclusion|NNP We|PRP have|VBP found|VBN that|WDT fibroblasts|NNS cultured|JJ from|IN normal|JJ and|CC IPF|NNP human|JJ lungs|NNS have|VBP a|DT significant|JJ and|CC similar|JJ induction|NN of|IN the|DT COX-2|NNP enzyme|NN when|WRB stimulated|VBN with|IN IL-1β|NNP ,|, but|CC that|IN IPF|NNP fibroblasts|NNS produced|VBD more|JJR thromboxane|NN and|CC had|VBD a|DT significantly|RB lower|JJR prostacyclin|NN :|: thromboxane|NN ratio|NN .|. 
We|PRP hypothesize|NN that|IN the|DT lower|JJR PGI|NNP 2|CD :|: TXA|NNP 2|CD ratio|NN seen|VBN in|IN HF-IPF|NNP may|MD be|VB a|DT phenotypic|JJ alteration|NN that|WDT plays|VBZ a|DT role|NN in|IN the|DT pathogenesis|NNS of|IN IPF|NNP .|. 

Abbreviations|NNP COX|NNP =|SYM cyclooxygenase|NN ;|: HF|NNP =|SYM human|JJ fibroblasts|NNS ;|: NL|NNP =|SYM normal|JJ lungs|NNS ;|: IPF|NNP =|SYM idiopathic|JJ pulmonary|JJ fibrosis|NNS ;|: IFN|NNP =|SYM interferon|NN ;|: IL|NNP =|SYM interleukin|NN ;|: PG|NNP =|SYM prostaglandin|NN ;|: TX|NNP =|SYM thromboxane|NN ;|: PGI|NNP 2|CD =|SYM prostacyclin|NN .|. 

