Epidermal Growth Factor Like-domain 7 and miR-126 are abnormally expressed in diffuse Systemic Sclerosis fibroblasts

Noemi Panzera,Piero Ruscitti,Paola Cipriani,Francesco Del Galdo,Vasiliki Liakouli,Paola Di Benedetto,Georgia Mavria,Ilenia Pantano,Filomena Esteves,Roberto Giacomelli,Onorina Berardicurti,Francesco Carubbi

doi:10.1038/s41598-019-39485-8

Abstract

Systemic sclerosis (SSc) is characterized by microangiopathy with impaired reparative angiogenesis and fibrosis. Epidermal Growth Factor Like-domain 7 (EGFL7), firstly described in endothelial cells plays a pivotal role in angiogenesis. Fibroblasts (FBs) are involved in vascular remodeling, under physiological and pathological conditions. In this study, we investigated: (i) the expression of EGFL7 and its miR-126 in patients affected by diffuse cutaneous SSc (dcSSc); (ii) the ability of Transforming Growth Factor-beta (TGF-β) to modulate EGFL7 expression; (iii) the ability of EGFL7 to modulate COL1A1 expression and proliferation/migration, and (iv) the functional role of EGFL7 on angiogenesis. Patients were divided in 2 subsets: patients fulfilling the classification criteria in less than one year from Raynaud’s Phenomenon onset (Early Onset Subset–EOS), and all the others (Long Standing Subset–LSS). We show that EGFL7 expression is increased in EOS dcSSc skin and cultured FBs. EGFL7 is inducible by TGF-β on Healthy Controls (HC) FBs but not in SSc-FBs. EGFL7 decreases COL1A1 expression in EOS SSc-FBs while EGFL7 silencing up-regulates COL1A1 expression. EGFL7 promotes migration/invasion of EOS SSc-FBs but not proliferation. Finally, SSc-FBs, partially inhibit angiogenesis in organotypic coculture assays, and this is reversed by treatment with human recombinant (rh)EGFL7. We conclude that EGFL7 and its specific microRNA miR-126 may be involved in the pathogenesis of SSc vasculopathy and fibrosis.

Highlights

Systemic sclerosis (SSc) is an autoimmune disease characterized by a widespread microangiopathy, autoimmunity and abnormal fibrosis of the skin and internal organs[1]
Epidermal Growth Factor Like-domain 7 (EGFL7) expression is increased in EOS diffuse cutaneous SSc (dcSSc) skin biopsies compared to Healthy Controls (HC) and downregulated in LSS dcSSc
A similar pattern of strong EGFL7 expression was detected in EOS dcSSc skin, a number of vessels were identified that displayed weaker EGFL7 immunostaining in endothelial cells (ECs) (Fig. 1B)

Summary

Introduction

Systemic sclerosis (SSc) is an autoimmune disease characterized by a widespread microangiopathy, autoimmunity and abnormal fibrosis of the skin and internal organs[1]. Expression is up-regulated during reparative angiogenesis[8,9,10] when EGFL7 modulates ECM rigidity and promotes cell migration and invasion through inhibition of mature elastic fibers[11], preventing premature vessel stabilization and allowing capillary sprouting[6]. FBs are involved in physiological and pathological angiogenesis through secretion of different ECM-related molecules, such as collagens, fibronectin, heparan sulfate and proteoglycans[15,16,17], playing an active role in the organization of the provisional matrix that supports angiogenic growth. In a recent study it was shown that the binding site of miR-126 lies within the 3′-UTR of EGFL7; and that the EGFL7 gene is down-regulated in human lung cancer cell lines[28]

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Conclusion