Fig 1.
Comparison of the apical glycocalyx (GCX) signal of permeabilized and non-permeabilized human lung microvascular endothelial cells (HLMVECs) exposed to 10 dynes/cm² of fluid shear stress revealed no statistically significant differences between HLMVECs of the same fluid shear stress (FSS) exposure time.
Sum projections (enface view of A-F) and accompanying orthogonal views (directly below enface view, A-F) of the apical GCX (green) and nuclei (blue) in non-permeabilized (A-C) and permeabilized (D-F) HLMVECs after static culture (A,D), 30 minutes (B,E), and 12 hours (C,F) of exposure to 10 dynes/cm2 FSS. No significant differences were found between GCX thickness (µm) (G), coverage (%) (H), and integrated intensity (fold-change (F.C.) (I) of non-permeabilized and permeabilized HLMVECs.
Fig 2.
Apical and basal glycocalyx (GCX) expression in permeabilized human lung microvascular endothelial cells (HLMVECs) after exposure to 10 dynes/cm2 of fluid shear stress (FSS).
Sum projection of the apical (A-C) and basal (D-F) GCX (green) and nuclei (blue) (A-C) after static culture (A,D), 30 minutes (B,E), and 12 hours (C,F) of exposure to physiologically normal (10 dynes/cm2) FSS. Apical (green) and basal (purple) GCX thickness (µm) (G), coverage (%) (H), and integrated intensity (fold-change (F.C.) (I) are reported as the mean ± standard error of the mean.
Fig 3.
Apical and basal glycocalyx (GCX) expression in permeabilized human lung microvascular endothelial cells (HLMVECs) after exposure to 0.5 dynes/cm2 of fluid shear stress (FSS).
Sum projection of the apical (A-C) and basal (D-F) GCX (green) and nuclei (blue) (A-C) after static culture (A,D), 30 minutes (B,E), and 12 hours (C,F) of exposure to pathophysiologically low (0.5 dynes/cm2) FSS. Apical (green) and basal (purple) GCX thickness (µm) (G), coverage (%) (H), and integrated intensity (fold-change (F.C.) (I) are reported as the mean ± standard error of the mean.
Fig 4.
Apical and basal glycocalyx (GCX) expression in permeabilized human lung microvascular endothelial cells (HLMVECs) after exposure to 30 dynes/cm2 of fluid shear stress (FSS).
Sum projection of the apical (A-C) and basal (D-F) GCX (green) and nuclei (blue) (A-C) after static culture (A,D), 30 minutes (B,E), and 12 hours (C,F) of exposure to physiologically high (30 dynes/cm2) FSS. Apical (green) and basal (purple) GCX thickness (µm) (G), coverage (%) (H), and integrated intensity (fold-change (F.C.) (I) are reported as the mean ± standard error of the mean.
Fig 5.
Comparison of apical and basal glycocalyx (GCX) expression in permeabilized human lung microvascular endothelial cells (HLMVECs) across fluid shear stress (FSS) magnitudes.
Sum projections (top image) and accompanying orthogonal view (bottom image) of the apical (A-C) and basal (D-F) GCX (green) and nuclei (blue) after 12 hours of exposure to 10 dynes/cm2 (physiologically normal)(B,E), 0.5 dynes/cm2 (pathophysiologically low)(A,D),and 30 dynes/cm2 (elevated)(C,F)FSS. Apical GCX thickness (µm), coverage (%), and integrated intensity (fold-change (F.C.) (G-I) are compared between FSS rates after 30 minutes and 12 hours of exposure. FSS comparisons are repeated for basal GCX thickness (µm), coverage (%), and integrated intensity (fold-change (F.C.) (J-L).
Fig 6.
Z-stack processing to separate full thickness z-stack into apical and basal glycocalyx (GCX) sum projections and orthogonal views for quantification.
The upper progression of images represents an animated version of the step of the image analysis pathway below shown as a real image from the pipeline. All nuclei completely contained within the field of view (FOV) of the full thickness confocal Z-stack (A) are identified through thresholding a basic maximum projection generated of only the nuclei channel of the Z-stack. The identified nuclei bodies are overlaid with the confocal Z-stack as shown in the lower confocal Z-stack (A). A minimum bounding box is drawn around each nucleus and used to crop the original Z-stack into sub-stacks of the GCX, and nuclei signal as shown in panel B. From each sub-stack, an orthogonal view is generated (C) in both the GCX and nucleus channel shown merged here (C). Within the nuclei orthogonal view, the Z-centroid of the nucleus is identified (C). The GCX orthogonal view of the sub-stack is then split into two orthogonal views, and apical and basal GCX orthogonal view, as shown in panel E. This process is repeated on each nucleus identified initially (A) generating a list of nucleus Z-centroid locations and apical and basal GCX orthogonal views. Measurements of apical and basal GCX thickness are made only in the apical and basal orthogonal views extracted from this process. The average Z location of all nuclei centroids is used to split the Z-stack at the Z-stack slice that corresponds to the average center of the nuclei heights into an apical and a basal sub-stack. These sub-stacks are used to generate sum-projections of the GCX signal (D) for analysis of GCX expression metrics of coverage and integrated intensity (E).Created in https://BioRender.com.