Ilenia Pantano

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.

Impaired endothelium-mesenchymal stem cells cross-talk in systemic sclerosis: a link between vascular and fibrotic features.

IntroductionTo assess if an impaired cross-talk between endothelial cells (ECs) and perivascular/multipotent mesenchymal stem cells (MSCs) might induce a perturbation of vascular repair and leading to a phenotypic switch of MSC toward myofibroblast in Systemic Sclerosis (SSc).MethodsWe investigated different angiogenic and profibrotic molecules in a tridimentional matrigel assay, performing co-cultures with endothelial cells (ECs) and bone marrow derived MSCs from patients and healthy controls (HC). After 48 hours of co-culture, cells were sorted and analyzed for mRNA and protein expression.ResultsECs-SSc showed a decreased tube formation ability which is not improved by co-cultures with different MSCs. After sorting, we showed: i. an increased production of vascular endothelial growth factor A (VEGF-A) in SSc-MSCs when co-cultured with SSc-ECs; ii. an increased level of transforming growth factor beta (TGF-β) and platelet growth factor BB (PDGF-BB) in SSc-ECs when co-cultured with both HC- and SSc-MSCs; iii. an increase of TGF-β, PDGF-R, alpha smooth muscle actin (α-SMA) and collagen 1 (Col1) in both HC- and SSc-MSCs when co-cultured with SSc-ECs.ConclusionWe showed that during SSc, the ECs-MSCs crosstalk resulted in an altered expression of different molecules involved in the angiogenic processes, and mainly SSc-ECs seem to modulate the phenotypic switch of perivascular MSCs toward a myofibroblast population, thus supporting the fibrotic process.

Impaired Cav-1 expression in SSc mesenchymal cells upregulates VEGF signaling: a link between vascular involvement and fibrosis

BackgroundSystemic sclerosis (SSc) is characterized by vascular alteration and fibrosis, the former probably leading to fibrosis via the ability of both endothelial cells and pericytes to differentiate toward myofibroblast. It is well known that vascular endothelial growth factor A (VEGF-A, hereafter referred to as VEGF) may induce a profibrotic phenotype on perivascular cells. Caveolin-1 (Cav-1) is involved in the regulation of VEGF signaling, playing a role in the transport of internalized VEGF receptor 2 (VEGFR2) toward degradation, thus decreasing VEGF signaling. In this work, we assessed the levels of Cav-1 in SSc bone marrow mesenchymal stem cells (SSc-MSCs), a pericyte surrogate, and correlate these results with VEGF signaling, focusing onpotential pathogenic pathways leading to fibrosis.ResultsWe explored the VEGF signaling assessing: (1) Cav-1 expression; (2) its co-localization with VEGFR2; (3) the activity of VEGFR2, by IF, immunoprecipitation, and western blot. In SSc-MSCs, Cav-1 levels were lower when compared to healthy controls (HC)-MSCs. Furthermore, the Cav-1/VEGFR2 co-localization and the ubiquitination of VEGFR2 were impaired in SSc-MSCs, suggesting a decreased degradation of the receptor and, as a consequence, the tyrosine phosphorylation of VEGFR2 and the PI3-kinase-Akt pathways were significantly increased when compared to HC. Furthermore, an increased connective tissue growth factor (CTGF) expression was observed in SSc-MSCs. Taken together, these data suggested the upregulation of VEGF signaling in SSc-MSCs. Furthermore, after silencing Cav-1 expression in HC-MSCs, an increased CTGF expression in HC-MSCs was observed, mirroring the results obtained in SSc-MSCs, and confirming the potential role that the lack of Cav-1 may play in the persistent VEGF signaling .ConclusionsDuring SSc, the lower levels of Cav-1 may contribute to the pathogenesis of fibrosis via an upregulation of the VEGF signaling in perivascular cells which are shifted to a profibrotic phenotype.

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.

Searching for a good model for systemic sclerosis: the molecular profile and vascular changes occurring in UCD-200 chickens strongly resemble the early phase of human systemic sclerosis

Introduction Vascular injury and endothelial cell (EC) apoptosis are the earliest events in systemic sclerosis (SSc), before the onset of fibrosis, and stromal cell-derived factor 1 (SDF-1), vascular endothelial growth factor (VEGFA), endothelin-1 (ET-1) and platelet-derived growth factors (PDGF-BB) represent the key molecules to study the link between vascular injury and fibrosis during SSc. The University of California at Davis line 200 (UCD-200) chickens display the same hallmarks of human SSc: vascular occlusion, perivascular lymphocytic infiltration and fibrosis of skin and internal organs. In this study we assessed both cytokines and growth factors involved in the early phases of the UCD-200 chickens’ skin lesions, to determine whether these animals might represent an appropriate experimental model to study the pathogenesis of SSc. Material and methods Immunofluorescence analysis was performed on human SSc skin, human healthy control (hHC) skin, UCD-200 combs and HC H.B15 chicken (cHC) combs, using anti-SDF-1, CXCR4, VEGFA, VEGF receptor 1 (VEGFR1), VEGF receptor 2 (VEGFR2), ET-1, ET receptor A (ETAR), ET receptor B (ETBR), PDGF-BB, and PDGF receptor (PDGFR) antibodies. The plasma concentrations of SDF-1, VEGFA, ET-1 and PDGF-BB were determined by ELISA. Results All the molecules analyzed showed higher levels in SSc patients and UCD-200 chickens than in hHC and cHC. Furthermore, the levels of the assessed molecules paralleled the severity of comb involvement. Conclusions The molecular similarities between avian and human SSc, observed in this study, suggest that the UCD-200 chickens are an interesting model for translational approaches to SSc.

FRI0437 Decreased Expression of Angiopoetin 1 on Perivascular Mesenchymal Stem Cells from SSC Patients Induces an Anti Angiogenetic Effect, when Co-Cultured with Endothelial Cells

Background Systemic Sclerosis (SSc) is characterized by vascular alteration with a progressive loss of capillaries resulting in insufficient blood flow and chronic peripheral hypoxia, associated to a failure of reparative angiogenesis, followed by progressive fibrosis of skin and internal organs[1]. An impaired production of angiogenic molecules is involved in this dysfunctional angiogenesis [2]. Tie2 is a trans-membrane receptor, exclusively expressed on the surface of endothelial cell (EC). Tie2 modulates the vessels quiescence, or alternatively, induces angiogenesis, via the interaction with its ligands, Ang1 and Ang2. Of note, activation of endothelial Tie2 signalling, by Ang1, which is produces by perivascular cells, enhances ECs barrier integrity and endothelial-pericyte interaction. On the contrary, Ang2, produced by ECs, normally acts as an Ang1 antagonist [3]. Objectives The aim of the present study was to assess if an impaired cross-talk between ECs and perivascular mesenchymal stem cells (MSCs), during SSc, may affect the normal interaction among Ang1, Ang2 and Tie2 thus contributing to the impaired angiogenesis. In this work, bone marrow derived MSCs were used as pericytes surrogate, considering that, perivascular cells share surface markers and differentiative ability with bone marrow MSCs, and MSCs express pericyte markers and cooperate with endothelial cells to form a vascular network, supporting the concept that pericytes are members of the adult multipotent MSCs family [4]. Methods We investigated Ang1, Ang2 and their receptor performing co-cultures with ECs and bone marrow MSCs obtained from patients and healthy controls (HC). After 48 hours, cells were sorted and analysed for molecular assays. Furthermore, we investigated, by ELISA assay, the proteins released in the supernatants. Finally, we silenced Ang-1 expression in HC-MSCs by Ang1-siRNA. Results At molecular level, SSc-MSCs, cultured alone, expressed lower amount of Ang1 when compared to HC-MSCs. After co-culture, a significant decreased of Ang1 mRNA levels was observed in the SSc-MSCs/SSc-ECs. On the contrary, SSc-ECs expressed higher levels of Ang2 and Tie2 in each co-culture condition, when compared to the expressions of cells cultured alone. The WB and ELISA assays mirrored the results observed in gene expression. HC-MSCs transfected with Ang1-siRNA lacked the ability to support the formation of tube like structure. Conclusions In this work we provided evidence that an imbalance of Ang1/Ang2 molecules and a decreased expression of their receptor, Tie2, during ECs-perivascular MSCs interplay, may modulate vessel stability, and vascular tube formation, thus contributing to the angiogenic alteration observed during SSc. References Cipriani P et al. Autoimmun Rev 2011,10:641-646. Cipriani P et al. Arthritis Res Ther 2013,16:442. Fukuhara S et al. Histol Histopathol 2010,25:387–396. Cipriani P et a. Angiogenesis 2013,16:595-607. Disclosure of Interest None declared

Decreased Expression of Angiopoetin 1 on Perivascular Mesenchymal Stem Cells from Ssc Patients Induces an Anti Angiogenetic Effect, when Co-cultured with Endothelial Cells

Introduction: The Angiopoietin (Ang)/Tie2 system plays crucial roles in vascular functions, regulating endothelialpericyte interaction and promoting vascular stabilization. We assessed if an impaired cross-talk, in Systemic Sclerosis (SSc), between endothelial cells (ECs) and perivascular mesenchymal stem cells (MSCs) may affect the normal interaction among Ang1, Ang2 and Tie2 thus contributing of the impaired angiogenesis in SSc. Methods: We investigated Ang1, Ang2 and their receptor performing co-cultures with ECs and bone marrow MSCs obtained from patients and healthy controls (HC). After 48 hours, cells were sorted and analysed for molecular assays. Furthermore, we investigated, by ELISA assay, the proteins released in the supernatants. Finally, we silenced Ang-1 expression in HC-MSCs by siRNA-Ang1. Results: At molecular level, SSc-MSCs, cultured alone, expressed lower amount of Ang1 when compared to HC-MSCs. After co-culture, a significant decreased of Ang1 mRNA levels was observed in the SSc-MSCs/SSc-ECs. On the contrary, SSc-ECs expressed higher levels of Ang2 and Tie2 in each co-culture condition, when compared to the expressions of cells cultured alone. The WB and ELISA assays mirrored the results observed in gene expression. HC-MSCs transfected with siRNA-Ang1 lacked the ability to support the formation of tube like structure. Conclusions: In this work we provided evidence that an imbalance of Ang1/Ang2 molecules and a decreased expression of their receptor, Tie2, during ECs-perivascular MSCs interplay, may modulate vessel stability, and vascular tube formation, thus contributing to the angiogenic alteration observed during SSc.

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