title: Systemic sclerosis endothelial cells recruit and activate dermal fibroblasts by induction of a connective tissue growth factor (CCN2)/transforming growth factor β-dependent mesenchymal-to-mesenchymal transition.
abstract: OBJECTIVE
  Clinical evidence suggests that the vascular abnormalities of systemic sclerosis (SSc) precede the onset of fibrosis, but molecular cues accounting for a possible vascular connection of SSc fibrosis have been elusive, although they have been searched for intensively. Since we had previously shown that connective tissue growth factor (CCN2), endowed with fibroblast-oriented activities, was overexpressed by endothelial cells (ECs) from SSc patients, we undertook this study to investigate its role and mechanisms in fibroblast activation.
  
  
  METHODS
  Normal fibroblasts were challenged with conditioned medium of normal microvascular ECs (MVECs) and MVECs obtained from SSc patients with the diffuse form of the disease. Fibroblast invasion was studied using the Boyden chamber Matrigel assay. Fibroblast activation was evaluated by Western blotting and immunofluorescence of α-smooth muscle actin (α-SMA), vimentin, and type I collagen. Matrix metalloproteinase (MMP) production was evaluated by zymography and reverse transcription-polymerase chain reaction. Signal transduction and activation of the small GTPases RhoA and Rac1 were studied by Western blotting. Inhibition of SSc MVEC conditioned medium-dependent fibroblast activation was obtained by anti-CCN2 antibodies and the transforming growth factor β (TGFβ) antagonist peptide p17.
  
  
  RESULTS
  SSc MVEC CCN2 stimulated fibroblast activation and invasion. Such activities depended on CCN2-induced overexpression of TGFβ and its type I, II, and III receptors combined with overproduction of MMP-2 and MMP-9 gelatinases. All of these effects were reversed by the TGFβ antagonist peptide p17. Motility increase required Rac1 activation and RhoA inhibition and was inhibited by an MMP inhibitor. These features connoted a mesenchymal style of cell invasion. Since fibroblast activation also fostered overexpression of α-SMA, vimentin, and type I collagen, the CCN2-dependent increase in fibroblast activities recapitulated the characteristics of a mesenchymal-to-mesenchymal transition.
  
  
  CONCLUSIONS
  SSc MVECs recruit and activate dermal fibroblasts by induction of a CCN2/TGFβ-dependent mesenchymal-to-mesenchymal transition.
authors.affiliation: Department of Experimental Pathology and Oncology, University of Florence, Florence, Italy, and Istituto Tumori Giovanni Paolo II, Bari, Italy.
authors.forename: Simona
authors.initials: S
authors.lastname: Serratì
authors.forename: Anastasia
authors.initials: A
authors.lastname: Chillà
authors.forename: Anna
authors.initials: A
authors.lastname: Laurenzana
authors.forename: Francesca
authors.initials: F
authors.lastname: Margheri
authors.forename: Elisa
authors.initials: E
authors.lastname: Giannoni
authors.forename: Lucia
authors.initials: L
authors.lastname: Magnelli
authors.forename: Paola
authors.initials: P
authors.lastname: Chiarugi
authors.forename: Javier
authors.initials: J
authors.lastname: Dotor
authors.forename: Esperanza
authors.initials: E
authors.lastname: Feijoo
authors.forename: Laura
authors.initials: L
authors.lastname: Bazzichi
authors.forename: Stefano
authors.initials: S
authors.lastname: Bombardieri
authors.forename: Bashar
authors.initials: B
authors.lastname: Kahaleh
authors.forename: Gabriella
authors.initials: G
authors.lastname: Fibbi
authors.forename: Mario
authors.initials: M
authors.lastname: Del Rosso
mesh_terms: D015153:Blotting, Western; D003094:Collagen; D055513:Connective Tissue Growth Factor / Q000378:metabolism*; D004338:Drug Combinations; D042783:Endothelial Cells / Q000378:metabolism*; D005260:Female; D005347:Fibroblasts / Q000187:drug effects* / Q000378:metabolism / Q000473:pathology; D005455:Fluorescent Antibody Technique; D006801:Humans; D007797:Laminin; D008297:Male; D008648:Mesoderm / Q000473:pathology; D011509:Proteoglycans; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012595:Scleroderma, Systemic / Q000378:metabolism* / Q000473:pathology; D015398:Signal Transduction; D012867:Skin / Q000378:metabolism* / Q000473:pathology; D016212:Transforming Growth Factor beta / Q000378:metabolism* / Q000494:pharmacology
chemical_list: C526317:CCN2 protein, human; D004338:Drug Combinations; D007797:Laminin; D011509:Proteoglycans; D016212:Transforming Growth Factor beta; C059485:matrigel; D055513:Connective Tissue Growth Factor; D003094:Collagen