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

Simona Serratì, Anastasia Chillà, Anna Laurenzana, Francesca Margheri, Elisa Giannoni, Lucia Magnelli, Paola Chiarugi, Javier Dotor, Esperanza Feijoo, Laura Bazzichi, Stefano Bombardieri, Bashar Kahaleh, Gabriella Fibbi, Mario Del Rosso

Research output: Contribution to journalArticle

23 Citations (Scopus)

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. Conclusion SSc MVECs recruit and activate dermal fibroblasts by induction of a CCN2/TGFβ-dependent mesenchymal-to-mesenchymal transition.

Original languageEnglish
Pages (from-to)258-269
Number of pages12
JournalArthritis and Rheumatism
Volume65
Issue number1
DOIs
Publication statusPublished - Jan 2013

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Connective Tissue Growth Factor
Systemic Scleroderma
Transforming Growth Factors
Endothelial Cells
Fibroblasts
Skin
Vimentin
Conditioned Culture Medium
Collagen Type I
Blood Vessels
Fibrosis
Western Blotting
Gelatinases
Matrix Metalloproteinase Inhibitors
Monomeric GTP-Binding Proteins
Matrix Metalloproteinase 2
Matrix Metalloproteinase 9
Matrix Metalloproteinases
Reverse Transcription
Cues

ASJC Scopus subject areas

  • Immunology
  • Immunology and Allergy
  • Rheumatology
  • Pharmacology (medical)

Cite this

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. / Serratì, Simona; Chillà, Anastasia; Laurenzana, Anna; Margheri, Francesca; Giannoni, Elisa; Magnelli, Lucia; Chiarugi, Paola; Dotor, Javier; Feijoo, Esperanza; Bazzichi, Laura; Bombardieri, Stefano; Kahaleh, Bashar; Fibbi, Gabriella; Del Rosso, Mario.

In: Arthritis and Rheumatism, Vol. 65, No. 1, 01.2013, p. 258-269.

Research output: Contribution to journalArticle

Serratì, S, Chillà, A, Laurenzana, A, Margheri, F, Giannoni, E, Magnelli, L, Chiarugi, P, Dotor, J, Feijoo, E, Bazzichi, L, Bombardieri, S, Kahaleh, B, Fibbi, G & Del Rosso, M 2013, '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', Arthritis and Rheumatism, vol. 65, no. 1, pp. 258-269. https://doi.org/10.1002/art.37705
Serratì, Simona ; Chillà, Anastasia ; Laurenzana, Anna ; Margheri, Francesca ; Giannoni, Elisa ; Magnelli, Lucia ; Chiarugi, Paola ; Dotor, Javier ; Feijoo, Esperanza ; Bazzichi, Laura ; Bombardieri, Stefano ; Kahaleh, Bashar ; Fibbi, Gabriella ; Del Rosso, Mario. / 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. In: Arthritis and Rheumatism. 2013 ; Vol. 65, No. 1. pp. 258-269.
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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. Conclusion SSc MVECs recruit and activate dermal fibroblasts by induction of a CCN2/TGFβ-dependent mesenchymal-to-mesenchymal transition.",
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AU - Serratì, Simona

AU - Chillà, Anastasia

AU - Laurenzana, Anna

AU - Margheri, Francesca

AU - Giannoni, Elisa

AU - Magnelli, Lucia

AU - Chiarugi, Paola

AU - Dotor, Javier

AU - Feijoo, Esperanza

AU - Bazzichi, Laura

AU - Bombardieri, Stefano

AU - Kahaleh, Bashar

AU - Fibbi, Gabriella

AU - Del Rosso, Mario

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N2 - 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. Conclusion SSc MVECs recruit and activate dermal fibroblasts by induction of a CCN2/TGFβ-dependent mesenchymal-to-mesenchymal transition.

AB - 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. Conclusion SSc MVECs recruit and activate dermal fibroblasts by induction of a CCN2/TGFβ-dependent mesenchymal-to-mesenchymal transition.

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