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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 (ET-1) 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 TA-derived 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 (ET B 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 ET B 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., Competing Interests: Competing interests: None declared., (© Article author(s) (or their employer(s) unless otherwise stated in the text of the article) 2017. All rights reserved. No commercial use is permitted unless otherwise expressly granted.)

Objectives: To study the epidemiology and mortality in patients with biopsy-proven giant cell arteritis (GCA) in southern Sweden., Methods: The study area was the County of Skåne. Patients with a positive temporal artery biopsy between 1997 and 2010 were identified using a regional register and a structured review of all histopathology reports. Standardised mortality ratios (SMR) were calculated using data for the Swedish population as the reference., Results: There were 840 patients with biopsy-proven GCA (626 women). The annual incidence rate per 100,000 inhabitants aged ≥50 years was 14.1 (95% CI 13.1 to 15.0); 7.7 (6.7 to 8.7) for men and 19.6 (18.1 to 21.1) for women, without seasonal variations. The incidence increased with age, with estimates of 2.0, 11.8, and 31.3 per 100,000 in the age groups 50-60, 61-70, 71-80 years, respectively (p<0.001). The age-standardised and sex-standardised incidence rate decreased from 15.9/100,000 in 1997-2001 to 13.3/100,000 in 2007-2010 (p=0.026). Two hundred and seventy-nine patients (207 women) died during the observation period. Mortality was significantly increased over the first 2 years after GCA diagnosis (SMR 1.52 (95% CI 1.20 to 1.85)), but not with longer follow-up. The estimated excess mortality was greater in women and in patients aged ≤70 years at diagnosis., Conclusions: In this large population-based study of biopsy-proven GCA from southern Sweden, the incidence of GCA may have decreased over time. Short-term mortality was increased, in particular among those diagnosed at ≤70 years of age, but long-term survival was not impaired., (Published by the BMJ Publishing Group Limited. For permission to use (where not already granted under a licence) please go to http://group.bmj.com/group/rights-licensing/permissions.)

Background: Search for therapeutic targets in giant-cell arteritis (GCA) is hampered by the scarcity of functional systems. We developed a new model consisting of temporal artery culture in tri-dimensional matrix and assessed changes in biomarkers induced by glucocorticoid treatment., Methods: Temporal artery sections from 28 patients with GCA and 22 controls were cultured in Matrigel for 5 days in the presence or the absence of dexamethasone. Tissue mRNA concentrations of pro-inflammatory mediators and vascular remodelling molecules was assessed by real-time RT-PCR. Soluble molecules were measured in the supernatant fluid by immunoassay., Results: Histopathological features were exquisitely preserved in cultured arteries. mRNA concentrations of pro-inflammatory cytokines (particularly IL-1β and IFNγ), chemokines (CCL3/MIP-1α, CCL4/MIP-1β, CCL5/RANTES) and MMP-9 as well as IL-1β and MMP-9 protein concentrations in the supernatants were significantly higher in cultured arteries from patients compared with control arteries. The culture system itself upregulated expression of cytokines and vascular remodelling factors in control arteries. This minimised differences between patients and controls but underlines the relevance of changes observed. Dexamethasone downregulated pro-inflammatory mediator (IL-1β, IL-6, TNFα, IFNγ, MMP-9, TIMP-1, CCL3 and CXCL8) mRNAs but did not modify expression of vascular remodelling factors (platelet derived growth factor, MMP-2 and collagens I and III)., Conclusions: Differences in gene expression in temporal arteries from patients and controls are preserved during temporal artery culture in tri-dimensional matrix. Changes in biomarkers elicited by glucocorticoid treatment satisfactorily parallel results obtained in vivo. This may be a suitable model to explore pathogenetic pathways and to perform preclinical studies with new therapeutic agents.

Objective: To investigate functional expression of NKG2D on CD4 and CD8 T-cells in patients with giant cell arteritis (GCA) and polymyalgia rheumatica (PMR)., Methods: Peripheral blood was drawn from patients with GCA (n=16), PMR (n=78) and healthy controls (HC, n=64). Tissue samples were obtained from GCA patients and controls. Proliferation and cytokine production assays were performed using CFSE and intracellular IFN-γ or TNF-α staining, respectively, and flow cytometry analysis. Immunofluorescence and immunohistology were applied to analyse the presence of NKG2D-expressing T-cells and NKG2D-ligands in temporal arteries, respectively. mRNA levels of NKG2D-ligands were determined by RT-PCR., Results: In both GCA and PMR patients, NKG2D was preferentially expressed on senescent CD4CD28(-) and CD8CD28(-), as well as on CD8CD28 T-cells. Frequencies of senescent T-cells were increased in GCA and PMR patients compared to HC. In GCA tissue samples, infiltrating T-cells were predominately CD28(-). NKG2D expressing T-cells concentrated around the vasa vasorum of the adventitia. Antigenic stimulation induced rapid up-regulation of NKG2D on CD4CD28(-) and CD4CD28 T-cells, whereas TNF-α and interleukin-15 enhanced NKG2D expression on senescent CD4 and CD8 T-cells only. NKG2D cross-linkage augmented anti-CD3 triggered proliferation, IFN-γ and TNF-α production of CD8 T-cells. In CD4CD28(-) T-cells, NKG2D ligation resulted in increased IFN-γ production only. NKG2D ligands were expressed in temporal arteries from GCA patients, particularly in the adventitial and medial layers of affected vessels., Conclusions: NKG2D is functionally expressed on CD4CD28(-) and CD8 T-cells in GCA and PMR. NKG2D-ligands are present in temporal arteries and may co-stimulate NKG2D expressing T-cells.

Background: Interleukin 17A (IL-17A) exerts pivotal proinflammatory functions in chronic inflammatory and autoimmune diseases., Objective: To investigate IL-17A expression in temporal artery lesions from patients with giant-cell arteritis (GCA), and its relationship with disease outcome., Methods: Fifty-seven patients with biopsy-proven GCA were prospectively evaluated, treated and followed for 4.5 years (52-464 weeks). Relapses, time (weeks) required to achieve a maintenance prednisone dose <10 mg/day, and time (weeks) to complete prednisone withdrawal were prospectively recorded. IL-17A mRNA was measured by real-time quantitative RT-PCR in temporal arteries from all patients and 19 controls. IL-17 protein expression was assessed by immunohistochemistry/immunofluorescence., Results: IL-17A expression was significantly increased in temporal artery samples from GCA patients compared with controls (6.22±8.61 vs 2.50±3.9 relative units, p=0.016). Surprisingly, patients with strong IL-17A expression tended to experience less relapses, and required significantly shorter treatment periods (median 25 vs 44 weeks to achieve <10 mg prednisone/day, p=0.0079). There was no correlation between IL-17A and RORc or RORα expression suggesting that these transcription factors may not exclusively reflect Th17 differentiation, and that cells other than Th17 cells might contribute to IL-17 expression in active patients. Accordingly, FoxP3(+)IL-17A(+) cells were identified in lesions by confocal microscopy and were dramatically reduced in specimens from treated patients., Conclusions: IL-17A expression is increased in GCA lesions, and is a predictor of response to glucocorticoid treatment. The contribution of FoxP3+ cells to IL-17A production in untreated patients suggests that induced-Tregs may facilitate disease remission when proinflammatory cytokine production is downregulated by glucocorticosteroids.

Objective: To study the expression of interleukin (IL)-33 and to evaluate its relationship with macrophage polarisation in artery biopsy specimens from patients with giant cell arteritis (GCA)., Methods: IL-33, ST2, p-STAT-6 and perivascular IL-1 receptor-associated kinase 1 (p-IRAK1) tissue distribution was evaluated by immunohistochemistry. Inducible nitric oxide synthase and CD163 were also used by immunohistochemistry to evaluate the M1 and M2 polarisation, respectively. Quantitative gene expression analysis of IL-33, T-helper (Th)2-related transcription factor STAT6, Th2 cytokines (IL-4, IL-5, IL-25) and interferon (IFN)-γ was performed in artery biopsy samples obtained from 20 patients with GCA and 15 controls. Five additional patients who had received prednisone when the temporal artery biopsy was performed were also enrolled., Results: IFN-γ and IL-33 were significantly overexpressed in the inflamed arteries of GCA patients. IL-33 overexpression was not accompanied by a concomitant increase of Th2 cytokines. Neovessels scattered through the inflammatory infiltrates were the main sites of IL-33 expression. The expression of IL-33 receptor ST2 and of p-IRAK1 was also increased in GCA patients. Arteries from glucocorticoid-treated patients had a lower expression of IL-33. IL-33 was accompanied by the expression of p-STAT6 and a clear M2 macrophages polarisation., Conclusions: A role for IL-33 in the inflammation of GCA patients is supported by these findings.

Objective: Approximately 15-20% of patients with giant-cell arteritis (GCA) develop ischaemic complications often preceded by transient ischaemia. The expression of the endothelin (ET) system in GCA lesions was investigated to assess its relationship with the development of ischaemic complications., Methods: Plasma ET-1 was quantified by immunoassay in 61 patients with biopsy-confirmed GCA and 16 healthy donors. ET-1, endothelin-converting enzyme (ECE-1) and endothelin receptor (ET(A)R and ET(B)R) messenger RNA were measured by real-time quantitative reverse transcriptase-PCR in temporal arteries from 35 of these patients and 19 control arteries. Proteins were measured by immunoassay and Western blot., Results: ET-1 concentration was increased at the protein level in temporal artery samples from GCA patients compared with controls (0.98 (SEM 0.32) vs 0.28 (SEM 0.098) fmol/mg, p = 0.028). ECE-1, ET(A)R and ET(B)R/actin ratios (Western blot) were also significantly higher in GCA patients. Intriguingly, mRNA expression of ET-1, ECE-1 and both receptors was significantly reduced in GCA lesions compared with control arteries. When investigating mechanisms underlying these results, platelet-derived growth factor and IL-1beta, present in GCA lesions, were found to downregulate ET-1 mRNA in cultured human temporal artery-derived smooth muscle cells. Glucocorticoid treatment for 8 days did not result in significantly decreased endothelin tissue concentration (0.87 (SEM 0.2) vs 0.52 (SEM 0.08); p = 0.6). Plasma endothelin concentrations were higher in patients with ischaemic complications (1.049 (SEM 0.48) vs 1.205 (SEM 0.63) pg/ml, p = 0.032)., Conclusions: The endothelin system is increased at the protein level in GCA lesions creating a microenvironment prone to the development of ischaemic complications. Recovery induced by glucocorticoids is delayed, indicating persistent exposure to endothelin during initial treatment.

Objectives: Gelatinases (MMP2 and MMP9) are expressed in giant-cell arteritis (GCA) and are thought to play a role in vessel disruption. However, their activation status and enzymatic activity have not been evaluated. Our aim was to investigate the distribution and proteolytic activity of gelatinases in GCA lesions at different stages., Methods: Expression of MMP2, MMP9, MMP2-activator MMP14 and their natural inhibitors TIMP1 and TIMP2 was determined by real-time PCR and immunohistochemistry in temporal artery sections from 46 patients and 12 controls. MMP activation status and enzymatic activity were assessed by gelatin and film in situ zymography., Results: Vascular smooth muscle cells from normal specimens constitutively expressed pro-MMP2 and its inhibitor TIMP2 with no resulting proteolytic activity. In GCA MMP2, MMP9 and MMP14 were strongly expressed in their active form by infiltrating leucocytes. Inflamed arteries also expressed TIMP1 and TIMP2. However, the MMP9/TIMP1 and MMP2/TIMP2 ratios were higher in patients compared with controls, indicating an increased proteolytic balance in GCA which was confirmed by in situ zymography. Maximal gelatinase expression and activity occurred at the granulomatous areas surrounding the internal elastic lamina (IEL). Myointimal cells also expressed MMPs and exhibited proteolytic activity, suggesting a role for gelatinases in vascular remodelling and repair., Conclusions: GCA lesions show intense expression of gelatinases. Activators and inhibitors are regulated to yield enhanced gelatinase activation and proteolytic activity. Distribution of expression and proteolytic activity suggests that gelatinases have a major role not only in the progression of inflammatory infiltrates and vessel destruction but also in vessel repair.

Objective: To examine the localisation of monocyte chemoattractant protein 1 (MCP-1) in the inflamed vessel wall in temporal arteritis (TA) and to measure MCP-1 in plasma both in patients with TA and patients with polymyalgia rheumatica (PMR)., Methods: By immunohistochemical techniques MCP-1 was localised to the vessel wall in patients with TA. In TA, PMR, and healthy controls MCP-1 was quantified by enzyme linked immunosorbent assay (ELISA) in plasma., Results: MCP-1 was localised to the majority of mononuclear cells, some smooth muscle cells, and giant cells in the arterial biopsy specimens from 12 patients with histologically verified TA. In all sections, including the vasa vasorum, the endothelium stained positive. In the intima 73% (range 57-91%), in the media 49% (range 32-67%), and in the adventitia 74% (range of 62-91%) of all cells stained positive. In plasma MCP-1 was significantly raised in untreated TA (n=33) and untreated PMR (n=27) compared with healthy controls (n=12). Untreated TA plasma levels of MCP-1 (mean 391 pg/ml (range 82-778 pg/ml)) were similar to untreated PMR plasma levels (mean 402 pg/ml (range 29-1153 pg/ml)), and no significant difference was found between the two groups of patients. In both patients with TA and patients with PMR no correlation was found between the plasma level of MCP-1 and the erythrocyte sedimentation rate, haemoglobin concentration, and CD4/CD8 ratio., Conclusions: These results show that MCP-1 plays a part in the disease processes of TA and PMR.

The possible role of the eosinophil and its cytotoxic granule proteins in the vascular lesions seen in temporal arteritis was elucidated. Sixteen sections of biopsy specimens from arteria temporalis showing giant cell arteritis were stained for eosinophil cationic protein (ECP) by polyclonal antibodies and the immunoperoxidase method. Activated eosinophils were identified by monoclonal antibodies linked to alkaline phosphatase. Activated eosinophils and secreted ECP were seen in all layers of the inflamed vessels and were most evident in necrotic lesions and thrombi. Only a small number of granulocytes seen in the adventitia were immunoreactive for cathepsin G, and no extracellular deposits of this neutrophil granule protein were seen. A few immunoreactive eosinophils were found in the adventitia in two of five negative temporal artery biopsy specimens from patients with polymyalgia rheumatica. All eight coronary artery biopsy specimens with atherosclerotic lesions showed no activated eosinophils or secreted ECP. These findings indicate that eosinophils are involved in the vascular lesion in temporal arteritis and suggest that cytotoxic eosinophil granule proteins may contribute to the necrotic lesions and the development of thrombi.