(G-277) Macrophages Mediate Endothelial-to-Mesenchymal Transition Following Exposure to Elevated Shear Stress

Introduction::

Discrete Subaortic Stenosis (DSS) is a pediatric cardiovascular disease occurring in the left ventricular outflow tract (LVOT). DSS is characterized by a fibro-membranous tissue that obstructs proper blood flow, resulting in elevated blood velocity across the aorta, thus increasing shear stress on the surrounding cardiac tissue. DSS can lead to a variety of issues, such as arrythmia, hypertrophy, and death. Moreover, DSS has a high rate of recurrence, and its causes are largely unknown. It has previously been shown that shear stress promotes endothelial-to-mesenchymal transition (endMT); however, constant hemodynamic shear stress can induce endothelial cell denudation, exposing underlying macrophages to shear. Macrophages are known to respond to various mechanical stimuli by altering their morphology, function, and polarization. Consequently, we hypothesize that macrophages could be promoting endMT under elevated shear stress, further exacerbating fibrosis.

Materials and Methods::

Human peripheral blood mononuclear cells were harvested from donor buffy coats through density gradient centrifugation. Following a 24-hour incubation, floating lymphocytes were aspirated and the adherent monocytes were incubated for 5 days with macrophage colony stimulating factor, resulting in differentiation into M0 macrophages. Shear stress was applied to macrophages in 60mm dishes using the cone-and-plate system. The cone was spun by a magnetic stir plate, providing uniform shear stress to all cells. 15 dynes/cm^2 and 35 dynes/cm^2 were chosen as the physiological and pathological shear stress levels, respectively. No shear stress (static) was used as a control. Following shear exposure for either 3 hours or 24 hours, conditioned media was collected from the samples. Conditioned media was analyzed by the Luminex Core (Baylor College of Medicine) for released cytokines. Additionally, human aortic endothelial cells (HAECs) were cultured with 70% conditioned media, 25% EGM-2 media, and 5% FBS for 6 hours. Controls included cells exposed to 70% fresh RPMI media, in place of the conditioned RPMI media. Following exposure, HAECs were analyzed by RT-qPCR.

Results, Conclusions, and Discussions::

The cytokine Luminex panel (Figure 1) shows that macrophages became generally more pro-inflammatory and began releasing more pro-inflammatory cytokines with the application of shear. However, the HAEC RT-qPCR data shows that pro-inflammatory markers generally decreased over time with shear (Figures 2, 3, 4), while SNAI1 expression increased at early time points (Figure 5). SNAI1 plays a critical role in the activation of the endMT pathway in endothelial cells by functioning as a transcription factor. These results suggest that shear-induced pro-inflammatory macrophages promote endMT. The high-shear conditions of the LVOT in DSS may induce a shift towards a pro-inflammatory phenotype in macrophages, resulting in the upregulation of the endMT pathway in cardiac endothelial cells, resulting in increased fibrous tissue.

 

Future work will consist of more conditioned media transfer studies onto HAECs followed by RT-qPCR to quantify expression levels of other endMT markers such as SLUG. Longer time points of exposure, such as 24 hours, will also be investigated. Additionally, conditioned media from shear-exposed HAECs will be applied to macrophages to assess cell-cell crosstalk. Furthermore, future studies will need to investigate if regulating the endMT pathway in patients with DSS could lower the severity of the disease, halt disease progression, and reduce the rate of recurrence.

Acknowledgements (Optional): : Reasearch was funded through NIH [R01HL140305]. Thank you to the Grande-Allen lab, especially my bench mentor Elysa Jui, for the continued support.

References (Optional): : Massé DD, Front. Cardiovasc. Med., 2018, 5, 1-9; Wu KQ, Am. J. Pathol., 2017, 187(9), 2102-2111.