Rapid stromal remodeling by short-term VEGFR2 inhibition increases chemotherapy delivery in esophagogastric adenocarcinoma.

Anti-angiogenic agents combined with chemotherapy is an important strategy for the treatment of solid tumors. However, survival benefit is limited, urging the improvement of combination therapies. We aimed to clarify the effects of vascular endothelial growth factor receptor 2 (VEGFR2) targeting on hemodynamic function and penetration of drugs in esophagogastric adenocarcinoma (EAC). Patient-derived xenograft (PDX) models of EAC were subjected to long-term and short-term treatment with anti-VEGFR2 therapy followed by chemotherapy injection or multi-agent dynamic contrast-enhanced (DCE-) MRI and vascular casting. Long-term anti-VEGFR2-treated tumors showed a relatively lower flow and vessel density resulting in reduced chemotherapy uptake. On the contrary, short-term VEGFR2 targeting resulted in relatively higher flow, rapid vasodilation, and improved chemotherapy delivery. Assessment of the extracellular matrix (ECM) revealed that short-term anti-angiogenic treatment drastically remodels the tumor stroma by inducing nitric oxide synthesis and hyaluronan degradation, thereby dilating the vasculature and improving intratumoral chemotherapy delivery. These previously unrecognized beneficial effects could not be maintained by long-term VEGFR2 inhibition. As the identified mechanisms are targetable, they offer direct options to enhance the treatment efficacy of anti-angiogenic therapy combined with chemotherapy in EAC patients.

NIK-IKK complex interaction controls NF-κB-dependent inflammatory activation of endothelium in response to LTßR ligation.

NF-κB-inducing kinase (NIK; also known as MAP3K14) is a central regulator of non-canonical NF-κB signaling in response to stimulation of TNF receptor superfamily members, such as the lymphotoxin-ß receptor (LTßR), and is implicated in pathological angiogenesis associated with chronic inflammation and cancer. Here, we identify a previously unrecognized role of the LTßR-NIK axis during inflammatory activation of human endothelial cells (ECs). Engagement of LTßR-triggered canonical and non-canonical NF-κB signaling promoted expression of inflammatory mediators and adhesion molecules, and increased immune cell adhesion to ECs. Sustained LTßR-induced inflammatory activation of ECs was NIK dependent, but independent of p100, indicating that the non-canonical arm of NF-κB is not involved. Instead, prolonged activation of canonical NF-κB signaling, through the interaction of NIK with IκB kinase α and ß (also known as CHUK and IKBKB, respectively), was required for the inflammatory response. Endothelial inflammatory activation induced by synovial fluid from rheumatoid arthritis patients was significantly reduced by NIK knockdown, suggesting that NIK-mediated alternative activation of canonical NF-κB signaling is a key driver of pathological inflammatory activation of ECs. Targeting NIK could thus provide a novel approach for treating chronic inflammatory diseases.

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 (LTßR) 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.

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.

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 ß receptor (LTßR)-induced activation of the non-canonical nuclear factor-κB (NF-κB) pathway via NF-κB-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-κB in this process. METHODS: Spheroids consisting of RAFLSs and ECs were stimulated with RASF, the LTßR ligands LTß 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), LTßR-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). LTß and LIGHT induced significant sprouting (P < 0.05), as did bFGF/VEGF (P < 0.01). siNIK pre-treatment of ECs led to reductions in LTßR-induced vessel formation (P < 0.05). LTßR-Ig not only blocked LTß- or LIGHT-induced sprouting, but also RASF-induced sprouting (P < 0.05). The NIK inhibitor blocked angiogenesis induced by LTß, 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-κB 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.

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 ß (LTß) receptor signaling is critical for the formation of lymph nodes and high endothelial venules. FDCs arise from perivascular platelet-derived growth factor receptor ß(+) precursor cells (preFDCs) that require specific group 3 innate lymphoid cells (ILC3s) and LTß for their expansion. Previously, we showed that RA ST contains ECs that express NF-κB-inducing kinase (NIK), which is pivotal in LTß-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 ß(+) 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.

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.