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Cellular and Molecular Bioengineering

Cellular and Molecular Bioengineering - Poster Session B

Poster I5 - Epigenetic Responses to Fluid Shear Stress: Mechanistic Insights into Atherosclerosis

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

Introduction:  Atherosclerosis, a leading cause of cardiovascular disease, is characterized by the accumulation of fats, cholesterol, and other substances within and on the artery walls, forming plaque [1] (Figure 1). This plaque buildup narrows arteries, restricting blood flow and increasing fluid shear stress (FSS), significantly altering cellular mechanosensation. This aberrant mechanical stimuli affects mechanotransduction, the process where cells convert mechanical signals into biochemical activity, including increases in intracellular calcium concentration [2]. More specifically, such disruptions impact the translocation of mechanosensitive proteins into the nucleus and ultimately influence gene transcriptional regulation. In this study, we examined how cellular mechanosensation during applied FSS influences Yes-associated protein 1 (YAP) nuclear translocation, inducing epigenetic changes via signaling cascades that alter gene transcription and expression [3]. While previous flow experiments have been mostly focused on mechanosensation in cells, few have focused specifically on how mechanical perturbation regulates chromatin organization in cells [5]. To this end, in this study we further examined transcriptional activation and repression through histone modifications H3K9ac and H3K27me3, respectively, through immunofluorescence imaging. Additionally, we examined histone modifications with cell contractility modulation through drug treatment, which plays a role in mechanosensation. Furthermore, super-resolution imaging was used to quantify chromatin condensation status to decipher between transcriptionally active and inactive genomic regions [4]. Understanding the mechanisms regulating epigenetics under FSS is crucial for elucidating the pathophysiology of atherosclerosis and other cardiovascular diseases and developing novel treatments.

Materials and

Methods:  In this study, we utilized a microfluidic device (µ-Slide I Luer Glass Bottom 0.6, Ibidi) coupled with a mechanical syringe pump to control the force, duration, and flow direction of the culture medium HeLa cells were cultured in Eagle's Minimum Essential Medium supplemented with 10% Fetal Bovine Serum and 1% Penicillin Streptomycin. The device was coated with fibronectin before cell seeding and incubated for 24 hours. Following cell attachment, warmed media was pumped at a force of either 1 dyne/cm² (low) or 10 dyne/cm² (high) for 15 minutes. Immunofluorescence (IF) imaging was used to quantify the nuclear translocation of YAP and the expression levels of histone modifications H3K27me3 (a marker of gene suppression) and H3K9ac (a marker of gene activation). Super-resolution imaging (i.e., STORM) was employed to examine organizational structure changes of histone H2B in the nucleus.
To alter cellular contractility, we pre-treated cells with lysophosphatidic acid (LPA) at 50 μM for one hour before the application of FSS which is known to increase cell contractility and YAP nuclear localization [6]. This approach aimed to understand the effects of contractility on intracellular mechanotransduction and epigenetic regulation under FSS conditions. All statistical analyses were performed using one-way ANOVA with Bonferroni's post-hoc testing.

Results, Conclusions, and Discussions: YAP translocation decreased at low FSS and increased at high FSS as expected (Figure 2). H3K27me3 expression significantly decreased with high FSS compared to both low and control groups (Figure 3a). H3K9ac expression decreased under both low and high FSS compared to control (Figure 3b). Applying FSS for a longer duration could determine if flow time affects epigenetic regulation. STORM revealed no difference in histone H2B density (Figure 4). While the high FSS conditions altered the expression of histone modification markers, it did not affect histone H2B organization, possibly due to insufficient flow duration to allow chromatin rearrangements.
Next, we further examined how changes in cellular contractility impact FSS-modulated histone modification expressions. For this, we conducted flow on LPA-treated cells at high FSS. IF of H3K27me3 showed a decrease in baseline intensity for all LPA-treated groups compared to untreated groups, including at both FSS conditions (Figure 5a). However, high FSS caused an increase in H3K27me3 with LPA treatment only (Figure 5a). IF of H3K9ac showed a decrease in mean intensity for all groups compared to the untreated control (Figure 5b). In the future, STORM could be used to explore changes in cell contractility and organization of histone H2B.
High FSS enhances YAP nuclear translocation and influences histone modifications, linking mechanical stimuli to epigenetic regulation. The observed changes suggest differential responses of H3K27me3 and H3K9ac modifications. YAP nuclear localization highlights FSS’s activation of G-coupled protein receptors, which are also chemically activated by LPA [6]. This chemical cue has a similar effect to applied FSS in the regulation of epigenetic state and mechanotransduction. Expression of histone modification markers was altered with FSS, suggesting that increases in shear stress can lead to altered transcriptional regulation as seen in cardiovascular disease.
Future studies could employ chromatin immunoprecipitation with sequencing (ChIP-Seq) to quantify gene activation or repression at specific loci, revealing local changes in histone activity by FSS. Additionally, exploring more histone modification markers could elucidate the role of mechanosensation in other epigenetic contexts. These findings increase our understanding of the pathophysiology of cardiovascular diseases like atherosclerosis, where altered hemodynamic forces play a role.

Acknowledgements (Optional):  NSF CMMI 1548571