Systemic scleroderma (SSc) is a systemic connective tissue disease affecting skin and internal organs. The pathogenesis of SSc is characterized by inflammation, vasculopathy and fibrosis. No agent has been proven effective in the treatment of SSc. There is a lack of suitable biomarkers for monitoring the disease activity or the response to the treatment of SSc. Therefore our aim was to analyse the extracellular levels of S100A4, Hsp90 (Heat shock protein 90) and IL-35 (interleukin-35) in SSc. S100A4 and Hsp90 have been initially studied in tumours; in some of them considered as suitable prognostic markers and candidates for future therapies. We have recently described the profibrotic role of S100A4 and Hsp90 in the pathogenesis of SSc. Our results showed that inactivation of S100A4 and Hsp90 effectively prevented the development of experimental skin fibrosis. This was consequently confirmed by the analysis of S100A4 and Hsp90 in the peripheral blood of patients with SSc, where significant associations with disease activity and organ involvement were detected. IL-35 may become another potential biomarker of SSc. We detected increased expression of IL-35 in the affected skin, dermal fibroblasts and in serum of patients with SSc. Moreover, the main profibrotic mediator transforming growth factor...

Introduction: Digital ulcers (DUs) are responsible for much of the pain and disability associated with systemic sclerosis (SSc), and are a biomarker of internal organ involvement and poor prognosis. DUs are often used as the primary end-point in SSc clinical trials, although the reliability of rheumatologists in grading DUs is poor to moderate at best. Fingertip DUs are believed to be ischaemic in aetiology, whereas, extensor DUs are thought to occur due to mechanical factors and recurrent microtrauma. Treatments for DUs are often poorly tolerated due to systemic vasodilation. The overarching aim was to investigate the definition and objective measurement of SSc-related DUs, their pathophysiology, and a new light treatment. Method: Five studies were undertaken. (1) A web-based study in which photographs of digital lesions were graded, all either with or without clinical context. (2) A pilot study to assess the feasibility and tolerability of high-frequency ultrasound (HFUS) imaging to measure DUs. (3) A retrospective study examining whether thermographic abnormalities are associated with DUs. (4) A double-blind, randomised, crossover, controlled study of glyceryl trinitrate (GTN) to explore the pathophysiology of DUs in SSc. (5). A feasibility study of a novel light (red, infrared and blue) device to treat SSc-related DUs. Results: (1) 51 rheumatologists graded ≥ 4500 images. The clinical context (without vs with, weighted kappa statistic) did not significantly improve the intra- (0.32,0.36) or inter-rater (0.64,0.71) reliability. (2) HFUS was performed on 15 DUs and was well tolerated and feasible in the majority. DU measurement was possible in most (n=13) DUs, the mean DU depth and width were 0.99mm and 5.74mm, respectively. (3) Patients (n=138) with abnormal (compared to normal) thermography were more likely (adjusted odds ratio = 2.84) to develop future DUs, including multiple episodes. (4) 16 DUs were studied; the microvessels of the DU centre were responsive to GTN, with an increase in perfusion, with a similar effect in both fingertip and extensor DUs. There was less of a clear signal in the DU periphery. (5) Light treatment was safe, feasible and well tolerated (46 light treatments administered in 8 patients, one studied on three separate occasions). There was a significant improvement (change in visual analogue score per visit) in DUs as assessed by both patient (-7.1, P = < 0.001) and clinician opinion (-5.2, P = < 0.001). DU perfusion (measured by LDI) significantly increased post-treatment. Conclusion: The reliability of DU grading did not improve with clinical context. HFUS was feasible and well tolerated, and measurement was possible in most DUs. Our data suggests that many DUs might have an ischaemic drive, including extensor DUs. A novel light treatment was safe, feasible and well tolerated, with a tentative suggestion of treatment efficacy.

Systemic sclerosis (SSc; scleroderma) is a chronic multisystem autoimmune disease that includes prominent skin involvement in all patients and is characterized by fibrosis, inflammation, and microvascular injury of the skin and internal organs. Clinical trial design for patients with systemic sclerosis (SSc) has been confounded by the heterogeneity of disease progression and lack of objective outcome measures. This has hampered identification of therapies for patients who have frequently fatal fibrotic complications. Direct pulmonary complications are the leading cause of death in SSc. For clinical trials in patients with diffuse cutaneous SSc, identification of a pharmacodynamic biomarker associated with clinical improvement would allow for alternative approaches to trial design. Furthermore, identification of a diagnostic biomarker for SSc complicated by pulmonary arterial hypertension (SSc-PAH) would provide a reliable non-invasive method for diagnosis of pulmonary arterial hypertension. Through the combination of high throughput technologies and clinical information we have identified three preliminary biomarkers for SSc: i) Two pharmacodynamic biomarkers for diffuse skin disease (dcSSc), one in using mRNA from skin biopsies and one using proteomic profiles from sera; ii) a serum based proteomic classifier for the screening and diagnostic evaluation of pulmonary arterial hypertension in systemic sclerosis. We show these biomarkers can be applied to assess changes in skin disease in dSSc patients over time and with further development could be used to supplement or replace the physical examination assessment (Modified Rodnan Skin Score, MRSS) as an outcome measure in clinical trials for dcSSc patients. Routine use of these biomarkers in SSc clinical trial design could expand treatment options for a patient population that currently has few if any treatment options that slow progression of disease. Furthermore we identified a serum biomarker for the major SSc pulmonary complication, SSc-PAH. This diagnostic SSc-PAH biomarker has the potential to be used as a screening tool in order to reduce the need for unnecessary invasive diagnostic procedures. This non-invasive screening method could lead to early diagnosis thus improving patient survival and aid in clinical management of a complication for which there are several treatments but which is still frequently fatal. / 2018-06-15T00:00:00Z

This thesis forms part of an ongoing long-term project to investigate the suitability of Doppler optical coherence tomography (OCT) as a measurement tool to investigate skin thickness and blood flow in patients with systemic sclerosis. There is a discussion of the characterisation of an electro-optic phase modulator for use in a Doppler OCT imaging system which is being built for the purpose of clinical studies. In addition to this the development of software for the same system is described. The work includes a comparison of two methods of obtaining Doppler information that were tested with the system; a phase resolved method and a correlation mapping method. Initial structural and Doppler images obtained using the system are presented. In addition to this the development of semi-automated software to measure skin thickness from both OCT and high frequency ultrasound images is discussed. The results of a study, for which this software was developed, into skin thickness measurements using both techniques in both patients with systemic sclerosis and healthy controls are presented. Both OCT and high frequency ultrasound were able to measure a statistically significant difference in epidermal thickness at multiple locations on the body. Finally, the modification of a freely available Monte Carlo simulation for light propagation in multi-layered tissue (MCML) to enable the simulation of structural and Doppler OCT images is covered. The simulation was able to extract the magnitude of the simulated flow accurately to within an order of magnitude, and after a simple filter was applied to eliminate fluctuations in the data the structure of the Doppler image closely matched what was modelled.

Scleroderma (SSc) is an autoimmune disease characterized by dysfunctional immunity, vasculopathy, and fibrosis of the skin and internal organs. There is a poor prognosis for SSc patients and effective therapeutics have not yet been developed. Many mouse models have been developed, but most fail to recapitulate all of the symptoms seen in SSc patients. In this study we characterize a Fli1flox/flox mouse with CAG Cre under the beta-actin promoter. Based on what has been previously described in mice with deletion of Fli1 in specific cell types, we predicted that global postnatal deletion of Fli1 will result in systemic fibrosis, vasculopathy, and inflammation in these mice. The penetrance of a phenotype was highly variable; however, mice that developed a phenotype displayed disorganized vascular networks, fibrosis and proinflammatory cytokines and chemokines in the skin and lungs after 4 and 8 weeks of Fli1 deficiency. Increased macrophage and dendritic cell populations were observed in the lungs after 8 weeks. Fli1flox/flox CAG Cre mice exhibited hair loss after 5 months of Fli1 deletion. Since our experiments focus mainly on the lungs and skin, more experiments are required to characterize these mice to determine if they can be used as a novel animal model of SSc.

The perivascular adventitia (PA) senses and responds to injuries in blood vessels and the tissues they feed. Cells in the PA form the outermost vascular layer, joining the circulatory system to other organs. Housing hematopoietic, mesenchymal and neuronal cells allows flexible adventitial responses to diverse perturbations. However, the PA response can also be pathogenic. Thickening of the adventitia may drive ischemia and hypertension. It can also be a niche for local lymphocyte priming in diseases such as idiopathic pulmonary arterial hypertension. Despite their importance, PA contributions to skin diseases were understudied. The hypothesis that contrasting two cutaneous diseases, scleroderma and discoid lupus erythematosus (DLE), would illuminate discrete PA alterations was explored. Vascular changes are prominent, but distinct, in both diseases. Studying perivascular adventitial changes in these diseases may yield insights into both dermal and vascular pathologies. PA fibroblasts in healthy human skin were phenotypically distinct from the surrounding dermal fibroblasts. In both scleroderma and DLE, PA fibroblasts expanded and expressed surface markers not observed in healthy skin including vascular cell adhesion molecule 1 (VCAM1), podoplanin (PDPN) and the p75 low affinity nerve growth factor receptor (NGFR). Elaborated networks of PA fibroblasts in DLE expressed VCAM1 and enmeshed dense, T cell-rich infiltrates. Transcriptional analyses indicated positive correlations between VCAM1, T cell chemoattractants and interleukin (IL)-15, which promotes their survival. Activated PA fibroblasts in DLE likely create a supportive niche for T cells infiltrating the skin. In contrast, enlarged PA fibroblast networks in scleroderma expressed NGFR in the absence of leukocyte infiltrates. This PA fibroblast phenotype was shared among reparative and pathologic scarring, and four dermal tumors. NGFR is a mesenchymal stem cell (MSC) marker, and expanded NGFR+ mesenchymal cells were immediately adjacent to cluster of differentiation (CD)34+ and CD73+ PA MSC. Expression of NGFR by PA fibroblasts is likely associated with reparative responses. Different stimuli induced VCAM1 and NGFR on cultured human dermal fibroblasts, supporting these as discrete activation states. In conclusion, these studies demonstrated the responsive and plastic nature of human dermal PA mesenchymal cells, and pointed to connections with vascular alterations in skin diseases.

Systemic sclerosis (SSc), or scleroderma, is a complex, rare disease of unknown etiology. Macrophages constitute a large portion of the immune cell infiltrate in the skin of patients with SSc, and are an important target of study. Particularly, the M2 macrophage has been implicated scleroderma and other fibrotic diseases as a key contributor to fibrotic processes. However, the definition of an M2 macrophage appears to change with context, and is poorly elucidated in different species. With varying characterizations between species and disease models, there is a need to establish some consensus on how to identify this macrophage in an uniform manner across species. We used a bioinformatic approach to identify a unique gene signature for the M2 macrophage phenotype, which is shared between human and mouse systems. We were able to confirm a 7-gene subset of this theorized signature using human and mouse in vitro systems. In addition, we selected one of the identified genes, Clec7a, and characterized its expression at the protein level on different macrophage phenotypes, across several human and mouse models. Our data show that Clec7a is a more selective marker of murine M2 macrophages than current reference markers, and is useful in human models as well. Using our M2-specific gene signature, we also identified a potential inhibitor of the signature and showed its effects on M2 marker expression. Finally, we showed some preliminary work into Clec7a expression in skin tissue from patients with scleroderma. Overall, our data suggest that Clec7a may be a valuable addition to the panel of markers used to characterize M2 macrophages and distinguish between macrophage phenotypes, and perhaps provide clarity into the development and function of the M2 macrophage. Better understanding of the M2 macrophage would ultimately be useful to the study of fibrotic diseases such as scleroderma, wherein this macrophage phenotype may be a viable target for antifibrotic therapy. / Thesis / Master of Science (MSc)

Yes / Objectives Systemic sclerosis (SSc) is characterised by tissue fibrosis and vasculopathy with defective angiogenesis. Transforming growth factor beta (TGF-β) plays a major role in tissue fibrosis, including downregulation of caveolin-1 (Cav-1); however, its role in defective angiogenesis is less clear. Pigment epithelium-derived factor (PEDF), a major antiangiogenic factor, is abundantly secreted by SSc fibroblasts. Here, we investigated the effect of TGF-β and Cav-1 on PEDF expression and the role of PEDF in the ability of SSc fibroblasts to modulate angiogenesis. Methods P EDF and Cav-1 expression in fibroblasts and endothelial cells were evaluated by means of immunohistochemistry on human and mouse skin biopsies. PEDF and Cav-1 were silenced in cultured SSc and control fibroblasts using lentiviral short-hairpin RNAs. Organotypic fibroblast–endothelial cell cocultures and matrigel assays were employed to assess angiogenesis. Results P EDF is highly expressed in myofibroblasts and reticular fibroblasts with low Cav-1 expression in SSc skin biopsies, and it is induced by TGF-β in vitro. SSc fibroblasts suppress angiogenesis in an organotypic model. This model is reproduced by silencing Cav-1 in normal dermal fibroblasts. Conversely, silencing PEDF in SSc fibroblasts rescues their antiangiogenic phenotype. Consistently, transgenic mice with TGF-β receptor hyperactivation show lower Cav-1 and higher PEDF expression levels in skin biopsies accompanied by reduced blood vessel density. Conclusions O ur data reveal a new pathway by which TGF-β suppresses angiogenesis in SSc, through decreased fibroblast Cav-1 expression and subsequent PEDF secretion. This pathway may present a promising target for new therapeutic interventions in SSc. / NIHR CDF; EULAR ODP

La Sclérodermie Systémique (ScS) est une maladie auto-immune rare et complexe, cliniquement divisée en deux sous-groupes : la ScS cutanée diffuse (dc-ScS) et la ScS cutanée limitée (lc-ScS). Les anticorps anti-topoisomérase et anti-centromère (ATA et ACA) sont respectivement les marqueurs de chaque sous-groupe. Beaucoup d'études ont analysé les gènes des Antigènes leucocytaires humains (HLA), fort facteur de risque, dans plusieurs groupes ethniques de patients atteints de ScS. La plupart des allèles de susceptibilité HLA-DRB1 (*11:01, *11:04, *15:01, *08:02…) et DQB1 (*03:01, *03:02, *06:01 and *06:02) ont en commun une séquence d'acides aminés, respectivement 67FLEDR71 et 71TRAELDT77 sur leurs chaînes &#946;. Pour la première fois, nous évaluons le risque relatif conféré par une ou deux doses de 67FLEDR71 et/ou une ou deux doses de 71TRAELDT77 chez des patients Caucasiens Français atteints de SSc. Nous montrons que le motif 67FLEDR71 est fortement associé à la dc-ScS et encore plus à la production d'ATA, alors que l'association de 71TRAELDT77 est plus faible dans les deux sous-groupes. Notre groupe a montré récemment dans la polyarthrite rhumatoïde (PR) que les patients qui n'ont pas les allèles de susceptibilité à la PR peuvent acquérir ces allèles via les cellules fœtales et/ou maternelles, aussi appelé Microchimérisme (Mc). Nous avons testé la même hypothèse dans la ScS. Contrairement à la PR, nos résultats n'ont pas démontré un tel transfert de susceptibilité via le Mc dans la SSc. Finalement, la présence d'auto-anticorps, liée à un génotype HLA, est précieuse au diagnostic et /ou pronostic de la maladie (ex : ACA ou ATA). / Systemic sclerosis (SSc) is a rare and complex autoimmune disease clinically divided into two subgroups: diffuse cutaneous (dcSSc) and limited cutaneous (lcSSc). Anti-topoisomerase and anti-centromere antibodies (ATA and ACA) are respectively markers of each subset. Many studies have analyzed Human Leukocyte Antigen (HLA) genes, the strongest genetic risk factor, in several ethnic groups in patients with SSc. Most common HLA-DRB1 (*11:01, *11:04, *15:01, *08:02…) and HLA-DQB1 (*03:01, *03:02, *06:01 and *06:02) susceptibility alleles have in common an amino acid sequence, respectively 67FLEDR71 and 71TRAELDT77 on their &#946; chains. For the first time, we evaluate the relative risk conferred by one or two 67FLEDR71 and/or one or two 71TRAELDT77 motives among Caucasian French patients with SSc. We show that 67FLEDR71 motif is highly associated with dcSSc and even more with ATA production, whereas 71TRAELDT77 association is weaker in both subgroups. Our group has recently shown in Rheumatoid Arthritis (RA) that patients who lack the RA HLA susceptibility that it could be transferred to patients via fetal and/or maternal cells, also called microchimerism (Mc). We test the same hypothesis in SSc. Contrary to RA, our results fail to demonstrate such transfer of HLA susceptibility via Mc in SSc. Finally linked to a particular HLA genotype is the presence of autoantibodies, helpful for disease diagnosis and/or prognosis (i.e. ACA or ATA). Patients who do not have these markers are difficult to diagnose or classify. By ProtoArray® technique, we identify 6 new auto-antigens in the plasmas of SSc patients.

Introdução: A capilaroscopia periungueal (CPU) tem sido relacionada ao dano a órgãos-alvo na esclerose sistêmica (ES). No entanto, estudos relativos à gravidade das alterações da CPU com a mortalidade evidenciam resultados discrepantes. Este estudo tem como objetivo verificar associação da gravidade da microangiopatia periférica na CPU com o risco de morte na ES. Pacientes e métodos: Cento e setenta pacientes com ES foram prospectivamente avaliados e acompanhados em média de 9,3 anos. Além da avaliação clínica, os pacientes foram submetidos à sorologia, testes de função pulmonar, ecocardiograma e tomografia computadorizada pulmonar de alta resolução (TCAR). A perda capilar na CPU foi avaliada pelo escore avascular (EA), que varia de 0 a 3. O número médio de ectasias, megacapillaries e hemorragias por dedo também foi registrado. O modelo de Cox proporcional Uni e Multivariado foi utilizado para analisar a associação do escore avascular com o risco de morte através da associação Hazard ratio (HR). Resultados: Por análise de Cox univariada, o escore avascular associado à mortalidade foi estatisticamente significativo (HR = IC 1.54, 95%: 1.13-2.09, p = 0.006), mas outras variáveis capilaroscópicas (número de ectasias, megacapillaries e hemorragias por dedo) não foram (p> 0,40 para todos os testes). Após o ajuste para escore de pele, idade, sexo, origem étnica, e sinais de isquemia digital, a associação entre o escore avascular perdeu significância estatística (HR = 1.23, IC: 0.84-1.81, p = 0.276). Também não houve associação significativa do escore avascular quando ajustado para uma combinação de resultados de exames complementares. Conclusões: A gravidade da desvascularização na CPU está relacionada a um maior risco de mortalidade na ES; No entanto, a associação não é estatisticamente significativa após ajuste para outras variáveis clínicas e laboratoriais. Apesar do fato de não mostrar uma associação independente do EA com a mortalidade, consideramos que é útil na avaliação de pacientes com diagnóstico de esclerose sistêmica, uma vez que pode dar uma visão geral e confiável da gravidade da doença. / Introduction: The nailfold capillaroscopy (NCF) has been related to end-organ damage in Systemic sclerosis (SSc). However, studies relating the severity of NCF alterations with mortality have rendered mixed results. This study aims to verify the association of the severity of peripheral microangiopathy in the NFC with the risk of death in SSc. Patients and methods: One hundred and seventy SSc patients were prospectively evaluated and followed a mean of 9.3 years. Besides clinical evaluation, patients underwent serology, pulmonary function tests, Doppler echocardiography, and pulmonary high resolution computed tomography (HRCT). Capillary loss on NCF was evaluated using the avascular score (AS), ranging from 0 to 3. The mean number of ectasias, megacapillaries and hemorrhages per finger was also recorded; univariate and multivariate Cox proportional models were used to analyze the association of the AS with the risk of death using hazard ratios (HR). Results: By univariate Cox analysis, the AS was statistically significantly associated with mortality (HR = 1.54, 95% CI: 1.13 to 2.09, p = 0.006), but other capillaroscopic variables (number of ectasias, megacapillaries and hemorrhages per finger) were not (p> 0.40 for all tests). After adjustment for skin score, age, gender, ethnic background, and signs of digital ischemia, the association between the AS weakened and lost statistical significance (HR = 1.23, CI: 0.84 to 1.81, p=0.276). There was also no significant association of the avascular score when adjusted for a combination of results of complementary tests. Conclusions: The severity of devascularization on NCF is related to a higher risk of mortality in SSc; however, the association disappeared after adjustment for other clinical and laboratory variables. Despite the fact that we were not able to show an independent association of the AS with mortality, we consider it useful in the evaluation in patients diagnosed with systemic sclerosis, since it can give a general view and reliable view of the severity of the disease.