Table 1.
Hemodynamic parameters in 3-D straight tube models.
Fig 1.
Glycocalyx establishment and degradation on HAAECs.
(A) Representative confocal microscope images of HAAECs cultured statically and immunolabelled for HS. Images were acquired at 40x magnification (scale bar = 50μm). Top: Control; Bottom: Degraded. (B) Quantification of glycocalyx degradation from confocal images (n = 3, *p<0.05, error bars represent SEM).
Fig 2.
HS expression with degradation and 24 hours of LSS exposure in 3D cell culture models.
(A) Representative confocal microscope images of HAAECs immunolabelled for HS and exposed to 10 dyne/cm2 LSS for 24 hours (t = 24h) or fixed directly after degradation (t = 0). Images were acquired at 10x magnification (scale bar = 50μm). (B) Quantification of HS expression after 24 hours of flow and degradation (n = 3, *p<0.05, error bars represent SEM).
Fig 3.
Morphological response of HAAECs with degradation and 24 hours of LSS in 3D cell culture models.
(A) Representative light microscope images of HAAECs stained with crystal violet. Images were acquired at 40x magnification (scale bars = 50μm). (B) Quantification of cell morphology based on the shape index and normalized to static control (n = 3, *p<0.05, error bars represent SEM).
Fig 4.
NB4 cell adhesion in 3-D cell culture models with degradation and flow.
(A) Representative light microscope images of NB4 cells adhered to HAEECs in models. Images were acquired at 10x magnification (scale bars = 25μm). (B) Quantification of adherent NB4 cells (n = 3, *p<0.05, error bars represent SEM).
Fig 5.
NB4 adhesion in static culture with degradation.
HAAECs were cultured statically and NB4 cells were added statically following degradation (n = 3, p = 0.23, error bars represent SEM).
Fig 6.
ICAM-1 protein expression with flow and degradation.
(A) Representative western blot images showing ICAM-1 with GAPDH as the loading control. (B) Quantification of western blots using densitometry (n = 4, *p<0.05, error bars represent SEM). (C) Representative confocal microscope images of HAAECs immunolabelled for ICAM-1. Images were acquired at 10x magnification (scale bar = 50μm). (D) Quantification of ICAM-1 on the surface of HAAECs (n = 3, *p<0.05, error bars represent SEM). Perfusions were performed for 24 hours at an inlet SS of 10 dynes/cm2. (E) Representative light microscope images of NB4 cells adhered to HAEECs in models under control and ICAM-1 antibody blocking conditions under flow. Images were acquired at 10x magnification (scale bars = 25μm). (F) Quantification of adherent NB4 cells (n = 3, **p<0.01, error bars represent SEM).
Fig 7.
NF-κB response with flow and degradation.
(A) Representative images from confocal microscopy of NF-κB (green) and nuclear counterstain (red) following exposure to different flow times. Images were acquired at 10x magnification (scale bar = 50μm). (B) Representative histogram showing a cell with nuclear NF-κB localization (overlap of green and red signals) versus a cell without NF-κB in the nucleus (no green overlap). These graphs were used to quantify the number of cells with NF-κB in the nucleus. (C) Quantification of the number of cells with nuclear NF-κB over time in either control or degraded conditions (n = 3, *p<0.05, error bars represent SEM).
Fig 8.
IκB and eNOS protein expression with flow and degradation.
(A and C) Representative western blot images showing IκB and eNOS with GAPDH as the loading control. (B and D) Quantification of western blots using densitometry (n = 4, *p<0.05, error bars represent SEM). Perfusions were performed for 24 hours at 10 dynes/cm2. (E) Representative western blot images showing ICAM-1 with GAPDH as the loading control in the presence of the NO inhibitor, L-NAME. (F) Quantification of western blots using densitometry (n = 3, *p<0.05, error bars represent SEM). (G) Quantification of adherent NB4 cells with and without L-NAME and under flow conditions (n = 3, *p<0.05, error bars represent SEM).
Fig 9.
Model for the molecular adhesion pathway altered with degradation.
Flow diagram illustrating how glycocalyx degradation interrupts the negative feedback loop regulating NF-κB activity. When the glycocalyx is degraded, NO levels are inhibited resulting in increased NF-κB activity. This results in the over-stimulation/activation of ECs evidenced by an increase in ICAM-1 and leukocyte adhesion.