Systemic sclerosis associated interstitial lung disease: a conceptual framework for subclinical, clinical and progressive disease.

OBJECTIVES: To establish a framework by which experts define disease subsets in systemic sclerosis associated interstitial lung disease (SSc-ILD). METHODS: A conceptual framework for subclinical, clinical and progressive ILD was provided to 83 experts, asking them to use the framework and classify actual SSc-ILD patients. Each patient profile was designed to be classified by at least four experts in terms of severity and risk of progression at baseline; progression was based on 1-year follow-up data. A consensus was reached if ≥75% of experts agreed. Experts provided information on which items were important in determining classification. RESULTS: Forty-four experts (53%) completed the survey. Consensus was achieved on the dimensions of severity (75%, 60 of 80 profiles), risk of progression (71%, 57 of 80 profiles) and progressive ILD (60%, 24 of 40 profiles). For profiles achieving consensus, most were classified as clinical ILD (92%), low risk (54%) and stable (71%). Severity and disease progression overlapped in terms of framework items that were most influential in classifying patients (forced vital capacity, extent of lung involvement on high resolution chest CT [HRCT]); risk of progression was influenced primarily by disease duration. CONCLUSIONS: Using our proposed conceptual framework, international experts were able to achieve a consensus on classifying SSc-ILD patients along the dimensions of disease severity, risk of progression and progression over time. Experts rely on similar items when classifying disease severity and progression: a combination of spirometry and gas exchange and quantitative HRCT.

Expert consensus on the management of systemic sclerosis-associated interstitial lung disease.

BACKGROUND: Systemic sclerosis (SSc) is a rare, complex, connective tissue disorder. Interstitial lung disease (ILD) is common in SSc, occurring in 35-52% of patients and accounting for 20-40% of mortality. Evolution of therapeutic options has resulted in a lack of consensus on how to manage this condition. This Delphi study was initiated to develop consensus recommendations based on expert physician insights regarding screening, progression, treatment criteria, monitoring of response, and the role of recent therapeutic advances with antifibrotics and immunosuppressants in patients with SSc-ILD. METHODS: A modified Delphi process was completed by pulmonologists (n = 13) and rheumatologists (n = 12) with expertise in the management of patients with SSc-ILD. Panelists rated their agreement with each statement on a Likert scale from - 5 (complete disagreement) to + 5 (complete agreement). Consensus was predefined as a mean Likert scale score of ≤ - 2.5 or ≥ + 2.5 with a standard deviation not crossing zero. RESULTS: Panelists recommended that all patients with SSc be screened for ILD by chest auscultation, spirometry with diffusing capacity of the lungs for carbon monoxide, high-resolution computed tomography (HRCT), and/or autoantibody testing. Treatment decisions were influenced by baseline and changes in pulmonary function tests, extent of ILD on HRCT, duration and degree of dyspnea, presence of pulmonary hypertension, and potential contribution of reflux. Treatment success was defined as stabilization or improvement of signs or symptoms of ILD and functional status. Mycophenolate mofetil was identified as the initial treatment of choice. Experts considered nintedanib a therapeutic option in patients with progressive fibrotic ILD despite immunosuppressive therapy or patients contraindicated/unable to tolerate immunotherapy. Concomitant use of nintedanib with MMF/cyclophosphamide can be considered in patients with advanced disease at initial presentation, aggressive ILD, or significant disease progression. Although limited consensus was achieved on the use of tocilizumab, the experts considered it a therapeutic option for patients with early SSc and ILD with elevated acute-phase reactants. CONCLUSIONS: This modified Delphi study generated consensus recommendations for management of patients with SSc-ILD in a real-world setting. Findings from this study provide a management algorithm that will be helpful for treating patients with SSc-ILD and addresses a significant unmet need.

Myocarditis in connective tissue diseases: an often-overlooked clinical manifestation.

To discuss what is currently known about myocarditis in the context of major connective tissue diseases, including Systemic lupus erythematosus, Rheumatoid Arthritis, Sjogren, Dermato-myositis and Polymyositis, Systemic Sclerosis, and Mixed connective tissue disease. Variability exists between studies regarding the incidence of myocarditis in connective tissue diseases, which is hypothesized to be the result of its subclinical course in most cases. Extensive gaps of knowledge exist in the field of pathophysiology. Although endomyocardial biopsy remains to be the gold standard for diagnosis, the advancement in non-invasive modalities such as cardiac MRI, echocardiography, and nuclear medicine has allowed for earlier and more frequent detection of myocarditis. A lack of treatment guidelines was found across the different connective tissue diseases. Most of the literature available revolved around myocarditis in the context of Systemic lupus erythematosus. Numerous recent studies were published that contributed to advancements in diagnosis and treatment however, there remains a lack of diagnostic and treatment guidelines.

Epigenetic down-regulation of microRNA-126 in scleroderma endothelial cells is associated with impaired responses to VEGF and defective angiogenesis.

Impaired angiogenesis in scleroderma (SSc) is a critical component of SSc pathology. MicroRNA-126 (miR-126) is expressed in endothelial cells (MVECs) where it regulates VEGF responses by repressing the negative regulators of VEGF, including the sprouty-related protein-1 (SPRED1), and phosphoinositide-3 kinase regulatory subunit 2 (PIK3R2). MVECs were isolated from SSc skin and matched subjects (n = 6). MiR-126 expression was measured by qPCR and in situ hybridization. Matrigel-based tube assembly was used to test angiogenesis. MiR-126 expression was inhibited by hsa-miR-126 inhibitor and enhanced by hsa-miR-126 Mimic. Epigenetic regulation of miR-126 expression was examined by the addition of epigenetic inhibitors (Aza and TSA) to MVECs and by bisulphite genomic sequencing of DNA methylation of the miR-126 promoter region. MiR-126 expression, as well as EGFL7 (miR-126 host gene), in SSc-MVECs and skin, was significantly down-regulated in association with increased expression of SPRED1 and PIK3R2 and diminished response to VEGF. Inhibition of miR-126 in NL-MVECs resulted in reduced angiogenic capacity, whereas overexpression of miR-126 in SSc-MVECs resulted in enhanced tube assembly. Addition of Aza and TSA normalized miR-126 and EGFL7 expression levels in SSc-MVECs. Heavy methylation in miR-126/EGFL7 gene was noted. In conclusion, these results demonstrate that the down-regulation of miR-126 results in impaired VEGF responses.

ANCA in systemic sclerosis, when vasculitis overlaps with vasculopathy: a devastating combination of pathologies.

In patients with SSc, the coexistence of ANCA-associated vasculitis (SSc-AAV) has been reported to be associated with a severe disease course, including significant pulmonary and renal involvement. The presence of ANCA is not uncommon in patients with SSc, and therefore clinicians must maintain a high index of clinical suspicion about SSc-AAV. p-ANCA and anti-myeloperoxidase antibodies are the most common antibodies observed. Patients typically present with clinical features of microscopic polyangiitis or renal-limited vasculitis. There are multiple areas of potential interaction in the pathogenesis of SSc and AAV, which can exacerbate/compound vascular disease. In addition, similar patterns of major internal organ involvement (e.g. lung and kidneys) are seen in both conditions. We highlight a diagnostic approach to SSc-AAV and the paucity of data to inform management. As such, SSc-AAV is typically treated as per isolated AAV, which can potentially be hazardous in patients with SSc (e.g. due to the association between high-dose steroid and scleroderma renal crisis). We propose that this rare clinical entity warrants rigorous investigation, including definition of a therapeutic strategy to ameliorate the potentially devastating combination of pathologies in SSc-AAV.

Molecular mechanisms underlying rheumatoid arthritis and cancer development and treatment.

Given recent advances in cancer immune therapy, specifically use of checkpoint inhibitors, understanding the link between autoimmunity and cancer is essential. Rheumatoid arthritis (RA) affects about 1% of the population, and early diagnosis is key to prevent joint damage. Management consists of disease-modifying antirheumatic drugs that alter normal immunologic pathways, which could affect malignancy growth and survival. Prolonged immune dysregulation and the resulting inflammatory response associated with development of RA may also lead to increased cancer development risk. RA has long been associated with increased risk of non-Hodgkin's lymphoma [1] and further evidence supports relationship to lung cancer [2]. This review will address the mechanisms behind cancer development and progression in RA patients, biomarkers and assess cancer risk and early detection.

Evaluation of topical econazole nitrate formulations with potential for treating Raynaud's phenomenon.

The purpose of this work was to design and characterize a topical formulation of econazole nitrate (EN) with potential for treating Raynaud's phenomenon (RP). Four topical dosage forms (F1_topical solution, F2_HPMC or hydroxypropyl methylcellulose dispersion, F3_VersaBase® cream, and F4_Lipoderm® Activemax™ Cream) containing 3% w/w EN were prepared and characterized for drug content, pH, viscosity, spreadability, drug crystallinity, stability, and in vitro permeation using Franz cells across pig ear skin, and results were compared to the 1% marketed EN cream. All four formulations had acceptable physical and visual characteristics required for topical application, with 3% w/w EN. The order of amount of drug permeated from highest to lowest was F2 (10.27%) > F4 (2.47%) > F1 (2.28%) > F3 (1.47%) > marketed formulation (0.22%). Formulation F2 showed better penetration of the drug into the stratum corneum, epidermis, and dermis layers. The drug concentration in the stratum corneum and epidermis was approximately 10-20 times higher with F2 compared to the marketed formulation. All formulations were found to be stable for up to 6 months. All four EN formulations were found to be better than the 1% marketed cream. Formulation F2_HPMC dispersion could be further explored as a treatment option for RP.

Tendonitis and Tendon Rupture After Treatment With Rituximab: A Case Series.

CLINICAL DATA: Rituximab is a mouse/human chimeric anti-CD20 IgG1 monoclonal antibody used to treat cancer and autoimmune conditions. Side effects of rituximab include fever, rash, cytopenia and hypotension, back pain, arthralgia, and myalgia. Here, we report on 3 patients who developed moderate to severe tendonitis after the second infusion of rituximab. THERAPEUTIC CHALLENGE: We report 3 patients who developed tendonitis after the second infusion of rituximab. These patients were undergoing treatment for connective tissue diseases. All 3 patients received 2 rituximab infusions, 2 weeks apart. The 3 cases developed clinical tendonitis that was confirmed by magnetic resonance imaging in 2 cases. INTERPRETATION: This is the first case series reporting new onset tendonitis in patients with connective tissue diseases after rituximab therapy. All 3 cases developed tendonitis 1 week after receiving the second dose of rituximab. Clinical features of tendonitis resolved 3-4 months in all cases. The underlying pathogenic mechanism by which rituximab causes tendonitis is not clear, but tendonitis and tendon rupture have been reported after using other medications such as quinolones. The tendon damage was progressive leading to tendon rupture in 1 patient, highlighting the importance of early recognition. It is plausible that there is a cause-effect relation between tendonitis and administration of rituximab in our 3 cases, since none of these cases had previous history of tendonitis; however, more data are needed to confirm this observation.

Scleroderma dermal microvascular endothelial cells exhibit defective response to pro-angiogenic chemokines.

OBJECTIVES: Angiogenesis plays a critical role in SSc (scleroderma). The aim of this study was to examine the expression of growth-regulated protein-γ (Gro-γ/CXCL3), granulocyte chemotactic protein 2 (GCP-2/CXCL6) and their receptor CXCR2 in endothelial cells (ECs) isolated from SSc skin and determine whether these cells mount an angiogenic response towards pro-angiogenic chemokines. The downstream signalling pathways as well as the pro-angiogenic transcription factor inhibitor of DNA-binding protein 1 (Id-1) were also examined. METHODS: Skin biopsies were obtained from patients with dcSSc. ECs were isolated via magnetic positive selection. Angiogenesis was measured by EC chemotaxis assay. RESULTS: Gro-γ/CXCL3 and GCP-2/CXCL6 were minimally expressed in both skin types but elevated in SSc serum. Pro-angiogenic chemokine mRNA was greater in SSc ECs than in normal ECs. SSc ECs did not migrate to vascular endothelial growth factor (VEGF), Gro-γ/CXCL3, GCP-2/CXCL6 or CXCL16. The signalling pathways stimulated by these chemokines were also dysregulated. Id-1 mRNA in SSc ECs was lower compared with normal ECs, and overexpression of Id-1 in SSc ECs increased their ability to migrate towards VEGF and CXCL16. CONCLUSION: Our results show that SSc ECs are unable to respond to pro-angiogenic chemokines despite their increased expression in serum and ECs. This might be due to the differences in the signalling pathways activated by these chemokines in normal vs SSc ECs. In addition, the lower expression of Id-1 also decreases the angiogenic response. The inability of pro-angiogenic chemokines to promote EC migration provides an additional mechanism for the impaired angiogenesis that characterizes SSc.

Epigenetics, the holy grail in the pathogenesis of systemic sclerosis.

The objective of this review is to present evidence that supports the central role of epigenetic regulation in the pathogenesis of SSc. SSc is a complex autoimmune disease characterized by immune activation, fibrosis of the skin and internal organs and obliterative vasculopathy affecting predominantly the microvessels. Remarkable progress has been made in the past few years emphasizing the importance of epigenetic modifications in the pathogenesis of many disorders, including SSc. Current evidence demonstrates alterations in DNA methylation, histone code modifications and changes in microRNA (miRNA) expression levels in SSc cells. Recent reports have described the differential expression of numerous regulatory miRNAs in SSc, mainly in SSc fibroblasts, a number of which are important in TGF-ß pathways and downstream signalling cascades. While studies to date have revealed the significant role of epigenetic modifications in the pathogenesis of SSc, the causal nature of epigenetic alterations in SSc pathogenesis remains elusive. Additional longitudinal and comprehensive epigenetic studies designed to evaluate the effect of environmental epigenetic factors on disease pathogenesis are needed.

Endothelial dysfunction in systemic sclerosis.

PURPOSE OF REVIEW: Microvascular endothelial cells (MVECs) injury is a critical early event in the pathogenesis of systemic sclerosis (SSc). We aim to provide the reader with an update regarding the role of MVECs in the pathogenesis of SSc and the mechanisms for MVECs dysfunction in the disease. RECENT FINDINGS: Recent evidence confirms the central role for MVECs in the pathogenesis of SSc, and suggests further mechanisms for MVECs injury. The impact of MVECs perturbations in SSc goes beyond the initiation of the vascular disease to include activation of fibroblasts through the release of cytokines and growth factors like connective tissue growth factor that induce an active and aggressive form of fibroblasts. Moreover, recent studies highlighted a more prominent role for epigenetic factors in the pathogenesis of SSc, and suggested defects in the function of progenitor endothelial cells in SSc. Recent reports helped to shed light on the role of antiendothelial cell antibodies in the pathogenesis of SSc, especially purified subsets of these antibodies like anti-ICAM-1 antibodies, and also reported possible mechanisms for defective vascular endothelial growth factor signaling. SUMMARY: It is clear that MVECs dysfunction is a key element in the pathogenesis of SSc, but the initial triggers for MVEC dysfunction remain uncharacterized.

International consensus criteria for the diagnosis of Raynaud's phenomenon.

Vasoconstriction accompanied by changes in skin color is a normal physiologic response to cold. The distinction between this normal physiology and Raynaud's phenomenon (RP) has yet to be well characterized. In anticipation of the 9th International Congress on Autoimmunity, a panel of 12 RP experts from 9 different institutes and four different countries were assembled for a Delphi exercise to establish new diagnostic criteria for RP. Relevant investigators with highly cited manuscripts in Raynaud's-related research were identified using the Web of Science and invited to participate. Surveys at each stage were administered to participants via the on-line SurveyMonkey software tool. The participants evaluated the level of appropriateness of statements using a scale of 1 (extremely inappropriate) through 9 (extremely appropriate). In the second stage, panel participants were asked to rank rewritten items from the first round that were scored as "uncertain" for the diagnosis of RP, items with significant disagreement (Disagreement Index > 1), and new items suggested by the panel. Results were analyzed using the Interpercentile Range Adjusted for Symmetry (IPRAS) method. A 3-Step Approach to diagnose RP was then developed using items the panelists "agreed" were "appropriate" diagnostic criteria. In the final stage, the panel was presented with the newly developed diagnostic criteria and asked to rate them against previous models. Following the first two iterations of the Delphi exercise, the panel of 12 experts agreed that 36 of the items were "appropriate", 12 items had "uncertain" appropriateness, and 13 items were "inappropriate" to use in the diagnostic criteria of RP. Using an expert committee, we developed a 3-Step Approach for the diagnosis of RP and 5 additional criteria for the diagnosis of primary RP. The committee came to an agreement that the proposed criteria were "appropriate and accurate" for use by physicians to diagnose patients with RP.

Systemic sclerosis endothelial cells recruit and activate dermal fibroblasts by induction of a connective tissue growth factor (CCN2)/transforming growth factor ß-dependent mesenchymal-to-mesenchymal transition.

OBJECTIVE: Clinical evidence suggests that the vascular abnormalities of systemic sclerosis (SSc) precede the onset of fibrosis, but molecular cues accounting for a possible vascular connection of SSc fibrosis have been elusive, although they have been searched for intensively. Since we had previously shown that connective tissue growth factor (CCN2), endowed with fibroblast-oriented activities, was overexpressed by endothelial cells (ECs) from SSc patients, we undertook this study to investigate its role and mechanisms in fibroblast activation. METHODS: Normal fibroblasts were challenged with conditioned medium of normal microvascular ECs (MVECs) and MVECs obtained from SSc patients with the diffuse form of the disease. Fibroblast invasion was studied using the Boyden chamber Matrigel assay. Fibroblast activation was evaluated by Western blotting and immunofluorescence of α-smooth muscle actin (α-SMA), vimentin, and type I collagen. Matrix metalloproteinase (MMP) production was evaluated by zymography and reverse transcription-polymerase chain reaction. Signal transduction and activation of the small GTPases RhoA and Rac1 were studied by Western blotting. Inhibition of SSc MVEC conditioned medium-dependent fibroblast activation was obtained by anti-CCN2 antibodies and the transforming growth factor ß (TGFß) antagonist peptide p17. RESULTS: SSc MVEC CCN2 stimulated fibroblast activation and invasion. Such activities depended on CCN2-induced overexpression of TGFß and its type I, II, and III receptors combined with overproduction of MMP-2 and MMP-9 gelatinases. All of these effects were reversed by the TGFß antagonist peptide p17. Motility increase required Rac1 activation and RhoA inhibition and was inhibited by an MMP inhibitor. These features connoted a mesenchymal style of cell invasion. Since fibroblast activation also fostered overexpression of α-SMA, vimentin, and type I collagen, the CCN2-dependent increase in fibroblast activities recapitulated the characteristics of a mesenchymal-to-mesenchymal transition. CONCLUSION: SSc MVECs recruit and activate dermal fibroblasts by induction of a CCN2/TGFß-dependent mesenchymal-to-mesenchymal transition.

2013 classification criteria for systemic sclerosis: an American College of Rheumatology/European League against Rheumatism collaborative initiative.

OBJECTIVE: The 1980 American College of Rheumatology (ACR) classification criteria for systemic sclerosis (SSc) lack sensitivity for early SSc and limited cutaneous SSc. The present work, by a joint committee of the ACR and the European League Against Rheumatism (EULAR), was undertaken for the purpose of developing new classification criteria for SSc. METHODS: Using consensus methods, 23 candidate items were arranged in a multicriteria additive point system with a threshold to classify cases as SSc. The classification system was reduced by clustering items and simplifying weights. The system was tested by 1) determining specificity and sensitivity in SSc cases and controls with scleroderma-like disorders, and 2) validating against the combined view of a group of experts on a set of cases with or without SSc. RESULTS: It was determined that skin thickening of the fingers extending proximal to the metacarpophalangeal joints is sufficient for the patient to be classified as having SSc; if that is not present, 7 additive items apply, with varying weights for each: skin thickening of the fingers, fingertip lesions, telangiectasia, abnormal nailfold capillaries, interstitial lung disease or pulmonary arterial hypertension, Raynaud's phenomenon, and SSc-related autoantibodies. Sensitivity and specificity in the validation sample were, respectively, 0.91 and 0.92 for the new classification criteria and 0.75 and 0.72 for the 1980 ACR classification criteria. All selected cases were classified in accordance with consensus-based expert opinion. All cases classified as SSc according to the 1980 ACR criteria were classified as SSc with the new criteria, and several additional cases were now considered to be SSc. CONCLUSION: The ACR/EULAR classification criteria for SSc performed better than the 1980 ACR criteria for SSc and should allow for more patients to be classified correctly as having the disease.

Epigenetic repression of bone morphogenetic protein receptor II expression in scleroderma.

Germline mutations in the bone morphogenetic protein type II receptor (BMPRII) gene play an essential role in the pathogenesis of familial pulmonary arterial hypertension (FPAH). In view of the histological similarities between scleroderma (SSc) and FPAH arterial lesion, we examined the expression levels of BMPRII in SSc microvascular endothelial cells (MVEC). Oxidative stress and serum starvation were used to examine apoptotic responses of MVECs. BMPRII expression levels were determined by RT-PCR and by Western blot. Epigenetic regulation of BMPRII expression was examined by the addition of epigenetic inhibitors to MVECs cultures, by methylation-specific PCR, and by sequence analysis of DNA methylation pattern of the BMPRII promotor region. SSc-MVECs were more sensitive to apoptotic signals than were normal-MVECs. A significant decrease in BMPRII expression levels in SSc-MVECs was noted, whereas no significant differences in the expression levels of BMPRIA and BMPRIB were observed. Similar reduction in expression levels was noted in SSc skin biopsies. The expression level of BMPRII in SSc-MVECs was normalized by the addition of 2-deoxy-5-azacytidine and trichostatin A to cell cultures. Extensive CpG sites methylation in the BMPRII promoter region was noted in SSc-MVECs with no detectable site methylation in control-MVECs. SSc-MVECs are more sensitive to apoptotic triggers than are control-MVECs. The enhanced apoptosis may be related to epigenetic repression of BMPRII expression as apoptosis of control-MVECs can be augmented by knocking down BMPRII expression. The role of BMPRII underexpression in the pathogenesis of SSc vasculopathy is suggested and should be investigated further.

2013 classification criteria for systemic sclerosis: an American college of rheumatology/European league against rheumatism collaborative initiative.

OBJECTIVE: The 1980 American College of Rheumatology (ACR) classification criteria for systemic sclerosis (SSc) lack sensitivity for early SSc and limited cutaneous SSc. The present work, by a joint committee of the ACR and the European League Against Rheumatism (EULAR), was undertaken for the purpose of developing new classification criteria for SSc. METHODS: Using consensus methods, 23 candidate items were arranged in a multicriteria additive point system with a threshold to classify cases as SSc. The classification system was reduced by clustering items and simplifying weights. The system was tested by (1) determining specificity and sensitivity in SSc cases and controls with scleroderma-like disorders, and (2) validating against the combined view of a group of experts on a set of cases with or without SSc. RESULTS: It was determined that skin thickening of the fingers extending proximal to the metacarpophalangeal joints is sufficient for the patient to be classified as having SSc; if that is not present, seven additive items apply, with varying weights for each: skin thickening of the fingers, fingertip lesions, telangiectasia, abnormal nailfold capillaries, interstitial lung disease or pulmonary arterial hypertension, Raynaud's phenomenon, and SSc-related autoantibodies. Sensitivity and specificity in the validation sample were, respectively, 0.91 and 0.92 for the new classification criteria and 0.75 and 0.72 for the 1980 ACR classification criteria. All selected cases were classified in accordance with consensus-based expert opinion. All cases classified as SSc according to the 1980 ACR criteria were classified as SSc with the new criteria, and several additional cases were now considered to be SSc. CONCLUSIONS: The ACR/EULAR classification criteria for SSc performed better than the 1980 ACR criteria for SSc and should allow for more patients to be classified correctly as having the disease.

Systemic Sclerosis (SSc) After COVID-19: A Case Report.

Since the start of the global pandemic caused by coronavirus disease 2019 (COVID-19), there have been numerous reports of autoimmune and rheumatological disorders developing after infection with SARS-CoV-2. To date, there has been only one reported case of systemic sclerosis (SSc) developing after SARS-CoV-2 infection. Here, we present another case of SSc developing after infection with SARS-CoV-2. A 48-year-old female with past medical history of anxiety and depression presented to the rheumatology clinic after being referred for further evaluation of abnormal labs, Raynaud's phenomenon, and other concerning symptoms. Shortly after hospitalization for COVID-19 pneumonia, she began experiencing symptoms that included fatigue, xerostomia, dysphagia, bilateral lower extremity weakness, dyspnea with exertion, unintentional weight loss, and diffuse skin hyperpigmentation. Labs ordered shortly before presentation were significant for antinuclear antibody (ANA) titer > 1:1280. Physical exam was remarkable for puffy fingers, sclerodactyly of the fingers, diffuse skin hyperpigmentation, and abnormal nailfold capillaries. Anti-RNA polymerase III, anti-Scl-70, anti-centromere, anti-SSA, anti-SSB, anti-Smith, and anti-Smith/RNP antibodies were all negative. BNP, aldolase, and serum myoglobin levels were within normal limits while creatine phosphokinase level was slightly decreased. Pulmonary function testing showed reduced diffusion capacity with normal lung mechanics and volumes. High-resolution CT scan of the chest showed interstitial lung disease, with findings suggestive of nonspecific interstitial pneumonia. Transthoracic echocardiogram showed mild elevation of right ventricular systolic pressure, but pulmonary hypertension was not found on right heart catheterization. Esophagogastroduodenoscopy (EGD) with biopsy performed for evaluation of esophageal dysphagia showed sliding hiatal hernia, irregular Z-line, and gastric hyperemia. Biopsy of the distal esophagus was consistent with Barrett's esophagus. The patient was diagnosed with SSc according to the 2013 American College of Rheumatology/European League Against Rheumatism (ACR-EULAR) classification criteria for SSc. She is currently being treated with mycophenolate mofetil, amlodipine, methotrexate, and prednisone.

Genome-wide DNA methylation pattern in systemic sclerosis microvascular endothelial cells: Identification of epigenetically affected key genes and pathways.

Background: The etiology of systemic sclerosis is not clear, but there is evidence suggesting a critical role for epigenetic alterations in disease pathogenesis and clinical expression. We sought, in this study, to characterize the genome-wide DNA methylation signature in systemic sclerosis microvascular endothelial cells. Methods: We performed a genome-wide DNA methylation study in microvascular endothelial cells derived from seven diffuse cutaneous systemic sclerosis patients compared to seven age-, sex-, and ethnicity-matched healthy controls. We paired matched samples on Illumina HumanMethylation450 (three diffuse cutaneous systemic sclerosis microvascular endothelial cells and three controls), and reproduced the results in an independent set of matched patient and controls using Illumina Infinium MethylationEPIC (four diffuse cutaneous systemic sclerosis patients and four controls) to identify differentially methylated genes. Results: We identified 71,353 differentially methylated CpG sites in systemic sclerosis microvascular endothelial cells using Infinium MethylationEPIC microarray in the first group (0.081% of representative probes) and 33,170 CpG sites in the second group using HumanMethylation450 microarray (0.073% of representative probes) in diffuse cutaneous systemic sclerosis microvascular endothelial cells. Among the two groups of subjects, we identified differential methylation of 2455 CpG sites, representing 1301 genes. Most of the differentially methylated CpG sites were hypermethylated (1625 CpG), corresponding to 910 genes. Common hypermethylated genes in systemic sclerosis microvascular endothelial cells include NOS1, DNMT3A, DNMT3B, HDAC4, and ANGPT2. We also identified hypomethylation of IL17RA, CTNNA3, ICAM2, and SDK1 in systemic sclerosis microvascular endothelial cells. Furthermore, we demonstrate significant inverse correlation between DNA methylation status and gene expression in the majority of genes evaluated. Gene ontology analysis of hypermethylated genes demonstrated enrichment of genes involved in angiogenesis (p = 0.0006). Pathway analysis of hypomethylated genes includes genes involved in vascular smooth muscle contraction (p = 0.014) and adherens junctions (p = 0.013). Conclusion: Our data suggest the presence of significant genome-wide DNA methylation aberrancies in systemic sclerosis microvascular endothelial cells, and identify novel affected genes and pathways in systemic sclerosis microvascular endothelial cells.

Modulation of the angiogenic phenotype of normal and systemic sclerosis endothelial cells by gain-loss of function of pentraxin 3 and matrix metalloproteinase 12.

OBJECTIVE: Studies have shown that in systemic sclerosis (SSc) endothelial cells, overproduction of matrix metalloproteinase 12 (MMP-12) and pentraxin 3 (PTX3) is associated with defective angiogenesis. This study was undertaken to examine whether overexpression of the relevant molecules could inhibit angiogenesis of normal microvascular endothelial cells (MVECs), and whether silencing of these molecules in SSc MVECs could restore the lost angiogenic properties of the cells in vitro and in vivo. METHODS: Transient transfection of MVECs with human MMP12 and PTX3 was performed by electroporation. Silencing of MMP12 and PTX3 was obtained by treatment with small interfering RNA, and treatment effects were validated by Western blotting with specific antibodies and a fluorimetric assay. In vitro cell migration and capillary morphogenesis were studied on Matrigel substrates. In vivo angiogenesis was studied using a Matrigel sponge assay in mice. RESULTS: Transfection of MMP12 and PTX3 in normal MVECs resulted in loss of proliferation, invasion, and capillary morphogenesis in vitro, attributed to truncation of the urokinase-type plasminogen activator receptor by MMP12 and to the anti-fibroblast growth factor 2/anti-vascular endothelial growth factor activity of PTX3. These effects were particularly evident in mixed populations of transfected normal MVECs (50% transfected with MMP12 and 50% with PTX3). Silencing of the same molecules in SSc MVECs increased their invasion in Matrigel. Single-gene silencing did not increase the capillary morphogenesis of SSc MVECs, whereas double-gene-silenced cells showed a burst of capillary tube formation. Culture medium of silenced SSc MVECs stimulated angiogenesis in assays of Matrigel sponge invasion in mice. CONCLUSION: Overexpression of either MMP12 or PTX3 in normal MVECs blunts their angiogenic properties. Loss of function of MMP12 and PTX3 in SSc MVECs restores the ability of the cells to produce capillaries in vitro and induces vascularization in vivo on a Matrigel sponge.

Similarities between COVID-19 and systemic sclerosis early vasculopathy: A "viral" challenge for future research in scleroderma.

OBJECTIVE: To review similarities between COVID-19 and systemic sclerosis (SSc) early vasculopathy to provide novel insights into both diseases. METHODS: A narrative review of the literature supplemented with expert opinion. RESULTS: There is clear evidence that the endothelium is at the centre stage in SSc and COVID-19, with endothelial cell activation/injury and dysfunction creating the crucial evolving step in the pathogenesis of both diseases. The angiotensin system has also been implicated in the early stages of both COVID-19 and SSc. Autoptic studies provide novel insights into the effects of SARS-CoV-2 on the endothelium. Normal endothelium and endothelial dysfunction in COVID-19 and SSc are discussed. It is debated whether SARS-CoV-2 infection triggers autoimmunity with production of autoantibodies which is of mechanistic interest because other viral illnesses are potentially involved in endothelial dysfunction and in SSc pathogenesis. CONCLUSION: COVID-19 is due to a direct assault of SARS-CoV-2 on the vascular system as an acute infection, whereas SSc remains a chronic/sub-acute autoimmune disease of largely unknown etiology Further study and exploration of the SARS-CoV-2 pathogenic mechanisms might provide further useful milestones in the understanding of the early SSc pathogenesis.