Fluid shear stress-dependent modulation of the basal endothelial glycocalyx
Data files
Jan 09, 2026 version files 2.98 GB
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Code.zip
35.87 KB
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Images.zip
2.98 GB
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Raw_Data.zip
14.23 KB
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README.md
7.64 KB
Abstract
The vascular endothelial glycocalyx is a major regulator of endothelium function, serving as a vital mechanotransducer and barrier. Shear stress due to blood flow exerted on the apical endothelial cell (EC) and glycocalyx has been the primary focus of the field due to their direct interaction. Recently, it has been demonstrated that the basal glycocalyx, exposed to the basement membrane, is sensitive to apical fluid shear stress and necessary for promoting mechanotransduction pathways key to the EC response to blood flow. Here, we demonstrate the fluid shear stress-dependent modulation of the basal endothelial glycocalyx, showing a notably different expression pattern and overall increased expression compared to the apical glycocalyx. The dataset found here includes the raw confocal z-stacks collected of fluorescently tagged human microvascular endothelial cells after fluid shear stress exposure and the associated raw data from quantitative analysis of the immunofluorescent signal. Our findings, coupled with prior evidence linking the basal GCX to cytoskeletal dynamics, underscore its potential role in vascular barrier integrity and mechanotransduction.
Dataset DOI: 10.5061/dryad.c59zw3rn1
Description of the data and file structure
The following folders contain raw images, code, and data files relating to our study: "Fluid shear stress-dependent modulation of the basal endothelial glycocalyx," published in PLOS ONE.
The raw z-stacks collected and analyzed are within the "Images" folder. The images are separated first by the shear rate the HLMVECs experienced (static (0), 0.5, 10, or 30 dynes/cm2), and then secondly by the exposure time (0.5 or 12 hours), and permeabilization status for the static and 10 dynes/cm2 groups.
Within the "Code" folder is the ImageJ macro used to separate the z-stacks collected into apical and basal sum projections ("BasalWGA_AB_StackProjGen") and the Cell Profiler pipelines used to segment and quantify glycocalyx signal in terms of integrated intensity, coverage, and thickness. The raw numerical output of the CellProfiler piplines relating to the images and data presented here is present within the "Raw_Data" folder.
Files and variables
File: Images.zip
Description: Raw z-stacks of HLMVECs tagged with WGA post flow exposure.
Naming convention: Raw z-stacks were collected on a Leica STELLARIS 8 laser scanning confocal microscope using a plan-apochromat 63 x 1.40 NA oil objective. Z-stack were collected with a slice thickness of 0.1 µm and field of view of 184.7 x 184.7 µm. Each z-stack was captured as a two chanel 8-bit image series with resolution of 1024 x 1024 pixels. Chanel one was used to collect the nuclei signal and chanel two the glycocalyx signal. Within the images folder there are 4 sub-folders, one for each shear stress exposure group (static (0), 0.5, 10, or 30 dynes/cm2). Within the 0.5 dynes/cm2 and 30 dynes/cm2 folders there are two sub-folders labeled "0.5_hours" and "12_hours" which correspond to the shear stress exposure time. All 0.5 dynes/cm2 and 30 dynes/cm2 samples were permeabilized, therefore there are 2 only sub-folders within these folders. Within the 10 dynes/cm2 and static folders there are both "_perm" and "_nonperm" folders for each timepoint, corresponding to the permeabilization status of the HLMVECs. The non-permeabilized HLMVECs (_nonperm) exposed to static and 10 dynes/cm2 shear conditions samples were compared to the permeabilized HLMVECs samples (_perm) of the same shear stress exposure conditions in Figure 1. The non-permeabilized HLMVEC data was also presented within Supplemental Figure 1. All samples within the final folders are the raw z-stacks collected. Each z-stack is labeled WGA, as they were immunofluorescent tagged with wheat germ agglutinin (WGA) followed by a short hand label describing the shear rate and exposure time and sample number. 30F and 12F correspond to the exposure times of 0.5 hours and 12 hours, and 10D, 0.5D, and 30D correspond to the shear rates of 0.5, 10, or 30 dynes/cm2 respectively, and finally a number denoting the sample number within that exposure group. For example a sample labeled "WGA 12F05D - 2" would be the second sample of the 12 hours of exposure to 0.5 dynes/cm2 of shear stress group.
File: Code.zip
Description: The Code.zip folder contains the ImageJ and CellProfiler executables. Version 4.2.6. of CellProfiler and ImageJ version 1.54i was used to complete this anaysis.
The ImageJ macro, named "BasalWGA_AB_StackProjGen.IMJ", is used to separate the raw z-stacks from the "Images" folder into apical and basal sum projections and generate the orthogonal views used to quantify glycocalyx thickness. When run, the macro will prompt you to select a directory pointing to the folder housing the raw z-stacks you would like to process and an output folder where all output images will be stored. The macro will output the apical and basal sum projections (.tif) of the nuceli and glycocalyx chanels labeled. Therefore, for each input z-stack there will be 4 output sum-projections labeled as follows. First with the last character of the z-stack name, in this case that is the sample number, "#", followed by wether the projection is of the glycocalyx chanel, "GCX", or the nuclei chanel, "NUC_", and finally wether the projection is an apical projection "A_Proj" or basal projection "B_Proj". For example processing the image labeled "WGA 12F05D - 2" would result in the output of 4 sum projections labeled: 2_GCX_A_Proj, 2_GCX_B_Proj, 2_Nuc_A_Proj, 2_Nuc_B_Proj.
For this project all z-stacks were collected in the positive Z direction and the computational method requires this consistency to properly label the images as apical and basal. If the z-stacks are collected in the -Z direction than the apical and basal labels should be inversed and all images processed in one batch must be consistently collected in the same Z direction.
Orthogonal views, used to measure apical and basal glycocalyx thickness are generated at each nuclei as described in the Methods section of the manuscript. At each nuclei completely contained within the image field of view an apical and basal orthogonal view is captured. The orthogonal views are named first by the sample number (last character of the z-stack name) and then the nuclei number followed by "GCX_A_Orth" or "GCX_B_Orth" for apical and basal orthogonal views resepctively. Therefore an output image labeled 3_9_GCX_A_Orth" would correspond to the apical orthongoal view of sample 3, nuceli 9.
The "GCX_coverage_and_II" pipeline will accept all intermediate images output by "BasalWGA_AB_StackProjGen" macro and identify pairs of apical and basal sum projections generated from one z-stack and output the coverage and integrated intensity measurements for the apical and basal sum projections. The "GCX_Thickness" pipeline will also accept all intermediate images output by "BasalWGA_AB_StackProjGen" macro and sort the orthogonal views into apical and basal images and report a measurement of GCX thickness for each image.
File: Raw_Data.zip
Description: The Raw_Data folder contains all raw numerical outputs from Cell Profiler pipelines analyzed in the manuscript. From the "GCX_coverage_and_II" pipeline each each apical and basal image pair, identified by the "GCX_A_Proj" and "GCX_B_Proj" naming convenstion, one output line will be added to the raw data output where the integrated intensity and coverage of the apical and basal images will be output. These measures are represented by the headers: "AreaOccupied_AreaOccupied_Comb_Apical_GCX", "AreaOccupied_AreaOccupied_Comb_Basal_GCX","Mean_Comb_Apical_GCX_Intensity_IntegratedIntensity_GCX_A", and "Mean_Comb_Apical_GCX_Intensity_IntegratedIntensity_GCX_B" within the "XX_cov_II" raw data files. Coverage is expressed as the number of positive pixels, the total number of pixels in the sum projections is 1048576 which can be used to convert the raw coverage metrics to the percentages reported in the manuscript.
The "GCX_Thickness" pipeline will similarly isolate all orthogonal views corresponding to the apical and basal GCX from the naming convenstion of the output images of the ImageJ macro and output the mean glycocalyx thicknesss in microns (um) across each image. The raw data output file contains 3 colulmns including "ImageNumber" relating to the nuceli number, "Mean_Apical_Math_ApicalLength" for the mean apical glycocalyx thickness (um) over the nuceli, "Mean_Basal_Math_BasalLength" relating to the mean basal glycocalyx thickness (um) under the nuceli.