Pro-fibrotic effect of the susceptible gene PCSK5 in vascular fibrosis of Takayasu arteritis via TGF-ß and SMAD3 signaling pathway activation.

BACKGROUND: Vascular fibrosis directly causes vascular thickening in Takayasu arteritis (TAK), in which sustained transforming growth factor beta (TGF-ß) activation is critical. Understanding TGF-ß activation regulation and blocking it might yield a therapeutic effect in TAK. Proprotein convertase subtilisin/kexin type 5 (PCSK5) rs6560480 (T/C) is associated with TAK development. In this study, we assessed the association between the PCSK5 rs6560480 genotype and PCSK5 expression in TAK and explored its molecular role in TGF-ß activation and vascular fibrosis development. METHODS: In TAK patients, PCSK5 and TGF-ß expression in plasma and aortic tissue was examined by ELISA and immunohistochemical staining, and PCSK5 rs6560480 was genotyped. The correlation between PCSK5 and extracellular matrix (ECM) expression was examined by Western blotting (WB) and immunohistochemistry staining. Detection by co-immunoprecipitation was performed to detect the interaction between PCSK5 and TGF-ß in adventitial fibroblasts (AAFs). Downstream signaling pathways were detected by WB and validated with appropriate inhibitors. Potential immunosuppressive agents to inhibit the effects of PCSK5 were explored in cell culture and TAK patients. RESULTS: Patients with PCSK5 rs6560480 TT patients had significantly higher PCSK5 levels and more thickened vascular lesions than patients with PCSK5 rs6560480 CT. PCSK5 expression was significantly increased in alpha smooth muscle actin (α-SMA)-positive myofibroblasts in TAK vascular lesions. Overexpressing PCSK5 facilitated TGF-ß and downstream SMAD2/3 activation and ECM expression in AAFs and aorta in in-vitro culture. The mechanistic study supported that PCSK5 activated precursor TGF-ß (pro-TGF-ß) to the mature form by binding the pro-TGF-ß cleavage site. Leflunomide inhibited PCSK5 and pro-TGF-ß binding, decreasing TGF-ß activation and ECM expression, which was also partially validated in leflunomide-treated patients. CONCLUSION: The findings revealed a novel pro-fibrotic mechanism of PCSK5 in TAK vascular fibrosis via TGF-ß and downstream SMAD2/3 pathway activation. Leflunomide might be anti-fibrotic by disrupting PCSK5 and pro-TGF-ß binding, presenting a new TAK treatment approach.

Neutrophil extracellular trap formation in anti-neutrophil cytoplasmic antibody-associated and large-vessel vasculitis.

Levels of neutrophil extracellular traps (NETs) were measured in plasma of healthy controls (HC, n = 30) and patients with granulomatosis with polyangiitis (GPA, n = 123), microscopic polyangiitis (MPA, n = 61), Takayasu's arteritis (TAK, n = 58), and giant cell arteritis (GCA, n = 68), at times of remission or activity and correlated with levels of the platelet-derived thrombospondin-1 (TSP-1). Levels of NETs were elevated during active disease in patients with GPA (p < 0.0001), MPA (p = 0.0038), TAK (p < 0.0001), and GCA (p < 0.0001), and in remission for GPA, p < 0.0001, MPA, p = 0.005, TAK, p = 0.03, and GCA, p = 0.0009. All cohorts demonstrated impaired NET degradation. Patients with GPA (p = 0.0045) and MPA (p = 0.005) had anti-NET IgG antibodies. Patients with TAK had anti-histone antibodies (p < 0.01), correlating with presence of NETs. Levels of TSP-1 were increased in all patients with vasculitis, and associated with NET formation. NET formation is a common process in vasculitides. Targeting NET formation or degradation could be potential therapeutic approaches for vasculitides.

Mast cells drive pathologic vascular lesions in Takayasu arteritis.

BACKGROUND: Takayasu arteritis (TAK) is a large vessel vasculitis resulting in artery wall remodeling with segmental stenosis and/or aneurysm formation. Mast cells (MCs) are instrumental in bridging cell injury and inflammatory response. OBJECTIVES: This study sought to investigate the contribution of MCs on vessel permeability, angiogenesis, and fibrosis in patients with TAK. METHODS: MC activation and their tissue expression were assessed in sera and in aorta from patients with TAK and from healthy donors (HDs). In vivo permeability was assessed using a modified Miles assay. Subconfluent cultured human umbilic vein endothelial cells and fibroblasts were used in vitro to investigate the effects of MC mediators on angiogenesis and fibrogenesis. RESULTS: This study found increased levels of MC activation markers (histamine and indoleamine 2,3-dioxygenase) in sera of patients with TAK compared with in sera of HDs. Marked expression of MCs was shown in aortic lesions of patients with TAK compared with in those of noninflammatory aorta controls. Using Miles assay, this study showed that sera of patients with TAK significantly increased vascular permeability in vivo as compared with that of HDs. Vessel permeability was abrogated in MC-deficient mice. MCs stimulated by sera of patients with TAK supported neoangiogenesis (increased human umbilic vein endothelial cell proliferation and branches) and fibrosis by inducing increased production of fibronectin, type 1 collagen, and α-smooth muscle actin by fibroblasts as compared to MCs stimulated by sera of HD. CONCLUSIONS: MCs are a key regulator of vascular lesions in patients with TAK and may represent a new therapeutic target in large vessel vasculitis.

FABP3 overexpression promotes vascular fibrosis in Takayasu's arteritis by enhancing fatty acid oxidation in aorta adventitial fibroblasts.

OBJECTIVE: To identify the role of fatty acid binding protein 3 (FABP3) in vascular fibrosis in Takayasu's arteritis (TAK) and to explore the underlying molecular mechanism. METHODS: The expression of FABP3 and extracellular matrix proteins (ECMs) were detected in aorta tissues from TAK patients (n = 12) and healthy controls (n = 8) by immunohistochemistry. The concentration of serum proteins was determined by ELISA. CCK8 and Ki67 staining were used to measure aorta adventitial fibroblast (AAF) proliferation. Widely targeted lipidomic profiling was used to screen for associated metabolic pathways. Changes in ECMs and fatty acid oxidation (FAO)-related enzymes were determined by RT-qPCR and Western blot. The interactions between FABP3 and these enzymes were explored with a co-immunoprecipitation (Co-IP) assay. RESULTS: The expression of FABP3 was increased in the thickened adventitia of TAK patients and was positively correlated with the serum expression of ECMs. FABP3 knockdown inhibited AAF proliferation and ECM production, whereas FABP3 overexpression enhanced these processes. Further analysis revealed that FABP3 upregulation promoted carnitine palmitoyltransferase 1A and carnitine/acylcarnitine carrier protein (CACT) expression, two key enzymes in FAO, as well as adenosine triphosphate (ATP) levels. FABP3 and CACT were co-localized in the adventitia and bound to each other in AAFs. Etomoxir reversed the enhanced FAO, ATP production, AAF proliferation and ECM production mediated by FABP3 upregulation. Treatment with 60 g/day curcumin granules for 3 months reduced the level of serum FABP3. Curcumin also inhibited vascular fibrosis by reducing FABP3-enhanced FAO in AAFs. CONCLUSION: Elevated FABP3 expression accelerated vascular fibrosis in TAK, which was likely mediated by promoting FAO in AAFs.

T-614 inhibits human aortic adventitial fibroblast proliferation and promotes interleukin-8 production in vitro.

OBJECTIVES: The effect and underlying mechanism of T-614 (iguratimod) on Takayasu's arteritis (TA) are unknown. Here, we report the effects of T-614 on cell proliferation and interleukin-8 (IL-8) production in human aortic adventitial fibroblasts (HAAFs) in vitro and explore its initial benefit in terms of vascular wall inflammation and remodeling for patients with TA. METHODS: HAAFs were cultured with 0, 5, 50, 100, or 250 µg/ml T-614 in the absence or presence of tumor necrosis factor-α (TNF-α) in vitro. Cell viability was determined by a modified MTT assay. Supernatant IL-8 levels were measured by enzyme-linked immunosorbent assays. RESULTS: In the presence of TNF-α, compared to that in the control group, cell viability of HAAFs significantly decreased in the 50, 100, and 250 µg/ml T-614 treatment groups (OD value: P < 0.01, P < 0.001, P < 0.001, respectively; survival fraction (SF): P < 0.05, P < 0.001, P < 0.001, respectively). However, there was no significant difference in cell viability between TNF-α-stimulated and unstimulated groups at the same concentration of T-614. In the absence or presence of TNF-α, T-614 suppressed HAAF cell viability dose-dependently (OD value: r = -0.915, P = 0.000; r = -0.926, P = 0.000, respectively; SF: r = -0.897, P = 0.000; r = -0.885, P = 0.000, respectively). Compared to that in the control group, in the absence of TNF-α, IL-8 levels in the 5 and 100 µg/ml T-614-treated groups were significantly higher (P < 0.05); in the presence of TNF-α, IL-8 levels in the 5, 50, and 100 µg/ml T-614-treated groups were significantly higher (P < 0.001, P < 0.001, P < 0.01, respectively). Further, there was a negative correlation between supernatant IL-8 levels and T-614 concentration in groups stimulated with TNF-α (r = -0.670, P = 0.000), but there was no significant correlation between these parameters in groups that were not stimulated with TNF-α. CONCLUSIONS: In the absence or presence of TNF-α, T-614 can inhibit HAAF proliferation and promote IL-8 production in vitro; therefore, it could be used to prevent adventitial thickening of the aorta and improve vascular remodeling in inflammatory environments in vitro and might provide a new immunotherapeutic intervention for TA.

Cytokines, growth factors and proteases in medium and large vessel vasculitis.

Giant cell arteritis and Takayasu arteritis are autoimmune vasculitides that cause aneurysm formation and tissue infarction. Extravascular inflammation consists of an intense acute phase response. Deeper understanding of pathogenic events in the vessel wall has highlighted the loss of tissue protective mechanisms, the intrusion of immune cells into "forbidden territory", and the autonomy of self-renewing vasculitic infiltrates. Adventitial vasa vasora critically control vessel wall access and drive differentiation of tissue-invasive T cells. Selected T cells establish tissue residency and build autonomous, self-sufficient inflammatory lesions. Pathogenic effector T cells intrude and survive due to failed immune checkpoint inhibition. Vasculitis-sustaining T cells and macrophages provide a broad portfolio of effector functions, involving heterogeneous populations of pro-inflammatory T cells and diverse macrophage subsets that ultimately induce wall capillarization and intimal hyperplasia. Redirecting diagnostic and therapeutic strategies from control of extravascular inflammatory markers to suppression of vascular inflammation will improve disease management.

Autophagy promotes aortic adventitial fibrosis via the IL-6/Jak1 signaling pathway in Takayasu's arteritis.

BACKGROUND: Autophagy is a ubiquitous and evolutionarily conserved self-rescue process. Studies have shown that autophagy is involved in the pathogenesis of multiple diseases; however, whether autophagy is associated with the pathogenesis of Takayasu's arteritis (TA), a large vessel idiopathic inflammatory disease characterized by vascular fibrosis, remains unclear. Moreover, although IL-6 is believed to be a direct target for TA treatment, anti-IL-6 treatment could not block TA-associated fibrosis in some cases, which impairs the aortic function of patients and can result in death. Thus, identify the mechanisms associated with TA is extremely important. Based on the relationship between autophagy and IL-6, we investigated the role of autophagy in the vascular fibrosis of TA induced by IL-6. METHODS: Autophagy proteins (LC3 and Atg3), IL-6, and markers of fibrosis (collagen 1 and α-SMA) were detected in tissues with TA lesions via immunochemistry, immunofluorescence, and Western blot, respectively. Different stages of autophagy were analyzed by the specific inhibitors, 3-methyladenosine (early stage), hydroxychloroquine sulfate (late stage), and bafilomycin A1 (late stage). Autophagosomes were detected using electron microscopy and a viral-vector transfection assay. The fibrosis profiles induced by IL-6-dependent autophagy was assessed with an ELISA. RESULTS: The expression of autophagy, IL-6, and fibrosis markers were elevated and correlated with each other in the adventitia tissues of TA patients. Furthermore, exogenous IL-6/IL-6Rα could significantly increase autophagy and fibrosis in vitro. An autophagy inhibitor was found to significantly block both autophagy and fibrosis induced by IL-6. Finally, IL-6 was found to significantly promote autophagy-induced fibrosis through the activation of the Jak1 pathway. CONCLUSIONS: IL-6-induced autophagy plays an important role in vascular fibrosis of TA. Targeting autophagy pathways might represent a novel therapeutic option for the treatment of TA.

The C-Reactive Protein/Albumin Ratio and Complete Blood Count Parameters as Indicators of Disease Activity in Patients with Takayasu Arteritis.

BACKGROUND This study aimed to evaluate the ratio of C-reactive protein (CRP) to albumin, inflammatory markers, and parameters from the complete blood count (CBC) in patients with Takayasu arteritis and the association with disease activity. MATERIAL AND METHODS A retrospective study included thirty-two patients with Takayasu arteritis and 32 healthy controls. Clinical and demographic characteristics of patients with Takayasu arteritis were recorded at baseline, before medication and on remission. Similar data were obtained for the controls at recruitment. Remission was defined as more than six months of stable disease without new vascular lesions in patients who previously had active disease. Kerr's criteria were used to define active Takayasu arteritis. RESULTS In patients with Takayasu arteritis, the erythrocyte sedimentation rate (ESR), CRP, CRP/albumin ratio, red cell distribution width (RDW), neutrophil-lymphocyte ratio (NLR), platelet-lymphocyte ratio (PLR), and monocyte-lymphocyte ratio (MLR) were significantly higher, and albumin and MPV were significantly lower compared with controls. The ESR, CRP, CRP/albumin ratio, NLR, PLR, and MLR were decreased in remission, whereas MPV was increased. CRP and the CRP/albumin ratio were positively correlated and albumin and MPV were negatively correlated with disease activity. The CRP/albumin ratio had the highest correlation with disease activity in Takayasu arteritis. The CRP/albumin ratio, RDW, NLR, PLR, and MLR were positively correlated with CRP and ESR. CONCLUSIONS The CRP/albumin ratio, RDW, NLR, PLR, MLR, and MPV were markers of remission of active disease, and the CRP/albumin ratio, total albumin, and MPV were markers of disease activity in Takayasu arteritis.

Discrepancies between vascular and systemic inflammation in large vessel vasculitis: an important problem revisited.

A lack of absolute correlation between systemic inflammation parameters and ongoing vascular disease activity is an important problem in some patients with large vessel vasculitis, especially Takayasu arteritis (TAK). Systemic and vascular wall inflammation in TAK are obviously interrelated, but sometimes they may act independently. There are clear discrepancies between these two types of inflammation, including cytokine patterns and responses to treatment. Vascular and systemic inflammation may also be discordant in two subgroups of giant cell arteritis. The first subgroup is mainly characterized by severe systemic inflammation mostly associated with IL-6-driven immunity, while in the second subgroup there is less systemic inflammation but prominent neuro-ophthalmic ischaemic complications characterized mostly by IFN-γ-mediated effects. Although no definite boundaries exist, it may be suggested that the IL-6/Th17/IL-17 pathway primarily drives systemic inflammation while the IL-12/Th1/IFN-γ pathway dominates in vascular wall inflammation both in TAK and giant cell arteritis. Immunosuppressive treatment of TAK (especially corticosteroids) initially suppresses systemic inflammation, while longer treatment duration is required for the suppression of vascular inflammation. Therefore, evaluating only the systemic inflammation may be misleading. Vascular wall inflammation is currently evaluated using expensive imaging methods, which are not feasible for repetitive use. Although pentraxin-3 is superior to erythrocyte sedimentation rate and CRP, we need more reliable biomarkers to reflect vascular wall inflammation in patients with TAK.

mTOR pathway is activated in endothelial cells from patients with Takayasu arteritis and is modulated by serum immunoglobulin G.

Objectives: Takayasu arteritis (TA) and GCA are large-vessel vasculitides characterized by vascular remodelling involving endothelial cells (ECs) and vascular smooth muscle cells. Mammalian target of rapamycin (mTOR) pathway has been involved in vascular remodelling. We hypothesized that the mTOR pathway was involved in the pathogenesis of large-vessel vasculitis. Methods: We used IF analysis on aortic and temporal artery biopsies from patients with TA and GCA to assess the involvement of the mTOR pathway and searched for antibodies targeting ECs in serum by IIF and cellular ELISA. We evaluated in vitro the effect of purified IgG from patients on mTOR pathway activation and cell proliferation. Results: IF analyses on tissues revealed that both mTORC1 and mTORC2 are activated specifically in ECs from TA patients but not in ECs from GCA patients and healthy controls (HCs). Using IIF and ELISA, we observed higher levels of antibodies binding to ECs in TA patients compared with GCA patients and HCs. Using western blot, we demonstrated that purified IgG from TA patients caused mTORC1 activation in ECs, whereas this effect was not observed with purified IgG from GCA patients or HCs. Purified IgG from TA patients induced a significant EC proliferation compared with to GCA and HC IgG, and this effect was decreased after EC exposure with sirolimus, a specific mTOR inhibitor and PI3K inhibitor. Conclusion: Our results suggest that antibodies targeting ECs drive endothelial remodelling in TA through activation of the mTOR pathway, but not in GCA. Inhibition of the mTOR pathway could represent a therapeutic option in TA.

Pro-fibrotic effect of IL-6 via aortic adventitial fibroblasts indicates IL-6 as a treatment target in Takayasu arteritis.

OBJECTIVES: This study aimed to clarify potential mechanism of IL-6 involved in adventitial fibrosis via adventitial fibroblast in Takayasu arteritis (TAK). METHODS: Immunohistochemistry and double-labelled immunofluorescence were performed on vascular tissue from patients with TAK and controls. Human aorta adventitial fibroblast (AAF) was cultured and stimulated with interleukine 6 (IL-6)/IL-6 receptor (IL-6R). Real-time PCR, western blot, enzyme-linked immunosorbent assays, chromatin immunoprecipitation (ChIP) and reporter assay were conducted in vitro experiments to determine effect of IL-6/IL-6R on AAF. RESULTS: The expression of IL-6, IL-6R, collagen I, collagen III, fibronectin, α-smooth muscle actin (α-SMA), and transforming growth factor (TGF-ß) in TAK arteries was significantly higher than that in the normal arteries. Co-localisation of α-SMA and IL-6 and a positive correlation between their expression were observed in local lesions. In vitro experiments, collagen I, collagen III, fibronectin, α-SMA, and TGF-ß expression increased significantly after stimulation and this fibrogenesis of AAFs was induced in TGF-ß-dependent and -independent manners. Additionally, phosphorylation of JAK2, STAT3 and Akt was significantly enhanced both in IL-6/IL-6R-treated AAFs in vitro and in TAK adventitial α-SMA positive cells. When AAFs were pretreated with inhibitors against JAK2, STAT3, and Akt, fibrosis was significantly reduced. Furthermore, IL-6/IL-6R promoted mRNA expression of IL-6 and MCP-1 in AAFs. Finally, according to ChIP and reporter assay results, STAT3 was the main transcriptional factor in the fibrosis of AAFs induced by IL-6/IL-6R. CONCLUSIONS: IL-6/IL-6R induces fibrogenesis of AAFs via the JAK2/STAT3 and JAK2/Akt pathways, which provides theoretical evidence for IL-6 as a treatment target in TAK.

18F-FDG PET reveals unique features of large vessel inflammation in patients with Takayasu's arteritis.

PURPOSE: The object of this study was to assess whether 18F-fluorodeoxyglucose PET/CT (FDG PET/CT) provides novel information in patients with Takayasu's arteritis (TA) in addition to that provided by current activity assessment, to analyse the effects of possible confounders, such as arterial grafts, and to verify whether PET/CT could be informative in lesions <4 mm thick. METHODS: We studied 30 patients with TA, evaluated from October 2010 to April 2014 by both PET/CT and magnetic resonance imaging (MRI). All arterial lesions were evaluated by PET both qualitatively (positive/negative) and semiquantitatively (maximum standardized uptake value, SUVmax), and the thickness of lesions in the MRI field of view was evaluated. In a per-patient analysis, the relationships between the PET data and acute-phase reactants and NIH criteria for active TA were evaluated. In a per-lesion analysis, the relationships between the PET features of each lesion and MRI morphological data were evaluated. The effects of the presence of arterial grafts were also evaluated. RESULTS: Increased FDG uptake was seen in 16 of 30 patients (53%) and in 46 of 177 vascular lesions (26%). Significant periprosthetic FDG uptake was seen in 6 of 7 patients (86%) with previous vascular surgery and in 10 of 11 of grafts (91%). Graft-associated uptake influenced the PET results in three patients (10%) and the SUVmax values in five patients (17%). Of 39 lesions with significant FDG uptake, 15 (38%) were <4 mm thick. Lesion thickness was correlated with lesion SUVmax in FDG-avid lesions only. FDG arterial uptake was not associated with systemic inflammation or NIH criteria. CONCLUSIONS: PET/CT reveals unique and fundamental features of arterial involvement in TA. PET/CT may be useful in the assessment of local inflammatory and vascular remodelling events independent of systemic inflammation during follow-up, even in lesions in which the arterial wall is <4 mm. The presence of arterial grafts is a potential confounder. Prospective studies are required to correlate PET findings with relevant clinical outcomes.

Vitamin D Levels in Takayasu's Arteritis and a Review of the Literature on Vasculitides.

OBJECTIVE: Takayasu's arteritis (TAK) is a chronic, large-vessel vasculitis. Vitamin D, as a steroidal hormone, has recently been shown to have immunoregulatory and immunosuppressive effects. Low vitamin D levels are demonstrated in various autoimmune disorders. The aim of this study is to investigate vitamin D levels in patients with TAK. A comprehensive review of vitamin D levels in systemic vasculitides (SVs) is also performed. METHODS: The study included 36 patients with TAK, 28 patients with Behçet's disease (BD) as disease control and 30 sex-matched healthy controls. Plasma 25-hydroxy vitamin D (25(OH) vit D) levels were measured with high-performance liquid chromatography. "Deficiency" was defined as 25(OH) vit D levels below 25 nmol/l and "insufficiency" as below 50 nmol/l. RESULTS: Plasma 25(OH) vit D levels were significantly lower in TAK patients (16.93 ± 10.62 nmol/l) than healthy controls (64.63 ± 21.82 nmol/l). Vitamin D level in BD patients (38.8 ± 20.9 nmol/l) is lower than healthy controls but higher than TAK patients. The frequency of vitamin D deficiency was 83.3% in patients with TAK compared to 3.3% in healthy controls. Plasma 25(OH) vit D levels were same between clinically active and inactive patients. In literature review, very few studies were found to investigate vitamin D in SVs. CONCLUSION: We observed a high prevalence of vitamin D deficiency in patients with TAK. As various immune effects of vitamin D on mononuclear cells and arterial endothelium is shown, vitamin D deficiency can be a predisposing factor for immune activation in SV. We therefore suggest monitorization and replacement of vitamin D status in all TAK and other SV patients.

NMR-Based Serum Metabolomics Discriminates Takayasu Arteritis from Healthy Individuals: A Proof-of-Principle Study.

Takayasu arteritis (TA) is a debilitating, systemic disease that involves the aorta and large arteries in a chronic inflammatory process that leads to vessel stenosis. Initially, the disease remains clinically silent (or remains undetected) until the patients present with vascular occlusion. Therefore, new methods for appropriate and timely diagnosis of TA cases are needed to start proper therapy on time and also to monitor the patient's response to the given treatment. In this context, NMR-based serum metabolomic profiling has been explored in this proof-of-principle study for the first time to determine characteristic metabolites that could be potentially helpful for diagnosis and prognosis of TA. Serum metabolic profiling of TA patients (n = 29) and healthy controls (n = 30) was performed using 1D (1)H NMR spectroscopy, and possible biomarker metabolites were identified. Using projection to least-squares discriminant analysis, we could distinguish TA patients from healthy controls. Compared to healthy controls, TA patients had (a) increased serum levels of choline metabolites, LDL cholesterol, N-acetyl glycoproteins (NAGs), and glucose and (b) decreased serum levels of lactate, lipids, HDL cholesterol, and glucogenic amino acids. The results of this study are preliminary and need to be confirmed in a prospective study.

Serum cytokine profiles in Takayasu's arteritis: search for biomarkers.

OBJECTIVES: Assessment of disease activity is one of the major difficulties in patients with Takayasu arteritis (TAK) during follow-up. To date, no biomarker is universally accepted to be a surrogate for active disease in TAK. In this study, we aimed to investigate levels of various pro-and anti-inflammatory molecules including serum granulocyte-macrophage colony-stimulating factor (GM-CSF), interleukin (IL)-6, IL-8, IL-10, IL-18 and IL-23 in patients with TAK. METHODS: The study included 51 patients (age: 40.6±12.2 years, F/M: 45/6) with TAK and 42 age- and sex-matched healthy controls (age: 38.1±7.4 years, F/M: 38/4). All patients fulfilled the criteria of the American College of Rheumatology (ACR). TAK patients were evaluated by physician's global assessment (PGA; active/inactive) and ITAS2010 (Indian Takayasu Arteritis Clinical Activity Score) in terms of clinical activity in baseline and follow-up visits. Commercial enzyme linked immuno-sorbent assay (ELISA) kits were used for measurements of serum cytokine levels. RESULTS: At baseline, 21 (41.2%) patients were active according to PGA and 8 (15.7%) according to ITAS2010. Serum IL-6, IL-8 and IL-18 levels were significantly higher in patients with TAK, whereas GM-CSF, IL-10, IL-23 levels were similar to healthy controls. IL-8 significantly decreased in the follow-up, associated with a decrease of clinical activity, whereas IL-23 level significantly increased. When assessed by ITAS2010 active patients had significantly higher IL-18 levels. CONCLUSIONS: We found significantly increased IL-6, IL-8 and IL-18 levels in patients with TAK compared to healthy controls. Only IL-18 level was significantly higher in active patients assessed by ITAS2010. IL-18 was also the only cytokine in our study that correlated with CRP. These findings suggest that cytokines associated with neutrophilic, pro-inflammatory responses such as IL-6, IL-8 and IL-18 can be potential biomarkers for the assessment of disease activity in TAK and warrant further studies in larger series.

Patients with Takayasu's arteritis having persistent acute-phase response usually have an increased major vessel uptake by 18F-FDG-PET/CT.

OBJECTIVES: Although not uniformly accepted, an increased uptake by 18F-fluorodeoxyglucose (FDG)-positron emission tomography (PET)/computed tomography (CT) in large vessels is accepted to be a sign of active disease in Takayasu's arteritis (TAK). We aimed to investigate the value of 18F-FDG-PET/CT for clinical assessment in a subset of TAK patients having a persistent acute-phase response (APR) without any signs or symptoms of clinical disease activity. METHOD: We studied 14 patients (mean age: 38.6 ± 13.9 years, Female/Male: 11/3, and disease duration: 5.7 ± 5 years). Patients were clinically inactive (according to the definition of activity by Kerr et al.), while categorized as having "persistent" disease activity by physician's global assessment due only to APR. 18F-FDG uptake was graded using a four-point scale from grade 0 (no uptake present) to grade 3 (high grade: uptake higher than that of liver). Any uptake in major vessels with a grade ≥ 2 was accepted to be "active." RESULTS: Mean erythrocyte sedimentation rate was 50.8 ± 13.2 mm/hour and mean C-reactive protein level was 28.5 ± 22.1 mg/L. Active vasculitic lesions were observed by 18F-FDG-PET/CT in 9 of 14 (64.3%) patients. The median number of active vascular lesions was 2 (range: 1-5). A step-up treatment change was decided in 8 patients according to 18F-FDG-PET/CT results. CONCLUSION: We observed increased 18F-FDG uptake in the majority of TAK patients with an increased APR, but clinically silent disease. 18F-FDG-PET/CT showed the presence and localization of active inflammation in the aorta and its branches. Although specificity for observed lesions is not clear, 18F-FDG-PET/CT imaging may influence physician's assessment of clinical activity and treatment choices in TAK.

PD-1 Deficiency in Regulatory T Cells May be Involved in the Pathogenesis of Takayasu's Arteritis.

OBJECTIVES: This study aims to investigate whether PD-1 expressions are abnormal in patients with TAK. METHODS: PD-1 expression was analyzed by flow cytometry. Serum cytokines IL-10, IL-7, IL-2, IL-15, CCL2, CCL3, and CXCL10 were detected using a cytokine cytometric bead array. Immunohistochemistry staining analysis was used to test PD-1 and programmed death-ligand 1 (PD-L1) expression in the aorta of three patients with TAK and three patients with atherosclerosis as controls. RESULTS: The mean fluorescence intensity of PD-1 in CD4+PD-1+ cells was decreased in patients with TAK and the frequency of CD4+Foxp3-PD-1+ cells among CD4+T cells was also decreased in peripheral blood relative to healthy controls (P < .05). The percentage of CD4+CD25+Foxp3+PD-1+ cells in the CD4+CD25+T cell population was lower in patients with TAK than in healthy control and was lower in active TAK group (P < .05). Comparing PD-1 and PDL-1 expression in aorta tissue showed that patients with TAK tended to have lower levels than patients with atherosclerosis, but the difference was not significant (P > .05). Patients with TAK had higher serum levels of IL-10, IL-7, CCL2, and CCL3 (P < .05). CONCLUSIONS: Abnormal expression of PD-1 in serum and aorta tissue of patients with TAK may contribute to TAK pathogenesis. KEY POINTS: PD-1 expression in both peripheral blood and aorta tissue of TAK patients decreased relative to healthy controls, indicating that PD-1 might be involved in TAK pathogenesis.

Metabolic inflammatory volume and total inflammatory glycolysis: novel parameters to evaluate PET-CT disease activity in Takayasu arteritis.

OBJECTIVES: To evaluate diagnostic accuracy for active Takayasu arteritis (TAK) for two novel 18F-fluorodeoxyglucose PET-CT parameters, the inflammatory volume (MIV) and total inflammatory glycolysis (TIG), to quantitate volume of metabolically-active arterial tissue. METHODS: From a cohort of TAK (n = 36, 35 immunosuppressive-naïve), images of PET-CTs were reviewed for mean and maximum standardized uptake value (SUVmean and SUVmax), target-to-blood pool ratio (TBR), target-to-liver ratio (TLR), and PET Vasculitis Activity Score (PETVAS). Regions of interest were drawn to semiautomatically calculate MIV in areas of 18F-fluorodeoxyglucose uptake ≥ 1.5 SUVmean after excluding physiological tracer uptake. TIG was calculated by multiplying MIV with SUVmean. PET-CT parameters, ESR, CRP, and clinical disease activity scores were compared against the gold standard of physician global assessment of disease activity (PGA, active/inactive). RESULTS: Using dichotomized cut-offs for active TAK at SUVmax (≥ 2.21), SUVmean (≥ 1.58), TBR (≥ 2.31), TLR (≥ 1.22), PETVAS (various cut-offs), ESR (≥ 40 mm/hour), and CRP (≥ 6 mg/L), the novel indices MIV (≥ 1.8) and TIG (≥ 2.7) performed similar [area under the receiver operating characteristics curve (AUC) 0.873 for both] to SUVmax (AUC 0.841) and SUVmean (AUC 0.851), and better than TBR (AUC 0.773), TLR (AUC 0.773), PETVAS [≥ 5.5 (AUC 0.750), ≥ 10 (AUC 0.636), ≥ 15 (AUC 0.546)], ESR (AUC 0.748), or CRP (AUC 0.731). MIV and TIG had similar agreement with PGA or CRP as with SUVmax or SUVmean, and better agreement than TBR, TLR, or PETVAS cut-offs. CONCLUSIONS: MIV and TIG performed similarly, therefore, are viable alternatives to existing PET-CT parameters to assess TAK disease activity in this preliminary report. Key Points • MIV and TIG performed similar to SUVmax and SUVmax for disease activity assessment in TAK. • MIV and TIG distinguished active TAK better than TBR, TLR, PETVAS cut-offs, ESR, or CRP. • MIV and TIG had better agreement with PGA or CRP than TBR, TLR, or PETVAS cut-offs.

A novel molecular mechanism of vascular fibrosis in Takayasu arteritis: macrophage-derived GPNMB promoting adventitial fibroblast extracellular matrix production in the aorta.

Takayasu arteritis (TAK) is a chronic large vessel disease characterized by aortic fibrotic thickening, which was mainly mediated by activation of aorta adventitial fibroblasts (AAFs). Our previous genetic study demonstrated that TAK-associated locus IL6 rs2069837 regulated glycoprotein non-metastatic melanoma protein B (GPNMB) expression. Thus, this study aimed to investigate the pathogenic role of GPNMB in TAK. Through pathological staining, we find that GPNMB was mainly expressed in vascular adventitia and positively correlated with adventitial extracellular matrix (ECM) expression in TAK vascular lesion. Specifically, GPNMB was increased in adventitial CD68+ macrophages, which were closely located with CD90+ adventitial fibroblasts. In in-vitro cell culture, THP-1-derived macrophages with GPNMB overexpression promoted ECM expression in AAFs. This effect was also confirmed in aortic tissue or AAFs culture with GPNMB overexpression or active GPNMB protein stimulation. Mechanistically, Co-IP assay and siRNA or inhibitor intervention demonstrated that integrin αVß1 receptor mediated GPNMB effect on AAFs, which also activated downstream Akt and Erk pathway in AAFs. Furthermore, we showed that leflunomide treatment inhibited GPNMB-mediated fibrosis in AAFs, as well as GPNMB expression in macrophages, which were also partially validated in leflunomide-treated patients. Taken together, these data indicated that macrophage-derived GPNMB promotes AAFs ECM expression via the integrin αVß1 receptor and Akt/Erk signaling pathway and leflunomide might play an anti-fibrotic role in TAK by interfering with the macrophage-derived GPNMB/AAFs axis. This study provides evidence that targeting GPNMB is a potential therapeutic strategy for treating vascular fibrosis in TAK.

Detection of IS6110 and HupB gene sequences of Mycobacterium tuberculosis and bovis in the aortic tissue of patients with Takayasu's arteritis.

BACKGROUND: Takayasu's arteritis (TA) is a chronic inflammatory disease affecting the large arteries and their branches; its etiology is still unknown. In individuals suffering from TA, arterial inflammation progresses to stenosis and/or occlusion, leading to organ damage and affecting survival. Relation of TA with Mycobacterium tuberculosis has been known, but there have been only a few systematic studies focusing on this association. The IS6110 sequence identifies the Mycobacterium tuberculosis complex and the HupB establishes the differences between M. tuberculosis and M. bovis. Our objective was to search the presence of IS6110 and HupB genes in aorta of patients with TA. METHODS: We analyzed aorta tissues embedded in paraffin from 5760 autopsies obtained from our institution, we divided the selected samples as cases and controls; CASES: aortic tissues of individuals with Takayasu's arteritis. Control positive: aortic tissues (with tuberculosis disease confirmed) and control negative with other disease aortic (atherosclerosis). RESULTS: Of 181 selected aorta tissues, 119 fulfilled the corresponding criteria for TA, TB or atherosclerosis. Thus 33 corresponded to TA, 33 to tuberculosis (TB) and 53 to atherosclerosis. The mean age was 22 ± 13, 41 ± 19, and 57 ± 10, respectively. IS6110 and HupB sequences were detected in 70% of TA tissues, 82% in tuberculosis, and in 32% with atherosclerosis. Important statistical differences between groups with TA, tuberculosis versus atherosclerosis (p = 0.004 and 0.0001, respectively) were found. CONCLUSION: We identified a higher frequency of IS6110 and HupB genes in aortic tissues of TA patients. This data suggests that arterial damage could occur due to previous infection with M. tuberculosis.