Wound Healing Society

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Role Of Mrtf-a And Mrtf-b In Post-operative Intra-abdominal Adhesion Formation
Paul McGaha1, Cullen McCarthy1, Phillip Bonney2, James Griffith1, Eric Howard1, James Tomasek1, Jason Lees1, William Berry1.
1University of Oklahoma Health Sciences Center, Oklahoma City, OK, USA, 2University of Southern California, Los Angeles, CA, USA.

Background: Post-operative intra-abdominal adhesions occur in over 90% of patients who undergo abdominal surgery. Intra-abdominal adhesion scar tissue can cause significant morbidity and mortality. Myocardin-related transcription factors-A and -B (MRTFs) regulate myofibroblast function in cutaneous wound healing. Similar to cutaneous wounds, intra-abdominal adhesions are enriched in myofibroblasts and collagens. We hypothesize that myofibroblast-mediated formation of intra-abdominal adhesions require MRTFs. Methods: Intra-abdominal adhesion tissue was collected from patients undergoing a secondary surgery. Half the tissue was fixed and embedded in paraffin. Sections were immunostained for MRTF-A, MRTF-B, smooth muscle α-actin (SMαA), and collagen type III. Cells were isolated and cultured from the other half of tissue using Liberase TM. Cytoplasmic and nuclear extracts from patients’ cultured cells were subjected to Western-immunoblotting for MRTF-A, MRTF-B, Tubulin, and Lamin A/C. Patients’ cultured cells were infected with lentiviral vectors expressing either shRNA targeting MRTFs or non-targeting shRNA. Lysates from infected cells were collected and subjected to Western-immunoblotting for MRTF-A, MRTF-B, SMαA, SM22α, collagen type III, and GAPDH. Contractile properties of infected cells were analyzed in a stressed-relaxed collagen lattice contraction assay. Results: MRTFs are highly expressed in adhesion tissue and correlated with expression of SMαA and collagen type III. MRTFs were found localized in the nucleus in all cultured patient adhesion cells and correlated with the expression of the pro-contractile genes (SMαA, SM22α) and pro-fibrotic collagen type III. shRNA mediated depletion of MRTFs reduced expression of these myofibroblast genes. Lastly, depletion of MRTFs reduced stressed-relaxed collagen lattice contraction. Conclusion: Presence of MRTFs correlates with the presence of myofibroblasts in adhesion tissue, while depletion of MRTFs in cultured patient adhesion fibroblasts decreases the functional characteristics of myofibroblasts. Animal studies are currently underway to directly test whether inhibition of MRTFs prevents the formation of myofibroblasts and of intra-abdominal adhesions. Therapeutic approaches to inhibit MRTFs may be useful in preventing post-operative adhesion formation.


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