Endothelin-1 promotes vascular smooth muscle cell migration across the artery wall: a mechanism contributing to vascular remodelling and intimal hyperplasia in giant-cell arteritis

Background: Giant-cell arteritis (GCA) is an inflammatory disease of large/medium-sized arteries, frequently involving the temporal arteries (TA). Inflammation-induced vascular remodelling leads to vaso-occlusive events. Circulating endothelin-1 (ET1) is increased in patients with GCA with ischaemic complications suggesting a role for ET-1 in vascular occlusion beyond its vasoactive function. Objective: To investigate whether ET-1 induces a migratory myofibroblastic phenotype in human TAderived vascular smooth muscle cells (VSMC) leading to intimal hyperplasia and vascular occlusion in GCA. Methods and results: Immunofluorescence/confocal microscopy showed increased ET-1 expression in GCA lesions compared with control arteries. In inflamed arteries, ET-1 was predominantly expressed by infiltrating mononuclear cells whereas ET receptors, particularly ET-1 receptor B (ETB R), were expressed by both mononuclear cells and VSMC. ET-1 increased TA-derived VSMC migration in vitro and α-smooth muscle actin (αSMA) expression and migration from the media to the intima in cultured TA explants. ET-1 promoted VSMC motility by increasing activation of focal adhesion kinase (FAK), a crucial molecule in the turnover of focal adhesions during cell migration. FAK activation resulted in Y397 autophosphorylation creating binding sites for Src kinases and the p85 subunit of PI3kinases which, upon ET-1 exposure, colocalised with FAK at the focal adhesions of migrating VSMC. Accordingly, FAK or PI3K inhibition abrogated ET-1-induced migration in vitro. Consistently, ET-1 receptor A and ETB R antagonists reduced αSMA expression and delayed VSMC outgrowth from cultured GCA-involved artery explants. Conclusions: ET-1 is upregulated in GCA lesions and, by promoting VSMC migration towards the intimal layer, may contribute to intimal hyperplasia and vascular occlusion in GCA

Expression and function of IL12/23 related cytokine subunits (p35, p40, and p19) in giant-cell arteritis lesions: contribution of p40 to Th1- and Th17-mediated inflammatory pathways

Background: Giant-cell arteritis (GCA) is considered a T helper (Th)1- and Th17-mediated disease. Interleukin (IL)-12 is a heterodimeric cytokine (p35/p40) involved in Th1 differentiation. When combining with p19 subunit, p40 compose IL-23, a powerful pro-inflammatory cytokine that maintains Th17 response. Objectives: The aims of this study were to investigate p40, p35, and p19 subunit expression in GCA lesions and their combinations to conform different cytokines, to assess the effect of glucocorticoid treatment on subunit expression, and to explore functional roles of p40 by culturing temporal artery sections with a neutralizing anti-human IL-12/IL-23p40 antibody. Methods and results: p40 and p19 mRNA concentrations measured by real-time RT-PCR were significantly higher in temporal arteries from 50 patients compared to 20 controls (4.35 ± 4.06 vs 0.51 ± 0.75; p < 0.0001 and 20.32 ± 21.78 vs 4.17 ± 4.43 relative units; p < 0.0001, respectively). No differences were found in constitutively expressed p35 mRNA. Contrarily, p40 and p19 mRNAs were decreased in temporal arteries from 16 treated GCA patients vs those from 34 treatment-naïve GCA patients. Accordingly, dexamethasone reduced p40 and p19 expression in cultured arteries. Subunit associations to conform IL-12 and IL-23 were confirmed by proximity-ligation assay in GCA lesions. Immunofluorescence revealed widespread p19 and p35 expression by inflammatory cells, independent from p40. Blocking IL-12/IL-23p40 tended to reduce IFNγ and IL-17 mRNA production by cultured GCA arteries and tended to increase Th17 inducers IL-1β and IL-6. Conclusion: IL-12 and IL-23 heterodimers are increased in GCA lesions and decrease with glucocorticoid treatment. p19 and p35 subunits are much more abundant than p40, indicating an independent role for these subunits or their potential association with alternative subunits. The modest effect of IL-12/IL-23p40 neutralization may indicate compensation by redundant cytokines or cytokines resulting from alternative combinations

Blocking interferon γ reduces expression of chemokines CXCL9, CXCL10 and CXCL11 and decreases macrophage infiltration in ex vivo cultured arteries from patients with giant cell arteritis

BACKGROUND: Interferon γ (IFNγ) is considered a seminal cytokine in the pathogenesis of giant cell arteritis (GCA), but its functional role has not been investigated. We explored changes in infiltrating cells and biomarkers elicited by blocking IFNγ with a neutralising monoclonal antibody, A6, in temporal arteries from patients with GCA. METHODS: Temporal arteries from 34 patients with GCA (positive histology) and 21 controls were cultured on 3D matrix (Matrigel) and exposed to A6 or recombinant IFNγ. Changes in gene/protein expression were measured by qRT-PCR/western blot or immunoassay. Changes in infiltrating cells were assessed by immunohistochemistry/immunofluorescence. Chemotaxis/adhesion assays were performed with temporal artery-derived vascular smooth muscle cells (VSMCs) and peripheral blood mononuclear cells (PBMCs). RESULTS: Blocking endogenous IFNγ with A6 abrogated STAT-1 phosphorylation in cultured GCA arteries. Furthermore, selective reduction in CXCL9, CXCL10 and CXCL11 chemokine expression was observed along with reduction in infiltrating CD68 macrophages. Adding IFNγ elicited consistent opposite effects. IFNγ induced CXCL9, CXCL10, CXCL11, CCL2 and intracellular adhesion molecule-1 expression by cultured VSMC, resulting in increased PBMC chemotaxis/adhesion. Spontaneous expression of chemokines was higher in VSMC isolated from GCA-involved arteries than in those obtained from controls. Incubation of IFNγ-treated control arteries with PBMC resulted in adhesion/infiltration by CD68 macrophages, which did not occur in untreated arteries. CONCLUSIONS: Our ex vivo system suggests that IFNγ may play an important role in the recruitment of macrophages in GCA by inducing production of specific chemokines and adhesion molecules. Vascular wall components (ie, VSMC) are mediators of these functions and may facilitate progression of inflammatory infiltrates through the vessel wall

Interleukin-6 (IL-6)/IL-6 receptor and persistence of inflammation in Giant Cell Arteritis. Effects of IL-6 receptor blockade with tocilizumab

[eng] Giant cell arteritis (GCA) is a chronic granulomatous vasculitis affecting large- and medium-sized vessels. This disease can lead to different symptoms related to vascular or systemic inflammation, such as fever and visual loss, and its exact etiology remains to be elucidated. Current treatment of GCA patients is based on glucocorticoids administration. However, not all patients respond properly to this treatment and the disadvantages associated to glucocorticoids promotes the search for new therapeutic alternatives. Blockade of IL-6 signaling with tocilizumab (TCZ), a humanized monoclonal antibody against IL-6 receptor (IL-6R), represents a newly promising alternative, supported by the results of two recently published clinical trials. However, beyond its implication in the acute phase response, the role of IL-6 in the pathogenesis of GCA and vascular inflammation is still unknown. GCA patients treated with TCZ also showed a decrease of acute-phase proteins, which are usually used to monitor disease activity. Therefore, the utilization of this monoclonal antibody remarks the urgency to find alternative biomarker not directly related with IL-6 signaling to monitor GCA patients treated with TCZ. Considering all this information, the aim of this doctoral thesis was to better understand the role of IL-6 in GCA pathogenesis as well as the impact of IL-6R blockade with TCZ. In addition, we aimed to test the potential of osteopontin (OPN) as a biomarker of disease activity in patients treated with this monoclonal antibody. The results from the present study show that IL-6 and IL-6R are remarkably increased in temporal artery lesions from GCA patients compared with control arteries. Co-culture experiments suggest that vascular smooth muscle cells (VSMC) may be an important source of IL-6. IL-6R was found upregulated in GCA lesions, particularly at the granulomatous areas. Co-culture experiments supported this result since IL-6R protein expression was increased in mononuclear cells when co-cultured with VSMC. Contrary to what was observed in tissue, serum levels of sIL-6R showed no differences between GCA patients and controls. The artery culture model was used to better understand the impact of TCZ. IL-6R blockade resulted in a significant decrease in the mRNA expression of STAT3 and SOCS3 after 5 days in culture. However, phosphorylation levels of STAT3 were not modified by TCZ treatment. Co-culture results suggest that under inflammatory conditions the inhibitory effect of TCZ on STAT3 activation may be partially compensated by alternative mechanisms. IL-6R blockade with TCZ also decreased CCL2 and increased the expression of CXCL9 and CXCL10 in cultured temporal arteries. Based on in vitro results, IL-6R blockade may promote an upregulation of CXCL9 and CXCL10 expression in mononuclear cells, that may explain the increased expression observed in cultured arteries. The upregulation of this chemokines may be due to an increase in STAT1 expression and activation after TCZ treatment. IL-6R blockade with TCZ also induced a reduction in the adhesion and migratory capacity of mononuclear cells. These results suggest that IL-6R blockade with TCZ may contribute to decrease tissue inflammation by limiting the advent of new inflammatory cells. Further research is needed to better understand the molecules involved in TCZ modulation of these processes. TCZ treatment of cultured arteries did not affect OPN expression in GCA lesions. Consistently, while levels of C-reactive protein (CRP) were virtually undetectable after IL- 6R blockade, serum concentration of OPN was similar in patients on glucocorticoid or TCZ maintained remission. All together, these data suggest that sOPN could be a useful biomarker of disease activity for TCZ treated patients. However, the role of sOPN needs to be further explored in larger studies with longitudinal cohorts.[cat] L’arteritis de cèl·lules gegants (ACG) és una malaltia inflamatòria crònica d’etiologia desconeguda que afecta les arteries de mitjà i gran calibre. El tractament actual es basa en l’administració de glucocorticoides tot i que presenten efectes adversos i molts pacients experimenten recaigudes. Aquest fet promou la recerca de teràpies alternatives o complementaries. Recentment, s’han publicat els resultats de dos assajos clínics on s’ha vist que un nou fàrmac anomenat tocilizumab (TCZ), un anticòs que bloqueja el receptor de la IL-6 (IL-6R), podria ser una bona alternativa terapèutica per al pacients amb ACG. No obstant, el paper de la IL-6 en la patogènesi de l’ACG és encara desconegut. A més, l’ús del TCZ ha posat de manifest la necessitat de buscar biomarcadors alternatius als clàssicament utilitzats per monitoritzar els pacients, ja que el tractament amb aquest anticòs redueix l’expressió de les proteïnes de fase aguda, les quals són induïdes per la IL-6. Els objectius de la present tesi doctoral han estat per tant, entendre millor el paper de la IL-6 en l’ACG, així com estudiar l’impacte del bloqueig del IL-6R amb TCZ. Tanmateix, s’ha analitzat el potencial de l’osteopontiona (OPN) com a biomarcador alternatiu en pacients tractats amb aquest anticòs. Els resultats del present estudi mostren que tant la IL-6 com el seu receptor es troben incrementats en les lesions de pacients amb ACG. El bloqueig del IL-6R amb TCZ té un efecte clar sobre l’expressió de les quimiocines CCL2, CXCL9 i CXCL10. A més, els resultats suggereixen que el tractament amb TCZ podria contribuir a disminuir la inflamació en els teixits al prevenir l’arribada de noves cèl·lules inflamatòries. En relació al possible paper de l’OPN com a biomarcador en pacients tractats amb TCZ es va veure que els nivells de OPN en sèrum eren similar als dels pacients tractats amb glucocorticoides. Per contra, els nivells de proteïna C reactiva eren pràcticament indetectables en el grup de pacients tractats amb l’anticòs. En conjunt, els resultats mostren que l’OPN podria ser un bon biomarcador de l’activitat de la malaltia en pacients tractats amb TCZ

Expression and Function of IL12/23 Related Cytokine Subunits (p35, p40, and p19) in Giant-Cell Arteritis Lesions: Contribution of p40 to Th1- and Th17-Mediated Inflammatory Pathways

BackgroundGiant-cell arteritis (GCA) is considered a T helper (Th)1- and Th17-mediated disease. Interleukin (IL)-12 is a heterodimeric cytokine (p35/p40) involved in Th1 differentiation. When combining with p19 subunit, p40 compose IL-23, a powerful pro-inflammatory cytokine that maintains Th17 response.ObjectivesThe aims of this study were to investigate p40, p35, and p19 subunit expression in GCA lesions and their combinations to conform different cytokines, to assess the effect of glucocorticoid treatment on subunit expression, and to explore functional roles of p40 by culturing temporal artery sections with a neutralizing anti-human IL-12/IL-23p40 antibody.Methods and resultsp40 and p19 mRNA concentrations measured by real-time RT-PCR were significantly higher in temporal arteries from 50 patients compared to 20 controls (4.35 ± 4.06 vs 0.51 ± 0.75; p &lt; 0.0001 and 20.32 ± 21.78 vs 4.17 ± 4.43 relative units; p &lt; 0.0001, respectively). No differences were found in constitutively expressed p35 mRNA. Contrarily, p40 and p19 mRNAs were decreased in temporal arteries from 16 treated GCA patients vs those from 34 treatment-naïve GCA patients. Accordingly, dexamethasone reduced p40 and p19 expression in cultured arteries. Subunit associations to conform IL-12 and IL-23 were confirmed by proximity-ligation assay in GCA lesions. Immunofluorescence revealed widespread p19 and p35 expression by inflammatory cells, independent from p40. Blocking IL-12/IL-23p40 tended to reduce IFNγ and IL-17 mRNA production by cultured GCA arteries and tended to increase Th17 inducers IL-1β and IL-6.ConclusionIL-12 and IL-23 heterodimers are increased in GCA lesions and decrease with glucocorticoid treatment. p19 and p35 subunits are much more abundant than p40, indicating an independent role for these subunits or their potential association with alternative subunits. The modest effect of IL-12/IL-23p40 neutralization may indicate compensation by redundant cytokines or cytokines resulting from alternative combinations

Expression and function of IL12/23 related cytokine subunits (p35, p40, and p19) in giant-cell arteritis lesions: contribution of p40 to Th1- and Th17-mediated inflammatory pathways

Background: Giant-cell arteritis (GCA) is considered a T helper (Th)1- and Th17-mediated disease. Interleukin (IL)-12 is a heterodimeric cytokine (p35/p40) involved in Th1 differentiation. When combining with p19 subunit, p40 compose IL-23, a powerful pro-inflammatory cytokine that maintains Th17 response. Objectives: The aims of this study were to investigate p40, p35, and p19 subunit expression in GCA lesions and their combinations to conform different cytokines, to assess the effect of glucocorticoid treatment on subunit expression, and to explore functional roles of p40 by culturing temporal artery sections with a neutralizing anti-human IL-12/IL-23p40 antibody. Methods and results: p40 and p19 mRNA concentrations measured by real-time RT-PCR were significantly higher in temporal arteries from 50 patients compared to 20 controls (4.35 ± 4.06 vs 0.51 ± 0.75; p < 0.0001 and 20.32 ± 21.78 vs 4.17 ± 4.43 relative units; p < 0.0001, respectively). No differences were found in constitutively expressed p35 mRNA. Contrarily, p40 and p19 mRNAs were decreased in temporal arteries from 16 treated GCA patients vs those from 34 treatment-naïve GCA patients. Accordingly, dexamethasone reduced p40 and p19 expression in cultured arteries. Subunit associations to conform IL-12 and IL-23 were confirmed by proximity-ligation assay in GCA lesions. Immunofluorescence revealed widespread p19 and p35 expression by inflammatory cells, independent from p40. Blocking IL-12/IL-23p40 tended to reduce IFNγ and IL-17 mRNA production by cultured GCA arteries and tended to increase Th17 inducers IL-1β and IL-6. Conclusion: IL-12 and IL-23 heterodimers are increased in GCA lesions and decrease with glucocorticoid treatment. p19 and p35 subunits are much more abundant than p40, indicating an independent role for these subunits or their potential association with alternative subunits. The modest effect of IL-12/IL-23p40 neutralization may indicate compensation by redundant cytokines or cytokines resulting from alternative combinations

table_4_Expression and Function of IL12/23 Related Cytokine Subunits (p35, p40, and p19) in Giant-Cell Arteritis Lesions: Contribution of p40 to Th1- and Th17-Mediated Inflammatory Pathways.PDF

Background<p>Giant-cell arteritis (GCA) is considered a T helper (Th)1- and Th17-mediated disease. Interleukin (IL)-12 is a heterodimeric cytokine (p35/p40) involved in Th1 differentiation. When combining with p19 subunit, p40 compose IL-23, a powerful pro-inflammatory cytokine that maintains Th17 response.</p>Objectives<p>The aims of this study were to investigate p40, p35, and p19 subunit expression in GCA lesions and their combinations to conform different cytokines, to assess the effect of glucocorticoid treatment on subunit expression, and to explore functional roles of p40 by culturing temporal artery sections with a neutralizing anti-human IL-12/IL-23p40 antibody.</p>Methods and results<p>p40 and p19 mRNA concentrations measured by real-time RT-PCR were significantly higher in temporal arteries from 50 patients compared to 20 controls (4.35 ± 4.06 vs 0.51 ± 0.75; p < 0.0001 and 20.32 ± 21.78 vs 4.17 ± 4.43 relative units; p < 0.0001, respectively). No differences were found in constitutively expressed p35 mRNA. Contrarily, p40 and p19 mRNAs were decreased in temporal arteries from 16 treated GCA patients vs those from 34 treatment-naïve GCA patients. Accordingly, dexamethasone reduced p40 and p19 expression in cultured arteries. Subunit associations to conform IL-12 and IL-23 were confirmed by proximity-ligation assay in GCA lesions. Immunofluorescence revealed widespread p19 and p35 expression by inflammatory cells, independent from p40. Blocking IL-12/IL-23p40 tended to reduce IFNγ and IL-17 mRNA production by cultured GCA arteries and tended to increase Th17 inducers IL-1β and IL-6.</p>Conclusion<p>IL-12 and IL-23 heterodimers are increased in GCA lesions and decrease with glucocorticoid treatment. p19 and p35 subunits are much more abundant than p40, indicating an independent role for these subunits or their potential association with alternative subunits. The modest effect of IL-12/IL-23p40 neutralization may indicate compensation by redundant cytokines or cytokines resulting from alternative combinations.</p

table_2_Expression and Function of IL12/23 Related Cytokine Subunits (p35, p40, and p19) in Giant-Cell Arteritis Lesions: Contribution of p40 to Th1- and Th17-Mediated Inflammatory Pathways.PDF

Background<p>Giant-cell arteritis (GCA) is considered a T helper (Th)1- and Th17-mediated disease. Interleukin (IL)-12 is a heterodimeric cytokine (p35/p40) involved in Th1 differentiation. When combining with p19 subunit, p40 compose IL-23, a powerful pro-inflammatory cytokine that maintains Th17 response.</p>Objectives<p>The aims of this study were to investigate p40, p35, and p19 subunit expression in GCA lesions and their combinations to conform different cytokines, to assess the effect of glucocorticoid treatment on subunit expression, and to explore functional roles of p40 by culturing temporal artery sections with a neutralizing anti-human IL-12/IL-23p40 antibody.</p>Methods and results<p>p40 and p19 mRNA concentrations measured by real-time RT-PCR were significantly higher in temporal arteries from 50 patients compared to 20 controls (4.35 ± 4.06 vs 0.51 ± 0.75; p < 0.0001 and 20.32 ± 21.78 vs 4.17 ± 4.43 relative units; p < 0.0001, respectively). No differences were found in constitutively expressed p35 mRNA. Contrarily, p40 and p19 mRNAs were decreased in temporal arteries from 16 treated GCA patients vs those from 34 treatment-naïve GCA patients. Accordingly, dexamethasone reduced p40 and p19 expression in cultured arteries. Subunit associations to conform IL-12 and IL-23 were confirmed by proximity-ligation assay in GCA lesions. Immunofluorescence revealed widespread p19 and p35 expression by inflammatory cells, independent from p40. Blocking IL-12/IL-23p40 tended to reduce IFNγ and IL-17 mRNA production by cultured GCA arteries and tended to increase Th17 inducers IL-1β and IL-6.</p>Conclusion<p>IL-12 and IL-23 heterodimers are increased in GCA lesions and decrease with glucocorticoid treatment. p19 and p35 subunits are much more abundant than p40, indicating an independent role for these subunits or their potential association with alternative subunits. The modest effect of IL-12/IL-23p40 neutralization may indicate compensation by redundant cytokines or cytokines resulting from alternative combinations.</p

table_3_Expression and Function of IL12/23 Related Cytokine Subunits (p35, p40, and p19) in Giant-Cell Arteritis Lesions: Contribution of p40 to Th1- and Th17-Mediated Inflammatory Pathways.PDF

Background<p>Giant-cell arteritis (GCA) is considered a T helper (Th)1- and Th17-mediated disease. Interleukin (IL)-12 is a heterodimeric cytokine (p35/p40) involved in Th1 differentiation. When combining with p19 subunit, p40 compose IL-23, a powerful pro-inflammatory cytokine that maintains Th17 response.</p>Objectives<p>The aims of this study were to investigate p40, p35, and p19 subunit expression in GCA lesions and their combinations to conform different cytokines, to assess the effect of glucocorticoid treatment on subunit expression, and to explore functional roles of p40 by culturing temporal artery sections with a neutralizing anti-human IL-12/IL-23p40 antibody.</p>Methods and results<p>p40 and p19 mRNA concentrations measured by real-time RT-PCR were significantly higher in temporal arteries from 50 patients compared to 20 controls (4.35 ± 4.06 vs 0.51 ± 0.75; p < 0.0001 and 20.32 ± 21.78 vs 4.17 ± 4.43 relative units; p < 0.0001, respectively). No differences were found in constitutively expressed p35 mRNA. Contrarily, p40 and p19 mRNAs were decreased in temporal arteries from 16 treated GCA patients vs those from 34 treatment-naïve GCA patients. Accordingly, dexamethasone reduced p40 and p19 expression in cultured arteries. Subunit associations to conform IL-12 and IL-23 were confirmed by proximity-ligation assay in GCA lesions. Immunofluorescence revealed widespread p19 and p35 expression by inflammatory cells, independent from p40. Blocking IL-12/IL-23p40 tended to reduce IFNγ and IL-17 mRNA production by cultured GCA arteries and tended to increase Th17 inducers IL-1β and IL-6.</p>Conclusion<p>IL-12 and IL-23 heterodimers are increased in GCA lesions and decrease with glucocorticoid treatment. p19 and p35 subunits are much more abundant than p40, indicating an independent role for these subunits or their potential association with alternative subunits. The modest effect of IL-12/IL-23p40 neutralization may indicate compensation by redundant cytokines or cytokines resulting from alternative combinations.</p

Serum osteopontin: a biomarker of disease activity and predictor of relapsing course in patients with giant cell arteritis. Potential clinical usefulness in tocilizumab-treated patients

Background: Osteopontin (OPN) is a glycoprotein involved in Th1 and Th17 differentiation, tissue inflammation and remodelling. We explored the role of serum OPN (sOPN) as a biomarker in patients with giant cell arteritis (GCA). Methods: sOPN was measured by immunoassay in 76 treatment-naïve patients with GCA and 25 age-matched and sex-matched controls. In 36 patients, a second measurement was performed after 1 year of glucocorticoid treatment. Baseline clinical and laboratory findings, as well as relapses and glucocorticoid requirements during follow-up, were prospectively recorded. sOPN and C reactive protein (CRP) were measured in 32 additional patients in remission treated with glucocorticoids or tocilizumab (interleukin 6 (IL-6) receptor antagonist). In cultured temporal arteries exposed and unexposed to tocilizumab, OPN mRNA expression and protein production were measured by reverse transcription polymerase chain reaction (RT-PCR) and immunoassay, respectively. Results: sOPN concentration (ng/mL; mean±SD) was significantly elevated in patients with active disease (116.75±65.61) compared with controls (41.10±22.65; p<0.001). A significant decline in sOPN was observed in paired samples as patients entered disease remission (active disease 102.45±57.72, remission 46.47±23.49; p<0.001). sOPN correlated with serum IL-6 (r=0.55; p<0.001). Baseline sOPN concentrations were significantly higher in relapsing versus non-relapsing patients (relapsers 129.08±74.24, non-relapsers 90.63±41.02; p=0.03). OPN mRNA expression and protein production in cultured arteries were not significantly modified by tocilizumab. In tocilizumab-treated patients, CRP became undetectable, whereas sOPN was similar in patients in tocilizumab-maintained (51.91±36.25) or glucocorticoid-maintained remission (50.65±23.59; p=0.49). Conclusions: sOPN is a marker of disease activity and a predictor of relapse in GCA. Since OPN is not exclusively IL-6-dependent, sOPN might be a suitable disease activity biomarker in tocilizumab-treated patients