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Cardiovascular Engineering

Cardiovascular Engineering - Poster Session B

Poster H14 - Elastic Fiber Fragmentation Promotes Macrophage Migration in Thoracic Aortic Aneurysms

Thursday, October 24, 2024
2:45 PM - 3:45 PM EST
Location: Exhibit Hall E, F & G

Introduction:  Thoracic aortic aneurysm (TAA) is a critical cardiovascular condition characterized by dilation of the aorta, that may eventually dissect or rupture [4]. Despite its prevalence, there are limited drug therapy treatments that prevent or restore the mechanical changes associated with TAA [1]. Common histopathological changes in TAAs include fragmentation of elastic fibers, loss of smooth muscle cells, and increased infiltration of inflammatory cells across the aortic wall [2]. Elastic fiber fragmentation and inflammatory cell infiltration may be linked as the compromised elastic fiber network may allow easier penetration of cells into the wall and also release elastic fiber fragments that serve as chemoattractants to inflammatory cells. The positive feedback between elastic fiber fragmentation and increased inflammatory cells that can secrete proteases that further degrade elastic fibers may contribute to TAA expansion and increase the risk of dissection or rupture [3]. This study aims to determine whether elastic fiber fragmentation indeed facilitates migration of inflammatory cells, specifically macrophages, using the transwell migration assay. The results from these studies may inform the development of therapies to slow or prevent inflammatory cell migration and TAA progression.

Materials and

Methods:  We cultured RAW 264.7 macrophages (ATCC) in pure DMEM supplemented with 10% fetal bovine serum (FBS), L-glutamine, pen-strep, and sodium pyruvate until the cells reached 70-80% confluency. Subsequently, we serum-starved the cells for 12 hours before initiating the transwell migration assay. As this was a new assay for the laboratory, initial work involved troubleshooting and optimizing the migration assay for chemoattractant, pore size, cell concentration, incubation time, and fixation method to ensure reliable results. The final protocol involved seeding 200,000 cells onto the upper chambers of 8 µm pore-size transwell inserts (Corning) in pure DMEM, adding various experimental medias to the lower chamber as chemoattractants, incubating the cells for 24 hours, fixing the cells with 4% paraformaldehyde, staining the cells with DAPI to show the nuclei, and then imaging the cells that had migrated from the upper to the lower chamber. Quantitative analysis of migrated cells was performed using ImageJ software and R Studio. The various experimental medias included pure DMEM (negative control), complete DMEM + FBS (positive control), and media with degraded and non-degraded mouse aorta (to determine if degradation released chemoattractants). For the degraded and non-degraded aortas, we dissected ascending aortas from wild type mice and incubated them in 500 µL of DMEM with elastase solution or pure DMEM for at least an hour.

Results, Conclusions, and Discussions: Results
After optimizing the experimental conditions for the transwell assay, we determined that pure DMEM could serve as a negative control with little cell migration (Fig. 1a) and that complete DMEM + FBS could serve as a positive control with abundant cell migration (Fig. 1b). There was a significant 6.2-fold increase in cells migrating to the lower chamber for complete DMEM + FBS compared to pure DMEM (Fig. 1c). We then tested our experimental medias containing either degraded or non-degraded aortas. We determined that media with non-degraded aorta was not a chemoattractant with little cell migration (Fig. 2a), similar to pure DMEM, while media with degraded aorta acted as a chemoattractant with abundant cell migration (Fig. 2b), similar to complete DMEM with FBS. There was a significant 39.7 fold increase in cells migrating to the lower chamber for degraded aorta compared to non-degraded aorta (Fig. 2c).

Discussion
Our results show that we were able to develop a transwell migration assay for a macrophage cell line and identify positive and negative controls. We then determined that media with elastase-degraded aorta was a chemoattractant for macrophages, while media with non-degraded aorta was not. These results indicate that the process of degrading elastic fibers with elastase releases chemokines that are recognized by macrophages and promote migration. The results support the assertion that degraded elastic fibers in TAA may facilitate inflammatory cell infiltration into the aortic wall that contributes to TAA progression [3]. Future work will include performing additional experiments to confirm robustness and repeatability, testing aorta from mouse models of TAA instead of elastase-degraded, and identifying factors that may block the migration response (i.e. antibodies to fragmented elastin) that may be used as therapies for TAA.

Acknowledgements (Optional):