Background Vascular injuriy is an early event in scleroderma (SSc). It precedes fibrosis and involves small vessels (1). The cellular changes in early lesions are loss of endothelial cells, proliferation of pericytes and vascular smooth muscle cells, and precence of immune cells in the perivascular space. We recently demonstrated that IgG isolated from sera of SSc scleroderma patients induced NOX4-derived reactive oxygen species (ROS) in smooth muscle cells (SMCs) through the activation of PDGFRα receptor (2). Modulation of ROS generation led to influence migration, proliferation and collagen production in SMCs. Mammalian target of rapamycin (mTOR) is a serine/threonine kinase that acts as a major activator of cell growth and proliferation. mTOR has also been shown to play a role in vascular SMCs proliferation and when dysregulated has been implicated in vascular remodeling (3).
Objectives The aim of this study was to investigate the role of mTOR as a downstream signaling of SSc IgG in SMCs activation.
Methods IgG were isolated from sera of SSc patients by affinity chromatography. SMCs from human pulmonary artery were purchased from Lonza. Total RNA was isolated, reverse-transcribed, and quantitative real-time PCR reactions were performed using SYBR-Green Master Mix. To analyzed protein expression, cells were lysed and subjected to western blot with specific antibodies. For immunocytochemistry, cells were fixed in PFA, permeabilized and labelled with specific antibodies to be evaluated through fluorescence microscopy. Migration was performed using the scratch test.
Results SMCs were treated with various concentrations of rapamycin (1-10 nM), inhibitor of mTOR, or the same amount of vehicle (DMSO) for 48 hours, and then incubated with PDGF (15 ng/ml) or SSc IgG (200 μg/ml) for further 24 hours. Rapamycin was able to reverse the effects of PDGF and SSc IgG. Inhibition of mTOR led to a significant decrease in proliferation and migration rate (40%), and collagen expression (54%) in SMCs exposed to SSc IgG compared to untreated cells.
Conclusions Our data demonstrate that mTOR is a crucial downstream signaling of SSc IgG effects through PDGFRα pathway, and is involved in the modulation of migration, proliferation and collagen expression in SMCs. These findings support the recent observation that mTOR is largely involved in collagen regulation in SSc dermal fibroblasts (4), suggesting an important role of this serine/threonine kinase in the signaling pathways involved in the development of SSc.
Gabrielli et al. N Engl J Med 2009;360:1989-2003.
Amico et al. Ann Rheum Dis 2011;70(S3):491.
Augusto et al. C J Cardiol 2014;30:482-484.
Tamaki et al. J Dermatol Science 2014;74:251-259.
Disclosure of Interest None declared
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