D. S. Foster1,2, C. D. Marshall1,2, G. Gulati1, R. E. Jones1, A. Titan1,2, A. Nguyen1, A. Salhotra1, R. C. Ransom1, S. Mascharak1, R. E. Brewer1, J. A. Norton1,2, C. J. Kin2, A. A. Shelton2, M. T. Longaker1 1Stanford University,Hagey Laboratory For Pediatric Regenerative Medicine,Stanford, CA, USA 2Stanford University,General Surgery,Palo Alto, CA, USA
Introduction: Intra-abdominal adhesions are a common cause of small bowel obstruction after surgery and cost the U.S. health care system $5 billion annually. Fibroblasts are activated in the context of abdominal surgery and deposit fibrotic scar tissue. Despite the prevalence of adhesions, no effective therapeutics exist for prevention or treatment.
Methods: Human adhesion tissues, along with control peritoneum from patients without surgical history, were harvested from patients undergoing elective operations at Stanford Hospital (Fig. 1A). Adhesion-forming and quiescent fibroblasts were fluorescence-activated cell sorting (FACS)-isolated using an unbiased, lineage-negative approach (Fig. 1B-C). These cells were screened for expression of known fibroblast surface markers. Bulk RNA-seq was conducted; gene expression was compared between adhesion and control cohorts, as well as with bulk RNA-seq data acquired from mouse abdominal adhesions and control specimens. Adhesion specimens were fixed, sectioned and stained. Histological data was compared with control and mouse tissue specimens. All experiments were approved by Stanford University’s IRB and APLAC, as appropriate.
Results: Human adhesion-forming fibroblasts show significantly increased cell surface expression of PDGFRa compared with non-adhesion fibroblasts (Fig. 1D). PDGFRa is also highly-expressed on the surface of adhesion-forming fibroblasts in mice. Histologically, PDGFRa+ fibroblasts appear to closely associate with collagen fibers (Fig. 1E). Adhesion fibroblasts also express the cell surface marker CD26 in both human (Fig. 1D) and mice. These markers are also associated with scar-forming fibroblasts in skin. Bulk RNA-seq of human adhesion fibroblasts shows significant upregulation of Col24a1 expression compared to control, which is a member of the collagen gene family involved in type 1 collagen regulation and is also highly expressed by mouse adhesion fibroblasts. In addition, multiple genes involved in the epithelial-mesenchymal transition (EMT) pathway are upregulated by both human and mouse adhesion fibroblasts (eg. SPP1, SDC1, Comp, CDH2) (data not shown). These present potential therapeutic targets.
Conclusion: This research identifies PDGFRa-expressing fibroblasts as major contributors in abdominal adhesion formation and provides in depth tissue level and gene expression characterization of adhesion fibroblasts in humans. These data provide insight into key signaling processes responsible for adhesion formation as well as identifying a set of potential therapeutic targets common to human and mice. These targets can be validated preclinically and suggest promise for the development of therapeutics that could change the course of this challenging disease.
