Systemic sclerosis sera affect fibrillin-1 deposition by dermal blood microvascular endothelial cells: therapeutic implications of cyclophosphamide

Abstract

Introduction: Systemic sclerosis (SSc) is a connective tissue disorder characterized by endothelial cell injury, autoimmunity and fibrosis. The following three fibrillin-1 alterations have been reported in SSc. (1) Fibrillin-1 microfibrils are disorganized in SSc dermis. (2) Fibrillin-1 microfibrils produced by SSc fibroblasts are unstable. (3) Mutations in the FBN1 gene and anti-fibrillin-1 autoantibodies have been reported in SSc. Fibrillin-1 microfibrils, which are abundantly produced by blood and lymphatic microvascular endothelial cells (B-MVECs and Ly-MVECs, respectively), sequester in the extracellular matrix the latent form of the potent profibrotic cytokine transforming growth factor β (TGF-β). In the present study, we evaluated the effects of SSc sera on the deposition of fibrillin-1 and microfibril-associated glycoprotein 1 (MAGP-1) and the expression of focal adhesion molecules by dermal B-MVECs and Ly-MVECs.

Methods: Dermal B-MVECs and Ly-MVECs were challenged with sera from SSc patients who were treatment-naïve or under cyclophosphamide (CYC) treatment and with sera from healthy controls. Fibrillin-1/MAGP-1 synthesis and deposition and the expression of αvβ₃ integrin/phosphorylated focal adhesion kinase and vinculin/actin were evaluated by immunofluorescence and quantified by morphometric analysis.

Results: Fibrillin-1 and MAGP-1 colocalized in all experimental conditions, forming a honeycomb pattern in B-MVECs and a dense mesh of short segments in Ly-MVECs. In B-MVECs, fibrillin-1/MAGP-1 production and αvβ₃ integrin expression significantly decreased upon challenge with sera from naïve SSc patients compared with healthy controls. Upon challenge of B-MVECs with sera from CYC-treated SSc patients, fibrillin-1/MAGP-1 and αvβ₃ integrin levels were comparable to those of cells treated with healthy sera. Ly-MVECs challenged with SSc sera did not differ from those treated with healthy control sera in the expression of any of the molecules assayed.

Conclusions: Because of the critical role of fibrillin-1 in sequestering the latent form of TGF-β in the extracellular matrix, its decreased deposition by B-MVECs challenged with SSc sera might contribute to dermal fibrosis. In SSc, CYC treatment might limit fibrosis through the maintenance of physiologic fibrillin-1 synthesis and deposition by B-MVECs.

Figure 1

Systemic sclerosis sera affect deposition of fibrillin-1 and microfibril-associated glycoprotein 1 by dermal blood microvascular endothelial cells. Fibrillin-1 and microfibril-associated glycoprotein 1 (MAGP-1) deposition by dermal blood microvascular endothelial cells (B-MVECs) was evaluated after challenge with systemic sclerosis (SSc) and healthy control (CTR) sera. (A) Representative immunofluorescent images for fibrillin-1 (green) and MAGP-1 (red). The two molecules colocalize and are deposited in a honeycomb pattern, with empty spaces in which endothelial cells are still visible. 4′,6-diamidino-2-phenylindole (DAPI)-stained nuclei are blue in merged images. Original magnification, ×40. (B) Quantification of fibrillin-1 deposition by measurement of optical density in arbitrary units (a.u.). Data are means ± SEM. Fibrillin-1 deposition was significantly reduced in B-MVECs challenged with naïve SSc sera (all naïve SSc, naïve limited SSc (lSSc) and naïve diffuse SSc (dSSc)) vs. healthy controls (**P < 0.001). Fibrillin-1 was significantly higher in cyclophosphamide (CYC)-treated lSSc than in controls (*P < 0.05). No significant difference was found between all CYC-treated SSc and CYC-treated dSSc vs. healthy controls.

Figure 2

Lack of effect of systemic sclerosis sera on deposition of fibrillin-1 and microfibril-associated glycoprotein 1 by dermal lymphatic microvascular endothelial cells. Fibrillin-1 and microfibril-associated glycoprotein 1 (MAGP-1) deposition by dermal lymphatic microvascular endothelial cells (Ly-MVECs) was evaluated after challenge with systemic sclerosis (SSc) and healthy control (CTR) sera. (A) Representative immunofluorescent images for fibrillin-1 (green) and MAGP-1 (red). The two molecules colocalize and are deposited in thin, short strands, only rarely forming honeycombs. 4′,6-diamidino-2-phenylindole (DAPI)-stained nuclei are blue in merged images. Original magnification, ×40. (B) Quantification of fibrillin-1 deposition by measurement of optical density in arbitrary units (a.u.). Data are means ± SEM. No significant differences vs. healthy controls were found in any of the conditions assayed. CYC, cyclophosphamide; dSSc, diffuse SSc; lSSc, limited SSc.

Figure 3

Expression of focal adhesion molecules in dermal blood microvascular endothelial cells and lymphatic microvascular endothelial cells. Representative immunofluorescent images of focal adhesion molecules in blood microvascular endothelial cells (B-MVECs) (A) and lymphatic microvascular endothelial cells (Ly-MVECs) (B) cultured with healthy sera are shown. Representative immunofluorescent photomicrographs show double-labeling for α_vβ₃ integrin (green) (a), phosphorylated focal adhesion kinase (FAK) (red) (b) and a merged image (c). Representative immunofluorescent photomicrographs show double-labeling for phalloidin (green) (d), vinculin (red) (e) and a merged image (f). 4′,6-diamidino-2-phenylindole (DAPI)-stained nuclei are blue in merged images. In both B-MVECs and Ly-MVECs, α_vβ₃ integrin (a) and phosphorylated FAK (b) are co-localized. α_vβ₃ integrin (a), phosphorylated FAK (b) and vinculin (e) are visible as small dots distributed all over the cell surface. Phalloidin-stained actin filaments (d) are arranged in long strands running parallel to cell borders. Original magnification, ×40.

Figure 4

Systemic sclerosis sera affect α_vβ₃ integrin and vinculin expression in dermal blood microvascular endothelial cells. Quantification of α_vβ₃ integrin (A) and vinculin (B) in dermal blood microvascular endothelial cells (B-MVECs) challenged with systemic sclerosis (SSc) and healthy control (CTR) sera. Data are means ± SEM of optical density in arbitrary units (a.u.). (A) The expression of α_vβ₃ integrin was significantly lower in cells challenged with sera from naïve SSc patients (all naïve SSc, naïve limited SSc (lSSc) and naïve diffuse SSc (dSSc)) than in cells treated with sera from healthy controls (**P < 0.001). No significant differences were found between cyclophosphamide (CYC)-treated SSc patients (all SSc, lSSc and dSSc) and healthy controls, whereas expression of α_vβ₃ integrin in all CYC-treated SSc was significantly higher than that in all naïve SSc (^§P < 0.001). (B) The expression of vinculin was significantly higher in cells challenged with the sera of naïve dSSc than in cells treated with sera from healthy controls (**P < 0.001). No significant differences were found in any of the other conditions tested.