B J Cook1, S Hasthorpe, J M Hutson. 1. F. Douglas Stephens Surgical Research Laboratory, Royal Children's Hospital Research Institute, Melbourne, Australia.
Abstract
BACKGROUND/ PURPOSE: Recent evidence has suggested that calcitonin gene-related peptide (CGRP), which is released from the genitofemoral nerve, may trigger fusion of the patent processus vaginalis in children with inguinal hernia. The purpose of this study was to determine whether CGRP triggers the release of mesenchymal factors leading to subsequent fusion of the processus vaginalis. METHODS: The response of cultured epithelial cells derived from the patent processus vaginalis was analysed by a novel in vitro culture system. Epithelial cells lining fresh hernial sacs (removed at inguinal herniotomy) were detached enzymatically and cultured for 72 hours on Micropore filters, in the presence of either 100 ng/mL hepatocyte growth factor (HGF), 7.4 x 10(-6) mol/L CGRP (amino acids 1 to 37), 7.4 x 10(-6) mol/L CGRP (8 to 37) antagonist, 10% fetal calf serum (FCS), or serum-free medium (SFM) alone. Transformation from an epithelial cell morphology to motile mesenchymal fibroblast-like cells was assessed by an average migration score (AMS), ranging from 0 with no sign of migration, to 3 with greater than 75% of cells migrating. Confocal microscopy was used to record changes in expression of epithelial (cytokeratin) and mesenchymal (vimentin) markers, as well as actin. Beta-catenin also was examined because it is part of the molecular complex that links cadherins to actin resulting in cell-cell adhesion. RESULTS: Epithelial and mesenchymal markers underwent either down-regulation or up-regulation as epithelial cell sheets broke apart and individual cells commenced migration. The AMS after 72 hours of culture was 0.22 with SFM (control); with FCS the score was 1.4 (P < .01). The AMS score with CGRP (1 to 37) was 0.55 (P = .165) and with its analogue, CGRP (8 to 37), which is a competitive inhibitor, 0.67 (P = .309). Neither was significant. HGF caused a significant increase in the AMS to 1.56 (P = .01). CONCLUSION: Both HGF and FCS (which contains various undefined peptides and growth factors) produced transformation of hernial sac epithelial cells, whereas CGRP and its inactive analogue did not. CGRP receptors are localised to mesenchymal fibroblasts within the processus vaginalis connective tissue, suggesting that CGRP could act indirectly via HGF, which, in turn, promotes fusion of the processus vaginalis. In the future, a nonsurgical treatment of inguinal herniae in children might be possible by the local administration of agents which promote fusion.
BACKGROUND/ PURPOSE: Recent evidence has suggested that calcitonin gene-related peptide (CGRP), which is released from the genitofemoral nerve, may trigger fusion of the patent processus vaginalis in children with inguinal hernia. The purpose of this study was to determine whether CGRP triggers the release of mesenchymal factors leading to subsequent fusion of the processus vaginalis. METHODS: The response of cultured epithelial cells derived from the patent processus vaginalis was analysed by a novel in vitro culture system. Epithelial cells lining fresh hernial sacs (removed at inguinal herniotomy) were detached enzymatically and cultured for 72 hours on Micropore filters, in the presence of either 100 ng/mL hepatocyte growth factor (HGF), 7.4 x 10(-6) mol/L CGRP (amino acids 1 to 37), 7.4 x 10(-6) mol/L CGRP (8 to 37) antagonist, 10% fetal calf serum (FCS), or serum-free medium (SFM) alone. Transformation from an epithelial cell morphology to motile mesenchymal fibroblast-like cells was assessed by an average migration score (AMS), ranging from 0 with no sign of migration, to 3 with greater than 75% of cells migrating. Confocal microscopy was used to record changes in expression of epithelial (cytokeratin) and mesenchymal (vimentin) markers, as well as actin. Beta-catenin also was examined because it is part of the molecular complex that links cadherins to actin resulting in cell-cell adhesion. RESULTS: Epithelial and mesenchymal markers underwent either down-regulation or up-regulation as epithelial cell sheets broke apart and individual cells commenced migration. The AMS after 72 hours of culture was 0.22 with SFM (control); with FCS the score was 1.4 (P < .01). The AMS score with CGRP (1 to 37) was 0.55 (P = .165) and with its analogue, CGRP (8 to 37), which is a competitive inhibitor, 0.67 (P = .309). Neither was significant. HGF caused a significant increase in the AMS to 1.56 (P = .01). CONCLUSION: Both HGF and FCS (which contains various undefined peptides and growth factors) produced transformation of hernial sac epithelial cells, whereas CGRP and its inactive analogue did not. CGRP receptors are localised to mesenchymal fibroblasts within the processus vaginalis connective tissue, suggesting that CGRP could act indirectly via HGF, which, in turn, promotes fusion of the processus vaginalis. In the future, a nonsurgical treatment of inguinal herniae in children might be possible by the local administration of agents which promote fusion.