Role of the notch signalling in the endothelial dysfunction of systemic sclerosis

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ROLE OF THE NOTCH SIGNALLING IN THE ENDOTHELIAL DYSFUNCTION OF SYSTEMIC SCLEROSIS

FILIPE MANUEL PEREIRA SEGURO DE OLIVEIRA PAULA

A thesis submitted in partial fulfilment of the requirements for the Doctoral Degree in Medicine, in the specialty Clinical Research

at Faculdade de Ciências Médicas | NOVA Medical School of NOVA University Lisbon

March, 2022

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ROLE OF THE NOTCH PATHWAY IN THE VASCULAR DYSFUNCTION OF SYSTEMIC SCLEROSIS

Filipe Manuel Pereira Seguro de Oliveira Paula

Supervisor:

José Delgado Alves, MD, PhD Associate Professor of Medicine at NOVA Medical School

A thesis submitted in partial fulfilment of the requirements for the Doctoral Degree in Medicine, in the specialty Clinical Research

March, 2022

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to Cláudia, my love, my light

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Abstract

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

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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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Resumo

Introdução: A esclerose sistémica (ES) é uma doença crónica e debilitante, para a qual a terapêutica existente tem uma eficácia muito limitada. Afectando indivíduos de qualquer idade, incide maioritariamente em mulheres jovens e resulta em elevada morbilidade e morte prematura, relacionadas com disfunção vascular e fibrose tecidular disseminadas. A sua fisiopatologia é ainda pouco clara, concentrando-se o conhecimento actual fundamentalmente em mecanismos gerais da activação fibroblástica e numa disfunção endotelial parcamente caracterizada, com alterações microanatómicas incluindo exuberantes dilatações capilares denominadas megacapilares.

A via de sinalização Notch é um mecanismo de comunicação intercelular evolutivamente conservado, que está presente virtualmente em todos os órgãos e tecidos, tanto durante a morfogénese como na vida adulta, no estado saudável e na patologia. O seu papel na biologia vascular é crucial para o estabelecimento e manutenção de uma rede capilar microvascular funcional. Neste contexto, medeia e regula também a interacção entre o endotélio e células perivasculares, como pericitos e fibroblastos. Está já demonstrado na literatura que a sobreactivação artificial da sinalização Notch em células endoteliais resulta numa série de distúrbios da fisiologia vascular normal que recapitulam em larga medida a fisiopatologia da ES.

Esta tese foi desenvolvida tendo por base a hipótese de que existirá nos doentes com ES um factor sérico ainda não caracterizado que produz uma activação da sinalização Notch no endotélio vascular.

Métodos: Analisaram-se diferenças na expressão genética de componentes da via Notch (receptores, ligandos e genes alvo) e respectivo estado de activação em células endoteliais da microcirculação in vitro, após exposição a soro de doentes com ES (n=22) ou de controlos (n=10), através de qRT-PCR, imunofluorescência e western blot. Averiguou-se a dependência do efeito sobre o tempo e sobre a concentração através da utilização de várias concentrações de soro e diferentes tempos de incubação.

Utilizou-se imunoglobulina G purificada a partir dos diferentes soros para caracterizar o factor sérico. O impacto na expressão e activação dos receptores endoteliais de VEGF foi também avaliado por RT-PCR e western blot. Foi testada a associação entre os níveis de expressão de Hey2 (um dos genes alvo da via) e

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várias características clínicas dos doentes, terapêutica actual e alterações microvasculares identificadas em videocapilaroscopia do leito ungueal.

Resultados principais: O soro de doentes com ES induziu de forma reprodutível a sobre-expressão e o aumento da localização intranuclear de Hey2. Este efeito teve início entre as 4h e as 6h de exposição ao soro, teve um pico às 8h, e entrou em plateau a partir das 12h, mantendo-se pelo menos até às 24h. O efeito correlacionou-se positivamente com a concentração do soro, sendo detectável mesmo com concentração de 1,9%. Associou-se também a um aumento da quantidade e da localização intranuclear da forma activada de Notch-1, e o efeito foi pelo menos parcialmente revertido com a administração de DAPT, um inibidor da gama-secretase (complexo enzimático necessário à activação dos receptores Notch).

A utilização de imunoglobulina G purificada a partir de soro de doentes com ES reproduziu as alterações na expressão de Hey2. Esteve também associado a uma redução da sinalização VEGF, que corresponde ao efeito previsível de uma sobreactivação da via Notch. O nível de expressão de Hey2 não diferiu entre fenótipos clínicos da ES, órgãos envolvidos, positividade para autoanticorpos previamente conhecidos, ou com a terapêutica. O grau de sobreexpressão de Hey2 induzido pelo soro de cada sujeito demonstrou uma forte associação com a presença de megacapilares na videocapilaroscopia.

Conclusões: Foi caracterizado um novo mecanismo fisiopatológico da ES, em que um autoanticorpo não previamente caracterizado existe nos doentes independentemente do fenótipo clínico, que resulta num aumento da sinalização Notch canónica através do receptor Notch-1 em células endoteliais da microcirculação, induzindo uma sobre-expressão e localização intranuclear de Hey2. Estes resultados sugerem também que a sobre-expressão de Hey2 está subsequentemente relacionada com níveis reduzidos de sinalização VEGF e com a ocorrência de megacapilares. Isto poderá corresponder a um passo a montante na fisiopatologia da ES, que poderá levar a novos alvos terapêuticos e a novas estratégias diagnósticas.

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Figure 17 - Western blot of protein extracts from microvascular endothelial cells exposed to control or SSc serum, stained for the activated form of Notch-1 ... 51 Figure 18 - Immunofluorescence for the activated form of Notch-1 of microvascular endothelial cells exposed to control or SSc serum ... 51 Figure 19 - Effect of the gamma-secretase inhibitor DAPT on the effect of SSc serum on Hey2 expression ... 52

Figure 20 - SDS-PAGE of immunoglobulin G after extraction from serum and dialysis, stained with Coomassie blue ... 56 Figure 21 - Effect of immunoglobulin G derived from control or SSc sera on Hey2 expression in microvascular endothelial cells ... 57

Figure 22 - Comparison of Hey2 expression levels of microvascular endothelial cells after exposure to SSc serum, according to autoantibody positivity ... 57 Figure 23 - Expression of different VEGF receptors on microvascular endothelial cells after being exposed for 12h to serum or immunoglobulin G from SSc patients ... 61

Figure 24 - Quantification of phosphorylated VEGFR-2 by western blot from lysates of microvascular endothelial cells exposed for 12h to control serum or SSc serum, and to immunoglobulin G derived from controls or SSc patients ... 62 Figure 25 - VEGF-A concentration in serum from controls and SSc patients used in the previous experiments, quantified by ELISA... 62

Figure 26 – Hey2 expression levels were non-significantly associated with increasing severity of interstitial lung disease (ILD) ... 66

Figure 27 - Hey2 expression level induced by the serum of SSc patients with and without each finding on nailfold videocapillaroscopy ... 68 Figure 28 - Results from a logistic regression model for the prediction of megacapillaries by Hey2 expression level ... 69 Figure 29 - Hey2 expression level induced by the serum of SSc patients currently taking or not taking each specific immunomodulator or vasoactive drug ... 70 Figure 30 - Final theory of this thesis in graphical form. ... 77

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List of tables

Table 1 - Primer sequences used in the qRT-PCR experiments. ... 24

Table 2 - General demographic variables of control and SSc subjects ... 31

Table 3 - SSc clinical and laboratory characteristics of SSc patients included in the analysis ... 32

Table 4 - Nailfold videocapillaroscopic findings in controls and SSc patients ... 33

Table 5 - Results from the unadjusted linear regression model for the prediction of the expression level of each gene based on disease status, and from the multivariate linear regression model adjusted for age ... 34

Table 6 - Inter-assay group-wise reproducibility of gene expression changes ... 39

Table 7 - Gene expression changes of components of the Notch pathway after exposure to SSc serum for 24h ... 43

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Abbreviations

ACEi Angiotensin converting enzyme inhibitors

ADAM A disintegrin and metalloproteinase

B2M Beta-2-microglobulin

bHLH basic helix-loop-helix

BMP Bone morphogenetic protein

BMP-RII Bone morphogenetic protein receptor II

BSA Bovine serum albumin

cDNA Complementary DNA

CSL CBF-1/RBP-Jk, Suppressor of Hairless [Su(H)],

Lag-1

DAPI 4′,6-diamidino-2-phenylindole

DAPT N-[N-(3,5-difluorophenacetyl)-L-alanyl]-S-

phenylglycine t-butyl ester

Dll1 Delta-like 1

Dll4 Delta-like 4

DMSO Dimethylsulphoxide

DSL Delta/Serrate ligands

EMT Epithelial-to-mesenchymal transition

EndoMT Endothelial-to-mesenchymal transition

FBS Foetal bovine serum

Fc Crystallizable fraction

GERD Gastro-oesophageal reflux disease

GSIs Gamma-secretase inhibitors

Hh Hedgehog

HPRT-1 hypoxanthine phosphoribosyl-transferase 1

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HRP Horseradish peroxidase

ILD Interstitial lung disease

mAbs monoclonal antibodies

MAML Mastermind-like proteins

NICD Notch intracellular domain

NRP-1 Neuropillin-1

OSA Obstructive sleep apnoea

PBS Phosphate-buffered saline

PBS-T Phosphate-buffered saline with 0.1% Tween-20

PDGF Platelet-derived growth factor

PDGFR Platelet-derived growth factor receptor

qRT-PCR Quantitative real-time polymerase chain reaction

rhDll4 Recombinant human delta-like 4

SDS Sodium dodecyl sulphate

SDS-PAGE sodium dodecyl sulphate polyacrylamide gel

electrophoresis

SSc Systemic sclerosis

TBS Tris-buffered saline

TBS-T Tris-buffered saline with Tween-20 0.1%

TEMED Tetramethylethylenediamine

TGF-β Transforming growth factor β

TNF-α Tumour necrosis factor α

TPE Therapeutic plasma exchange

VEGF-A Vascular endothelial growth factor A

VEGFR Vascular endothelial growth factor receptor

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Acknowledgments

“If I have seen further, it is by standing on the shoulders of Giants”, Sir Isaac Newton said. My giant was Prof. José Delgado Alves. This thesis follows an 8-year long journey, during which he has repeatedly opened many doors and pointed the way for me. To him I owe my scientific career and know-how. Thank you for believing in me.

I would like to express my most sincere gratitude to Dr. Marta Carapeto Amaral, for having pushed me in the right direction when I did not know what it was, and for all the confidence and support.

I would like to thank Prof. Emília Monteiro and Prof. Lino Gonçalves for all the strength and continuing dedication to advance science in Portugal. In this respect, the organization of the Clinical Scholar Research Program in Portugal of the Harvard Medical School was pivotal for me.

I wish to extend my special thanks to Prof. Leonor Parreira and Prof. António Cidadão, for having introduced me to translational science and the world of Notch signalling.

I am indebted to Dr. Frederico Baptista, for all the support and friendship.

I would also like to thank the medical staff of the Internal Medicine Department 4 of Fernando Fonseca Hospital, the place where I have learnt how to be a physician and who have always supported me in my endeavours. Thank you also to the medical staff of the Intensive Care Unit of Cascais Hospital, especially to Dr. Armindo Ramos, for all continued support and understanding during these years.

My sincere gratitude to my closest friends, Nuno, La Feria, Inês, Renata, Mafalda, Ângela, Rita, for keeping up with me and for still being there even when I disappear.

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To my parents, Vítor and Maria do Carmo, I can only say that without everything you gave me, I would not be anywhere near here. If I have pursued a path of idealism and love, it is because you taught me to.

Most importantly, I am grateful that during these 8 years my wife Cláudia has managed to stand up to all the challenges and the enormous personal and familial sacrifices my struggle with science has brought.

It was beside her that I have come up with the idea for this thesis, and it was beside her that I have finished it. This thesis is also yours. To my children, Pilar and António, I can only be grateful that they were always able to make me certain that they still loved me as if I was never absent.

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Scientific articles used in the preparation of this thesis

Seguro Paula F, Delgado Alves J. The role of the Notch pathway in the pathogenesis of systemic sclerosis: clinical implications. Expert Rev Clin Immunol. 2021;17:1257–1267.

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I. BACKGROUND

1.1 Introduction to systemic sclerosis (SSc)

Systemic sclerosis (SSc) is a chronic debilitating disease, with an estimated prevalence of around 50/100000, which involves multiple organs and often leads to premature death. It affects individuals of all ages, but the majority are females, diagnosed between 20 and 40 years of age. SSc is characterized by immunological dysregulation, vasculopathy and widespread fibrosis.(Clements and Furst 2004)

The immunological deregulation in SSc is characterized by a mild increase of the inflammatory response both at the local level (with inflammatory infiltrates in all affected organs and the persistence of an inflammatory milieu)(Van Praet et al. 2011) and at the systemic level, with increased concentrations in serum of several inflammatory mediators.(Gabrielli, Avvedimento, and Krieg 2009) Autoimmunity is also clearly involved. Several autoantibodies have been described in SSc, many of which are fairly specific for the disease and are correlated with disease subtypes and risk of organ involvement. Vasculopathy is present since the early stages, with the almost universal presence of Raynaud’s phenomenon, an exaggerated cold- induced vasospasm which can lead to ischemic damage and necrosis, especially of the digits. Endothelial cells are activated and increasingly apoptotic, leading to an enhanced thrombogenic risk.(Kavian and Batteux 2015; Asano and Sato 2015) Endothelium activation is associated with evident microanatomical vascular changes, such as media hyperplasia with proliferation of myofibroblasts and vascular smooth muscle cells in arterioles, and dilatations, microhaemorrhages and thrombosis in capillaries. Capillaries are reabsorbed and avascular, hypoxic regions appear. Normal neoangiogenesis does not take place, leading to aberrant capillary networks which are not entirely functional. In the lung, these overall disruption leads to pulmonary capillary hypertension which is the main cause of death.(Karassa and Ioannidis 2008)

The final manifestation which characterizes SSc is widespread fibrosis. Increased numbers of fibroblasts are evident in many tissues and heightened activation and deposition of extracellular matrix components

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takes place, especially of collagen type I and reticulin, compromising organ function.(Wei et al. 2011) Skin involvement is the most well described (hence the term “scleroderma” for which the disease was initially named) with a loss of elasticity and progressive thickening. These manifestations can become serious enough to prevent mouth opening, compromise feeding, and hinder the normal function of the hands and fingers. The lung is frequently involved, with extensive fibrosis leading to chronic respiratory failure, another main cause of death. The gastrointestinal tube can also be severely affected, with loss of peristalsis and absorptive capacity, which leads to chronic diarrhoea and the requirement of parenteral feeding in many patients.

The overall risk of death has been evaluated by a meta-analysis as almost 4-fold when compared to the general population.(Elhai et al. 2012) Therapeutic interventions have been directed to vascular spasm and to immunological activation, and only more recently to fibrosis. However, the modest efficacy of such interventions has been reflective of the current lack of understanding of the underlying pathophysiology, which remains elusive in many aspects. In fact, mortality associated with SSc has remained practically unchanged since the 1970’s.(Elhai et al. 2012)

1.2 Introduction to Notch signalling

Notch signalling is an ancient pathway in the evolution of living organisms. Its origins revolve around the emergence of multicellular life forms and is present in all metazoans. This reflects its importance to life:

it is a system used to coordinate the actions of neighbouring cells, both during development and adult life, and acts through direct contact between cells. During somitogenesis, where a spatial pattern emerges from an undifferentiated group of cells, Notch signalling is a central player in defining distinct cellular territories.(Takahashi et al. 2003) Pivotal roles have been described in many stages of development in almost every tissue and organ.(Lai 2004) In this regard, one can classify its function into two recognizable strategies:

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lateral inhibition and inductive signalling (Figure 1). Regarding lateral inhibition, Notch signalling acts to ensure that two adjacent cells will have different

behaviours or differentiation programs. In neural development, for example, each cell with its neural differentiation program set in place will express Notch ligands in the membrane, which will in turn activate Notch receptors and subsequent signalling in all neighbouring cells, blocking their differentiation into neurons and directing them to be glial cells.(Lai 2004) In turn, Notch activation in neighbouring cells will down-regulate the expression of Notch ligands, preventing them to activate Notch receptors in the central cell. In this way, it is ensured that

neurons are covered by glial cells. In inductive signalling, Notch signalling in one cell will induce the expression of ligands, which will engage with Notch receptors in the neighbouring cells, activating the Notch pathway and spatially perpetuating the loop. (Takahashi et al. 2003)

It is easily understood from the characteristics stated above that for it to work, its signalling must be restricted to cell-cell interactions. Accordingly, Notch ligands are all transmembrane proteins – every ligand needs to be “docked” onto a surface to be able to activate the respective receptors.(Chillakuri et al. 2012) In mammals there are 4 receptors (Notch 1-4) and 5 ligands (Delta-like (Dll)-1, -3 and -4, and Jagged- 1 and -2). During cell-cell contact (Figure 2), the ligands interact with the extracellular domains of the single-pass transmembrane receptor protein and generate a “pulling force” from the signalling cell through endocytosis of the ligand region, which exposes the receptor molecule to a proteolytic cleavage event (S2) by ADAM-2 or ADAM-17 metalloproteinases. Subsequently, a final proteolytic cleavage (S3) occurs in the cytoplasmic surface of the plasma membrane by the gamma-secretase complex, liberating the intracellular domain (Notch Intracellular Domain, NICD) into the cytoplasm. It then migrates to the

Figure 1 - Conceptual framework for the two modes of action of the Notch signalling pathway in development and adult life: lateral inhibition (top figure) and inductive signalling (bottom figure).

Baseline Notch signalling High Notch signalling Low Notch signalling

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nucleus, where it joins a protein complex together with a series of transcription factors collectively known as CSL (CBF-1/RBP-Jk, Suppressor of Hairless [Su(H)], Lag-1) and Mastermind-like (MAML) proteins,(Gordon, Arnett, and Blacklow 2008) which will bind to and activate the transcription of a series of target genes, which vary immensely with the cell type and context. The most well described target genes of the Notch pathway are Hairy and Enhancer-of-split-related basic helix-loop-helix (bHLH) transcription factors from two related gene families: Hes and Hey (Iso, Kedes, and Hamamori 2003), albeit not exclusively. This activation mechanism is called the canonical Notch pathway. There are several non- canonical pathways described, wherein other signalling pathways are able to activate the same gene repertoires, interacting directly with components of the Notch pathway.(Ayaz and Osborne 2014;

Andersen et al. 2013; Layden and Martindale 2014; Jin et al. 2013)

Figure 2 - Overview of the Notch signalling pathway. Notch receptors are transmembrane heterodimeric proteins produced by the cleavage of a single chain precursor in the Golgi apparatus by a furin-like protease (S1 cleavage). Then it is translocated to the plasma membrane, where it may interact with ligands, which are membrane bound proteins in a neighbour cell. Receptor activation and release of the downstream signalling molecule Notch Intracellular Domain (NICD) requires two more cleavage steps. The first (S2) is the critical regulatory step. The cleavage site is burrowed in a pocket by the tertiary structure of the receptor protein, which is exposed by a pulling mechanical force performed by the signal-sending cell through endocytosis of the ligand bearing plasma membrane region. Only then will it be available to S2 cleavage by ADAM metalloproteinase. The third cleavage by the gamma secretase complex releases NICD, which translocates to the nucleus and interacts with CSL and MAML to activate target genes.

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However, the artificial categorization of the behaviour and role of the Notch pathway in development and adult life is very difficult. The dynamics of receptor/ligand modulation, the intracellular pathways that follow and the target gene repertoire which is inducted are highly dependent on cell type and context.

Furthermore, target gene responses cannot be entirely predicted based on specific ligand-receptor combinations. Although specific receptors and ligands are characteristic of certain cell types in certain defined physiological circumstances, it has not been shown to date that different ligands elicit conceptually different responses from the same receptor or even that different receptors generate different responses. It is plausible that the differential effects of specific ligand-receptor combinations in each specific cell type and context is a consequence of the association with different intracellular molecules, as well as being based on its interaction with many other ubiquitous signalling pathways, thus increasing the level of complexity.(Kopan 2010)

One final aspect that needs recognition about the Notch pathway is its capability to be “fine-tuned”.

It is noteworthy that there is no signal amplification. One ligand activates one receptor (after which it will be internalized), which will generate one NICD molecule, which in turn will associate with one nuclear CSL complex and activate target genes. This positions the Notch pathway as a unique tool for the cell to react in specific ways to environmental stimuli with different intensities,(Shen, Huang, and Wang 2021) and to be easily regulated.

1.3 Notch in fibrogenesis

1.3.1 Fibroblast activation and differentiation

Fibrogenesis is a common end-result of chronic inflammation.(Rockey, Bell, and Hill 2015) It probably evolved as a means of increasing the tensile strength of the tissues torn apart by trauma or to contain chronic inflammatory stimuli which could not be resolved.(Thannickal et al. 2014) Many of the most debilitating features of SSc are direct results of pathologically exaggerated and widespread fibrosis, such as interstitial lung disease and skin fibrosis. Hence, most of the current knowledge on its

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pathophysiology relates to the mechanisms involved in fibroblast differentiation, activation and deposition of extracellular matrix.

The Notch pathway has a central role in this regard, which is now well established in the literature.(Condorelli et al. 2021; Zmorzyński et al. 2019; Beyer and Dees 2013; B. Hu and Phan 2016;

Beyer and Distler 2013) The skin in SSc patients has an increased expression of the Notch ligand Jagged- 1 and a diffuse activation of Notch signalling is observed, namely in resident fibroblasts, reflected by NICD accumulation in the nucleus.(Dees, Tomcik, et al. 2011) Indeed, the activation of Notch signalling in fibroblasts, usually by Jagged-1/Notch-3 interactions, involves at least Hes2 as the main target gene effector, inducing their differentiation into myofibroblasts which are the main effectors of fibrogenesis. Then, an increased deposition of collagen types I and III ensues.(Dees, Tomcik, et al. 2011) Interestingly, the pharmacological inhibition of ADAMS-17 (responsible for the first cleavage [S2] of the Notch receptor upon its activation) results in a regression of fibrosis in an SSc animal model.(Kavian et al. 2010)

Even though the contribution of Notch signalling to fibroblast activation and heightened fibrogenesis seems to be clear, it should not be analysed in an isolated fashion nor should that mechanism be considered specific for SSc. Transforming growth factor beta (TGF-β) is the main inducer of connective tissue remodelling during wound healing, as well as in many other fibrotic pathologic changes.(Meng, Nikolic- Paterson, and Lan 2016) Not surprisingly, it is more abundant in the skin of SSc patients, and fibroblasts can synthesize and secrete it, leading to a “TGF-β autocrine hypothesis” on SSc fibrosis.(Wei et al. 2011) In fact, fibroblasts isolated from the skin of SSc patients remain activated in vitro even after several culture passages, in a pure culture milieu,(Dees, Tomcik, et al. 2011) suggesting a perpetuation of the fibrogenic response based on autocrine factors. However, inhibition of TGF-β only partially reverts the pathological changes seen in SSc fibroblasts in vitro,(Y. Chen et al. 2006) supporting the notion that even though TGF-β may be sufficient to induce the full fibroblast activation program, once it is set in motion, other factors may perpetuate the response.

TGF-β and Notch signalling pathways have a reciprocal relationship in many models. In general, TGF-β enhances Notch signalling but the reverse seems to be more variable. In keratinocytes, TGF-β signalling directly increases Jagged-1 and Hes1 expression.(Zavadil et al. 2001) In myoblasts, the effector

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of the TGF-β/BMP signalling pathway Smad3 is able to interact with NICD in RBP-Jk binding sites, increasing Hes1 expression.(Blokzijl et al. 2003) At the same time, Notch signalling is able to modulate TGF-β signalling.(Sirin and Susztak 2012) This synergism between TGF-β and Notch, especially leading to fibroblast activation and acquisition of a contractile and profibrotic phenotype has been confirmed in several situations,(Condorelli et al. 2021) including in lung fibroblasts. Apart from TGF-β, all the signalling pathways which are classically related to fibrosis, including Wnt, PDGF and Hh are present in SSc skin and their interrelationship with the Notch pathway, although not completely elucidated, seems to exist.(Beyer and Distler 2013; LaFoya et al. 2016) As such, Notch involvement in fibrosis is but one piece in a series of intricate interactions of many signalling pathways, which may produce an auto-perpetuating response.

The mechanisms of fibrosis present in SSc are quite similar with other fibrosing diseases. In airway subepithelial fibrosis, such as that which occurs in asthma, Notch signalling induces fibroblasts in the lamina propria to produce collagen through an increased expression of Hes1.(M. Hu et al. 2014) In non-alcoholic steatohepatitis, increased Notch signalling in hepatocytes leads to activation of stellate cells through Jagged- 1/Notch-3 interaction, leading to liver fibrosis.(Zhu et al. 2018) In hypertrophic scars, including keloid disease, Notch signalling is increased in lesional skin.(Syed and Bayat 2012) In kidney fibrosis, Jagged-1 and Notch signalling have also been implicated.(Id et al. 2018) In fact, the Notch pathway, namely through the ligand Jagged-1, is implicated in the pathogenesis of many of the fibrotic skin diseases in addition to scleroderma, such as chronic graft-versus-host disease, eosinophilic fasciitis, nephrogenic systemic fibrosis or dystrophic epidermolysis bullosa.(Condorelli et al. 2021)

1.3.2 Epithelial-to-mesenchymal transition

Apart from being implicated in fibroblast activation and differentiation into myofibroblasts, Notch signalling is central to a process of transdifferentiation called epithelial-to-mesenchymal transition (EMT).

In different contexts, during development and adult life, epithelial cells (including the endothelium) may lose the expression of some epithelium-defining genes, such as several cytoskeleton proteins, E-cadherin and CD31, and acquire the expression of a different set of genes, typical of cells of mesenchymal origin such as vimentin, alpha-smooth muscle actin and even collagen type I.(Zmorzyński et al. 2019) This results

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in the acquisition of a different cellular behaviour, with the loss of direct contact with the basement membrane and the acquisition of motility through the extracellular matrix, therefore behaving essentially as fibroblasts. It has been proposed, rather logically, that this would be the main mechanism for generating increased numbers of fibroblasts in sites of injury and in fact EMT has been described in many organs and conditions. Notch signalling has been implicated in many of these situations, namely by regulating Snail transcription factors.(Lamouille, Xu, and Derynck 2014) In tubulointerstitial fibrosis of the kidney for example, EMT occurs in intercalated duct epithelial cells, leading to an increased number of fibroblasts and increased deposition of ECM in the interstitium.(Lebleu et al. 2014) Not surprisingly, the signalling pathways that have been implicated in EMT are the same that are classically related to fibrosis, namely TGF-β, Notch, Wnt, Hh and PDGF.

In conclusion, notwithstanding the uncontroversial role of the Notch signalling pathway in inducing and maintaining a fibrotic response, its contribution is difficult to separate from that of many other morphogen pathways and it does not seem to be specific for SSc. On the contrary, it is easily interpreted as a standard molecular mechanism to induce fibroblast activation and EMT, regardless of the underlying disease. It is worth considering that of all the conditions which induce a fibrotic response, from myocardial infarcts to diabetic nephropathy passing through other fibrotic skin diseases, none has the vascular and immunologic phenotype seen in SSc.

Although an increased expression of receptors, ligands, target genes and increased nuclear NICD has been described in fibroblasts, we are rather clueless to which mechanism increases Notch signalling, apart from the redundancy of the many signalling pathways involved in fibrosis. It has been pointed out that oxidative stress and inflammation are associated with an increase in ADAMS-17 activity which could presumptively activate the Notch pathway by facilitating the S2 cleavage of the receptor.(Kavian et al.

2010; Xu et al. 2011) However, a facilitation of ADAMS-17 activity is not expected to induce autonomous (ligand-independent) activation of Notch receptors. Also, inflammation and oxidative stress are very common in many diseases, none of which has the same manifestations of SSc. In the same scope, autoantibodies against endothelial cell antigens are present in SSc,(Kayser and Fritzler 2015) but none have been reported to target any of the components of the Notch pathway.

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1.4 Notch in endothelial dysfunction

EMT also occurs with endothelial cells, being then called EndoMT (endothelial-to-mesenchymal transition). Notch signalling in endothelial cells seems to induce EndoMT. During development, EndoMT is necessary to normal heart valve development, in a process which starts with the activation of the Notch pathway.(Chang et al. 2011)

Endothelial cells with a constitutively activated form of Notch-4 lose the normal cobblestone arrangement in vitro and fail to develop a monolayer, whilst losing the expression of von Willebrand factor, VE-Cadherin and other endothelial markers and upregulating myofibroblast markers such as alpha-smooth muscle actin and vimentin. They acquire a migrating behaviour, with chemotaxis towards PDGFb.(Noseda et al. 2004; J. Liu et al. 2014) In SSc, the same process has been documented in the skin.(Jimenez 2013)

Considering the widespread nature of the pathologic changes in SSc, the activation of the Notch pathway in fibroblasts and the presence of EndoMT induced by an activated Notch pathway, it seems reasonable to consider that the Notch pathway is pathologically activated also in the endothelial cells of the affected organs in SSc. If so, one possibility would be that an unknown factor is responsible for the activation of the Notch pathway both in fibroblasts and endothelial cells. This would lead to fibroblast activation and at the same time would induce EndoMT in capillaries, contributing further to fibrosis.

However, an increased Notch signalling in the endothelium implies several consequences to the homeostasis of the capillary network which must be considered. Indeed, the Notch pathway is well established as a core mechanism for the shaping of a sprouting microcirculatory network and for the maintenance of established capillaries.

During sprouting angiogenesis, an established capillary will be induced to generate new capillaries mostly by increased local concentrations of vascular endothelium growth factor A (VEGF-A). Endothelial cells are able to respond to VEGF-A because they express the receptor VEGFR-2.(Blanco and Gerhardt 2013) VEGF-A/VEGFR-2 interaction results in the activation of a series of intracellular pathways which will in turn activate a number of genes responsible for endothelial cell survival,(Gerber, Dixit, and Ferrara 1998) increased permeability(Dvorak et al. 1995) and the acquisition of a mobile chemotactic behaviour

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towards increased concentrations of VEGF-A in the extracellular matrix,(Matsumoto et al. 2005) which coincides with the most hypoxic areas.(Karamysheva 2008)

One main target of VEGFR-2 signalling is the Notch ligand DLL4 gene,(Lobov et al. 2007; Z. Liu et al. 2003) which will engage Notch receptors on neighbouring endothelial cells. However, Notch signalling in endothelial cells will hamper their ability to respond to VEGF-A because it will down-regulate VEGFR- 2, along with its co-receptor neuropillin-1,(Williams et al. 2006) and at the same time up-regulate the expression of VEGFR-1(Funahashi et al. 2010) along with its soluble form,(Harrington et al. 2008) which acts as a decoy. In turn, the neighbouring cells will not be able to sustain Dll4 expression (because VEGF- A signalling is hampered), which will not activate Notch signalling in the first cell (Figure 3). In this manner, small differences in VEGF-A exposure between contiguous endothelial cells will be amplified (Agrawal, Archer, and Schaffer 2009) so that the final result will be a single isolated cell with high expression and signalling activity of VEGFR-2, low expression levels of VEGFR-1 and low Notch signalling activity,

Figure 3 - Interaction between the Notch and VEGF pathways in endothelial cells. Standard arrows: transcriptional activity;

squared red arrows: inhibitory stimulus; round arrows: positive stimulus.

Notch-1/-4

DLL4

VEGFR-2 Dll4

Notch target genes

VEGFR-1 DLL4

Notch target genes

VEGFR-2

Dll4 Notch-1/-4

VEGFR-1

VEGF-A

Notch-1/-4

VEGFR-2 Dll4

VEGFR-1

Stalk cell

High Notch Low VEGF

DLL4 Notch target genes

DLL4

Notch target genes

VEGFR-2

Dll4 Notch-1/-4

VEGFR-1

Tip cell

Low Notch High VEGF

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which will digest the basement membrane surrounding the capillary (Funahashi et al. 2011) and will acquire a motile phenotype with weak intercellular junctions. This will be the tip cell, which will lead the sprouting capillary.(Hellström et al. 2007; Siekmann, Covassin, and Lawson 2008; Siekmann and Lawson 2007)

The following cells will have an activated Notch signalling, low VEGF-A signalling and will not be motile, exhibiting solid intercellular junctions thus ensuring the integrity of the emerging vessel whilst developing a lumen. These are the stalk cells. Stalk cells will produce a new basement membrane and recruit pericyte coverage through the secretion of PDGFb. Once pericytes are embedded in the basement membrane, they will develop a tight relationship with nearby endothelial cells, through “peg-socket”

contacts.(R. C. a Sainson and Harris 2008) Through this interaction, a reciprocal signalling activity will develop between both cellular types, where endothelial cells express Jagged-1 at their surface, which will engage Notch-3 receptors on pericytes, which in turn will promote their activation (Yang and Proweller 2011), survival and retroactively increase further Notch signalling on endothelial cells through Jagged- 1/Notch-1.

This is an oversimplistic view of the process, as Notch signalling can induce waves of activation/deactivation resulting in a cyclic phenotypic change between stalk and tip cell states.(Beets et al.

2013) Nonetheless, this sequence of events can mimic and explain reasonably well many experimental findings.

When a capillary network has an abnormally active Notch signalling, this dynamic equilibrium will be lost. Every endothelial cell will have a hampered VEGF-A sensitivity and they will all tend to acquire a stalk-cell phenotype, resulting in the production of a neoangiogenic circulation with sparse branching and dilated capillaries. This has been observed both in vitro and in vivo.(Li et al. 2007; Patel et al. 2005) Furthermore, an increased PDGFb production in stalk-cell predominant conditions due to Notch hyperactivity will increase pericyte recruitment, further induce vascular smooth muscle cells hyperplasia and increase production of extracellular matrix.(Schrimpf et al. 2014)

In this way, a pathologically activated endothelial Notch signalling might produce, as downstream events, much of the typical changes seen in patients with SSc, as observed by nailfold digital

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capillaroscopy.(Cutolo and Smith 2013) Additionally, that could explain the time course of SSc, where vascular manifestations precede the development of fibrosis.

As such, a second scenario might be hypothesized (Figure 4), where an unknown factor produces an increase in Notch signalling in the endothelium, which will disturb vascular homeostasis in such a way that there will be vasculopathy, EndoMT and increased numbers and activity of myofibroblasts and perivascular Notch activation, whilst producing a dilated, sparsely ramified capillary network in response to hypoxia.

This scenario is very appealing as it recapitulates the findings in SSc patients.

Raynaud’s phenomenon is more difficult to include in the predicted effects of an overactive Notch pathway, on the light of the available knowledge on the relationship between arteriolar vasoreactivity and Notch signalling, which is scant. The contractile apparatus of the vascular smooth muscle cells is dependent on canonical Notch signalling to effectively operate: inhibition of Notch signalling leads to both contraction and relaxation impairment.(Basu et al. 2013) As such, it is not clear what effect would an overactivation of Notch signalling have on the normal response to vasoactive stimuli.

Figure 4 - Conceptual framework for the role of the Notch pathway in SSc pathophysiology. Notch signalling is involved in endothelial cell dysfunction, which leads to clinical vasculopathy, and fibroblast activation which leads to collagen synthesis and fibrosis. The two processes might be interrelated by endothelial cell - fibroblast interactions, where Notch is a central player.

Depicted are the two hypothetical scenarios where the unknown trigger might act.

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1.5 Current applicability of the knowledge on the Notch pathway in systemic sclerosis

The recognition that an increased Notch signalling is consistently present in various clinical models of SSc opens some opportunities that can be exploited with clinical benefit.

1.5.1 Diagnosis, monitoring and prognosis

The utilization of a Notch activation status screening method to help diagnosing SSc would encounter many difficulties particularly regarding specificity. As stated above, the role of the Notch pathway in fibroblast differentiation and activation is not at all specific to SSc, being observed in many diseases which have a pro-fibrotic phenotype. Regarding the potential role of the Notch pathway in vasculopathy, one would expect that those pathologic changes in Notch signalling activity would be more specific, considering the uniqueness of the microvascular changes present in SSc. In fact, there are some particularities about the Notch pathway in the endothelium that could be used to increase the specificity of a diagnostic assay. For instance, the ligand Dll4 seems to be rather specific for the endothelium (it is also present only in the development of T-lymphocytes and in macrophages).(Vanderbeck and Maillard 2021)

Nevertheless, one would expect that if a method to screen a whole-body activation status of the Notch signalling pathway was developed, it would reflect disease activity in terms of fibrogenesis that could allow separating those patients with primary Raynaud’s phenomenon, a benign condition, from those with Raynaud’s phenomenon secondary to SSc, where one would expect an increased Notch activity in fibroblasts.

Concerning risk assessment, it is worthwhile to consider genome-wide association studies made in patients with SSc. No association with any component of the Notch pathway has ever been made until recently when a case-control study(Wojcierowska-litwin et al. 2020) of a limited number of SSc patients found an association between diffuse cutaneous SSc and single-nucleotide polymorphisms in the NOTCH3 gene, which is one of the main Notch receptors in vascular smooth muscle cells. Along with other methodologies, a more precise risk assessment could be made by including these variables in a potential

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model to predict the risk of a patient with Raynaud’s phenomenon developing SSc or the risk for a patient with the diagnosis of SSc evolving into a specific disease subtype, or even to predict its response to Notch- targeting therapies.

1.5.2 Therapeutics

Notch pathway inhibition can prevent the development and/or induce regression of fibrosis. The administration of DAPT^*, a gamma-secretase inhibitor, as well as the overexpression of an antisense construct of Notch-1, both inhibited the development of fibrosis in the bleomycin-induced fibrosis model of SSc as well as induced regression of fibrosis in the Tsk and HOCl mouse models.(Dees, Zerr, et al. 2011;

Kavian et al. 2010) Although there is plenty of support from basic science for the use of Notch pathway modulators in the treatment of SSc, and considerable data regarding pharmacological inhibition of the Notch pathway (mainly in oncology)(Majumder et al. 2021), little has been made in order to develop clinical trials in SSc.

The first class of drugs used in humans that directly targeted a component of the Notch pathway was the gamma-secretase inhibitors (GSIs) which were initially developed for Alzheimer’s disease, as the gamma-secretase complex is responsible for the cleavage of the beta-amyloid precursor protein into the neurotoxic beta-amyloid peptide. However, soon it was realized that the gamma-secretase complex is a multifunctional structure that is involved in the processing of a multitude of proteins, with that inhibition resulting in a large array of unintentional targets.(Güner, Lichtenthaler, and Dzne 2020) The adverse effects of GSIs were a consequence of the inhibition of all four Notch receptors, as the gamma-secretase-mediated S3 cleavage of the receptor after the ligand interaction is necessary for the release of the intracellular domain in all of them resulting in a pan-Notch inhibition. Of all the possible clinical limitations, the ones that proved to be dose-limiting both in Alzheimer’s and in cancer therapy, were the gastrointestinal complications. Notch-1 and Notch-2 are used in the intestinal crypts of Lieberkühn as a mechanism for directing basal cells into the enterocyte differentiation path.(Riccio et al. 2008) The standard differentiation

* N-[N-(3,5-Difluorophenacetyl)-L-alanyl]-S-phenylglycine t-butyl ester.

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setup is onto the goblet cell phenotype such that upon Notch-1/Notch2 inhibition, goblet cell hyperplasia results, which manifests clinically as diarrhoea. Additionally, nausea, anorexia, hyperglycaemia, hypophosphatemia, asthenia and cough were also reported in multiple trials in all major GSIs, which included LY3039478, nirogacestat, AL101, and RO4929097.(Moore et al. 2020) Interestingly, the gastrointestinal adverse effects of GSIs seem to be diminished by the co-administration of glucocorticoids,(Samon et al. 2012) which could be interesting in SSc, considering that many patients are already treated with low-dose regimens. Several drugs are currently being tested in clinical trials and an overarching concept which is not usually considered is that although the gamma-secretase complex is needed for every Notch receptor activation, its inhibition with different compounds does not result in the inhibition of the same ligand/receptor interactions, paving the way to develop ligand- or receptor-specific GSIs. Inhibitors of the ADAM-2/ADAM-17 metalloproteinases also target the activating cleavages of the Notch receptors. They are also very ubiquitous and have a large number of substrates but are still underdeveloped when compared to GSIs.

Monoclonal antibodies (mAbs) to block specific Notch receptors or ligands have also been considered.

These compounds have other advantages, including a good tolerability profile (with hypersensitivity reactions being relatively rare) and long half-lives(Keizer et al. 2010) but parenteral administration is required and are expensive. mAbs against the Notch system have been mainly developed to halt the growth of Notch-dependent tumours. Contrarily to their use in oncology, the effector activity of several mAbs is conferred by domains in the Fc region (antibody-dependent cytotoxicity, induced phagocytosis or complement damage) which are probably not required and will lead to undesirable side effects.

Nevertheless, the vast majority of mAbs in this context have been designed with a IgG2 scaffold which would probably reduce those effector functions.(Christopoulos et al. 2021)

The first in-human study using an anti-Notch mAb was with brontictuzumab,(Ferrarotto et al. 2018) but again the gastrointestinal side effects were considerable, especially with chronic use. Several others have been developed, mainly targeting the ligand-binding extracellular domains of the Notch receptors. It is noteworthy that one of them, by targeting specifically Notch-1, averted the gastrointestinal toxicity in a preclinical study, supposedly by leaving the Notch2 signalling intact.(Christopoulos et al. 2021) The same

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logic applies to a mAb against Notch-3 which would not produce goblet cell hyperplasia and diarrhoea, as those effects are dependent on Notch-1/Notch-2 signalling. However, gastrointestinal side-effects still existed, including nausea and abdominal pain in up to 25% of participants of a phase I clinical trial,(Rosen et al. 2020) despite being reported as well tolerated. Notch-3 inhibition could be helpful in patients with SSc as it is one of the two most important receptors in myofibroblasts and vascular smooth muscle cells.

mAbs directed against Notch ligands have also been developed with the most studies to date being directed against Dll4 due to its role in angiogenesis. Inhibition of Dll4 induces a endothelium-specific defect in Notch signalling, that will lead to a highly ramified vascular network which is paradoxically dysfunctional – many capillaries lack a lumen and have serious defects in pericyte covering, leading to haemorrhages and hypoxia.(Chiorean et al. 2015) This effect was beneficial in tumours, halting their growth and in some cases inducing regression of the tumour mass. However, a serious adverse consequence of these changes in the microcirculation seen with the anti-Dll4 mAbs demcizumab and enoticumab was the development of pulmonary hypertension(McKeage et al. 2018) which prohibits its use in SSc, as it is one of the most serious manifestations of the disease. A mAb against the ligand Jagged-1 could have an excellent applicability in SSc due to its central role in myofibroblast activation and endothelial cell- perivascular cell interactions. Even though it has only been tested in preclinical trials and despite being developed for oncologic applications, it showed a very interesting safety profile.(Masiero et al. 2019; Lafkas et al. 2015)

There are several other investigational approaches to modulate Notch signalling including interfering with nuclear effectors such as MAML proteins or changing the glycosylation patterns of Notch receptors and ligands, but data regarding these approaches is still scarce.

It must be kept in mind that no long-term follow-up has been performed with these drugs and there are concerns about oncogenicity. If it is true that Notch signalling is commonly an oncogenic pathway, such that inhibiting it would be protective, there are tissues where Notch inhibits carcinogenesis, such as the epidermis, where Notch inhibition in keratinocytes can result in proliferations of basal cells resembling basal cell carcinomas.(Rangarajan et al. 2001)

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Nevertheless, there are two concepts that should be entertained while translating data from oncology to potential therapeutic applications in SSc. One is the fact that the Notch pathway is very sensitive to dosage due to its unamplified nature. One should not assume that the magnitude of blockade of Notch signalling that is required to induce remission of a Notch signalling-dependent tumour will be the same required to modulate a fibrogenic response. The dosages required to block fibrinogenesis may be much smaller, in this way averting the dose-limiting adverse effects of Notch inhibition. Another important concept should be that a multi-drug/multi-target approach to inhibit such a redundant process as fibrogenesis could be of value. In fact, concomitant inhibition of Notch (with a pan-Notch inhibitor), Wnt and Hh morphogen pathways have produced very interesting results, with excellent antifibrotic effects and almost no toxicity in pre-clinical models.(Distler et al. 2014)

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1.6 Conclusion

Research about the Notch pathway has been full of controversial findings and several aspects are still obscure. At the biochemical level, the exact functioning of the interactions of Notch receptors with their ligands and even their activation, as well as the DNA binding dynamics are not entirely elucidated. In fact, we do not fully understand how this system is able to drive completely different functions in almost every tissue and every cell, and yet operate with a limited number of receptor and ligands, with the molecular basis for the differential effects of each specific combination being elusive. Furthermore, as is usually the case with “ancient molecular tools”, its intracellular behaviour is extremely intermingled with many other signalling pathways. All this generates a great uncertainty concerning Notch signalling, its precise role in the pathophysiology of SSc and how it can potentially be used both in diagnostics and therapeutics.

Notwithstanding the uncertainty, it is now established that Notch signalling is a key component of the physiologic regulation of fibroblast differentiation and activation. In SSc, it is well established that a hyperactive Notch pathway drives the differentiation of myofibroblasts and the deposition of extracellular matrix. Pharmacological inhibition of the Notch pathway is able to prevent the development of fibrosis and even to induce its regression in many preclinical models.

Endothelial dysfunction and vascular insufficiency with impaired angiogenesis and poor vascular maintenance are all features of SSc, and the Notch pathway is a central regulator of all these processes.

Importantly, the effects of an increased Notch signalling in endothelial cells, although not properly studied in SSc, could explain many features of the disease, potentially acting at an upstream target for new treatments. In this respect, it is noteworthy that a unified and holistic explanation for the manifestations of SSc is still lacking. A specific common upstream trigger for SSc is yet elusive. Vasculopathy is frequently assessed separately from fibrogenesis and the fact that it occurs before fibrosis in the vast majority of patients speaks to its importance. Even though Notch signalling is known to orchestrate many endothelial functions which are affected in patients with SSc, its status in the endothelium in this condition is largely unknown.

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1.7 Objective of this thesis

The general aim of this thesis is to characterize the Notch activation status in the microvascular endothelium in systemic sclerosis.

The specific aims are the following:

1) To characterize the changes in the expression of the components of the Notch pathway induced by the serum of SSc patients in the microvascular endothelium.

2) To characterize the mechanism by which such changes occur.

3) To identify the nature of the serum factor responsible for such changes.

4) To find whether the effect is confined to a specific subset of patients.

5) To find if there is an association between the changes in the activation of the Notch pathway and microvascular abnormalities in vivo.

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II. METHODS

2.1 Subjects, consent, ethics, clinical data and serum collection

SSc patients were selected from stored samples at the Biobank of the Systemic Immune-mediated Diseases Unit of Fernando Fonseca Hospital, Amadora, Portugal. Patients were selected if they fulfilled ACR/EULAR classification criteria for systemic sclerosis. Patients had to be over 18 years old and could not be pregnant. Controls were recruited through local advertisement in Fernando Fonseca Hospital.

Clinical data was collected from the databases associated with the Systemic Immune-mediated Diseases Unit of Fernando Fonseca Hospital. Informed consent was acquired for all participants prior to inclusion.

The study protocol was approved by the ethics committees of Fernando Fonseca Hospital and of NOVA Medical School, as well as by the National Commission for Data Protection (CNPD).

Blood collection was performed through antecubital vein puncture, left to clot in dry tubes with a clotting activator for 15 minutes, and centrifuged at 1000g for 15min. The supernatant (serum) was collected, aliquoted and stored at -80ºC until further use.

2.2 Cell culture

Dermal microvascular endothelial cells (Cell Applications ref. 100-05a) were handled in the standard way concerning cell culture maintenance practices. They were seeded on porcine skin gelatine (Sigma, ref.

G2500) at 1% in water, and maintained in Lonza’s EGM2-MV culture media (Lonza, refs. CC3156 + CC4147), supplemented with 30% foetal bovine serum (FBS) (Gibco Life Technologies ref. 10270) and antibiotics (Gibco Life Technologies ref. 15140-122). Detachment for passages was performed using 0,05%

trypsin-EDTA (Gibco Life Technologies, ref. 15400-054) for 2-3 minutes, neutralized with 30% FBS in phosphate buffer saline (PBS). At critical experiments, preservation of endothelial cell identity was

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confirmed by morphological assessment and through immunofluorescence staining for von Willebrand factor (Figure 5).

2.3 Serum exposure assays

Microvascular endothelial cells were cultured in 12-well plates (3,8cm²/well) or 25cm² flasks until 80% confluence. Culture medium was then changed to the same medium but FBS was replaced with the subject serum at the specified concentration. No serum deprivation prior to assays was performed. No medium change was done during the assays, which had a maximum duration of 24h before collection.

2.4 Modulation of the Notch pathway

To induce an overactivation of the canonical Notch pathway, microvascular endothelial cells were cultured on 1% gelatine containing recombinant human Dll4 peptide (rhDll4) (R&D ref. 1506) at 1μg/mL for a minimum of 6h before collection. The major limitation of this system is that to maintain a comparable timeframe for the activation of the pathway (in relation to serum assays), confluency cannot be achieved,

Figure 5 - Microvascular endothelial cell characterization: von Willebrand factor positivity by immunofluorescence microscopy was used as a marker of phenotypic integrity before critical experiments.

400x. Blue: DAPI nuclear counterstain; green: vonWillebrand factor.

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as the Notch pathway starts to be activated as soon as the cells are seeded. This compromises cell number and RNA/protein yields if they are collected sooner than 6h (this was experimentally determined). A soluble form of Dll4 could not be applied on the culture medium, as it would engage Notch receptors but would not be able to signal due to the lack of physical attachment to generate a mechanical force that exposes the cleavage site on the receptor.(Lahmar et al. 2008; Vas et al. 2004)

To inhibit the canonical Notch pathway, γ-secretase was inhibited by N-[N-(3,5-difluorophenacetyl)- L-alanyl]-S-phenylglycine t-butyl ester (DAPT) (Sigma ref. D5942), added to culture medium at a 2μM concentration. DAPT is solubilized in dimethylsulphoxide (DMSO). Due to concerns of an unspecific effect of DMSO in endothelial cells, DMSO was added at the same concentration to other conditions of the experiments where DAPT was used. To ensure full γ-secretase inhibition prior to the application of serum in serum assays, DAPT was applied for 1h before the actual serum assay started and included again in the medium of the assay until its completion.

2.5 Immunoglobulin G assays

Immunoglobulin G was purified from serum in Protein G-containing beaded resin 1mL columns (NAb protein G spin kit, ThermoScientific ref. 89979). Briefly, 150μL aliquots of serum were diluted in binding buffer (Tris-HCl 0,1M pH=8,0) to 1mL, centrifuged at 10000g for 15 minutes and the supernatant passed through a 0,45μm cellulose acetate filter to remove debris, with a further 500μL wash with binding buffer to maximize yield. Columns were washed with 6mL of binding buffer before the diluted serum was applied. After serial washes (a total of 15mL of binding buffer), 1mL of elution buffer (glycine 0,1M pH=2,7) was passed 4 times through the columns and collected as elution fractions 1-4, and immediately neutralized with 100μL of 1M Tris-HCl pH=8,5. Total protein content of the 4 elution fractions was determined by a Bradford assay. Elutions 2 and 3 had the majority of the protein (around 1mg/mL). The two were mixed together and used in subsequent assays. When used in culture medium directly, the purified immunoglobulin eluate was found to be cytotoxic inducing widespread apoptosis of endothelial cells (39% vs. 80% alive cells by haemocytometer count with Trypan blue 0,4% staining, and an apoptotic

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morphology by light microscopy), probably due to the Tris buffer. As such, eluates were dialyzed against phosphate buffer saline (PBS) in dialysis cassettes with a 20kDa molecular weight cut-off (ThermoScientific ref. 87735). Cell culture compatibility was confirmed in an exploratory experiment, with no morphologic changes of endothelial cells and a good viability (72% vs. 73% alive cells). Purity of the eluate was confirmed by SDS-PAGE using reducing conditions of eluates, followed by staining with Coomassie blue. Due to the relatively reduced concentration of immunoglobulin after purification, to reproduce the immunoglobulin concentration used in serum assays with 15% serum in the culture medium, culture medium had to be diluted down to 23% of its usual usage. However, this applied both to control and SSc- derived serum.

2.6 RNA purification and qRT-PCR

RNA was extracted and purified by a standard phenol-chloroform protocol. Cells were washed twice with PBS and homogenized in 100μL/cm² of a guanidine thiocyanate/phenol solution (TRI Reagent Sigma-Aldrich ref. T9424). Chloroform was added (27μL/100μL TRI reagent), thoroughly mixed and centrifuged at 12’000g for 20 minutes. The upper phase was separated into a new Eppendorf tube and RNA was precipitated by adding 66μL isopropanol per 100μL TRI reagent overnight at -80ºC, along with Pellet-Paint co-precipitant to maximize pellet visualization and increase RNA yield (Millipore ref. 69040).

The RNA pellet was cleaned with a 1mL/mL TRI Reagent of 70% ethanol in diethyl-pyrocarbonate (DEPC) treated water, dried and resuspended in 10μL DEPC water. RNA yield was determined by spectrophotometry (Nanodrop 2000 ThermoFisher) with a lot-specific correction to subtract Pellet-Paint absorbance overlap with that of RNA. It was stored at -80ºC until use.

Complementary DNA (cDNA) was synthesized using 500ng RNA with reverse transcriptase (Superscript II Life Technologies ref. 18064) using the recommended buffers and random primer concentrations. It was stored at -20ºC after a 1:2 dilution with DEPC-water.

Role of the notch signalling in the endothelial dysfunction of systemic sclerosis

Abstract

Resumo

TABLE OF CONTENTS

List of figures

List of tables

Abbreviations

Acknowledgments

Scientific articles used in the preparation of this thesis

I. BACKGROUND

1.1 Introduction to systemic sclerosis (SSc)

1.2 Introduction to Notch signalling

1.3 Notch in fibrogenesis

1.4 Notch in endothelial dysfunction

1.5 Current applicability of the knowledge on the Notch pathway in systemic sclerosis

1.6 Conclusion

1.7 Objective of this thesis

II. METHODS

2.1 Subjects, consent, ethics, clinical data and serum collection

2.2 Cell culture

2.3 Serum exposure assays

2.4 Modulation of the Notch pathway

2.5 Immunoglobulin G assays

2.6 RNA purification and qRT-PCR