Enhancement of the Functional Repertoire of the Rat Parietal Peritoneal Mesothelium In Vivo : Directed Expression of the Anticoagulant and Antiinflammatory Molecule Thrombomodulin

Abstract

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.

Overview summary

Ex vivo gene therapy allows the unprecedented opportunity to target changes in cell physiology to the tissue of pathology. The simplest method of targeting is the restricted expression of the new, therapeutic, gene product to the genetically modified cell. A class of proteins so far underrepresented in therapeutic models of ex vivo gene therapy is that of cell surface intrinsic membrane proteins. In the present work we demonstrate the modification of the peritoneal membrane to a new constitutive anticoagulant state by the expression of thrombomodulin on the mesothelial cell surface. We envision this strategy as a prototype for a variety of therapeutic interventions designed to target specific pathologies that occur at the peritoneal surface, such as the development of postsurgical adhesions, and the progressive fibrotic thickening of the peritoneal membrane often observed in the long-term peritoneal dialysis patient.