Session Title: Cell-cell Adhesion, Cell Trafficking and Angiogenesis
Session Type: Abstract Submissions (ACR)
Background/Purpose: In rheumatoid arthritis (RA) synovial tissue (ST) angiogenesis can be observed already in the earliest phase of disease, which may be critical in the switch from acute to chronic inflammation. The chemokine CXCL12, which is induced via the non-canonical nuclear factor-kB (NF-kB) pathway, plays an important role in angiogenesis, lymphocyte transendothelial migration, and the homing of endothelial progenitor cells. Therefore, the non-canonical pathway, with its key mediator NF-kB inducing kinase (NIK), may play an important role in pathological angiogenesis and the perpetuation of synovial inflammation in RA.
Objectives: To study the role of non-canonical NF-kB signaling in pathological angiogenesis in RA.
Methods: Expression of NIK and CXCL12 in RA ST was evaluated using immunofluorescence microscopy (IF). The angiogenic potential of endothelial cells (EC) was studied in vitro using the tube formation assay and siRNA-mediated gene silencing. Microvessel outgrowth was studied ex vivo by comparing WT and NIK-/-mice in the aortic ring assay. Physiological (developmental) angiogenesis was evaluated in these mice by isolectin B4 staining of the retina followed by confocal microscopy. Finally, the contribution of NIK to synovial angiogenesis was studied in vivo in antigen-induced arthritis (AIA).
Results: NIK was highly expressed in EC in RA ST and co-localized with the EC marker vWF in small (newly formed) blood vessels. NIK, p52 and CXCL12 were expressed both in EC in small blood vessels and in PNAd+ high endothelial venules. In vitro, EC treated with stimuli that induce non-canonical NF-kB signaling (i.e. lymphotoxin, LIGHT, CD40L) significantly enhanced tube formation 2,5-fold (p<0.05), which could be completely blocked by siRNA targeting NIK or IKKα, but not completely by IKKβ (canonical NF-kB pathway). Aortic rings from WT and NIK-/- mice showed normal TNF- and VEGF-induced microvessel outgrowth. In contrast, whereas non-canonical NF-kB stimuli induced microvessel outgrowth in WT mice (unstim 29.94 ± 6.08 vs. LT 159.1 ± 50.24 vs. LIGHT 110.3 ± 17.68 (mm^2) p<0.05), no microvessel outgrowth was observed in aortic rings from NIK-/- mice (unstim 28.74 ± 15.89 vs. LT 45.9 ± 16.71 vs. LIGHT 43.41 ± 15.73 (mm^2)). In line with this, NIK-/- mice exhibited normal developmental angiogenesis in the retina, but a 50% reduction in pathological angiogenesis in synovial inflammation (blood vessels in synovial tissue WT 20 ± 5.07 vs. NIK-/-10.2 ± 3.02).
Conclusion: NIK is preferentially expressed in EC in RA ST. Induction of non-canonical NF-kB signaling in EC resulted in enhanced angiogenesis in vitro, and siRNA-mediated selective blockade of this pathway abrogated these effects. Moreover, NIK-/-mice exhibited normal developmental and VEGF-induced angiogenesis, but reduced pathological angiogenesis in AIA. These findings point towards an important role of the non-canonical NF-kB pathway in pathological angiogenesis associated with chronic (synovial) inflammation. This could be exploited for the development of future new therapies for RA.
Acknowledgement: SWT was supported by a VENI grant and a Clinical Fellowship from the Netherlands Organisation for Scientific Research (ZON-MW).
A. R. Noort,
K. P. M. van Zoest,
D. V. Novack,
M. J. Siemerink,
P. P. Tak,
S. W. Tas,
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