(25b) Expression of Glycocalyx on Human Lung Microvascular Endothelial Cells Is Shear Stress and Time Dependent

PROBLEM

Endothelial Glycocalyx (GCX) are crucial to endothelial integrity. Endothelial cell pathological responses more often than not are reliant on shear and stretch forces that GXC are exposed to in blood vessels, making the study of GCX important for understanding the endothelial cell mechanical microenvironment. There are few studies replicating the microenvironment of the endothelial cell GCX. We hope to add to this body of knowledge by studying the expression of GCX in a replicated in vivo environment.

BACKGROUND

GCX are an extremely important determinant in vascular permeability and endothelial integrity. Vascular endothelial cells comprise the interior surface lining of blood vessels, forming the interface between the vessel walls and the circulating blood. Because of this location, they are subjected to shear stress, tensile, and compressive strain due to blood flow. These forces can regulate morphology, physiology, gene expression, and inflammatory response [1, 2]. This reliance on these forces can result in pathological responses to an altered mechanical state and lead to various diseases [2, 3].

The GCX is vasculoprotective and regulates vascular permeability and molecular interaction with endothelial receptors [4]. This layer is responsible for flow-mediated responses in the endothelial cells. GCX also plays a role in mediating blood-cell and plasma interactions with the endothelium. Deterioration of the GCX layer has been shown to lead to adverse effects on vasculature. When the GCX is damaged or compromised, these flow-mediated responses are impaired and the endothelial cells begin to exhibit pathological responses [5]. Studies have determined that GCX expression is vital for the endothelial layer to properly function and respond to stimuli and to serve as a protective barrier to pathogenic molecules [4].

In this study, we investigated the effects of shear stress on the expression of HLMVEC GCX, we also looked at the effect of shear stress on GCX subcomponent heparan sulfate. Heparan sulfate (HS) is the most abundant glycosaminoglycan of the GCX and an important structure for cell signaling and the inflammatory response [6]. Fragments of HS in the bloodstream have been shown to signal release of pro-inflammatory cytokines. These fragments are released when the endothelium or GCX are damaged. A lack of HS on the endothelial surface leads to endothelial damage [8].

SCOPE

The goal of this project is to understand the role of shear stress on the expression of HLMVEC GCX and its subcomponent HS. We hypothesized that the presence of shear stress would increase the expression of GCX and its major component HS compared to control conditions. In our experiments, we introduced shear stress to HLMVEC in a time-dependent manner and performed immunostaining and confocal microscopy to access GCX expression in control versus uniform flow conditions.

METHODS

We developed a parallel-plate flow chamber to introduce shear stress to HLMVEC. We used SolidWorks to confirm that the cells in the flow region would be exposed to a consistent shear stress. The flow rate was determined by equation 1 where σ is shear stress, in dynes/cm2, w is the width of the flow chamber, h is the height of the flow chamber, and v is the viscosity of the flow media.

HLMVEC were cultured on glass cover slips, for control and experimental conditions, using microvascular endothelial cell growth media (Sigma-Aldrich). The cells were seeded for two days prior and were assessed for confluence. Once confluent, the cells were transferred to the flow chamber, using experimental media, which was a mixture of microvascular endothelium cell growth media and bovine serum albumin (BSA) (Sigma-Aldrich). The flow chamber was placed in an incubator to ensure a controlled environment with a temperature of 37 °C and at 5% CO2. In the first set of experiments, the cells were exposed to uniform flow for 4 hours. Flow was later increased to 6 hours to develop a trend for the relationship between GCX expression and time. Following exposure to shear stress, HLMVEC were quickly washed with phosphate-buffered saline (PBS) and BSA, then fixed with a solution of 2% paraformaldehyde (PFA), 0.1% glutaraldehyde (Glu) in (PBS) for 30 minutes at room temperature. The blocking agent solution is a 1000:100 PBS too BSA ratio, cells were incubated for 30 minutes in solution, and after a PBS wash were stained with wheat germ agglutinin (WGA; Vector Labs) for 2 days, being incubated at 4°C to label the GCX. For secondary detection Alexa Fluor 488-conjugate at a concentration of 1:1000 was used. To stain for HS, the cells were fixed using the same procedure for WGA. After the initial fixing, samples were placed in a mixture of PBS and goat serum for blocking, then incubated for 30 minutes at 4°C. After, they were stained with primary HS antibody Ab-Heparan Sulfate (Amsbio). They were again incubated at 4°C, for two days. Then the samples were stained with Alexa Fluor 488 goat anti-mouse for 30 minutes, and incubated at 4°C. The WGA and HS samples were covered with VECTASHIELD antifade mounting medium (Vector Labs) with 4′,6-diamidino-2-phenylindole, or DAPI, to stain the cell nuclei. The samples were sealed with clear nail polish to prepare them for imaging. The stained cells were imaged using confocal microscopy. This allowed for different planes of the cell layers to be captured. The z projections collected during imaging were reconstructed for analysis. Coverage and thickness of the GCX was quantified using ImageJ FIJI. The coverage was determined by analyzing the composite images of each sample and measuring the percentage of the cell surface covered with WGA or HS. The thickness was quantified by measuring the GCX in the orthogonal view in ImageJ FIJI. The measurements of the thickness and total coverage from the control and uniform flow experiments for WGA were statistically analyzed using a one-way ANOVA on the fold change values. The student's t-test was used to statistically analyze differences in HS samples.

RESULTS

Simulations revealed that cells in the flow area can be exposed to shear stress of up to 12 dynes/cm^2 (Fig 1). For the purpose of this project the flow area cells were exposed to 10 dynes/cm^2 with an average flow velocity of 171.3 mL/min (Fig 1). The coverage fold change of GCX for the experimental group was 1.07±0.027 and 1.05±0.015, for 4 and 6 hours respectively. The thickness fold change was 1.18±0.051 and 1.35±0.071 for 4 and 6 hours respectively. The HS fold change was 1.17±0.63 for coverage and 1.14±0.036 for thickness. The morphological changes between control and experimental groups can be observed visually in green in the figures two and three. The data was found to be statistically significant through statistical analysis with the exception of the HS thickness. This is likely due to needing more samples.

DISCUSSION

The results show that HLMVEC GCX and HS expression is shear stress dependent. Our results provide insight into the effect of shear stress on GCX expression, demonstrating that consistent shear stress from blood flow is important for GCX health. The data trend also suggests that increasing the time of exposure to shear stress could correlate to the increase in GCX expression of HLMVEC.

The immediate future direction of this project is to increase the time of exposure. To confirm the relationship between exposure time and GCX expression, we plan to increase the time of exposure of HLMVECs to shear stress to 8 and 24 hours. This increase will further confirm the trend for the relationship between time and GCX expression. Additionally, we plan to perform these same experiments with a cyclic stretch inducing flow chamber and ultimately with both shear stress and cyclic stretch forces to further study the effects on GCX health.

REFERENCES

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[2] Russo, T., Stoll, D., Nader, H., et al. (2018). Mechanical stretch implications for vascular endothelial cells: Altered extracellular matrix synthesis and remodeling in pathological conditions. Life sciences. 213,214–225. https://doi.org/10.1016/j.lfs.2018.10.030

[3] Pedrigi, R., Papadimitriou, K., Kondiboyina, A., et al. (2017). Disturbed Cyclical Stretch of Endothelial Cells Promotes Nuclear Expression of the Pro-Atherogenic Transcription Factor NF-κB. Annals of biomedical engineering, 45(4),898–909. https://doi.org/10.1007/s10439-016-1750-z

[4] Koo, A., Dewey, C., García-Cardeña, G. (2013). Hemodynamic shear stress characteristic of atherosclerosis-resistant regions promotes glycocalyx formation in cultured endothelial cells. American journal of physiology. Cell physiology, 304(2),C137–C146. https://doi.org/10.1152/ajpcell.00187.2012

[5] Abassi, Z., Armaly, Z., Heyman, S. (2020). Glycocalyx Degradation in Ischemia-Reperfusion Injury. The American journal of pathology, 190(4),752–767. https://doi.org/10.1016/j.ajpath.2019.08.019

[6] Oshima, K., Haeger, S., Hippensteel, J., et al. (2018). More than a biomarker: the systemic consequences of heparan sulfate fragments released during endothelial surface layer degradation. Pulmonary circulation, 8(1),2045893217745786. https://doi.org/10.1177/2045893217745786