Oxidative Stress-Dependent Activation Of Collagen Synthesis Is Induced In Human Pulmonary Vascular Smooth Muscle Cells By Scleroderma Sera and Predicts Pulmonary Vascular Disease

Francesco Boin¹, Anna Maria Posadino², Annalisa Cossu², Roberta Giordo², Ami A. Shah¹, Gaia Spinetti³, Gian Luca Erre⁴, Costanza Emanueli⁵, Giuseppe Passiu⁴, Fredrick M. Wigley¹ and Gianfranco Pintus², ¹Division of Rheumatology, Johns Hopkins University School of Medicine, Baltimore, MD, ²Department of Biomedical Sciences, University of Sassari, Sassari, Italy, ³Istitituto di Ricovero e Cura a Carattere Scientifico (IRCCS), Multi Medica, Milan, Italy, ⁴Department of Clinical and Experimental Medicine, Rheumatology Unit, University of Sassari, Sassari, Italy, ⁵Laboratories of Vascular Pathology and Regeneration, University of Bristol, Bristol, United Kingdom

Meeting: 2013 ACR/ARHP Annual Meeting

Keywords: Scleroderma

Session Information

Title: Systemic Sclerosis, Fibrosing Syndromes and Raynaud's I

Session Type: Abstract Submissions (ACR)

Background/Purpose: Vascular disease is a dominant component of Systemic Sclerosis (SSc) pathogenesis. Hypoxia and oxidative stress have been linked to endothelial injury, intimal hyperplasia and progressive vessel occlusion in SSc and the aberrant function of vascular smooth muscle cells (VSMC) has been associated with the initiation and the amplification of this process. We hypothesize that in SSc patients with pulmonary vascular disease circulating factors may drive vascular damage by exerting pro-oxidant effects involving VSMC.

Methods: The generation of reactive oxygen species (ROS) and the activation of collagen synthesis was investigated in primary Human Pulmonary Vascular Smooth Muscle Cells (HPVSMC) after exposure to sera obtained from 19 SSc patients with pulmonary vascular disease (PVD) defined by echocardiographic criteria (right ventricular systolic pressure ≥ 45 mmHg), 17 patients with no PVD and 15 healthy donors (HD). Intracellular ROS levels were assessed using the general oxidative stress indicator dichlorodihy drofluorescein diacetate (H₂-DCFDA). Collagen type I (COL1A1) promoter activity was investigated using a GFP-based lentiviral vector, while collagen protein expression was assessed by ELISA in culture supernatants. In selected experiments, pretreatment of cells with diphenyleneiodonium (DPI), a general flavoprotein inhibitor, was performed.

Results: HPVSMC treated with SSc sera from PVD patients significantly increased intracellular ROS levels (248.8 ± 27.5 Relative Fluorescence Units – RFU)compared to no-PVD subjects (155.1 ± 10.5 RFU; p=0.008) and HD (124.9 ± 2.8 RFU; p=0.002). DPI effectively prevented the raise of intracellular ROS, implicating the involvement of Flavin Oxidases (FO) in this process. The pro-oxidant stimulus provided by SSc sera was associated with a parallel increase of both COL1A1 promoter activity (PVD-SSc sera 278.1 ± 24.2 RFU vs 194.4 ± 7.9 RFU; p=0.01 in no-PVD and 164.9 ± 5.1; p=0.002 in HD) and collagen protein expression in HPVSMC. Also this effect was abrogated by DPI pretreatment.

Conclusion: This study provides evidences that sera from SSc patients with pulmonary vascular disease drive pro-fibrotic responses mediated by Flavin Oxidases-derived ROS production in HPVSMC and suggests that antioxidant therapies should be further explored in the treatment of SSc vascular disease.

Disclosure:

F. Boin,
None;

A. M. Posadino,
None;

A. Cossu,
None;

R. Giordo,
None;

A. A. Shah,
None;

G. Spinetti,
None;

G. L. Erre,
None;

C. Emanueli,
None;

G. Passiu,
None;

F. M. Wigley,
None;

G. Pintus,
None.

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