SAT0293 EXOSOMES DERIVED FROM PLASMA OF SYSTEMIC SCLEROSIS (SSC) PATIENTS AND FROM SSC CULTURED FIBROBLASTS CONTAIN PRO-FIBROTIC MIRNA SIGNATURES AND COULD INDUCE MYOFIBROBLAST DIFFERENTIATION IN VITRO
Authors: C. Corallo, M. Cutolo, S. Soldano, E. Selvi, F. Bellisai, N. Giordano
Journal: Annals of the Rheumatic Diseases (2020)

Background: Exosomes generated great resonance in the last few years due to their important roles in different biological pathways and diseases, including systemic sclerosis (SSc) (1). They are lipid-like nanovesicles containing biomarkers, such as proteins, lipids, macromolecules and nucleic acids, including microRNA (miRNA) (2). Exosomes are implicated in intercellular communication by fusing and releasing their cargo into the target cells (3). Objectives: In the present study, we evaluated the potential of exosomes deriving from plasma of SSc patients or generating from cultured SSc fibroblasts to drive the fibrotic signaling in the disease. Methods: Exosomes were isolated from plasma of n=10 SSc patients and from n=10 control subjects. Exosomes were also purified from cell culture supernatants of SSc fibroblasts and of control fibroblasts. Exosome size and concentration were assessed by Nanosight Particle Tracking Analysis (NTA) and by transmission electron microscopy (TEM). The content of anti-fibrotic (let-7a, 146a, 200a, 223a) and pro-fibrotic (150, 155) miRNAs was assessed in all the plasma-derived and cell culture-derived exosome populations by semiquantitative real time PCR. Finally, isolated exosomes were used to stimulate control dermal fibroblasts in culture. Gene expressions (COL1A1, ACTA2 and TAGLN) were assessed by quantitative real time PCR (qRT-PCR) and protein levels (type-I-collagen, α-SMA and SM22) by immunofluorescence (IF). Results: Exosomes isolated from SSc plasma samples showed higher concentration (3.3x10 10 ±1.1x10 10 particles/mL) compared to those isolated from control plasma ones (1.5x10 10 ±0.4x10 10 particles/mL) (p&lt;0.01). The exosome size did not differ between SSc and control plasma samples and ranged from 50nm to 150nm. Similar results were obtained with exosomes generated from fibroblast cultures: the concentration was higher in SSc fibroblasts (1.1x10 10 ±0.2x10 10 particles/mL) than in control ones (0.4x10 10 ±0.1x10 10 particles/mL) (p&lt;0.05) with no significant differences in size distribution. The content of all anti-fibrotic (let-7a, 146a, 200a, 223a) miRNAs was decreased in exosomes coming from both SSc plasma samples and from SSc fibroblasts with respect to control plasma samples (p&lt;0.05) and to control fibroblasts (p&lt;0.05). On the contrary, the pro-fibrotic (150, 155) miRNAs were significantly upregulated in exosomes deriving from SSc plasma samples and from SSc fibroblasts, with respect to control plasma samples (p&lt;0.05) and to control fibroblasts (p&lt;0.05). Finally, only exosomes coming from SSc plasma samples or SSc fibroblast cultures were able to induce pro-fibrotic gene (COL1A1, ACTA2 and TAGLN) and protein (type-I-collagen, α-SMA and SM22) expression in control fibroblasts. No pro-fibrotic induction was seen in presence of exosomes isolated from control plasma samples or control fibroblast cultures. Conclusion: This study demonstrates that plasma from SSc patients contains higher concentration of exosomes compared to plasma from control subjects and SSc-derived exosomes contain specific pro-fibrotic miRNA signatures that can induce myofibroblast differentiation in vitro . These results suggest that exosomes could be fibrotic drivers towards non-affected areas in vivo , and they might represent novel targets for precision medicine treatments in SSc. References: [1]Zhu T, Wang Y, Jin H, Li L. The role of exosome in autoimmune connective tissue disease. Ann Med. 2019 Mar;51(2):101-108. [2]Wermuth PJ, Piera-Velazquez S, Rosenbloom J, et al. Existing and novel biomarkers for precision medicine in systemic sclerosis. Nat Rev Rheumatol. 2018 Jul;14(7):421-432. [3]Colletti M, Galardi A, De Santis M, et al. Exosomes in Systemic Sclerosis: Messengers Between Immune, Vascular and Fibrotic Components? Int J Mol Sci. 2019 Sep 4;20(18). pii: E4337. Disclosure of Interests: Claudio Corallo: None declared, Maurizio Cutolo Grant/research support from: Bristol-Myers Squibb, Actelion, Celgene, Consultant of: Bristol-Myers Squibb, Speakers bureau: Sigma-Alpha, Stefano Soldano: None declared, Enrico Selvi: None declared, Francesca Bellisai: None declared, Nicola Giordano: None declared