the notion that even slight deviations from physiology in Notch signalling still lead to pathologic changes, might mean that the concentration of the autoantibody must be lowered to minute quantities in order to achieve a significant therapeutic effect. In this respect, it is notable that the B-cell depleting rituximab has recently shown a good effect on skin thickening in a clinical trial.(Ebata et al. 2021) It is also interesting to consider the results of myeloablative autologous hematopoietic stem cell transplantation in systemic sclerosis, which although still investigational and with several shortcomings, has resulted in good clinical responses.(Sullivan et al. 2018; Van Laar et al. 2014) In this logical framework, therapeutic plasma exchange (TPE) should lead to a significant improvement in both vascular and fibrotic outcomes of SSc by removing autoantibodies from the plasma of patients. TPE has indeed been tried repeatedly as a treatment for SSc since the 1970’s with conflicting results. Different methodological approaches and mistakes in study design have contaminated the evidence. In a recent review however,(Harris et al. 2018) when considering the total accumulated data, TPE has produced significant treatment efficacy, both for skin fibrosis and Raynaud’s phenomenon, leading to healing of digital ulcers and various other manifestations. Furthermore, the effect was long-lasting – for up to 6 months after a single session. Until now, the mechanism for the efficacy of TPE in SSc has been elusive, due to the lack of identification of the suspected circulating factor which could be causing the disease.
Additionally, a potential feedback mechanism was described involving Hey2 and 1 and Notch-4 receptors. The mechanism was not explored, namely whether Notch-1 and Notch-Notch-4 receptor genes are directly repressed by Hey2 or indirectly via another pathway (e.g., VEGF signalling). However there has never been a particular focus on whether Notch signalling is subject to a feedback mechanism involving the expression of receptors in microvascular cells. Considering the implications to the understanding of the dynamic role that the Notch pathway has on microvascular physiology and angiogenesis, this should be clarified.
Also, it is interesting to note that in a previous work,(Seguro Paula et al. 2016) myself and others have shown that the presence of megacapillaries might not be such a specific finding of SSc. In fact, they are also found, albeit heterogeneously, amongst various metabolic and genetic myopathies. It would be interesting to test the sera of patients with such conditions to assess whether the same factor exists or if there may be
a different mechanism that produces megacapillaries, which would have different functional consequences to vascular biology, considering the disparity of the clinical phenotype between the two groups of diseases.
Finally, strengthening the findings with wider-population studies will be of utmost importance, especially for the association with clinical features. A particular challenge for this necessary step will be the logistics of the assay used. Using a serum-based assay with microvascular endothelial cells in culture, RNA purification, cDNA synthesis and qRT-PCR to produce the result will not be compatible with the upscaling of the protocol. The solution might pass through two different approaches. First, the use of a proxy for the endothelium-specific overactivation of the Notch pathway which is measurable in serum.
Being the Notch pathway completely cell-based, this will be predictably hard to do.
To date, it has not been developed any method to assess the Notch status in clinical practice apart from histopathologic analysis of biopsies from tumours where the Notch signalling pathway has a role in tumorigenesis: the only report with the quantification of plasma Dll4 by ELISA showed differences between patients and controls and was associated with metastatic disease in breast cancer.(Kontomanolis et al. 2014) However, the distributions overlapped significantly precluding its discriminatory power.
Moreover, the precise nature of what was being measured was not investigated. Juxtamembrane and transmembrane proteolytic cleavage of Notch ligands by ADAMs and gamma-secretase has been described in mammals with the release of their extracellular domains.(D’Souza, Miyamoto, and Weinmaster 2008) The activity of such soluble ectodomains is still obscure but it is suggested that they could behave as a decoy for they would not be able to transduce the signal.(Aho 2004; Nikopoulos et al. 2007; Qi et al.
1999) Another possibility would be that they could be measuring Dll4-containing exosomes, which have been described as a paracrine mechanism for the tip cell to activate Notch receptors in more distant cells.(Sheldon et al. 2010) If these soluble forms would diffuse into the intravascular compartment, they could then be measurable by simple immunometric assays on serum, such as ELISA. This would have the advantage of being a relatively endothelium-specific Notch ligand, but other possibilities are worth exploring, considering the endothelial cell culture serum assay as the gold-standard.
The other approach would pass through the identification of the specific autoantibody that leads to Notch-1 activation, which could then be easily measurable in serum. Immunoprecipitation studies might
clarify this issue, and anti-PDGFR antibodies are necessarily a candidate. A major limitation of this approach would be if the identified autoantibodies would have the same antigen specificity but different epitopes, leading to different (or no) biological effects and false-positive results. It is not certain that the antibody in question would target the Notch pathway components directly, although it would be a simpler, and thus more elegant, mechanism. Some questions arise due to the known dependence on physical attachment for the Notch ligands to activate their receptors. However, several other hypotheses without the need for physical attachment exist, including the stabilization of ligand-receptor contact or the inhibition of regulating ligands such as Jagged-1/2. Single-cell suspension studies on the effect of the antibody, such as with flow cytometry, could bring some light concerning the dependence of the effect of the pathogenic antibody on cell-cell contact.
The clinical implications of these findings are several-fold. Firstly, deepening our understanding of SSc is an essential step to revolutionize the way we approach these patients. The lack of disease-modifying therapeutic options is probably a consequence of our lack of understanding of the critical steps in its aetio-pathogenesis. The identification and dissection of the effect on Notch signalling by a SSc-associated antibody may pave the way to develop better diagnostic tools. Secondly, if an upstream step of the pathophysiology is identified which is common to a majority of the heterogeneous SSc-spectrum of diseases, this might support their unification under the same clinical strategy. Thirdly, the identification of a specific upstream disturbance in endothelial cells may lead to exciting new therapeutic targets. Apart from the optimization of the immunomodulatory treatments discussed above, drugs to modulate the Notch pathway have already been developed (cf. section 1.5.2). The overall limitation to these approaches relates to the ubiquitous nature of the Notch pathway, it being involved in several physiological processes, including oncorepressive functions. This has put a cap on the therapeutic window of these drugs. However, the development of low-dose combinations of drugs have shown exciting results, with agreeable levels of tolerability and maintaining efficacy in the inhibition of the canonical Notch signalling. In fact, as discussed in chapter I, using the same dosage protocols for these drugs as the ones needed to control lymphomas might be an erroneous extrapolation, as we might be excluding useful drugs for SSc from the therapeutic armamentarium on the basis of their toxicities with higher dosages than those which would potentially be needed.
To conclude, in the present work a novel mechanism in SSc pathophysiology was unveiled (Figure 30). SSc patients produce an autoantibody which induces an overactivation of endothelial Notch-1 receptors, increasing Hey2 expression and intranuclear localization via canonical signalling. This can directly cause the vascular dysfunction, dysangiogenesis, increased perivascular cell activation and fibrosis which characterize the disease. In agreement with the predicted effects on Notch signalling, it was associated with reduced VEGF signalling, and was strongly associated with the presence of megacapillaries in the nailfold microcirculation, one of the hallmarks of the disease. Furthermore, this is a trait which is found in SSc patients irrespectively of disease subtype or specific manifestations, and apparently indifferent to the use of currently available medical therapies, constituting an appealing therapeutic target.
Figure 30 - Final theory of this thesis in graphical form.