title: Stromal fibroblast-bone marrow-derived cell interactions: implications for myofibroblast development in the cornea.
abstract: The purpose of this study was to test the hypothesis that mouse corneal stromal fibroblast and bone marrow-derived cell interactions augment corneal myofibroblast generation and, if so, to study whether such interactions are mediated by paracrine or juxtacrine mechanisms. Mouse bone marrow-derived cells and mouse corneal stromal fibroblasts were obtained from both mice with green fluorescent protein (GFP) expressed in all cells and normal GFP- BL6 control mice. To study the interactions of the different cell types, GFP+ cells of one type were co-cultured with GFP- cells of the other type in Primaria plates (to monitor juxtacrine signaling) or Transwell System plates (to monitor paracrine effects mediated by soluble mediators). Both cell types were cultured at a cell density of 1 × 10(5) cells per ml. The percentage of alpha smooth muscle actin+ myofibroblasts was significantly higher (ANOVA, p<0.001) when bone marrow-derived cells and mouse corneal stromal fibroblasts were co-cultured compared to when bone marrow-derived cells and mouse corneal stromal fibroblasts were cultured alone (control). The in vitro studies using GFP+ corneal fibroblasts or GFP+ bone marrow-derived cells demonstrated conclusively that both cells types could transform into myofibroblasts. However, the percentage of alpha smooth muscle actinassds+ myofibroblasts generated from either cell type precursor was higher when both cells were co-cultured together (juxtacrine) as compared to when bone marrow-derived cells and mouse corneal stromal fibroblasts were co-culture in different compartments of Transwell System (paracrine). Thus, more alpha smooth muscle actin+ GFP+ myofibroblasts were generated from GFP+ corneal stromal fibroblasts when GFP- bone marrow-derived cells were present and more alpha smooth muscle actin+ GFP+ myofibroblasts were generated from GFP+ bone marrow-derived cells when GFP- corneal stromal fibroblasts were present. Polyclonal anti-human latency associated peptide (LAP) (transforming growth factor-β1) neutralizing antibody (a-LAP) and/or transforming growth factor-β type I receptor kinase inhibitor (LY-364947) inhibited the generation of alpha smooth muscle actin+ myofibroblasts from either precursor cell in Transwell System co-culture experiments. These data suggest that TGFβ is a paracrine modulator that regulates the generation of myofibroblasts from either corneal fibroblasts or bone marrow-derived cell precursors.
authors.affiliation: Cole Eye Institute, The Cleveland Clinic Foundation, Cleveland, OH 44195, USA.
authors.forename: V
authors.initials: V
authors.lastname: Singh
authors.forename: V
authors.initials: V
authors.lastname: Agrawal
authors.forename: M R
authors.initials: MR
authors.lastname: Santhiago
authors.forename: S E
authors.initials: SE
authors.lastname: Wilson
mesh_terms: D000199:Actins / Q000378:metabolism; D000818:Animals; D001854:Bone Marrow Cells / Q000166:cytology* / Q000378:metabolism; D002450:Cell Communication / Q000502:physiology*; D002469:Cell Separation; D054338:Cell Transdifferentiation / Q000187:drug effects / Q000502:physiology*; D018920:Coculture Techniques; D060527:Corneal Keratocytes / Q000166:cytology* / Q000378:metabolism; D003319:Corneal Stroma / Q000166:cytology*; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005260:Female; D019084:Fluorescent Antibody Technique, Indirect; D015870:Gene Expression / Q000235:genetics; D049452:Green Fluorescent Proteins / Q000235:genetics; D051379:Mice; D008810:Mice, Inbred C57BL; D008822:Mice, Transgenic; D058628:Myofibroblasts / Q000166:cytology* / Q000378:metabolism; D011720:Pyrazoles / Q000494:pharmacology; D011758:Pyrroles / Q000494:pharmacology; D015398:Signal Transduction; D014162:Transfection; D053773:Transforming Growth Factor beta1 / Q000378:metabolism; D011804:Quinolines
chemical_list: D000199:Actins; D004791:Enzyme Inhibitors; D049452:Green Fluorescent Proteins; D011720:Pyrazoles; D011758:Pyrroles; D053773:Transforming Growth Factor beta1; C558396:Acta2 protein, mouse; C506615:HTS 466284; D011804:Quinolines