Profibrotic Changes Following Tension Application in a Fetal Lamb Model of Long Gap Esophageal Atresia

Esophageal atresia is a common congenital anomaly, occurring in 1 in 3,500 live births. The Foker process has revolutionized the treatment of long gap esophageal atresia (LGEA). It is well established that the Foker process causes tension accelerated growth of the esophagus, but what occurs at the molecular level during tension accelerated growth is still unknown. We aimed to create tension accelerated growth in a fetal lamb model of LGEA in order to answer this question.

Following IACUC approval, time-dated fetal lambs (108 to 120 days of gestation) underwent thoracic esophagectomy. Both esophageal ends were ligated and sutured together to create an internal pexy under high tension. Lambs were delivered on postoperative day 2 (POD2) (n=7), POD6 (n=9) or term (n=5). The native esophagus collected at model creation served as control tissue. Specimens were bluntly separated into two layers: inner layer (IL) (epithelium, lamina propria, muscularis mucosa, submucosa) and outer layer (OL) (submucosa, muscle layer, adventitia). RNA sequencing (RNAseq), proteomics, immunohistochemistry, western blotting and real-time qRT-PCR were performed on the specimens. Mann-Whitney's or unpaired t-test were used for statistical analyses. Esophageal fibroblast cell lines established from human biopsy specimens were cultured and stimulated with TGF-beta for in vitro studies on collagen expression.

23 lambs underwent esophagectomy with tension suture placement at 108 to 120 days gestation. Histologic analysis of tension conditioned compared to control esophagus by trichrome staining demonstrated an increase in collagen deposition in tension conditioned esophagus compared to controls. High throughput bulk RNA sequencing and proteomic analysis were performed with a focus on pathways implicated in fibrosis. GSEA analysis of the inner layer demonstrates upregulation of TGFB signaling, extracellular matrix organization, and collagen deposition at all timepoints. Further analysis was performed to evaluate specific collagen subtypes contributing to this profibrotic phenotype, and COL8A1 and COL12A1 were both significantly upregulated in both RNA and proteomic analysis at all timepoints, with Western blotting confirming up regulation in stretched tissue. In order to evaluate the relationship between TGFB signaling and collagen deposition in the esophagus, we stimulated esophageal fibroblasts with TGFB, qRT-PCR was performed to evaluate the expression of COL8A1, COL12A1, and COL6A3. Expression of all three of these collagen subtypes was noted to be significantly upregulated at all timepoints following TGFB stimulation when compared to non-stimulated controls.

Tension accelerated growth can safely be achieved in a fetal ovine model of long gap esophageal atresia. Additionally, esophageal atresia can be modeled in the ovine fetus as early as 92 days gestation. Our results demonstrate that esophageal tissue subjected to sustained tension undergoes significant profibrotic changes, as evidenced by upregulation of TGFB signaling, alterations in extracellular matrix organization, and increased collagen deposition. While it is well documented that patients with LGEA have an increased risk of post operative esophageal strictures, these findings provide the first in vivo proof of the role of tension in conferring a profibrotic phenotype in the tension-lengthened esophagus.