Apoptosis inhibition in P. aeruginosa-induced lung injury influences lung fluid balance.
OBJECTIVE
Pseudomonas aeruginosa-induced lung injury is characterized not only by the alteration in lung fluid movement but also by apoptosis of lung epithelial and endothelial cells. We studied whether inhibition of apoptosis using a broad spectrum caspase inhibitor, benzyloxycarbonyl-Val-Ala-Asp fluoromethylketone (Z-VAD.fmk), would affect lung fluid balance in rat P. aeruginosa pneumonia.


METHODS
Z-VAD.fmk (3 mg/kg) was administered intravenously simultaneously with P. aeruginosa intratracheal instillation (0.5 ml/kg, 2 x 10(9) CFU/ml). Apoptosis was evaluated with the TUNEL technique, cytoplasmic oligonucleosome assay, and caspase 3 activation. To evaluate lung permeability, extravascular plasma equivalent (EPE) and lung wet to dry weight ratio (W/D) were measured 4 h after intratracheal instillation of P. aeruginosa.


RESULTS
We found an increase of lung apoptosis 4 h after P. aeruginosa instillation: cytoplasmic oligonucleosome assay increased from 3.17+/-0.78 to 26.82+/-4.67 ODx1000/mg of proteins/ml, Z-VAD.fmk administration decreased this parameter to 10.3+/-2.98 ODx1000/mg of proteins/ml. Caspase 3 levels followed the same pattern. Apoptosis involved both epithelial cells and endothelial cells. Endothelial permeability was increased after Pseudomonas instillation: W/D increased from 3.75+/-0.28 in the Co group to 4.42+/-0.23 in the Pn group; EPE was also higher in the Pn group compared with the Co group (0.125+/-0.04 and 0.002+/-0.01 ml, respectively). Both of these parameters were improved after Z-VAD.fmk administration; W/D decreased to 3.36+/-0.25 and EPE to 0.02+/-0.02 ml.


CONCLUSIONS
Apoptosis occurs in the early phase of P. aeruginosa pneumonia. Administration of Z-VAD.fmk significantly decreases DNA fragmentation and caspase 3 levels. This is associated with an improvement of endothelial permeability and lung fluid balance.
Laboratoire de Recherche en Pathologie Infectieuse, 2689 Lille, France.
Rozenn
R
Le Berre
Karine
K
Faure
Harold
H
Fauvel
Nathalie B
NB
Viget
Florence
F
Ader
Thierry
T
Prangère
Anne Marie
AM
Thomas
Xavier
X
Leroy
Jean-François
JF
Pittet
Philippe
P
Marchetti
Benoit P
BP
Guery
D000590:Amino Acid Chloromethyl Ketones / Q000494:pharmacology*; D000818:Animals; D017209:Apoptosis / Q000187:drug effects* / Q000502:physiology; D002199:Capillary Permeability / Q000187:drug effects; D004730:Endothelium, Vascular / Q000187:drug effects / Q000378:metabolism; D011552:Pseudomonas Infections / Q000503:physiopathology*; D011650:Pulmonary Alveoli / Q000187:drug effects / Q000378:metabolism; D011654:Pulmonary Edema / Q000503:physiopathology*; D051381:Rats; D017207:Rats, Sprague-Dawley; D012128:Respiratory Distress Syndrome / Q000503:physiopathology*; D018709:Statistics, Nonparametric
D000590:Amino Acid Chloromethyl Ketones; C096713:benzyloxycarbonylvalyl-alanyl-aspartyl fluoromethyl ketone