Nezam Altorok

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-β 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.

Epigenetics and systemic sclerosis

Systemic sclerosis (SSc) is an autoimmune connective tissue disease characterized by vascular injury, activation of the immune system, and diffuse tissue fibrosis. The precise etiology of SSc is undetermined, but there is evidence suggestive of a connection between environmental factors and SSc pathogenesis. In general, harmful environmental factors are sensed by the epigenetic regulatory mechanisms that alter host gene expression leading to the emergence of disease-specific phenotype. There are three epigenetic mechanisms involved in gene regulation: DNA methylation, histone modifications, and microRNAs. Although there is evidence that SSc phenotype could be, to a some degree, determined by genetic variants, it is clear now that non-genetic factors outweigh the genetic risk in SSc. Accordingly, the environment can trigger epigenetic regulation that in turn establishes a molecular framework linking environmental exposures to genetics, leading to the disease process, possibly in a genetically predisposed host. Although we have just begun to appreciate the potential role of epigenetics in SSc, many important and promising clues have been observed. In this review, we will summarize the work that has been done in the field of epigenetic regulation in SSc, and we will discuss possible factors and mechanisms that may lead to epigenetic dysregulation in SSc.

IMAGES IN CLINICAL MEDICINE. Diffuse Soft-Tissue Calcinosis

A 50-year-old-woman with dermatomyositis presented with progressive, diffuse subcutaneous nodules on the arms and legs. A radiograph of the left shoulder showed extensive soft-tissue calcification and an old fracture of the surgical neck of the humerus.

Patient-reported outcome instruments for assessing Raynaud’s phenomenon in systemic sclerosis: A SCTC Vascular Working Group Report

The episodic nature of Raynaud’s phenomenon in systemic sclerosis has led to a reliance on patient-reported outcome instruments such as the Raynaud’s Condition Score diary. Little is known about the utilization in routine clinical practice and health professional attitudes toward existing patient-reported outcome instruments for assessing systemic sclerosis-Raynaud’s phenomenon. Members of the Scleroderma Clinical Trials Consortium Vascular Working Group (nu2009=u200928) were invited to participate in a survey gauging attitudes toward the Raynaud’s Condition Score diary and the perceived need for novel patient-reported outcome instruments for assessing patient-reported outcome. Nineteen Scleroderma Clinical Trials Consortium Vascular Working Group members (68% response rate) from academic units based in North America (nu2009=u20099), Europe (nu2009=u20098), South America (nu2009=u20091) and Australasia (nu2009=u20091) took part in the survey. There was broad consensus that Raynaud’s Condition Score diary returns could be influenced by factors including seasonal variation in weather, efforts made by patients to avoid or ameliorate attacks of Raynaud’s phenomenon, habituation to Raynaud’s phenomenon symptoms, evolution of Raynaud’s phenomenon symptom characteristics with progressive obliterative microangiopathy, patient-coping strategies, respondent burden and placebo effect. There was consensus that limitations of the Raynaud’s Condition Score diary might be a barrier to drug development (79% of respondents agree/strongly agree) and that a novel patient-reported outcome instrument for assessing systemic sclerosis-Raynaud’s phenomenon should be developed with the input of both clinicians and patients (84% agree/strongly agree). Perceived potential limitations of the Raynaud’s Condition Score diary have been identified along with concerns that such factors might impede drug development programs for systemic sclerosis-Raynaud’s phenomenon. There is support within the systemic sclerosis community for the development of a novel patient-reported outcome instrument for assessing systemic sclerosis-Raynaud’s phenomenon.

Bacterial Endocarditis Complicated by Leukocytoclastic Vasculitis

A 29-year-old woman who underwent dental extraction 1 month before her presentation with painful purpuric skin lesions that progressed to necrotic ulcers over the buttocks and lower extremities (Figure 1A). Physical examination demonstrated new pansystolic murmur grade IV/VI at the apex of the heart and splinter hemorrhages. Initial laboratory testing was pertinent for elevated inflammatory markers, low C3 and C4 levels, positive Rheumatoid factor, positive p-ANCA (titer 1:5120) and positive anti-serine proteinase 3. Blood cultures grew Staphylococcus epidermidis. A transesophageal echocardiogram confirmed the presence of large vegetation on the mitral valve associated with severe mitral regurgitation (Figure 1B). Skin biopsy demonstrated smalland medium-sized vessels with reactive endothelium and surrounding acute inflammatory cells as depicted between the arrows in Figure 1C, on background of fibrinoid necrosis and focal extravasated red blood cells and nuclear dust, all suggestive of leukocytoclastic vasculitis. Endocarditis is one of the vasculitis mimickers that should remain in the differential diagnosis of systemic vasculitis because clinically and pathologically it may be hard to differentiate between the 2 entities, as both diseases can present with peripheral ischemic/embolic phenomena, splinter hemorrhages, low complements and positive rheumatoid factor. Moreover, positive testing for ANCA, anti-proteinase 3 and/ or anti-myeloperoxidase is not uncommon in endocarditis, which further complicates the clinical picture. Although the treatment for vasculitis is immunosuppressive therapy, the treatment for vasculitis driven by infective endocarditis is eradication of the infection. Therefore, it is important to recognize and distinguish vasculitis mimickers from true vasculitis to avoid unnecessary treatment with immunosuppressive agents.

Role of dual-energy computed tomography scan in diagnosis of gout.

T he patient is a 35-year-old man with a background of crystalproven gout. The image on the left side in the Figure represents a dual-energy computed tomography (DECT) scan, which demonstrate a tophaceous deposit (green color) and erosive changes with joint destruction of the right second metatarsophalangeal (MTP) joint (white arrow). There are also minimal tophi deposits in the first MTP joint, around the ankle, and in proximity to the Achilles tendon insertion (green color). The clinical image on the right side of the screen in the Figure shows the corresponding tophus swelling of the second MTP joint (black arrow). Dual-energy CTuses 2 x-ray tubes with DECT scanning (80 and 140 kVp) to simultaneously acquire 2 sets of images of the desired anatomic region. Chemical composition of the targeted material is determined by attenuation images acquired by the dual

Successful treatment of antisynthetase syndrome presenting as rhabdomyolysis with rituximab

Rhabdomyolysis is a syndrome of muscle necrosis with subsequent release of intracellular content into thexa0blood. There are various causes for rhabdomyolysis that include trauma, medications and rarely autoimmune conditions such as autoimmune myositis. Antisynthetase syndrome is an autoimmune condition characterized by positive antisynthetase antibody, myopathy, lung disease and arthritis. To our knowledge, rhabdomyolysis in antisynthetase syndrome has not been reported in thexa0literature. In this report, we present a patient who presented with features of rhabdomyolysis and was diagnosed with antisynthetase syndrome. This patient was treated with systemicxa0steroids with partial improvement, followed by rituximab, which led to significant improvement in his condition. In addition, we summarize all cases reported in the literature of inflammatory myopathy-associated rhabdomyolysis.

Epigenetics in Bone and Joint Disorders

There is growing evidence that implicates the epigenome in the development of bone and joint disorders. The complexity and dynamic nature of epigenetic regulations confound the investigation of these disorders. We will review current evidence that connects epigenetics to the development of autoimmune and musculoskeletal diseases. The pathogenesis of these disorders is likely to be related to modifications to epigenetics regulation by environmental signals in individuals with a certain genetic makeup. The epigenome is an attractive therapeutic target; however, successful epigenetic-based treatment will depend on a greater understanding of the molecular mechanisms controlling the epigenome and its alteration in rheumatic and bone disorders.

The isolation and characterization of systemic sclerosis vascular smooth muscle cells: enhanced proliferation and apoptosis resistance

Background Vascular dysfunction is a major pathologic component of systemic sclerosis (SSc). The role of vascular smooth muscle cells (vSMCs) in the development of vascular dysfunction is still unknown. In this study, we describe a method for isolation of dermal vSMCs from skin biopsies, and we outline their functional phenotype. Methods We obtained 4 mm punch-skin biopsies from three SSc patients and matched controls. After trypsin digestion, cells were cultured for 14 days. vSMCs were isolated by first depleting CD31+ cells (endothelial cells), followed by positive selection of CD146+ cells. The CD31- CD146+ cells were then cultured in media optimized for SMCs proliferation. We evaluated cell proliferation, viability and apoptosis in normal and in low serum culture conditions. Cytoplasmic and nuclear expression levels of β-catenin were also investigated. Results The CD31- CD146+ cell population expressed smooth muscle MYH11, Desmin and Vimentin but did not express NG2. Flow cytometry confirmed the high purity of CD31- CD146+ MYHC11+ cell population that was maintained for up to the eleventh passage. SSc-vSMCs exhibited increased cell proliferation and viability compared to control cells. Under serum starvation conditions, SSc-vSMCs exhibited more proliferative capacity, and resistance to apoptosis compared to control-vSMCs. Furthermore, a cytoplasmic to nuclear translocation of β-catenin was seen in SSc-vSMCs but not in control-vSMCs. Conclusions This is the first report of successful isolation and initial characterization of SSc-vSMCs. It is likely that increased proliferation of SSc-vSMCs in association with resistance to apoptosis can adversely impact the vascular lesion in SSc.

Chapter 12 – Epigenetics and Systemic Sclerosis

Epigenetic aberrancies are emerging as key pathogenic features in systemic sclerosis (SSc). This chapter presents the layers of evidence for epigenetic alterations in SSc. We discuss DNA methylation aberrancies, histone modifications, and altered microRNA (miRNA) expression in SSc, and the role of the epigenetic program in the pathogenesis of SSc. More recently, epigenome-wide DNA methylation studies have identified alterations of the epigenome in fibroblasts from patients with SSc, with an interesting divergence of the “methylome” between limited and diffuse cutaneous SSc subsets. In addition, there is evidence for intrinsic DNA methylation aberrancies in key pathways in the fibrosis process, such as the TGF-β and Wnt/β-catenin signaling pathways, that are strong stimuli for myofibroblast differentiation. There is also evidence for altered miRNA expression that has either profibrogenic or antifibrogenic properties. The mechanisms that initiate and perpetuate defects in the epigenetic program in SSc have not been fully elucidated, but we propose potential extrinsic factors such as occupational exposures and nutritional deficiencies, and intrinsic factors including cellular hypoxia and oxidative stress as possible mechanisms.