Lipopolysaccharide attenuates endothelial barrier function through a pp60 Src mediated inhibition of dimethylaminohydrolase (DDAH)

Acute lung injury is a severe hypoxemic respiratory insufficiency associated with alterations in lung structure and function. Previously, we found that decreases in the activity of DDAH and increased in asymmetric dimethylarginine (ADMA) contributes to the development of ALI in mice exposed to lipopolysaccharide (LPS). In this study, we elucidated the mechanisms involved in the attenuation of DDAH2 by LPS. Utilizing an electric cell impedance sensing apparatus, we found that overexpression of DDAH2 in human lung microvascular endothelial cells (HLMVEC) prevents the LPS (1 endotoxin unit/ml) induced decrease in transendothelial resistance. Further, we found that overexpression of a dominant negative mutant of pp60 src attenuated the LPS mediated decrease in DDAH activity and increase in ADMA levels. While, overexpression of a constitutively active mutant decreased DDAH activity and increased ADMA levels without altering DDAH2 protein levels. Further, LPS increased the interaction between DDAH2 and pp60 src and also increased its tyrosine phosphorylation. In the LPS treated mouse lung, we found that the decrease in DDAH activity correlated with an increase in pp60 src interaction and tyrosine phosphorylation of DDAH2. Finally, the overexpression of DDAH2 in murine lung endothelial cells, using a polyethyleneimine (PEI) derivative transfection reagent, led to an increase in DDAH activity, a decrease in ADMA levels, and the attenuation of the LPS mediated increase in the lung leak as measured by extravasation of Evans blue dye. The prevention of the pp60 src mediated decrease in DDAH activity may have clinical utility in the prevention of LPS induced ALI.