Angular difference in human coronary artery governs endothelial cell structure and function

Figures

• 

  Fig. 1. Increasing angular bifurcations of coronary artery creates regions with low WSS and alters biochemical signatures of EC experiencing D-Flow. a–f WSS contours of steady-state flow simulations in the human coronary artery with bifurcation angles of 30° (a), 40° (b), 50° (c), 60° (d), 70° (e) and 80° (f). g WSS or TAWSS levels under steady-state, pulsatile-rigid and pulsatile-FSI conditions in the D-Flow regions (outer walls of vessel branching point) 30, 40, 50, 60, 70 and 80° bifurcations. h–m TAWSS contours of pulsatile-rigid studies in the bifurcation region of branched coronary artery with angles 30° (h), 40° (i), 50° (j), 60° (k), 70° (l) and 80° (m). n, o Alterations in the protein level expressions eNOS (n) and ICAM1 (o), with varying angles of vessel bifurcations, in EA.hy926 exposed to 4 h flow in the 30, 40, 50, 60, 70 and 80° microchannels.

• 

  Fig. 2. Angular differences at the coronary artery branching points cause turbulent flow patterns, perturb velocity dispensation thereby forming WSS gradient. a–f Velocity vectors on steady-state simulations of 30° (a), 40° (b), 50° (c), 60° (d), 70° (e) and 80° (f) bifurcation angles. g Outlet fluid flow velocities (m/s) obtained from right/fixed arm and left/variable arm of human coronary artery with 30, 40, 50, 60, 70 and 80° branching points. h–m Velocity vectors of pulsatile-rigid simulations in coronary artery with bifurcation angle 30° (h), 40° (i), 50° (j), 60° (k), 70° (l), and 80° (m) at t = 0.415 s. n WSS or TAWSS in the D-Flow regions variable and fixed for steady-state and pulsatile-rigid simulations. o Based on the simulation studies performed on a bifurcated human right-coronary artery, various zone were identified and defined across the vessel. Inlet arm: steady laminar flow (S-Flow), right/fixed outer wall (bifurcation): disturbed flow in fixed arm (D-Fixed), left/variable outer wall (bifurcation): disturbed flow in variable arm (D-Variable), steady laminar flow outlet arms: right/fixed arm (S-Fixed) and left/variable arm (S-Variable). Similar regions were identified and…

• 

  Fig. 3. Exposure of EC to D-Flow in microchannels with varying angles of bifurcation leads to reduced eNOS expressions. a–c Immunofluorescence staining of EA.hy926 for eNOS (n = 3) after 4 h fluid flow exposure, using human coronary microchannels with 30° (a), 60° (b) and 80° (c) branching points. DAPI staining is shown in blue. eNOS fluorescence signal in individual EA.hy926 cells (dots) from three individual experiments. Fluorescence intensity AU values per individual cells are indicated together with the mean. Total number of cells, n ≥ 45. Values represent the mean ± SD. *p < 0.05, **p < 0.01 and ***p < 0.001, by one-way ANOVA. Magnification: ×20, Scale: 100 µm. d Tile scan images microchannels harbouring EC stained for eNOS. Magnification: ×10, Scale: 2000 µm.

• 

  Fig. 4. D-Flow exposure causes elevated expression of pro-inflammatory ICAM1 in EC. a–c Immunofluorescence staining of EA.hy926 for ICAM1 (n = 3) after 4 h fluid flow exposure, using human coronary microchannels with 30° (a), 60° (b) and 80° (c) branching points. DAPI staining is shown in blue. ICAM1 fluorescence signal in individual EA.hy926 cells (dots) from three individual experiments. Fluorescence intensity AU values per individual cells are indicated together with the mean. Total number of cells, n ≥ 45. Values represent the mean ± SD. *p < 0.05, **p < 0.01 and ***p < 0.001, by one-way ANOVA. Magnification: ×20, Scale: 100 µm. d Tile scan images microchannels harbouring EC stained for ICAM1. Magnification: ×10, Scale: 2000 µm.

• 

  Fig. 5. Varying angles of vessel bifurcation induce pro-atherogenic-endothelial dysfunction. Monocyte adhesion assay was performed to assess the impact of increasing angular bifurcations on EC function. Pre-stained THP-1 (human monocytes) were incubated with flow exposed (4 h) EA.hy926 for 30 mins. Adherent THP-1 (red dots) were counted in S-Flow and D-Flow regions of 30°, 60° and 80° microchannel.

• 

  Fig. 6. Increasing bifurcation angles of coronary artery incrementally impacts the EC morphology. FE-SEM (top) based structural and surface level analysis of EC exposed to D-Flow using 30°, 60° and 80° microchannels. Magnification: ×5000, Scale: 10 µm. Flow exposed (4 h; 30°, 60° and 80° microchannels) EA.hy926 cells stained for cell-cell contact markers CD31 (second) and CD144 (penultimate). DAPI staining is shown in blue. Circularity of such cells was assessed using ImageJ. Cell circularity AU values per individual cells are indicated together with mean. Total number of cells, n ≥ 30. Values represent the mean ± SD. *p < 0.05, **p < 0.01 and ***p < 0.001, by one-way ANOVA. Magnification: ×50/×63, Scale: 80 µm/20 µm. Cell cytoskeletal F-actin (bottom) staining for EA.hy926 exposed to D-Flow (D-variable or D-fixed regions) in 30°, 60° and 80° microchannels, using phalloidin. DAPI staining is shown in blue. Magnification: ×63, Scale: 20 µm.

• 

  Fig. 7. Differential angular branching points of a human coronary artery cause varying degrees of fluid flow turbulence consequently switching EC towards pro-atherogenic phenotypes. Vessel branch points are known to affect the WSS levels, cause endothelial dysfunction and thus are the prime spots of atherosclerotic plaque formation. Increasing angles of the branching points differentially impact the balance of genes responsible for EC homeostasis. A bifurcation angle of 30° exhibits no significant alterations in the WSS levels, velocity distribution and protein level expressions of eNOS and ICAM1. On the contrary, vessel branching at an angle of 60° displayed the lowest WSS levels at the outer walls of the bifurcation. EC in the D-Flow regions of such a vessel were found to have a round and flat morphology with high levels of ICAM1 and a concurrent reduction in eNOS expression, in comparison to S-Flow. The larger angular disturbance caused by the 80° branching, affected WSS to similar levels; however, a huge alteration was observed in the velocity dispensation across the bifurcation. These differential velocity gradients are responsible for generating special zones with varying WSS…