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Role of the notch signalling in the endothelial dysfunction of systemic sclerosis
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Introduction: Systemic sclerosis (SSc) is a chronic debilitating disease for which current treatments have
very limited efficacy. It affects individuals of all ages, especially young females, and results in a high
morbidity and premature death related to disseminated vascular dysfunction and tissue fibrosis. While its
pathophysiology is still incompletely understood, current knowledge focuses mainly on the general
mechanisms of fibroblast activation and a scarcely described endothelial dysfunction with microanatomical
changes, including extreme capillary dilatations (megacapillaries).
The Notch pathway is a conserved intercellular signalling mechanism which operates in virtually all
organs and tissues during morphogenesis and adult life during health and disease. Its role in vascular biology
is crucial for the establishment and maintenance of a functional microvascular capillary network. At the
same time, it mediates the interaction between endothelial cells and perivascular cells, including pericytes
and fibroblasts. The artificial overactivation of the Notch pathway in endothelial cells has been shown to
produce a set of derangements of normal physiology which overlaps significantly with the SSc
pathophysiology.
This thesis was developed from the hypothesis that a previously uncharacterized serum factor exists in
SSc patients which is able to activate the endothelial Notch signalling.
Methods: Changes in the Notch pathway components (receptors, ligands and target genes) and its
activation status in microvascular endothelial cells were studied in vitro after exposure to serum derived
from SSc patients (n=22) or controls (n=10), through RT-PCR, immunofluorescence, and western blot.
Time- and concentration-dependence of the effect were analysed by using various serum concentrations
and varying incubation time. Immunoglobulin G was purified from the sera and utilized to further
characterize the serum factor. The impact on the expression and activation of VEGF receptors was also
assessed by RT-PCR and western blot. An association of the Hey2 expression levels (one of Notch target
genes) with the clinical characteristics, current medication and microvascular changes assessed by nailfold
videocapillaroscopy of the patients was also tested.
Main findings: SSc serum induced a reproductible overexpression and increased intranuclear
localization of the Notch target gene Hey2 in microvascular endothelial cells in culture, when compared
with control serum. This effect started between 4h to 6h post-exposure to serum, peaked at 8h, and entered
a plateau phase from 12h until at least 24h. The effect was positively correlated with the serum
concentration used, being detectable at concentrations as low as 1,9%. This was associated with an increased
quantity and intranuclear localization of the activated form of the receptor Notch-1, and the effect was at
least partially reversed by DAPT, a gamma-secretase inhibitor (an enzymatic complex necessary for the
activation of Notch receptors). The use of purified immunoglobulin G from SSc serum reproduced the
changes in Hey2 expression. It was also associated with decreased VEGF signalling, which is the predicted
effect of an activated Notch pathway. The increase in Hey2 expression did not differ between SSc clinical
phenotype or pattern of organ involvement, known autoantibodies, nor with current medications. The
degree of overexpression of Hey2 induced by the serum of each subject was strongly associated with the
presence of megacapillaries in nailfold videocapillaroscopy.
Conclusions: A novel pathophysiological mechanism of SSc was uncovered, whereby a previously
unidentified autoantibody exists in SSc patients, independently of clinical phenotype, that is able to increase
canonical signalling in microvascular endothelial cells through the receptor Notch-1 and induce an
overexpression and increased intranuclear localization of Hey2. These changes were associated with an
impaired VEGF signalling. These results also suggest that Hey2 overexpression is subsequently related to
the development of megacapillaries. This might correspond to an upstream step in SSc pathogenesis which
can lead to new therapeutic targets and diagnostic tools.
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Palavras-chave
Systemic sclerosis