Alessandra Marrelli

Angiogenesis in rheumatoid arthritis: A disease specific process or a common response to chronic inflammation?

Angiogenesis is the formation of new blood vessels from existing vessels. During RA new blood vessels can maintain the chronic inflammatory state by transporting inflammatory cells to the site of inflammation and supplying nutrients and oxygen to the proliferating inflamed tissue. The increased endothelial surface area also creates an enormous capacity for the production of cytokines, adhesion molecules, and other inflammatory stimuli, simultaneously the propagation of new vessels in the synovial membrane allows the invasion of this tissue supporting the active infiltration of synovial membrane into cartilage and resulting in erosions and destruction of the cartilage. This angiogenic phenotype is promoted by several pro-angiogenic molecules, the most potent of which is vascular endothelial growth factor (VEGF). Although angiogenesis is recognized as a key event in the formation and maintenance the infiltration of synovial membrane during RA, it is unclear whether angiogenesis should be considered a specific feature of the disease or a common inflammation driven process. However the emergence of biological therapies, such as anti TNF blockade, has suggested that there are features of the inflammatory response that are not general but contextual to the specificity of the tissue where inflammation occurs, and point out the relevant role of tissue-resident stromal cells in determining the site at which inflammation occurs and the specific features of chronic inflammation such as that occurs in RA.

The IL1-like cytokine IL33 and its receptor ST2 are abnormally expressed in the affected skin and visceral organs of patients with systemic sclerosis

Background Early endothelial cell (EC) activation/damage and profibrotic Th2-associated cytokines play a pivotal role in systemic sclerosis (SSc). Interleukin 33 (IL33) is a novel member of the IL1 family that promotes Th2 responses and inflammation through the ST2 receptor. IL33 is also a chromatin-associated transcriptional regulator in ECs. Objective To investigate the role of the IL33/ST2 axis in SSc. Methods Skin biopsies were obtained from 30 patients with SSc (15 early/15 late stage) and 10 healthy subjects. Lung, kidney, heart, oesophagus, stomach, placenta biopsies and bronchoalveolar lavage cells from patients with SSc and controls were also analysed. IL33/ST2 expression was investigated by immunohistology, confocal immunofluorescence microscopy, western blotting and RT-PCR. Results In skin biopsies from control subjects, constitutive nuclear IL33 protein expression was found in dermal ECs and keratinocytes, while ST2 was weakly expressed in ECs and fibroblasts. In skin biopsies from patients with early SSc, IL33 protein was downregulated or absent in ECs and epidermis while IL33 mRNA was normally expressed or even upregulated. Moreover, ECs, perivascular infiltrating mast cells, CD68-positive macrophages, CD3-positive T cells, CD20-positive B cells and activated fibroblasts/myofibroblasts exhibited strong ST2 expression. In skin biopsies from patients with late SSc, IL33 was constitutively found in most ECs while ST2 immunostaining was weaker. In early SSc, the loss of endothelial IL33 protein and the overexpression of ST2 involved all affected organs. Dermal and pulmonary fibroblasts showed IL33 expression in SSc. Conclusion IL33 and ST2 are abnormally expressed in SSc. In early SSc, upon EC activation/damage IL33 may be mobilised from ECs to signal through ST2 in key profibrotic players such as inflammatory/immune cells and fibroblasts/myofibroblasts.

Angiogenic cytokines and growth factors in systemic sclerosis

Systemic sclerosis is an autoimmune connective tissue disorder characterized by a widespread microangiopathy, autoimmunity and fibrosis of the skin and of various internal organs. Microangiopathy is characterized by a reduced capillary density and an irregular chaotic architecture that lead to chronic tissue hypoxia. Despite the hypoxic conditions, there is no evidence for a sufficient compensative angiogenesis in SSc. Furthermore, vasculogenesis is also impaired. An imbalance between angiogenic and angiostatic factors might explain the pathogenetic mechanisms of SSc vasculopathy. As far as angiogenic factors are concerned, within the most important are vascular endothelial growth factor (VEGF) and its receptors, platelet derived growth factor (PDGF), transforming growth factor beta (TGF-β), fibroblast growth factor -2 (FGF-2), angiopoietin 1 (Ang-1), stromal cell-derived factor 1 (SDF-1/CXCL12), endothelin-1 (ET-1), monocyte chemoattractant protein -1 (MCP-1), urokinase type plasminogen activator receptors (uPAR) and kallikreins, vascular adhesion molecules. On the other hand, angiostatic factors include: endostatin, angiostatin, thrombospodin-1 (TSP-1), angiopoietin 2 (Ang-2). Our knowledge concerning the dysregulation of angiogenic homeostasis is largely incomplete and needs further research, for the future.

Scleroderma Mesenchymal Stem Cells display a different phenotype from healthy controls; implications for regenerative medicine

IntroductionVascular involvement is a key feature of Systemic sclerosis (SSc). Although the pericytes/endothelial cells (ECs) cross-talk regulates vessels formation, no evidences about the pericytes contribution to ineffective angiogenesis in SSc are available. Recent findings showed similarities between pericytes and Bone Marrow Mesenchymal Stem Cells (BM-MSCs). Due to difficulties in pericytes isolation, this work explores the possibility to use BM-MSCs as pericytes surrogate, clarifying their role in supporting neo-angiogenesis during SSc.MethodsTo demonstrate their potential to normally differentiate into pericytes, both SSc and healthy controls (HC) BM-MSCs were treated with TGF-β and PDGF-BB. The expression of pericytes specific markers (α-SMA, NG2, RGS5 and desmin) was assessed by qPCR, western blot, and immunofluorescence; chemioinvasion and capillary morphogenesis were also performed. Cell-sorting of BM-MSCs co-cultured with HC-ECs was used to identify a possible change in contractile proteins genes expression.ResultsWe showed that BM-MSCs isolated from SSc patients displayed an up-regulation of α-SMA and SM22α genes and a reduced proliferative activity. Moreover during SSc, both TGF-β and PDGF-BB can specifically modulate BM-MSCs toward pericytes. TGF-β was found interfering with the PDGF-BB effects. Using BM-MSCs/MVECs co-culture system we observed that SSc BM-MSCs improve ECs tube formation in stressed condition, and BM-MSCs, sorted after co-culture, showed a reduced α-SMA and SM22α gene expression.ConclusionsBM-MSCs from SSc patients behave as pericytes. They display a more mature and myofibroblast-like phenotype, probably related to microenvironmental cues operating during the disease. After their co-culture with HC-MVECs, SSc BM-MSCs underwent to a phenotypic modulation which re-programs these cells toward a pro-angiogenic behaviour.

Surface Expression of Fractalkine Receptor (CX3CR1) on CD4+/CD28− T Cells in RA Patients and Correlation with Atherosclerotic Damage

Abstract:  Rheumatoid arthritis (RA) is associated with accelerated atherosclerosis. Fractalkine, a CX3C/chemokine (CX3CL1), mediates monocytes–macrophage infiltration in activated endothelium, suggesting its specific role in atherosclerosis‐related inflammation. In this study, we evaluate the following in early RA patients: the expression of fractalkine receptor (CX3CR1) on CD4+/CD28− T cells, a subset involved in atherosclerotic infiltration; the correlation between this subset and validated markers of early atherosclerosis. CD4+ T cells were isolated by immunomagnetics beads in 50 early RA patients and 26 healthy controls (HC). After isolation, CD4+/CD28−/CX3CR1+ T lymphocytes were assessed by FACS analysis. Endothelial dysfunction was evaluated by both carotid intima‐media thickness (IMT) and flow‐mediated vasodilation (FMV). We observed: a higher expansion of CD4+/CD28− subset in RA patients when compared to HC (7.7%, 5.15–9.7 vs. 0.7%, 0.2–1.5, P < 0.01; respectively); this expansion directly correlated with increased IMT (0.91 mm, 0.5–1.3 vs. 0.7 mm, 0.2–1, P < 0.01; RA vs. C, respectively) and inversely correlated with FMV (3.5%, 1.7–7 vs. 9%, 3.5–11, P < 0.01; RA vs. C, respectively); the large majority of CD4+/CD28−, in RA, coexpressed CX3CR1 (93%, 67–99 vs. 30%, 10–48, P < 0.01; RA vs. C, respectively); this expansion significantly correlated with both the parameters of premature vascular damage and DAS 28. Our data suggest that CX3CL1/CX3CR1 axis might play a role in the induction and development of the endothelial dysfunction during RA.

Stem cells in autoimmune diseases: Implications for pathogenesis and future trends in therapy

In this review we report the recent progresses, available in the literature, concerning the biology and the potential therapeutic role of both mesenchymal stem cells (MSCs) and hematopoietic stem cells in autoimmune diseases. Mesenchymal stem cells (MSCs) are responsible for the normal turnover and maintenance of adult mesenchymal tissues and their pleiotropic nature allows them to sense and respond to an event in the local environment, be it injury or inflammation. Recently, MSCs have been shown to have immune-modulatory properties and immunosuppressive capacities, acting on different immune cells both in vitro and in vivo, in addition to an immunologically privileged phenotype. Moreover, several works suggest that MSCs are defective in autoimmune diseases. These aspects are now considered the most intriguing aspect of their biology, introducing the possibility that these cells might be used as effective therapy in autoimmune diseases. Autoimmune diseases represent a failure of normal immune regulatory processes as they are characterized by activation and expansion of immune cell subsets in response to non-pathogenic stimuli. As autoimmune diseases can be transferred, or alternatively, cured, by stem cell transplantation, a defect in the hemopoietic stem cell as a cause of autoimmune diseases may be postulated. The rationale for autologous hematopoietic stem cell transplantation (HSCT) in autoimmune diseases is the ablation of an aberrant or self-reactive immune system by chemotherapy and regeneration of a new and hopefully self-tolerant immune system from hematopoietic stem cells. In the past 15years, more than 1500 patients worldwide have received HSCT, mostly autologous, as treatment for a severe autoimmune disease and the majority were affected by multiple sclerosis, systemic sclerosis, systemic lupus erythematosus, rheumatoid arthritis, juvenile idiopathic arthritis and idiopathic cytopenic purpura.

Mesenchymal stem cells (MSCs) from scleroderma patients (SSc) preserve their immunomodulatory properties although senescent and normally induce T regulatory cells (Tregs) with a functional phenotype: implications for cellular-based therapy.

Systemic sclerosis (SSc) is a chronic disease, with early activation of the immune system. The aim of our work was to address how SSc–mesenchymal stem cells (MSCs), although senescent, might preserve specific immunomodulatory abilities during SSc. MSCs were obtained from 10 SSc patients and 10 healthy controls (HC). Senescence was evaluated by assessing cell cycle, β‐galactosidase (β‐Gal) activity, p21 and p53 expression; doxorubicin was used as acute senescence stimulus to evaluate their ability to react in stressed conditions. Immunomodulatory abilities were studied co‐culturing MSCs with peripheral blood mononuclear cells (PBMCs) and CD4+ cells, in order to establish both their ability to block proliferation in mixed lymphocyte reaction and in regulatory T cells (Tregs) induction. SSc–MSC showed an increase of senescence biomarkers. Eighty per cent of MSCs were in G0–G1 phase, without significant differences between SSc and HC. SSc–MSCs showed an increased positive β‐Gal staining and higher p21 transcript level compared to HC cells. After doxorubicin, β‐Gal staining increased significantly in SSc–MSCs. On the contrary, doxorubicin abolished p21 activation and elicited p53 induction both in SSc– and HC–MSCs. Interleukin (IL)‐6 and transforming growth factor (TGF)‐β‐related transcripts and their protein levels were significantly higher in SSc–MSCs. The latter maintained their immunosuppressive effect on lymphocyte proliferation and induced a functionally regulatory phenotype on T cells, increasing surface expression of CD69 and restoring the regulatory function which is impaired in SSc. Increased activation of the IL‐6 pathway observed in our cells might represent an adaptive mechanism to senescence, but preserving some specific cellular functions, including immunosuppression.

Cellular players in angiogenesis during the course of systemic sclerosis.

Vascular endothelial injury in Systemic Sclerosis (SSc) leads to pathological changes in the blood vessels that adversely impact the physiology of many organs, resulting in chronic tissue ischemia. The response to hypoxia induces complex cellular and molecular mechanisms in the attempt to recover endothelial cell function and tissue perfusion. The progressive losses of capillaries on one hand, and the vascular remodeling of arteriolar vessels on the other, result in insufficient blood flow, causing severe and chronic hypoxia. Hypoxia is a major stimulus of angiogenesis, leading to the expression of pro-angiogenic molecules, mainly of Vascular Endothelial Growth Factor (VEGF), which triggers the angiogenic process. Nevertheless, in SSc patients there is no evidence of adaptive angiogenesis. Failure of the angiogenic process in SSc largely depends on alteration in the balance between pro- and anti-angiogenic factors, as well as on functional alterations of the cellular players involved in the angiogenic and vasculogenic program. A decreased urokinase plasminogen activator (uPA) dependent invasion, proliferation, and capillary morphogenesis, was showed in SSc endothelial cells (EC). Although hematopoietic endothelial progenitor cells (EPC) count in the peripheral blood of SSc patients is still a matter of controversy, alterations in mobilization process, an excessive immune-mediated EPC destruction in the peripheral circulation or in the bone marrow, a progressive depletion of EPCs following homing to ischemic tissues under persistent peripheral vascular injury, an intrinsic functional impairment could lead to poor vasculogenesis. Human mesenchymal stem cells represent an alternative source of endothelial progenitor cells and it has been observed that their angiogenic potential is reduced in SSc. Targeting autologous stem and progenitor cells could be an ideal tool to counteract and repair dysfunctional angiogenesis.

The -670G>A polymorphism in the FAS gene promoter region influences the susceptibility to systemic sclerosis

Objective: To evaluate the role of the single-nucleotide polymorphism (SNP) at position −670 in the FAS gene promoter (FAS−670G>A) in influencing the susceptibility, clinical features and severity of systemic sclerosis (SSc). Methods: 350 white Italian SSc patients (259 with limited cutaneous SSc (lcSSc) and 91 with diffuse cutaneous SSc (dcSSc)) and 232 healthy individuals were studied. Patients were assessed for the presence of autoantibodies (anticentromere, anti-topoisomerase I (anti-Scl-70) antibodies), interstitial lung disease (ILD), pulmonary arterial hypertension and scleroderma renal crisis. FAS−670G>A SNP was genotyped by PCR restriction fragment length polymorphism assay. Serum levels of soluble FAS (sFAS) were analysed by ELISA. Results: A significant difference in FAS−670 genotype distribution was observed between SSc patients and healthy individuals (p = 0.001). The frequency of the FAS−670A allele was significantly greater in SSc than in controls (p = 0.001). No significant difference in genotype distribution and allele frequencies was observed between lcSSc and dcSSc, although a greater frequency of the FAS−670A allele was found in dcSSc. The FAS−670AA genotype significantly influenced the predisposition to SSc (OR 1.97, 95% CI 1.35 to 2.88, p = 0.001) and to both lcSSc (OR 1.84, 95% CI 1.23 to 2.75, p = 0.003) and dcSSc (OR 2.37, 95% CI 1.41 to 3.99, p = 0.001). FAS−670A allele frequency was greater, although not significantly, in anti-Scl-70 antibody-positive dcSSc and ILD dcSSc. sFAS was significantly higher in patients and controls carrying the FAS−670AA genotype compared with those carrying the FAS−670GG genotype (p = 0.003 in SSc, p = 0.004 in controls). Conclusion: The FAS−670A allele is significantly associated with susceptibility to SSc, suggesting a role for a genetic control of apoptosis in the pathogenesis of the disease.

THU0188 Lymphoproliferative complications in the course of primary sjægren’s syndrome: Prevalence and common features in a large cohort of patients:

Background Primary Sjögren’s syndrome (pSS) is the autoimmune disease most often associated with lymphoma. Mixed cryoglobulinemia as well as persistent parotid swelling are the well known pre-lymphomatous clinical conditions. Objectives To describe the prevalence of lymphoproliferative complications (defined as B-cell lymphoma or definite conditions predisposing to lymphoma, i.e, cryoglobulinemic vasculitis (CV) and major salivary gland swelling) in a cohort of pSS patients followed in five Rheumatologic Centres. Methods Demographic, clinical, laboratory and histopathologic data in 1170 pSS were retrospectively collected according to a standard protocol. Univariate analyses were performed. Results Prevalence of lymphoma in this SS cohort was 4.4% (51/1170), prevalence of CV (1) was 3.9% (33/850), and prevalence of salivary gland swelling and/or myoepithelial syaladenitis was 30.9% (362/1170). Salivary gland swelling and/or MESA, CV and lymphoma shared many laboratory features, i.e., positive rheumatoid factor (RF), hypocomplementemia and leucopenia, as well as a the presence of purpura as clinical hallmark of the circulating immune complexes (table 1). Interestingly, polyclonal hypergammaglobulinemia was strictly associated with salivary gland swelling, but it was not associated with CV or lymphoma; on the other hand, serum monoclonal component was significantly associated with CV or lymphoma, but not with salivary gland swelling and/or MESA. Table 1. Common association (P values) between clinical and laboratory features of pSS in patients with salivary gland swelling, cryoglobulinemic vasculitis or B-cell lymphoma Feature pSS with salivary gland swelling and/or MESA pSS with cryoglobulinemic vasculitis pSS with lymphoma 362/1170: 30.9% 33/850: 3.9% 51/1170: 4.4% Age (yrs) <0.0001 (for youngers) ns ns Sex ns ns <0.0001 (for males) Salivary gland swelling and/or MESA NA ns <0.0001 Cryoglobulinemic vasculitis 0.009 NA 0.001 Lymphoma <0.0001 <0.0001 NA Purpura 0.03 <0.0001 0.02 Rheumatoid factor positivity <0.0001 <0.0001 <0.0001 Low C3 0.003 <0.0001 0.02 Low C4 <0.0001 <0.0001 <0.0001 Leukopenia (<3000/mmc) 0.004 <0.0001 <0.0001 NA, not applicable; ns, not significant. Conclusions Salivary gland swelling and CV are confirmed as red flags for lymphoma evolution in pSS and are strictly linked (2). Polyclonal hypergammaglobulinemia is not a red flag for lymphoma in pSS. B-cell clonal proliferation from polyclonal to monoclonal likely occurs in the target tissue of pSS (e.g. salivary gland MALT tissue), predisposing to CV and/or to lymphoma. Cryoglobulinemia marks the emergence of abnormal RF-positive B-cell clones, but MALT lymphoma may also occur in the absence of cryoglobulinemia. References De Vita S et al. Ann Rheum Dis 2011 De Vita S, et al. Rheumatology 2012 Disclosure of Interest None Declared