Enhancement of the functional repertoire of the rat parietal peritoneal mesothelium in vivo: directed expression of the anticoagulant and antiinflammatory molecule thrombomodulin.
We have used our previously described ex vivo mesothelial cell (MC)-mediated gene therapy strategy (Gene Ther. 2:393-401, 1995) to modify the functional properties of the rat parietal peritoneal mesothelium in vivo by expression of a membrane-bound recombinant protein on the MC surface. Rat primary MCs were stably transfected (using strontium phosphate DNA coprecipitation) with a plasmid containing the gene for rat thrombomodulin (TM), a transmembrane glycoprotein that functions as an essential cofactor for the physiological activation of the anticoagulant protein C by the enzyme thrombin. As demonstrated by immunohistochemistry and by direct equilibrium binding with radiolabeled thrombin, genetically modified MCs expressed high levels of TM antigen on their surface in vitro. As judged by a thrombin-dependent protein C activation assay, such MC membrane-bound TM was biologically active. Once reseeded on the denuded parietal peritoneal surface of syngeneic recipients, these TM-transfected MCs continued to express TM antigen in vivo for at least 90 days. Moreover, the recombinant TM expressed on the reconstituted parietal mesothelium retained its ability to activate protein C in a thrombin-dependent manner. Our data indicate that MC-mediated expression of TM can be used to augment the anticoagulant properties of the parietal peritoneal surface. In general, our results suggest that ex vivo MC-mediated gene therapy can be used to deliver other therapeutic transmembrane proteins to the MC surface to enhance the functional repertoire of the parietal mesothelium in vivo.
Jackman
R W
RW
Department of Pathology, Beth Israel Deaconess Medical Center and Harvard Medical School, Boston, MA 02215, USA.
Stapleton
T D
TD
Masse
E M
EM
Harvey
V S
VS
Meyers
M S
MS
Shockley
T R
TR
Nagy
J A
JA
D000818:Animals; D000893:Anti-Inflammatory Agents / Q000378:metabolism; D000925:Anticoagulants / Q000378:metabolism; D015152:Blotting, Northern; D002417:Cattle; D004847:Epithelial Cells / Q000378:metabolism*; D005260:Female; D015870:Gene Expression; D018014:Gene Transfer Techniques; D005822:Genetic Vectors; D010529:Peritoneal Cavity / Q000166:cytology*; D010957:Plasmids; D011233:Precipitin Tests; D011817:Rabbits; D051381:Rats; D011916:Rats, Inbred F344; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D018180:Thrombomodulin / Q000235:genetics* / Q000378:metabolism; D014162:Transfection; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology
D000893:Anti-Inflammatory Agents; D000925:Anticoagulants; D011993:Recombinant Fusion Proteins; D018180:Thrombomodulin; D014409:Tumor Necrosis Factor-alpha