Chrissta X. Maracle

NF-κB-inducing kinase is a key regulator of inflammation-induced and tumour-associated angiogenesis

Angiogenesis is essential during development and in pathological conditions such as chronic inflammation and cancer progression. Inhibition of angiogenesis by targeting vascular endothelial growth factor (VEGF) blocks disease progression, but most patients eventually develop resistance which may result from compensatory signalling pathways. In endothelial cells (ECs), expression of the pro‐angiogenic chemokine CXCL12 is regulated by non‐canonical nuclear factor (NF)‐κB signalling. Here, we report that NF‐κB‐inducing kinase (NIK) and subsequent non‐canonical NF‐κB signalling regulate both inflammation‐induced and tumour‐associated angiogenesis. NIK is highly expressed in endothelial cells (ECs) in tumour tissues and inflamed rheumatoid arthritis synovial tissue. Furthermore, non‐canonical NF‐κB signalling in human microvascular ECs significantly enhanced vascular tube formation, which was completely blocked by siRNA targeting NIK. Interestingly, Nik−/− mice exhibited normal angiogenesis during development and unaltered TNFα‐ or VEGF‐induced angiogenic responses, whereas angiogenesis induced by non‐canonical NF‐κB stimuli was significantly reduced. In addition, angiogenesis in experimental arthritis and a murine tumour model was severely impaired in these mice. These studies provide evidence for a role of non‐canonical NF‐κB signalling in pathological angiogenesis, and identify NIK as a potential therapeutic target in chronic inflammatory diseases and tumour neoangiogenesis.

Targeting of proangiogenic signalling pathways in chronic inflammation

Angiogenesis is de novo capillary outgrowth from pre-existing blood vessels. This process not only is crucial for normal development, but also has an important role in supplying oxygen and nutrients to inflamed tissues, as well as in facilitating the migration of inflammatory cells to the synovium in rheumatoid arthritis, spondyloarthritis and other systemic autoimmune diseases. Neovascularization is dependent on the balance of proangiogenic and antiangiogenic mediators, including growth factors, cytokines, chemokines, cell adhesion molecules and matrix metalloproteinases. This Review describes the various intracellular signalling pathways that govern these angiogenic processes and discusses potential approaches to interfere with pathological angiogenesis, and thereby ameliorate inflammatory disease, by targeting these pathways.

Tertiary Lymphoid Structures in Rheumatoid Arthritis: NF-κB-Inducing Kinase-Positive Endothelial Cells as Central Players.

Tertiary lymphoid structures (TLSs) in chronic inflammation, including rheumatoid arthritis (RA) synovial tissue (ST), often contain high endothelial venules and follicular dendritic cells (FDCs). Endothelial cell (EC)-specific lymphotoxin β (LTβ) receptor signaling is critical for the formation of lymph nodes and high endothelial venules. FDCs arise from perivascular platelet-derived growth factor receptor β(+) precursor cells (preFDCs) that require specific group 3 innate lymphoid cells (ILC3s) and LTβ for their expansion. Previously, we showed that RA ST contains ECs that express NF-κB-inducing kinase (NIK), which is pivotal in LTβ-induced noncanonical NF-κB signaling. We studied the relation between NIK(+) ECs, (pre)FDCs, and ILC3s with respect to TLSs in RA ST. TLS(+) tissues exhibited a significantly increased expression of genes involved in noncanonical NF-κB signaling, including NIK, and immunohistochemical analysis revealed that NIK was almost exclusively expressed by ECs. ILC3s were present in human RA ST in very low numbers, but not differentially in TLS(+) tissues. In contrast, TLS(+) tissues contained significantly more NIK(+) ECs and perivascular platelet-derived growth factor receptor β(+) preFDCs, which correlated significantly with the quantity of FDCs. We established a strong link between NIK(+) ECs, (pre)FDCs, and the presence of TLSs, indicating that NIK(+) ECs may not only be important orchestrators of lymph node development but also contribute to the formation of TLSs in chronic inflammation.

Inhibitors of angiogenesis: Ready for prime time?

Angiogenesis plays a crucial role in the pathogenesis of inflammatory diseases, including rheumatoid arthritis (RA). Therefore, targeting neovascularization in RA may hold great therapeutic potential. Several mediating factors are involved in synovial angiogenesis, including growth factors, cytokines, chemokines, adhesion molecules, and matrix-remodeling enzymes. This review aims to summarize the current understanding of these contributing factors in RA, as well as to describe both the preclinical and clinical studies in which these factors are targeted in an attempt to ameliorate the symptoms associated with RA. In addition, we highlight methods to monitor synovial angiogenesis in patients and discuss possible future therapeutic approaches in RA, including the combination of existing immunosuppressive antirheumatic therapies and anti-angiogenic treatments to potentially maximize efficacy with limited toxicity.

Targeting non-canonical nuclear factor-κB signalling attenuates neovascularization in a novel 3D model of rheumatoid arthritis synovial angiogenesis

Objective. Angiogenesis is crucial in RA disease progression. Lymphotoxin &bgr; receptor (LT&bgr;R)-induced activation of the non-canonical nuclear factor-&kgr;B (NF-&kgr;B) pathway via NF-&kgr;B-inducing kinase (NIK) has been implicated in this process. Consequently, inhibition of this pathway may hold therapeutic potential in RA. We describe a novel three-dimensional (3D) model of synovial angiogenesis incorporating endothelial cells (ECs), RA fibroblast-like synoviocytes (RAFLSs) and RA synovial fluid (RASF) to further investigate the contributions of NF-&kgr;B in this process. Methods. Spheroids consisting of RAFLSs and ECs were stimulated with RASF, the LT&bgr;R ligands LT&bgr; and LIGHT, or growth factor bFGF and VEGF, followed by quantification of EC sprouting using confocal microscopy and digital image analysis. Next, the effects of anginex, NIK-targeting siRNA (siNIK), LT&bgr;R–Ig fusion protein (baminercept) and a novel pharmacological NIK inhibitor were investigated. Results. RASF significantly promoted sprout formation, which was blocked by the established angiogenesis inhibitor anginex (P < 0.05). LT&bgr; and LIGHT induced significant sprouting (P < 0.05), as did bFGF/VEGF (P < 0.01). siNIK pre-treatment of ECs led to reductions in LT&bgr;R-induced vessel formation (P < 0.05). LT&bgr;R–Ig not only blocked LT&bgr;- or LIGHT-induced sprouting, but also RASF-induced sprouting (P < 0.05). The NIK inhibitor blocked angiogenesis induced by LT&bgr;, LIGHT, growth factors (P < 0.05) and RASF (P < 0.01). Conclusion. We present a novel 3D model of synovial angiogenesis incorporating RAFLSs, ECs and RASF that mimics the in vivo situation. Using this system, we demonstrate that non-canonical NF-&kgr;B signalling promotes neovascularization and show that this model is useful for dissecting relative contributions of signalling pathways in specific cell types to angiogenic responses and for testing pharmacological inhibitors of angiogenesis.

Noncanonical NF-κB signaling in microvessels of atherosclerotic lesions is associated with inflammation, atheromatous plaque morphology and myocardial infarction

BACKGROUND AND AIMS Neovascularization is associated with atherosclerotic plaque instability and increased chance of myocardial infarction (MI). Patients with chronic inflammatory diseases (CID) have increased risk of atherosclerosis, and evidence demonstrates that NF-κB inducing kinase (NIK)-mediated noncanonical NF-κB signaling in endothelial cells (EC) is linked to inflammation and angiogenesis. Here, we hypothesized NIK may also be activated in EC of atherosclerotic lesion microvessels. METHODS Using cohorts of atherosclerotic lesions from coronary and carotid arteries, we quantified NIK expression in plaque microvessels and compared it to pathological markers, including inflammatory cell content, plaque characteristics and MI. Differences in gene transcripts were evaluated between stable and ruptured lesions. RESULTS NIK+EC were present in both coronary and carotid lesions. In CID patients, plaques with stenosis >40% had an increased number of NIK+EC and higher content of immune cells (p < .05) as compared to controls. Immune cells per NIK+EC were also greater in CID patients (p < .05), with pronounced differences as stenosis increased. In unstable lesions, NIK+EC were elevated as were EC expressing CXCL12 (p < .05). NIK+EC were increased in lesions with lipid content >40% (p < .05) and more abundant in coronary artery lesions implicated in MI (p < .05). These vessels also associated with atheromatous rather than fibrous plaque morphology (p < .05). Transcriptomic profiling demonstrated components of noncanonical NF-κB pathway were also upregulated in ruptured plaques (p < .05). CONCLUSIONS NIK+EC associate with chronic inflammation in advanced lesions and are linked to markers of local inflammation, lipid content, unstable plaque phenotype and development of MI. Therefore, targeting noncanonical NF-κB signaling may hold therapeutic potential for patients with atherosclerosis and cardiovascular disease.

Silencing NIK potentiates anti-VEGF therapy in a novel 3D model of colorectal cancer angiogenesis

Angiogenesis is essential for colorectal cancer (CRC) progression, as demonstrated by the beneficial clinical effects of therapeutics inhibiting VEGF signaling. However, alternative mechanisms of neovascularization can develop, resulting in treatment failure. Previously we demonstrated NF-κB-inducing kinase (NIK) contributes to pathological angiogenesis. Here, we investigate NIK as a therapeutic target in endothelial cells (EC) in CRC. To determine NIK expression levels in CRC tissues, we immunostained both primary colorectal tumors and tumors metastasized to the liver. Additionally, a 3D tumor-stromal cell interaction model was developed including EC, fibroblasts and CRC cells to study tumor angiogenesis. This model tested efficacy of NIK-targeting siRNA (siNIK) in EC alone or in combination with the anti-VEGF antibody, bevacizumab. Both primary CRC and liver metastases contained blood vessels expressing NIK. In patients receiving chemotherapy plus bevacizumab, immature NIK+ vessels (p < 0.05) were increased as compared to chemotherapy alone. Activation of NIK by lymphotoxin-beta receptor (LTβR) induced increases in pro-angiogenic mediators, including interleukin (IL)-6, IL-8, chemokine (C-X-C motif) ligand (CXCL)1 and CXCL5 in EC and fibroblasts, accompanied by sprouting in the 3D model, which was blocked by siNIK in EC. Treatment with bevacizumab plus siNIK in EC resulted in a synergistic effect and reduced VEGF and bFGF-induced sprouting (p < 0.05). Here, we demonstrate a role for NIK in CRC-associated angiogenesis. Targeting NIK in EC in combination with anti-VEGF antibody bevacizumab may hold therapeutic potential to increase efficiency in blocking tumor neovascularization, either to prevent treatment failure due to activation of accessory pathways such as NF-κB signaling or as a rescue treatment.

A7.14 Identification of new inhibitors of angiogenesis in a novel 3d model of rheumatoid arthritis synovial angiogenesis

Background/objective Angiogenesis contributes to rheumatoid arthritis (RA) pathogenesis, however, many models focus solely on endothelial cells (EC). We developed a 3D-spheroid model including both EC and RA fibroblast-like-synoviocytes (FLS) to study angiogenesis associated with RA and to test efficacy of several inhibitors targeting this process. Previous work demonstrated a role for the non-canonical NF-κB pathway and its main regulator, NF-kB inducing kinase (NIK) in pathological angiogenesis. Here we aim to use the 3D model to further characterise its contribution to neovascularization. Methods Human umbilical vein EC (HUVEC) were combined with RA FLS in spheroids and placed in a collagen gel. Spheroids were stimulated with RA synovial fluid (SF), growth factors (GF), LT or LIGHT. To establish NIK dependency in EC, EC were pre-transfected with a non-targeting or NIK-targeting siRNA before incorporation into spheroids. Pharmacological inhibitors were also added to determine their ability to abrogate EC sprouting induced by 10% RASF. Sprouting was imaged by confocal microscopy and quantified using the Leica QWin Plus software. Results Spheroids formed sprouts under all conditions with significant increases observed upon stimulation with LT, LIGHT, 10% RA SF (p < 0.05) and growth factors (p < 0.01), as compared to basal levels. LT and LIGHT induced sprout formation was NIK dependent as spheroids containing HUVEC transfected with NIK targeting siRNA had reductions in vessel formation (p < 0.05) as compared to controls. Anginex blocked sprout formation induced not only by growth factors, but also by that induced by RA SF (p < 0.05). Inhibitors of the non-canonical NF-kB pathway were able to attenuate cumulative sprout growth promoted by LT, LIGHT and RA SF (p < 0.05). Conclusion The 3D model is an effective tool for studying synovial angiogenesis in that it incorporates several elements essential to the process namely EC, RA FLS and immune cell factors found in RA SF. Using this system, we have further demonstrated a role for activation of the non-canonical NF-kB pathway, and its central regulator NIK, in neovascularization associated with RA. Moreover, we have shown this method to be useful for testing inhibitors of angiogenesis and found that targeting of non-canonical NF-kB signalling to be an effective method of blocking pathological angiogenesis.

AB0265 Non-Canonical NF-κB Signaling in Microvessels of Atherosclerotic Lesions in Coronary Arteries Is Associated with Inflammatory Cell Infiltration and Myocardial Infarction

Background Patients with chronic inflammatory diseases (CID) have higher risk of developing cardiovascular disease which may be in part due to increased systemic inflammatory burden. In atherosclerosis, extensive neovascularization is associated with plaque instability and increased chance of myocardial infarction (MI). We have established that non-canonical NF-κB signaling, and its central regulator NF-κB inducing kinase (NIK) in endothelial cells (EC) contributes to angiogenesis in synovial tissue of patients with various types of arthritis. Thus, we hypothesized that NIK+ EC may also contribute to neovascularization in atherosclerotic lesions. Objectives Evaluate the expression of NIK in microvessels in atherosclerotic lesions from patients with and without CID and determine if it is associated with inflammatory cell influx, systemic inflammation or involvement of coronary arteries in MI. Methods Atherosclerotic lesions from 11 individuals with CID along with 11 matched controls, isolated from parts of the coronary artery implicated in MI (CA+) or not (CA-), were immunohistochemically stained with antibodies against NIK, CD31/34 (EC), myeloperoxidase (neutrophils), CD45 (lymphocytes), CD68 (macrophages) and tryptase (mast cells). NIK vessel density (NIK+VD) and immune cell density (ICD) were subsequently calculated. Results NIK+EC were present in atherosclerotic lesions of all coronary arteries. NIK+VD significantly correlated with ICD of all characterized immune cells: leukocytes (r=0.5227; p<0.0001), macrophages (r=0.3397, p<0.0001), mast cells (r=0.4205, p<0.0001) and neutrophils (r=0.2129, p=0.0016). No significant differences were found in NIK+VD in CID patients versus healthy, however, influx of leukocytes and macrophages per NIK+ microvessel was significantly higher in CID lesions. Increased NIK+ microvessels were also noted in CA+ as compared to CA- tissues (p=0.0139) in both healthy and CID patients. Conclusions NIK+ microvessels are present in high numbers in atherosclerotic lesions and are strongly associated with the influx of inflammatory cells. Systemic inflammation is not a prerequisite for activation of this pathway in EC in atherosclerotic lesions, but our findings suggest that non-canonical NF-κB signaling is more pronounced in patients with CID resulting in the attraction of more immune cells that may further enhance progression of atherosclerotic lesions. Since activation of the non-canonical NF-κB pathway in EC induces angiogenesis and NIK+ vessels are increased in coronary arteries associated with MI, non-canonical NF-κB signaling in EC may drive neovascularization and plaque instability, thus increasing the chance of developing a (fatal) MI. Disclosure of Interest None declared

A6.31 Contributions of canonical and non-canonical NF-κB signalling to LTR-driven inflammatory activation of endothelium and interaction with leukocytes

Background and objectives Sites of chronic inflammation, such as rheumatoid arthritis synovial tissue, are often characterised by the formation of tertiary lymphoid structures, which show characteristic features of lymphoid organs, like high endothelial venules (HEV) and sometimes even true germinal centres. Ligation of the lymphotoxin (LT)-β receptor (LTβR) results in activation of both canonical and non-canonical NF-κB signalling in endothelial cells (ECs) and plays a crucial role in lymphoid neogenesis. Non-canonical NF-κB signalling in ECs promotes inflammation-induced angiogenesis and triggers the development of the cuboidal HEV appearance. However, the relative contribution of the individual pathways to the acquisition of leukocyte traffic-regulating properties by ECs is less well understood. We therefore aimed to identify the molecular pathways by which LTβR drives activation of ECs and interactions with leukocytes. Methods Primary human ECs were stimulated with LTβ or LIGHT to activate LTβR. Activation of downstream signalling pathways was assessed by western blot analysis. Levels of cytokines secreted by LTβ-treated ECs were measured in conditioned media using antibody arrays. To block canonical NF-κB signalling pathway, a small molecule inhibitor of IKKβ was used. Inactivation of non-canonical NF-κB signalling was obtained with a small molecule inhibitor of NIK, and siRNAs targeting NIK or NFκB2. EC interactions with leukocytes were determined by an adhesion assay, whereas EC monolayer permeability was assessed by a permeability assay. Results Treatment of ECs with LTβ or LIGHT activated both canonical and non-canonical NF-κB signalling pathways. Exposure of ECs to LTβ induced the expression of several inflammatory cytokines (IL-8, IL-6, CXCL1, CXCL5, MCP-1, GM-CSF, MIP3α), and this response was mediated mainly by the canonical NF-κB pathway. Consistent with the inflammatory activation of ECs, LTβR ligation induced adhesion of T cells and monocytes to activated endothelium in a predominantly canonical NF-κB-dependent manner. Of note, LTβ was more potent at inducing adhesive properties of ECs than LIGHT. Finally, LTβR ligation by LTβ induced permeability across EC monolayers. Conclusions LTβR ligation on ECs drives inflammation-induced angiogenesis and differentiation into HEV via the non-canonical NF-κB pathway, as well as inflammatory activation of the endothelium and leukocyte adhesion through canonical NF-κB signalling, implicating ECs as immune cell mobilizers during the formation of tertiary lymphoid structures in the course of autoimmune diseases. Further molecular dissection of these pathways may help to better understand the function of tertiary lymphoid structures and potentially to develop therapeutics that modulate the formation of these structures and reduce inflammation.