DOI: 10.5061/dryad.c59zw3rn1
title: Fluid shear stress-dependent modulation of the basal endothelial glycocalyx

description: 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.
description: # Fluid shear stress-dependent modulation of the basal endothelial
 glycocalyx Dataset DOI:
 [10.5061/dryad.c59zw3rn1](https://doi.org/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/cm^2^), and
 then secondly by the exposure time (0.5 or 12 hours), and permeabilization
 status for the static and 10 dynes/cm^2^ 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/cm^2^). Within the 0.5 dynes/cm^2^ and
 30 dynes/cm^2^ 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/cm^2^ and 30 dynes/cm^2^ samples
 were permeabilized, therefore there are 2 only sub-folders within these
 folders. Within the 10 dynes/cm^2^ 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/cm^2^ 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/cm^2^ 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/cm^2^ 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.