Background/Purpose:

Angiogenesis plays a crucial role in rheumatoid arthritis (RA) synovitis and is regarded as a switch from acute to chronic inflammation. Previously, we have demonstrated that NF-kappaB inducing kinase (NIK) mediated non-canonical NF-kappaB signaling contributes to pathological angiogenesis in endothelial cells (EC). However, many of the current in vitro models of angiogenesis focus solely on EC and do not include interactions with other cell types. RA synovial fibroblasts (RASF) are important contributors to angiogenesis in synovial inflammation, and thus a model including both RASF and EC would more accurately represent the pathophysiology of RA angiogenesis.

Objective:

To develop better in vitro models to study the interaction between RASF and EC and to further delineate the role of the non-canonical NF-kappaB pathway in pathological angiogenesis.

Methods:

First, we utilized a 2 dimensional (2D) model to evaluate the effects of non-canonical NF-kappaB signaling on angiogenesis. RASF and human umbilical cord EC (HUVEC) were co-cultured in the presence or absence of lymphotoxin (LT), LIGHT, or growth factors (bFGF/VEGF). EC were visualized through immunohistochemical staining of CD31, which was semi-quantitatively scored. Next, we developed a novel 3 dimensional (3D) model in which HUVEC and RASF were labeled with green or orange cell tracker dye, respectively, and incubated overnight to form spheroids. Subsequently, the spheroids were harvested and plated in a collagen solution, and medium with or without LT, LIGHT or growth factors (bFGF/VEGF) was added. After 48 hours, spheroids were fixed and imaged through confocal microscopy. Cumulative EC sprout length and the number of sprouts was quantified using Leica QWin Plus software.

Results:

The 2D culture model revealed an increase in CD31 positive area under all stimulation conditions, with LIGHT inducing almost a 2-fold increase (p<0.05). Confocal analysis of the 3D model showed that spheroids containing HUVEC and RASF formed sprouts under all conditions and that LT and LIGHT caused significant increases (p<0.05) in cumulative sprout length. Interestingly, the total number of sprouts formed by each spheroid also increased significantly. Preliminary results indicate that siRNA-mediated knock-down of NIK in EC leads to a clear reduction in cumulative sprout length, as well as in total sprout numbers.

Conclusion:

Both the 2D and 3D model demonstrate that activation of the non-canonical NF-kappaB pathway via LT and LIGHT enhances angiogenesis. However, in contrast to the 2D model, the 3D model allows visualization of actual blood vessel formation. Importantly, preliminary results demonstrate that the observed increase in angiogenesis is at least in part NIK-dependent. This suggests that NIK may be a novel therapeutic target to block pathological angiogenesis in RA, thereby halting disease progression. Of interest, it is also possible to incorporate immune cells in the 3D model to study their contributions to pathological angiogenesis in RA synovial inflammation, which makes this a valuable tool for future studies.

---

« Back to 2013 ACR/ARHP Annual Meeting

ACR Meeting Abstracts - https://acrabstracts.org/abstract/non-canonical-nf-kappa-b-signaling-enhances-angiogenesis-in-a-novel-3d-spheroid-model-of-rheumatoid-arthritis-synovial-inflammation/