Celestina Mazzotta

Vascular biomarkers and correlation with peripheral vasculopathy in systemic sclerosis.

Vascular disease is a hallmark of systemic sclerosis (SSc). It is present in every patient, being responsible both for the earliest clinical manifestations and the major life-threatening complications of the disease, and thus determining important morbidity and mortality. In SSc, progressive vascular injury leads to vascular tone dysfunction and reduced capillary blood flow, with consequent tissue ischemia and chronic hypoxia. These phenomena are often accompanied by abnormal levels of vascular factors. Microangiopathy in SSc may be easily assessed by nailfold videocapillaroscopy. The variety of derangements detected in the nailfold capillaries is accompanied by abnormal levels of different vascular mediators and appears to be the best evaluable predictor of the development of peripheral vascular complications, such as digital ulcers. The purpose of this review is to summarize in SSc the most relevant vascular biomarkers and the main associations between vascular biomarkers and capillaroscopic parameters and/or the presence of digital ulcers. Vascular biomarkers could become useful predictive factors of vascular damage in SSc, allowing an earlier management of vascular complications.

Decreased expression of neuropilin-1 as a novel key factor contributing to peripheral microvasculopathy and defective angiogenesis in systemic sclerosis

Objectives In systemic sclerosis (SSc), vascular involvement is characterised by vascular endothelial growth factor (VEGF)-A/VEGF receptor (VEGFR) system disturbances. Neuropilin-1 (NRP1), a receptor for both class-3 semaphorins (Sema3s) and VEGF-A, is required for optimal VEGF-A/VEGFR-2 signalling. Here, we investigated the possible involvement of Sema3A/NRP1 axis in SSc. Methods Circulating Sema3A and soluble NRP1 (sNRP1) were measured in patients with SSc and controls. NRP1 and Sema3A expression in skin biopsies was evaluated by immunofluorescence and western blotting. NRP1 expression was assessed in SSc and healthy dermal microvascular endothelial cells (SSc-MVECs and H-MVECs), and in SSc and control endothelial progenitor cell (EPC)-derived endothelial cells (ECs). The possible impact of transcription factor Friend leukaemia integration 1 (Fli1) deficiency on endothelial NRP1 expression was investigated by gene silencing. The binding of Fli1 to NRP1 gene promoter was evaluated using chromatin immunoprecipitation. Capillary morphogenesis was performed on Matrigel. Results Decreased sNRP1 levels in SSc were associated with active and late nailfold videocapillaroscopy patterns and digital ulcers. No difference in Sema3A was found between patients and controls. NRP1 was significantly decreased in SSc-MVECs both ex vivo and in vitro. NRP1 and Fli1 significantly decreased in H-MVECs challenged with SSc sera, while they were not different in SSc and control EPC-derived ECs. Fli1 occupied the NRP1 gene promoter and Fli1 gene silencing reduced NRP1 expression in H-MVECs. NRP1 gene silencing in H-MVECs resulted in a significantly impaired angiogenic capacity comparable to that of cells treated with SSc sera. Conclusion In SSc, NRP1 deficiency may be an additional factor in the perturbed VEGF-A/VEGFR-2 system contributing to peripheral microvasculopathy and defective angiogenesis.

Proangiogenic effects of soluble α-Klotho on systemic sclerosis dermal microvascular endothelial cells

BackgroundSystemic sclerosis (SSc) is characterized by endothelial cell (EC) apoptosis, impaired angiogenesis and peripheral microvasculopathy. Soluble α-Klotho (sKl) is a pleiotropic molecule with multiple effects on ECs, including antioxidant and vasculoprotective activities. On the EC surface, sKl interacts with vascular endothelial growth factor (VEGF) receptor-2 (VEGFR-2) and transient receptor potential canonical-1 (TRPC-1) cation channel to control EC homeostasis. Here, we investigated whether sKl might act as a protective factor to improve angiogenesis in dermal microvascular endothelial cells (MVECs) from SSc patients (SSc-MVECs).MethodsWound healing assay was performed on healthy dermal MVECs (H-MVECs) challenged with sera from healthy controls or SSc patients with or without the addition of sKl. Capillary morphogenesis on Matrigel was assessed in H-MVECs and SSc-MVECs at basal conditions and treated with sKl, as well as in H-MVECs challenged with healthy or SSc sera in presence or absence of sKl. The expression of α-Klotho, VEGF165b, VEGFR-2, TRPC-1, Ki67 and active caspase-3 in H-MVECs and SSc-MVECs was investigated by western blotting. Immunostaining for α-Klotho was performed in H-MVECs and SSc-MVECs, and in healthy and SSc skin sections.ResultsTreatment with sKl effectively counteracted the inihibitory effects of SSc sera on wound healing ability and angiogenic performance of H-MVECs. The addition of sKl significantly improved angiogenesis and maintained over time capillary-like tube formation in vitro by SSc-MVECs. Stimulation of SSc-MVECs with sKl resulted in the upregulation of the proliferation marker Ki67 in parallel with the downregulation of proapoptotic active caspase-3. The expression of α-Klotho was significantly lower in SSc-MVECs than in H-MVECs. The expression of TRPC-1 was also significantly decreased, while that of VEGFR-2 and VEGF165b was significantly increased, in SSc-MVECs compared with H-MVECs. Challenge with sKl either significantly increased TRPC-1 or decreased VEGF165b in SSc-MVECs. Ex vivo analyses revealed that α-Klotho immunostaining was almost absent in the dermal microvascular network of SSc skin compared with control skin.ConclusionsOur findings provide the first evidence that α-Klotho is significantly decreased in the microvasculature in SSc skin and that sKl administration may effectively improve SSc-MVEC functions in vitro by acting as a powerful proangiogenic factor.

Plexin-D1/Semaphorin 3E pathway may contribute to dysregulation of vascular tone control and defective angiogenesis in systemic sclerosis

IntroductionThe vascular and nervous systems have several anatomic and molecular mechanism similarities. Emerging evidence suggests that proteins involved in transmitting axonal guidance cues, including members of class III semaphorin (Sema3) family, play a critical role in blood vessel guidance during physiological and pathological vascular development. Sema3E is a natural antiangiogenic molecule that causes filopodial retraction in endothelial cells, inhibiting cell adhesion by disrupting integrin-mediated adhesive structures. The aim of the present study was to investigate whether in systemic sclerosis (SSc) Plexin-D1/Sema3E axis could be involved in the dysregulation of vascular tone control and angiogenesis.MethodsSema3E levels were measured by quantitative colorimetric sandwich ELISA in serum samples from 48 SSc patients, 45 subjects with primary Raynauds phenomenon (pRP) and 48 age-matched and sex-matched healthy controls. Immunofluorescence staining on skin sections from 14 SSc patients and 12 healthy subjects was performed to evaluate Sema3E and Plexin-D1 expression. Western blotting was used to assess Plexin-D1/Sema3E axis in human SSc and healthy dermal microvascular endothelial cells (SSc-MVECs and H-MVECs, respectively) at basal condition and after stimulation with recombinant human vascular endothelial growth factor (VEGF), SSc and healthy sera. Capillary morphogenesis on Matrigel was performed on H-MVECs treated with healthy, pRP or SSc sera in the presence of Sema3E and Plexin-D1 soluble peptides.ResultsSerum Sema3E levels were significantly higher both in pRP subjects and SSc patients than in controls. In SSc, Sema3E levels were significantly increased in patients with early nailfold videocapillaroscopy (NVC) pattern compared to active/late patterns and pRP, and in patients without digital ulcers versus those with ulcers. In SSc skin, Sema3E expression was strongly increased in the microvascular endothelium. Cultured SSc-MVECs showed higher levels of phosphorylated Plexin-D1 and Sema3E expression than H-MVECs, and stimulation with SSc sera increased phosphorylated Plexin-D1 and Sema3E in H-MVECs. The addition of Sema3E-binding Plexin-D1 soluble peptide significantly attenuated the antiangiogenic effect of SSc sera on H-MVECs.ConclusionsOur findings suggest that Plexin-D1/Sema3E axis is triggered in SSc endothelium and may have a role in the dysregulation of angiogenesis and vascular tone control by inducing neuro-vascular mechanism alterations clinically evident in particular in the early disease phases.

Evidence for a Derangement of the Microvascular System in Patients with a Very Early Diagnosis of Systemic Sclerosis

Objective. To investigate whether patients with a very early diagnosis of systemic sclerosis (VEDOSS) may already present circulating markers and in vitro signs of microvascular dysfunction. Methods. Serum samples were obtained from 55 patients with systemic sclerosis (SSc), 25 patients with VEDOSS, and 55 matched healthy controls (HC). Serum levels of pan-vascular endothelial growth factor (VEGF) and soluble neuropilin-1 (sNRP-1) were measured by ELISA. Human dermal microvascular endothelial cells (H-MVEC) were cultured and stimulated with SSc, VEDOSS, and HC sera. Protein expression of NRP-1 was analyzed by Western blotting, cell proliferation by 5′-bromodeoxyuridine assay, migration capacity by wound-healing assay, and capillary-like tube formation by Matrigel assay. Results. Serum levels of pan-VEGF were increased in patients with VEDOSS and SSc versus HC (p = 0.05 and p = 0.003, respectively). Serum levels of sNRP-1 were significantly reduced in patients with VEDOSS and SSc compared with controls (p = 0.012 and p = 0.027, respectively). NRP-1 expression was decreased in H-MVEC stimulated with VEDOSS sera (p < 0.001 vs HC). Proliferation was reduced in H-MVEC stimulated either with VEDOSS or SSc sera in comparison with HC sera (p = 0.015 and p = 0.043, respectively). Wound healing was compromised in H-MVEC stimulated with VEDOSS and SSc sera versus HC sera (p < 0.01 for both). Capillarogenesis was decreased in H-MVEC stimulated with VEDOSS sera (p < 0.01) and SSc sera (p < 0.001) compared with cells stimulated with HC sera. Conclusion. Similar to patients with SSc, patients with VEDOSS already present biological signs of endothelial dysfunction. Our data demonstrate that VEDOSS sera significantly modify endothelial cell behavior and impair the angiogenic potential of the microvascular system.

Angiostatic and angiogenic chemokines in systemic sclerosis: an overview

In systemic sclerosis (SSc), the dysregulation of several molecular pathways seem to have a role in the disease pathogenesis. Either angiogenesis and vasculogenesis are disturbed and impaired, and an imbalance between angiogenic and angiostatic factors may be involved in the genesis and maintenance of vasculopathy. Aberrant immune system activation and function involves both B and T cells, as well as many different chemokines and cytokines. Particularly, chemokines are central to the initiation and maintenance of inflammatory responses as well as angiogenesis and fibrosis. Increased expression of several chemokines as CXCL4 (platelet factor 4), CXCL8 (IL8), CXCL5 (ENA-78), CCL5 (RANTS), CXCL9 (MIG), CCL24, CXCL10 IP-10), CXCL12, CXCL16 (SRPSDX), CCL2 (MCP-1), CCL19 (MIP-3β/ELC), CCL24 (Eotaxin 2), suggests a complex mechanism by which many immune cell types, including T cells, macrophages and neutrophils are recruited to the skin in SSc patients. Many of these chemokines have redundant roles, possibly to ensure recruitment of specific cell types. Several studies have shown a synergistic effect of combinations of these chemokines in cell recruitment, emphasizing the importance of understanding global chemokine expressions. urthermore, chemokines can be detected in peripheral blood compared with cytokines or growth factors. The utility of cytokines as biomarkers has been investigated but longitudinal studies are necessary to clarify their clinical utility for the evaluation of disease activity, therapeutic effects on skin sclerosis or interstitial lung disease and risk stratification of SSc patients. An effective therapeutic agent, able to interfere with complex chemokine networks, is warranted to attenuate perivascular inflammation, dysregulated angiogenesis and the evolution of skin and internal organ fibrosis, is the most ambitious goal for the scientific research of the future.

AB0194 Ll-37: A New Marker for Interstitial Lung Disease (ILD) in Systemic Sclerosis (SSC)?

Background Fibrosis of the skin and visceral organs is the hallmark of SSc and ILD is the leading cause of SSc related morbidity and mortality [1]. LL-37 peptide is the only cathelicidin of the human antimicrobial peptide family with antimicrobial effects and an immunomodulatory activity [2]. LL-37 was shown to decrease with age [3]. Recent data defined its anti-fibrotic effects on dermal fibroblasts and anti-apoptotic effects on SSc dermal fibroblasts [4]. Objectives To investigate the association between SSc related ILD and circulating levels of LL-37. Methods SSc patients and healthy controls aged 18-80, without signs or symptoms of systemic infection. Clinical data, autoantibody panel and internal organ assessment results (echocardiogram, esophageal manometry, chest HRCT, Lung Function Tests, Capillaroscopy). The pulmonary involvement was defined as SSc related ILD appearance on HRCT scans like ground-glass, reticular and honeycomb pattern. For the measurement of the LL-37 (Hycult Biotech) levels from blood samples of the patients ELISA has been used. Statistical analysis was performed with association/correlation test as appropriate. Results 58 SSc patients were enrolled and divided into 1.patients with pulmonary involvement (n=30), 2. patients without pulmonary involvement (n=28). 28 healthy subjects were used as controls. LL-37 concentrations were remarkably lower in SSc patients with ILD vs SSc patients without ILD (1,3575 ng/ml vs 4,6175 ng/ml, p=0,035) and control subjects (1,3575 ng/ml vs 5,5300 ng/ml, p=0,009). In SSc patients without pulmonary involvement, LL-37 serum levels were not different from controls (p=0,812). No association was found between LL37 circulating levels and any of the other clinical, laboratory or instrumental parameter recorded. Conclusions Significantly lower levels of LL-37 were observed in patients with SSc-ILD. Our results suggest that reduction of the levels of the peptide may be associated to the development of ILD. It remains to be tested in larger SSc cohorts if the circulating levels of LL-37 might be used as an indirect marker of ILD. References Bussone G, Mouthon L. Interstitial lung disease in systemic sclerosis. Autoimmunity reviews. 2011;10(5):248-55. Vandamme D, Landuyt B, Luyten W, Schoofs L. A comprehensive summary of LL-37, the factotum human cathelicidin peptide. Cellular immunology. 2012;280(1):22-35. Alvarez-Rodriguez L, Lopez-Hoyos M, Garcia-Unzueta M, Amado JA, Cacho PM, Martinez-Taboada VM. Age and low levels of circulating vitamin D are associated with impaired innate immune function. Journal of leukocyte biology. 2012;91(5):829-38. Kim HJ, Cho DH, Lee KJ, Cho CS, Bang SI, Cho BK, et al. LL-37 suppresses sodium nitroprusside-induced apoptosis of systemic sclerosis dermal fibroblasts. Experimental dermatology. 2011;20(10):843-5. Disclosure of Interest None declared DOI 10.1136/annrheumdis-2014-eular.2948

SAT0229 A Novel Serum Test Based Algorithm To Aid in Very Early Diagnosis of Systemic Sclerosis (VEDOSS)

Background VEDOSS project relied on a specific set of inclusion criteria identifying patients in very early stage of systemic sclerosis (SSc) to be submitted to a tight follow up and unravel factors linked to the progression of the disease. The components of the ELF score (PIIINP, TIMP-1 and HA) have been shown to correlate with severity of skin and lung fibrosis in SSc. Objectives Our aim was to analyze the concentrations of ELF components in a subset of patients enrolled in the VEDOSS study and determine their potential diagnostic value. Methods Sera from 114 patients enrolled into the VEDOSS data base and 67 SSc controls fulfilling ACR/EULAR 2013 criteria (subsets: 33 diffuse; 34 limited) were obtained from 2 centres. Serum concentrations of ELF components were determined on Siemens Advia Centaur platform. Logistic regression analysis of the results was performed employing classification of SSc as state variable. Results Among the 114 patients enrolled from VEDOSS, 30 subjects had primary Raynauds phenomenon (PRP); 54 had Raynauds phenomenon (RP) and at least one of the following “red flags” (risk factors) for SSc: (Puffy fingers, ANA, SSc specific autoantibodies or SSc-specific capillaroscopic pattern) without fulfilling 2013 ACR/EULAR criteria (score <9). 30 VEDOSS patients fulfilled new ACR/EULAR criteria (score >9) despite having no evidence of skin involvement nor interstitial lung disease at lung high resolution CT or pulmonary arterial hypertension, left ventricular systolic impairment or renal involvement. All serum biomarkers concentrations correlated with age (p<0.05 for all). Logistic regression analysis using the three biomarkers as variables identified a specific model (SSc score) ranging from -3.92 to 5.77. The new score showed good ability to discriminate between patients with SSc (VEDOSS patients already classified as SSc and SSc controls) versus VEDOSS patients not yet classified as SSc (AOUC under ROC curve: 0.853, 95%CI 0.797–0.910). Within the VEDOSS database, SSc score showed a fair ability to discriminate between VEDOSS patients fulfilling SSc classification criteria, despite lack of skin internal organ involvement (lung, heart and kidneys), and those not fulfilling the criteria (AOUC =0.756, 95%CI 0.646–0.865). Indeed, patients fulfilling SSc classification criteria, had average score of 0.44 vs -0.86 of patients not fulfilling the criteria and the score remained significant even after correcting for age (p=0.026). Furthermore, SSc controls had significantly higher SSc score compared to VEDOSS patients already classified as SSc, confirmed after age correction (p<0.000). No difference in SSc score has been observed between PRP and VEDOSS patients not yet classified as SSc. Conclusions Our data indicate that the SSc score is a simple serum test based model that can be used in patients with RP to aid in the very early diagnosis of SSc. Furthermore, the identification of VEDOSS patients with a high SSc score test and already classified as SSc even in the absence of internal organ involvement can be used in intervention trials aimed to prevent further disease progression. Disclosure of Interest None declared

A6.28 The role of Plexin-D1/Semaphorin 3E pathway in the dysregulation of vascular tone control in systemic sclerosis (SSc)

Background and objectives The vascular and nervous systems have several anatomic and molecular mechanism similarities. Emerging evidence suggests that proteins involved in transmitting axonal guidance cues, including class III semaphorin families, also play a critical role in blood vessel guidance during physiological and pathological vessel development. Sema3E is a natural antiangiogenic molecule that causes filopodial retraction in endothelial cells, inhibiting cell adhesion by disrupting integrin-mediated adhesive structures. The aim of the present study was to investigate if Plexin-D1/Sema3E axis could be involved in the dysregulation of vascular tone control (RF) in SSc. Materials and methods Sema3E levels were measured by quantitative colorimetric sandwich ELISA in serum samples from 45 subjects with primary Raynaud’s phenomenon (pRP), 48 SSc patients and 48 age- and sex-matched healthy controls. Western blot was used to evaluate Plexin-D1/Sema3E axis in human SSc and healthy dermal microvascular endothelial cells (SSc-MVEC and H-MVEC, respectively) at basal condition, and after stimulation with recombinant human vascular endothelial growth factor (VEGF), SSc and healthy sera (both n = 3). Immunofluorescence staining on skin sections from 14 SSc patients and 12 healthy subjects was performed in order to evaluate Sema3E and Plexin-D1 expression. Results Serum Sema3E levels were significantly higher both in pRP subjects and SSc patients than in sera controls. In SSc, Sema3E levels were significantly increased in patients with early nailfold videocapillascopy pattern (NVC) compared to active/late pattern and pRP, and in patients without ulcers compared to those with digital ulcers. SSc-MVEC showed higher levels of phosphorylated Plexin-D1 and Sema3E expression compared with H-MVEC, and stimulation with SSc sera increased the expression of phosphorylated Plexin-D1 and Sema3E in H-MVEC. In SSc, Sema3E expression was increased in the dermis, particularly in the vascular endothelium. Conclusions Our findings suggest that Plexin-D1/Sema3E axis is triggered in SSc and may have a role in the dysregulation of vascular tone control by inducing neuro-vascular mechanism alterations clinically evident in particular in the early disease phases.

A6.29 In vitro protective effects of soluble klotho (SKL) protein on endothelial cells in systemic sclerosis (SSc)

Background and objectives Systemic Sclerosis (SSc) is an autoimmune connective tissue disease characterised by impairs angiogenesis, alterations of microcirculation, senescence and apoptosis of endothelial cells (ECs). A recent study demonstrated that soluble Klotho (sKl) protein interacts with the vascular endothelial growth factor receptor 2 (VEGFR-2) and with transient receptor potential canonical-1 (TRPC-1) by forming a complex on the surface of endothelial cells. This heterotrimeric complex is internalised in response to vascular endothelial growth factor (VEGF) stimulation, thus regulating VEGFR-2/TRPC-1–mediated Ca2+ influx, to maintain endothelial biological homeostasis. The aim of this study was to evaluate, if soluble Klotho might act as protective humoral factor on SSc Microvascular Endothelial Cells (MVECs) by inhibiting cellular senescence and inducing angiogenesis. Materials and methods Wound healing capacity was performed in healthy and SSc-MVECs under standard conditions and after sKl stimulation. Angiogenesis was evaluated by in vitro capillary morphogenesis on Matrigel in healthy and SSc-MVECs at basal condition, upon challenge with sKl, SSc and healthy sera, and sKl in combination with SSc and healthy sera. Western blot was performed in order to evaluate TRPC-1 and VEGFR-2 expression level in SSc and healthy MVECs at basal condition, after stimulation with sKl, SSc sera (n = 5), healthy sera (n = 5), and with sKl in combination with SSc and healthy sera before and after wound injury. Results Wound healing capacity significantly increased in healthy and SSc-MVECs after sKl challenge in respect to basal condition. Angiogenesis was significantly higher in SSc-MVECs upon challenge with sKl compared to both basal SSc and healthy MVEC. Moreover, angiogenesis was significantly increased in healthy MVECs challenged with SSc sera in combination with sKl respect to MVECs with SSc sera alone. TRPC-1 and VEGFR-2 expression level significantly increased under injury in both healthy and SSc-MVECs cultured with sKl respect to basal condition. Conclusions Our findings suggest that, under vascular injury conditions, sKl increases the wound healing ability and induces blood vessels formation in SSc-MVECs.