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High School Poster

HS Poster

Poster 43 - Compound C Induces Cytotoxic Extranuclear DAPI Staining in HAECs Under Static but Not Arterial Flow Conditions

Saturday, October 26, 2024
10:00 AM – 12:00 PM EST
Location: Exhibit Hall E, F & G

Introduction:

Introduction: Disruptions to physiologic flow patterns may induce pathologic responses in endothelial cells. Compound C, primarily used as an inhibitor of AMP dependent protein kinase (AMPK)[1] has been shown to induce apoptosis via upregulating ceramide production and causing Bcl-2-associated X protein (BAX) translocation to mitochondria[2]; however, the effects of Compound C in human endothelial cells have been less well characterized under varying mechanical conditions. Here we demonstrate that Compound C induces extranuclear DAPI staining, indicative of cytotoxicity in human aortic endothelial cells (HAECs) under static but not arterial flow conditions.

Materials and

Methods:

Methods: HAECs (ATCC) between passages 6 through 9 were grown to confluence under static conditions in complete endothelial growth medium (VascuLife VEGF kit, Lifeline) with 10% fetal bovine serum (FBS, Hyclone). Static experiments were conducted in polymer bottom chamber slides (IbiTreat µ-Slide 8 Well, Ibidi) while flow experiments were conducted in polymer bottom channel slides (IbiTreat µ-Slide VI 0.4, Ibidi) connected to a peristaltic pump (Cole Palmer). All cultures were conditioned with experimental media containing endothelial basal medium, 10% FBS, and 0.5% Bovine Serum Albumin[3] (BSA, Fisher) for 24 hours under static or flow conditions prior to experimental treatments. Arterial flow was replicated in flow channel slides at 10 dynes/ cm2 for 24 hours prior to treatments. Compound C (Dorsomorphin dihydrochloride, Abcam, ab144821) was used at 15 mM as demonstrated previously[1]. All cells were incubated at 37°C and 5% CO­2 for all experiments. Cells were fixed in 2% paraformaldehyde (PFA) with 0.1% glutaraldehyde for 30 minutes at room temperature as described previously[3]. Nuclei were stained using 4′,6-diamidino-2-phenylindole (DAPI) (1:10000, Invitrogen) at room temperature for 5 minutes. Samples were then immersed in 1X DPBS and imaged at 20X magnification using a Zeiss LSM 880 inverted confocal microscope system. Images were analyzed using FIJI (SciJava) software. Statistical analyses were performed with R software. Data are presented as mean ± standard deviation. Data were analyzed using the two-sample independent t-test. Statistical significance was determined for p < 0.05.

Results, Conclusions, and Discussions:

Results: HAECs treated with Compound C under arterial flow conditions expressed no extranuclear DAPI staining, which was significantly lower than HAECs treated with Compound C under static conditions which demonstrated 64±10.3% of cells with extranuclear DAPI staining (n = 4, p < 0.01, two-sample independent t-test, Figure 1). Control untreated HAECs did not demonstrate any extranuclear DAPI staining under static or flow conditions (Figure 1).

Discussion: Endothelial cells are physiologically exposed to hemodynamic shear stress, regulating their morphology and function[4-6]. Disruption to physiologic flow is often pathologic in endothelial cells as measured by their disrupted mechanotransduction pathways[7]. Compound C is a cell permeable compound commonly used a reversible inhibitor of AMPK. It has demonstrated cytotoxicity in MCF7 cell lines through inhibition of cell growth, upregulation of ceramide levels, and translocation of the BAX protein to mitochondria independent of AMPK. Similarly, Kim et. al demonstrated that the protective effects of laminar flow against endothelial apoptosis are independent of the AMPK pathway. Therefore, laminar flow protection against endothelial apoptosis induced by Compound C under static conditions is likely mediated through inhibition of BAX protein translocation or ceramide level accumulation in HAECs.

Conclusion: Arterial shear stress protects HAECs against cytotoxic extranuclear DAPI staining induced by Compound C under static conditions.

Acknowledgements (Optional):

References (Optional):

References:

[1] A. Young, W. Wu, W. Sun, H. Benjamin Larman, N. Wang, Y.S. Li, J.Y. Shyy, S. Chien, G. Garcia-Cardena, Flow activation of AMP-activated protein kinase in vascular endothelium leads to Kruppel-like factor 2 expression, Arterioscler Thromb Vasc Biol 29 (2009) 1902-1908. 10.1161/ATVBAHA.109.193540.

[2] J. Jin, T.D. Mullen, Q. Hou, J. Bielawski, A. Bielawska, X. Zhang, L.M. Obeid, Y.A. Hannun, Y.T. Hsu, AMPK inhibitor Compound C stimulates ceramide production and promotes Bax redistribution and apoptosis in MCF7 breast carcinoma cells, J Lipid Res 50 (2009) 2389-2397. 10.1194/jlr.M900119-JLR200.

[3] Y. Zeng, E.E. Ebong, B.M. Fu, J.M. Tarbell, The structural stability of the endothelial glycocalyx after enzymatic removal of glycosaminoglycans, PLoS One 7 (2012) e43168. 10.1371/journal.pone.0043168.

[4] A.M.W. Bartosch, R. Mathews, M.M. Mahmoud, L.M. Cancel, Z.S. Haq, J.M. Tarbell, Heparan sulfate proteoglycan glypican-1 and PECAM-1 cooperate in shear-induced endothelial nitric oxide production, Sci Rep 11 (2021) 11386. 10.1038/s41598-021-90941-w.

[5] A.M.W. Bartosch, R. Mathews, J.M. Tarbell, Endothelial Glycocalyx-Mediated Nitric Oxide Production in Response to Selective AFM Pulling, Biophys J 113 (2017) 101-108. 10.1016/j.bpj.2017.05.033.

[6] E.E. Ebong, S.V. Lopez-Quintero, V. Rizzo, D.C. Spray, J.M. Tarbell, Shear-induced endothelial NOS activation and remodeling via heparan sulfate, glypican-1, and syndecan-1, Integr Biol (Camb) 6 (2014) 338-347. 10.1039/c3ib40199e.

[7] M.J. Davis, S. Earley, Y.S. Li, S. Chien, Vascular mechanotransduction, Physiol Rev 103 (2023) 1247-1421. 10.1152/physrev.00053.2021.