Stretch and Shear Interactions Affect Intercellular Junction Protein Expression and Turnover in Endothelial Cells

Abstract

Complex hemodynamics play a role in the localization and development of atherosclerosis. Endothelial cells (ECs) lining blood vessel walls are directly influenced by various hemodynamic forces: simultaneous wall shear stress (WSS), normal stress, and circumferential stress/strain (CS) due to pulsatile flow, pressure, and diameter changes. ECs sense and transduce these forces into biomolecular responses that may affect intercellular junctions. In this study, a hemodynamic simulator was used to investigate the combined effects of WSS and CS on EC junctions with emphasis on the stress phase angle (SPA), the temporal phase difference between WSS and CS. Regions of the circulation with highly negative SPA, such as the coronary arteries and carotid bifurcation, are more susceptible to the development of atherosclerosis. At 5 h, expression of the tight junction protein zonula occludens-1 was significantly higher for the atheroprotective SPA = 0° compared to the atherogenic SPA = -180° while the apoptosis rate was significantly higher for SPA = -180° than SPA = 0°. This decrease in tight junction protein and increase in apoptosis and associated leaky junctions suggest a decreased junctional stability and a higher paracellular permeability for atherogenic macromolecules for the atherogenic SPA = -180° compared to SPA = 0°.

Figure 1. Simultaneous WSS and CS on ECs is characterized by SPA

A: Arterial blood vessel walls are composed of different layers, with focus here on the intima, the innermost single layer of ECs that is directly exposed to blood flow. The endothelium is very sensitive to the hemodynamic forces to which it is subjected. Wall shear stress (WSS) is the frictional stress that acts tangential to the apical surface of the ECs in response to blood flow. Pressure (P) is the normal stress that acts perpendicular to the ECs and is balanced by circumferential stress (hoop stress) which induces circumferential strain (CS) that is synchronous with diameter variation. B: The stress phase angle (SPA) characterizes the temporal phase difference between the oscillatory WSS and CS waveforms, with negative SPA indicating WSS leading CS.

Figure 2. Relative protein expression for ZO-1, occludin, and VE-cadherin

A: ZO-1 relative protein content for 1, 5, and 12 h via Western blotting. All data were normalized to SC (static control) and presented as mean ± standard error of the mean. + p < 0.05 compared to 1 h; * p < 0.05 between SPA cases (0° and −180°) ; ** p < 0.05 compared to SC; *** p < 0.05 compared to SS (steady shear stress); **** p < 0.05 compared to PC (pressurized control); n: 3–12. B: Occludin relative protein content for 5 and 12 h via Western blotting. All data were normalized to SC and presented as mean ± standard error of the mean. ++ p < 0.05 compared to 5 h; n: 8–14. C: VE-cadherin relative protein content for 1, 5, and 12 h via Western blotting. All data were normalized to SC and presented as mean ± standard error of the mean. + p < 0.05 compared to 1 h; ** p < 0.05 compared to SC; **** p < 0.05 compared to PC; n: 3–15.

Figure 3. Immunocytochemistry images for ZO-1

Labeled arrows indicated breaks in immunostaining between cells (B), holes between cells (H), and small holes at tricellular corners (T). A: Sample images for ZO-1 protein localization at 5 h via immunocytochemistry. For SPA = 0°, SPA = −180°, and SS (steady shear stress), the flow was in the direction from the bottom to the top of the page. Stretch was in the perpendicular direction for the two SPA cases. 40x images; SC is static control, PC is pressurized control. B: Sample images for ZO-1 protein localization at 12 h via immunocytochemistry. For SS, the flow was in the direction from the bottom to the top of the page. The 12-h SPA cases were not shown due to experiment complications. 40x images.

Figure 4. Immunocytochemistry images for VE-cadherin

Labeled arrows indicated breaks in immunostaining between cells (B), holes between cells (H), and small holes at tricellular corners (T). A: Sample images for VE-cadherin protein localization at 5 h via immunocytochemistry. For SPA = 0°, SPA = −180°, and SS (steady shear stress), the flow was in the direction from the bottom to the top of the page. Stretch was in the perpendicular direction for the two SPA cases. 40x images; SC is static control, PC is pressurized control. B: Sample images for VE-cadherin protein localization at 12 h via immunocytochemistry. For SS, the flow was in the direction from the bottom to the top of the page. The 12-h SPA cases were not shown due to experiment complications. 40x images.

Figure 5. 5-h image analysis for ZO-1 and VE-cadherin

A: Average number (over all the images taken for each sample) of breaks in ZO-1 staining between cells, holes between cells, and small holes at tricellular corners grouped for each experimental condition. All data were presented as mean ± standard error of the mean. B represents p < 0.05 compared to average number of breaks in junctions; n = 3; SPA cases are 0° and −180°, SS is steady shear stress, SC is static control, PC is pressurized control. B: Average number (over all the images taken for each sample) of breaks in VE-cadherin staining between cells, holes between cells, and small holes at tricellular corners grouped for each experimental condition. All data were presented as mean ± standard error of the mean. ** p < 0.05 compared to SC; **** p < 0.05 compared to PC; B represents p < 0.05 compared to average number of breaks in junctions; n: 3–4.

Figure 6. 5- and 12-h cell characterization

A: Angle of cell alignment relative to the direction of flow at 5 and 12 h showed no statistically significant differences for SPA = −180° compared to SPA = 0° via ZO-1 immunocytochemistry imaging analysis. Note that an angle of 0 indicates perfect alignment in the flow direction. All data were presented as mean ± standard error of the mean. **** p < 0.05 compared to PC (pressurized control); n: 4–9; SS is steady shear stress, SC is static control. B: Aspect ratio at 5 and 12 h showed no statistically significant differences for SPA = −180° compared to SPA = 0° via ZO-1 immunocytochemistry imaging analysis. All data were presented as mean ± standard error of the mean. p > 0.05 for all cases; n: 4–9. C: Circularity factor at 5 and 12 h showed no statistically significant differences for SPA = −180° compared to SPA = 0° via ZO-1 immunocytochemistry imaging analysis. Note that a circularity of 0 indicated an elongated polygon and 1 indicated a circle. All data were presented as mean ± standard error of the mean. p > 0.05 for all cases; n: 4–9.

Figure 7. SPA effects on apoptosis and mitosis

A: 5-h exposure to experimental conditions significantly increased apoptosis for SPA = −180° compared to SPA = 0° via immunocytochemistry. All data were presented as mean ± standard error of the mean. * p < 0.05 between SPA cases; ** p < 0.05 compared to SC (static control); *** p < 0.05 compared to SS (steady shear stress); **** p < 0.05 compared to PC (pressurized control); n: 5–6. B: 5- and 12-h exposure to experimental conditions did not significantly alter mitosis for each experimental case relative to time. All data were presented as mean ± standard error of the mean. **** p < 0.05 compared to PC; n: 4–12.