Vasiliki Liakouli

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.

Virtual skin biopsy by optical coherence tomography: the first quantitative imaging biomarker for scleroderma

Background Skin involvement is of major prognostic value in systemic sclerosis (SSc) and often the primary outcome in clinical trials. Nevertheless, an objective, validated biomarker of skin fibrosis is lacking. Optical coherence tomography (OCT) is an imaging technology providing high-contrast images with 4 μm resolution, comparable with microscopy (‘virtual biopsy’). The present study evaluated OCT to detect and quantify skin fibrosis in SSc. Methods We performed 458 OCT scans of hands and forearms on 21 SSc patients and 22 healthy controls. We compared the findings with histology from three skin biopsies and by correlation with clinical assessment of the skin. We calculated the optical density (OD) of the OCT images employing Matlab software and performed statistical analysis of the results, including intraobserver/interobserver reliability, employing SPSS software. Results Comparison of OCT images with skin histology indicated a progressive loss of visualisation of the dermal–epidermal junction associated with dermal fibrosis. Furthermore, SSc affected skin showed a consistent decrease of OD in the papillary dermis, progressively worse in patients with worse modified Rodnan skin score (p<0.0001). Additionally, clinically unaffected skin was also distinguishable from healthy skin for its specific pattern of OD decrease in the reticular dermis (p<0.001). The technique showed an excellent intraobserver and interobserver reliability (intraclass correlation coefficient >0.8). Conclusions OCT of the skin could offer a feasible and reliable quantitative outcome measure in SSc. Studies determining OCT sensitivity to change over time and its role in defining skin vasculopathy may pave the way to defining OCT as a valuable imaging biomarker in SSc.

The role of IL-1β in the bone loss during rheumatic diseases.

Several inflammatory diseases have been associated with increased bone resorption and fracture rates and different studies supported the relation between inflammatory cytokines and osteoclast activity. The main factor required for osteoclast activation is the stimulation by receptor activator of nuclear factor kappa-B ligand (RANKL) expressed on osteoblasts. In this context, interleukin- (IL-) 1β, one of the most powerful proinflammatory cytokines, is a strong stimulator of in vitro and in vivo bone resorption via upregulation of RANKL that stimulates the osteoclastogenesis. The resulting effects lead to an imbalance in bone metabolism favouring bone resorption and osteoporosis. In this paper, we review the available literature on the role of IL-1β in the pathogenesis of bone loss. Furthermore, we analysed the role of IL-1β in bone resorption during rheumatic diseases and, when available, we reported the efficacy of anti-IL-1β therapy in this field.

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.

Association of a functional polymorphism in the matrix metalloproteinase-12 promoter region with systemic sclerosis in an Italian population.

Objective. To investigate the possible implication of the matrix metalloproteinase-12 (MMP-12) gene in the genetic predisposition to systemic sclerosis (SSc) susceptibility and clinical phenotype. Methods. The MMP-12 rs2276109 A/G functional polymorphism was selected as a genetic marker and genotyped by polymerase chain reaction-restriction fragment length polymorphism assay in 513 unrelated subjects of Italian white ancestry: 250 patients with SSc [146 limited cutaneous SSc (lcSSc), 104 diffuse cutaneous SSc (dcSSc)] and 263 healthy individuals. Results. A significant difference was observed in MMP-12 rs2276109 genotype distribution between patients with SSc and controls (p = 0.0003), and between lcSSc and dcSSc (p = 0.003). The A allele frequency was significantly higher in patients with SSc than in controls (p = 0.0002), and higher in dcSSc than in lcSSc (p = 0.003). After adjustment for age and sex, the homozygosity for the A allele significantly influenced the predisposition to SSc and to dcSSc (OR 2.44, 95% CI 1.61–3.71, p < 0.0001; OR 4.69, 95% CI 2.36–9.33, p < 0.0001, respectively). A trend toward an association between the AA genotype and lcSSc was observed (p = 0.06). The homozygosity for the A allele was also significantly and independently associated with antitopoisomerase I antibody positivity (OR 6.39, 95% CI 2.18–18.76, p = 0.001) and interstitial lung disease (OR 2.94, 95% CI 1.25–6.95, p = 0.01). Conclusion. The MMP-12 rs2276109 gene polymorphism may contribute to susceptibility to SSc, and in particular to dcSSc and pulmonary fibrosis.

Methotrexate in Rheumatoid Arthritis: Optimizing Therapy Among Different Formulations. Current and Emerging Paradigms

BACKGROUND Methotrexate (MTX) is currently considered the drug of choice, among the disease-modifying antirheumatic drugs, for the treatment of rheumatoid arthritis (RA) because of its favorable risk/benefit ratio, good safety profile, and low costs. Despite MTXs widespread use and large experience accumulated over the many years since its introduction into clinical practice, specific guidelines have not been published. OBJECTIVE We report here the available research regarding the optimal dosage and route of MTX administration. METHODS MEDLINE and the Cochrane Library were systematically searched for articles published between 1990 and 2013, using terms related to RA and MTX. The search was conducted by using both MeSH terms and free text. The references of the retrieved studies were also screened manually for additional articles. RESULTS For the treatment of rheumatic diseases, the antimetabolite drug MTX can be administered weekly by different routes: oral, subcutaneous, or intramuscular. One of the goals of treatment is to minimize acute and chronic toxicity. A starting dose of 15 mg/week orally, escalating to 25 to 30 mg/week or the highest tolerable dose (with a subsequent switch to parenteral administration in cases of insufficient response), seems to be the optimal evidence-based strategy for MTX treatment of RA. Oral MTX is widely preferred because of its low costs and patient preferences; the bioavailability of parenteral MTX is higher, however. This is supported by data from observational studies, in which patients switching from parenteral to oral MTX at an equal dose had disease exacerbations. In several trials, the subcutaneous formulation of MTX was considered, by both physicians and patients, to be more advantageous in terms of discomfort and compliance. In addition, a significant proportion of patients reported that this formulation led to greater independence, with a resulting improvement in quality of life. CONCLUSIONS Although MTX treatment can be initiated by using the oral administration route, parenteral administration of MTX is indicated in those patients with poor compliance toward the oral form. The subcutaneous route seems to be more effective than the oral route for MTX administration based on the results of several studies, and this route may be preferred because of better usability and absence of pain at the infusion site.