Discussion

Independently of disease state, serum induced a strong overexpression of Hey2 on microvascular endothelial cells in culture. This effect has been previously described (Wöltje, Jabs, and Fischer 2015) and has been attributed to bone morphogenetic proteins (BMPs) content in serum and cell culture media, which increase Hey1 and Hey2 expression through a non-canonical Alk1-dependent manner. Indeed, a

Figure 19 - Effect of the gamma-secretase inhibitor DAPT on the effect of SSc serum on Hey2 expression.

Results expressed as geometric mean and standard deviation. ** p<0.01 from a Student’s t test.

* ^** *

**

critical mechanism for the oscillatory nature of Notch signalling in endothelial cells is precisely the BMP signalling, specifically BMP-9 and -10.(Beets et al. 2013) These molecules activate heterotetrameric cell surface receptors composed of two pairs of different serine/threonine kinase receptors of different families,(R. N. Wang et al. 2014) including Alk1. Alk1 activation generates phospho-SMAD1/5/8 complexes, which are able to directly activate the transcription of Hey1 and Hey2 in endothelial cells.

Although serum BMP-9 or -10 concentrations in systemic sclerosis have not been determined, studies in SSc have found impaired BMP signalling by increased TGFβ-induced recycling of BMP receptors(Gilbane et al. 2015) or epigenetic repression of its expression.(Y. Wang and Kahaleh 2013) Type 2 BMP receptors (BMP-RII) haploinsufficiency is a known aetiology for primary pulmonary hypertension, which share many morphological similarities to the pulmonary vasculature defects found in SSc (although in SSc no inactivating mutations have been found in BMP-RII(Morse et al. 2002) or Akt1(Selva-O’Callaghan et al.

2007)). Overall, the bulk of these data suggests that BMP signalling might be down-regulated, not up-regulated. However, increased circulating BMPs concentrations in SSc in response to a feedback mechanism due to low BMP signalling is still a possibility, which in this case would produce heightened BMP signalling in normal endothelial cells in culture, exaggerating the unspecific response to serum in SSc.

However, the time dynamics do not match: in (Wöltje, Jabs, and Fischer 2015), serum elicited a peak response in Hey1 and Hey2 expression levels at 2h exposure, which had waned completely 6h post-exposure. This is in stark contrast to the results obtained in this work, where control serum induced a peak Hey2 at 3h (the 2h time point was not determined), but persisted until almost 8h post-exposure, whilst SSc serum elicited an effect which lasted at least 24h. This suggests that the mechanism underlying the difference between control and SSc serum is unrelated to the one described for BMP-9/-10 in serum.

Alternatively, other non-canonical activating pathways of Notch target genes would be reasonable candidate mechanisms. A VEGF-dependent activation of Foxc1/2 transcription factors, which then directly control Dll4 and Hey2, has been postulated as a mechanism for VEGF-A - induced Dll4 overexpression.(Hayashi and Kume 2008) However, Dll4 expression levels did not differ between the control and SSc sera, and there was no correlation between Dll4 and Hey2 expression. Furthermore, the receptor Notch-1 is also reported to be over-expressed by VEGF-A,(Gao et al. 2013) and these results show the opposite.

To settle the question whether the effect would be mediated by a canonical or non-canonical Notch signalling, two approaches were undertaken: comparing the SSc-driven expression profile of the Notch components under analysis with the artificial modulation of the Notch pathway; and comparing quantities of the activated form of Notch receptors between experimental conditions.

In this experimental system, an activated canonical Notch signalling resulted in a selective overexpression of Hey2 amongst the target genes, which is the same pattern induced by SSc serum.

Conversely, by inhibiting the canonical Notch signalling, only the target gene Hes1 was repressed when compared to control cells. This suggests that Hes1 expression is dependent on a baseline Notch signalling but is not subject to modulation by a high Notch signalling strength. Conversely, these results suggest that Hey2 is sensitive to maximal increases in Notch signalling but is not dependent on a baseline Notch signalling strength. This concept is not new for Notch signalling dynamics: Dll4 has been shown to depend on a baseline Notch signalling to be expressed,(Caolo et al. 2010) even though it is repressed by a high Notch signalling strength (due to a downstream regulation of VEGF signalling, one of the major inducers of Dll4 expression in endothelial cells). In support of this notion, Dll4 was indeed repressed by DAPT, indicating its dependence upon a baseline level of Notch signalling.

Furthermore, canonical Notch signalling repressed the expression of Notch-1, in a similar fashion to the effects of SSc serum. This reinforces the hypothesis that Notch receptors -1 and -2 are subject to a negative feedback mechanism upon canonical activation of the pathway. In fact, canonical Notch inhibition by DAPT increased the expression of both receptors Notch-1 and Notch-4.

The discrepancy between the patterns produced by SSc serum and by an artificially induced canonical Notch signalling concern mainly Notch-4, which proved to be a reproducible effect of SSc serum.

Although the mechanism for this difference was not pursued, one explanation might reside on an overlap of the effects of inflammatory mediators, such as tumour necrosis factor alpha (TNFα). TNFα signalling has been shown to repress the expression of Notch-4 but not of Notch-1 in endothelial cells.(Quillard and Devallie 2010; R. Sainson et al. 2008) Interestingly, the other major effect of TNFα in endothelial cells is to increase Jagged-1 expression, which could also explain the significant, although not reproducible, increase of Jagged-1 with SSc serum.

In conclusion, the artificial overactivation of the canonical Notch signalling reproduced the changes in Hey2 and Notch-1 seen with SSc serum, providing circumstantial evidence that a canonical activation of the Notch receptors might be mechanistically involved in the effect seen with SSc serum.

The fact that the canonical Notch signalling was involved in the response to SSc serum was further confirmed by the increased total quantities and increased intranuclear localization of the activated form of Notch-1. As no increase in any receptor or ligand was induced by SSc serum – apart from the non-reproducible increase in Jagged-1, which is expected to hamper Notch signalling – it is unlikely that the activation of the Notch receptor should be a consequence rather than a cause for the gene expression changes, namely Hey2. Further support for this notion comes from the fact that the canonical Notch signalling inhibitor DAPT inhibited the effect of SSc serum on Hey2. Although it might not be clear if the effect of SSc serum is completely or partially dependent on the canonical Notch signalling, there were no significant differences on Hey2 expression levels between the unstimulated control cells and the cells exposed to SSc serum and DAPT, which suggests that the inhibition was virtually complete. As a final remark, it is noteworthy that the activated forms of other Notch receptors were not measured. At least for 4 and 2, which are, in order of relevance, the most important Notch receptors after Notch-1 in microvascular endothelial cells, one cannot exclude the concomitant activation of these receptors alongside Notch-1.

In conclusion, SSc serum seems to produce a lasting effect on the canonical Notch signalling in endothelial cells, through the activation of Notch-1 which then leads to the overexpression of Hey2.