Saverio Alvaro

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.

Monocytes from patients with rheumatoid arthritis and type 2 diabetes mellitus display an increased production of interleukin (IL)-1β via the nucleotide-binding domain and leucine-rich repeat containing family pyrin 3(NLRP3)-inflammasome activation: a possible implication for therapeutic decision in these patients

A better understanding about the mechanisms involved in the pathogenesis of type 2 diabetes mellitus (T2D) showed that inflammatory cytokines such as tumour necrosis factor (TNF) and interleukin (IL)‐1β play a pivotal role, mirroring data largely reported in rheumatoid arthritis (RA). IL‐1β is produced mainly by monocytes (MO), and hyperglycaemia may be able to modulate, in the cytoplasm of these cells, the assembly of a nucleotide‐binding domain and leucine‐rich repeat containing family pyrin (NLRP3)‐inflammosome, a cytosolic multi‐protein platform where the inactive pro‐IL‐1β is cleaved into active form, via caspase‐1 activity. In this paper, we evaluated the production of IL‐1 β and TNF, in peripheral blood MO of patients affected by RA or T2D or both diseases, in order to understand if an alteration of the glucose metabolism may influence their proinflammatory status. Our data showed, after 24 h of incubation with different glucose concentrations, a significantly increased production of IL‐1β and TNF in all evaluated groups when compared with healthy controls. However, a significant increase of IL‐1β secretion by T2D/RA was observed when compared with other groups. The analysis of relative mRNA expression confirmed these data. After 24 h of incubation with different concentrations of glucose, our results showed a significant increase in NLRP3 expression. In this work, an increased production of IL‐1β by MO obtained from patients affected by both RA and T2D via NLRP3‐inflammasome activation may suggest a potential IL‐1β targeted therapy in these patients.

IL-1β at the crossroad between rheumatoid arthritis and type 2 diabetes: may we kill two birds with one stone?

ABSTRACT Although in the past the prevention of joint destruction in rheumatoid arthritis (RA) was strongly emphasized, now a great interest is focused on associated comorbidities in these patients. Multiple data suggest that a large percentage of RA patients are affected by Type 2 Diabetes (T2D), whose incidence has reached epidemic levels in recent years, thus increasing the health care costs. A better knowledge about the pathogenesis of these diseases as well as the mechanisms of action of drugs may allow both policy designers and physicians to choose the most effective treatments, thus lowering the costs. This review will focus on the role of Interleukin (IL)-1β in the pathogenesis of both the diseases, the efficacy of IL-1 blocking molecules in controlling these diseases, and will provide information suggesting that targeting IL-1β, in patients affected by both RA and T2D, may be a promising therapeutic choice.

Increased Cardiovascular Events and Subclinical Atherosclerosis in Rheumatoid Arthritis Patients: 1 Year Prospective Single Centre Study.

Objectives Several studies showed the close relationship between Rheumatoid Arthritis (RA) and cerebro-cardiovascular events (CVEs) and subclinical atherosclerosis. In this study, we investigated the occurrence of CVEs and subclinical atherosclerosis during the course of RA and we evaluated the possible role of both traditional cardiovascular (CV) and disease related risk factors to predict the occurrence of new CVEs and the onset of subclinical atherosclerosis. Methods We designed a single centre, bias-adjusted, prospective, observational study to investigate, in a homogeneous subset of RA patients, the occurrence of new onset of CVEs and subclinical atherosclerosis. Statistical analyses were performed to evaluate the role of traditional CV and disease-related risk factors to predict the occurrence of new CVEs and subclinical atherosclerosis. Results We enrolled 347 RA patients prospectively followed for 12 months. An increased percentage of patients experienced CVEs, developed subclinical atherosclerosis and was affected by systemic arterial hypertension (SAH), type 2 diabetes mellitus and metabolic syndrome (MS), at the end of follow up. Our analysis showed that the insurgence of both SAH and MS, during the follow up, the older age, the CVE familiarity and the lack of clinical response, were associated with a significantly increased risk to experience CVEs and to develop subclinical atherosclerosis. Conclusions Our study quantifies the increased expected risk for CVEs in a cohort of RA patients prospectively followed for 1 year. The occurrence of both new CVEs and subclinical atherosclerosis in RA patients may be explained by inflammatory burden as well as traditional CV risk factors.

Jejunoileal bypass as the main procedure in the onset of immune-related conditions: the model of BADAS.

Bariatric surgery represents a common approach for the control of severe morbid obesity, reducing caloric intake by modifying the anatomy of the gastrointestinal tract. Following jejunoileal bypass, a large spectrum of complications has been described, with rheumatic manifestation present in up to 20% of cases. Although bowel bypass syndrome, also called blind loop syndrome, is a well-recognized complication of jejunoileal bypass, the same syndrome was recognized in patients who had not had intestinal bypass surgery, and the term the ‘bowel-associated dermatosis–arthritis syndrome’ (BADAS) was coined. The pathogenesis of BADAS is as yet poorly understood and only few data concerning this issue have been published in the literature. The aim of the present paper is to review the literature and to discuss putative pathogenic mechanisms of BADAS, focusing on the immune system.

The Emerging Role of IL-1 Inhibition in Patients Affected by Rheumatoid Arthritis and Diabetes

BACKGROUND Although in the past, prevention of the joint destruction and disability was strongly emphasised in Rheumatoid Arthritis (RA), at present, a growing body of evidence is focused at identifying the best management of associated comorbidities, such as Type 2 Diabetes (T2D). Recently, the hypothesis that blocking pro-inflammatory activity may be helpful in the treatment of some comorbidities has been proposed in RA patients. OBJECTIVE We reviewed the role of IL-1β during RA and T2D, the efficacy of IL-1 blocking agents in controlling both diseases and, possible, decreasing the concomitant enhanced atherosclerotic process. METHOD After literature search, the available evidence has been selected and commented in the text. RESULTS During RA, it is well known that different inflammatory cytokines, such as interleukin-1β (IL-1β), are pivotal pathogenic mediators and their role has been largely confirmed in clinical settings. Similarly, it has been shown that the excess of nutrients, secondary to over-nutrition, may activate the immune system, leading to an increased production of inflammatory cytokines, including IL-1β, suggesting new possible therapeutic targets. CONCLUSION Although further studies are needed to fully investigate the pathogenic interplay between inflammation and metabolic disorders, IL-1β has been implicated in both RA and T2D pathogenic mechanisms. Intriguingly, the potential role of anti-IL-1 drugs has been proposed in RA patients affected by T2D.

AB0051 Monocytes from Patients with Rheumatoid Arthritis and Type 2 Diabetes Mellitus Display an Increased Production of IL-1β Via the NLRP3-Inflammasome Activation. A Possible Implication for Therapeutic Decision in These Patients

Background A better understanding about the mechanisms involved in the pathogenesis of type 2 diabetes mellitus (T2DM) showed that inflammatory cytokines such as tumor TNF-α and IL-1β play a pivotal role, mirroring data largely reported in rheumatoid arthritis (RA) [1]. Both preclinical and clinical studies confirmed the usefulness of IL-1β antagonism therapy in both diseases [2]. IL-1β is mainly produced by monocytes/macrophages. Hyperglycaemia may be able to modulate, in the cytoplasm of these cells, the assembly of a cytosolic multiprotein platforms, composed by 3 molecules: NLRP3 sensor protein; ASC adaptor protein; caspase-1, the so called NLRP3-inflammasome, where, the inactive pro-IL-1β is cleaved into active form, via caspase-1 activity [3]. Objectives We evaluated the production of IL-1 β and TNF-α, in peripheral blood monocytes (MO) of patients affected by RA or T2DM or both diseases, in order to understand if, an alteration of the glucose metabolism may influence their pro-inflammatory status. Methods We enrolled 10 RA patients, 10 T2DM patients and 10 patients affected by both the diseases (T2DM/RA patients), and 5 healthy controls (HC). From these patients, MO were isolated from human peripheral blood mononuclear cells using CD14 selection. After that MO were incubated with 11 mmol/1 glucose (11G) and 33 mmol/1 glucose (33G), for 24 hours, and we evaluated by ELISA the concentration of both IL-1β and TNF-α released in cultured MO supernatants, the relative mRNA expression by qRT-PCR and the western blot analysis of NLRP3. Due to the non parametric distribution of our data the Mann-Whitney U test was used as appropriate for analyses. Statistical significance was expressed by a p value <0.05. Results Our data showed, after 24 hours of incubation with 11G, a significantly increase of IL-1β in supernatants of T2DM, RA, and T2DM/RA cultured MO, when compared with HC (p<0.01 for each comparison). Our data showed that after 24 hours of incubation with 33G a significant increase of IL-1β secretion by T2DM/RA MO of patients when compared with other groups (p<0.01 for each comparison). We observed after 24 hours of incubation with both 11G and 33G an increase of TNF-α in supernatants, in all groups when compared with HC (p<0.01 for each comparison). We did not observed significant differences in TNF-α secretion among T2DM, RA and T2DM/RA patients. The analysis of relative mRNA expression confirmed these data. Our results showed after 24 hours of incubation with both 11G and 33G a significant increase in relative NLRP3 mRNA expression when we compared T2DM/RA patients and the other groups (p<0.001 for each comparison). The results of western blot analyses for NLRP3 mirrored these data. Conclusions Our study showed an increased production of IL-1β by MO obtained from patients affected by both RA and T2DM via NLRP3-inflammasome activation suggesting a potential IL-1β target therapy. References Donath MY, et al. Type 2 diabetes as an inflammatory disease. Nat Rev Immunol. 2011;11:98-107. Eguchi K, et al. Macrophages and islet inflammation in type 2 diabetes. Diabetes Obes Metab. 2013;3:152-158. Schroder K, et al. The NLRP3 inflammasome: a sensor for metabolic danger? Science. 2010;327:296-300. Disclosure of Interest None declared

AB0226 Caveolin-1 orchestrates vascular endothelial growth factor (VEGF) signaling control of angiogenesis during systemic sclerosis (SSC)

Background MVECs damage is a central event in the pathogenesis of SSc leading to capillaries rarefaction and consequent ischemia. VEGF is the best characterized angiogenic growth factor; its increase after ischemia is a timed controlled event, acting only in the first stages of new blood vessels formation when it regulates ECs sprouting and proliferation. During SSc, VEGF lacks this tight control and is strongly up-regulated over time both at local and systemic levels. Caveolae, specialized plasma membrane microdomains, abundant in ECs, and caveolin-1 (cav-1), their principal residual protein, compartmentalize VEGF-induced signaling toward angiogenesis. Objectives Here we aimed to prove whether caveolae/cav-1 act as platform for organizing and compartmentalizing VEGF/VEGFR2 signaling in MVECs during SSc. In fact an abnormal cav-1 expression in microvascular endothelium, may contribute to angiogenic defective response during the disease. Methods After ethical approval skin was collected from 10 patients with SSc and used for immunofluorescence (IF), qPCR and MVECs isolation. Normal skin was obtained from healthy donors (HC) who underwent surgery for trauma. Cav-1 detection and its co-localization with VEGFR-2 was perfomed on skin sections and MVECS isolated from SSc patients and compared to HC by IF, immunoprecipitation and Western Blot (WB). qPCR analysis was used to assess mRNA cav-1 expression level both in SSc skin and MVECs. To determine whether VEGF treatment could affect cav-1/VEGFR2 colocalization in MVECs during SSC, all the above was assessed after culturing these cells in the presence of VEGF. Results We found that VEGFR-2 colocalizes with cav-1 in blood vessels within SSc skin, as already observed in HC skin. However semi-quantitative analysis of IF cav-1 expression level revealed lower expression in SSc MVECS with respect to HC skin. This result was confirmed by qPCR, showing cav-1 was down-regulated in MVECs isolated from SSc patients when compared to HC cells. When we assessed cav-1/VEGFR.2 co-localization within MVECS, we observed that VEGFR-2 was localized in the caveolae directly bound by cav-1. This observation was further confirmed by cav-1 immunoprecipitation and WB for VEGFR-2 in SSc MVECs, without differences from HC. Interestingly, after culturing these cells in the presence of VEGF, we found a disruption of cav-1/VEGFR-2 co-localization both in HC and SSc MVECs, without cav-1 mRNA levels re-modulation Conclusions During SSc cav-1 expression down-regulation within MVECs, could possibly explain the dysfunctional, ineffective VEGF angiogenic signaling. This mechanism might maintain the redundancy of deregulated VEGF abundance in a vicious loop, strongly contributing to not compensative angiogenic response during SSc. References Del Galdo Fet al Curr Opin Rheumatol. 2008 Nov;20(6). Feng L et al J Cell Physiol. 2011 Aug 9 [Epub ahead of print]. Liao WX et al Mol Endocrinol. 2009 Sep;23(9). Disclosure of Interest None Declared