(165ad) Characterization of Glycocalyx Components in Human Endothelial Cells

The endothelial cell glycocalyx (EC GCX) is a sugar-rich layer lining the endothelium which consists of glycosaminoglycan (GAG) chains, such as heparan sulfate (HS) and hyaluronic acid (HA)¹. These chains are attached to core proteins such as syndecan-1 (sdc1), glypican-1 (gpc1), and CD44 which line the endothelial cell membrane¹. Research has shown that EC GCX degradation can lead to atherosclerosis, which is one of the precursors to strokes, as well as other serious cardiovascular events^1,2. Studies conducted by our lab and others have shown the presence of a degraded EC GCX in the early stages of vascular diseases, particularly in atherosclerosis¹. Because cardiovascular disease is the leading cause of death worldwide, affecting over 126 million adults in the United States alone, there is incentive to develop treatment strategies which target this structure². Previous research has specifically targeted the HA and HS components of the EC GCX due to their significant composition within this structure³. Given that HA and HS are closely associated with their respective core proteins, understanding how these core proteins respond to flow conditions allows a crucial secondary confirmation of HA/HS presence. In this study we aim to establish the presence of the core proteins CD44 and sdc1 in Human Umbilical Vein Endothelial Cells (HUVECs) in static and uniform flow conditions. HUVECs were used because they are an established and commonly used cell line when studying endothelial cells in vitro. In addition to HUVECs, we plan to use Human Coronary Arterial Endothelial Cells (HCAECs), a cell line more geographically relevant (within the body) in the progression of atherosclerosis.

METHODS

HUVECs were plated at a density of 62,000 cells/cm² on 2.2 cm x 2.2 cm fibronectin-coated glass coverslips until 100% confluency. These cells were then exposed to a uniform flow environment and static environment for 0 or 24 hours. The flow environment was modeled using a custom parallel plate flow chamber¹. Following exposure to the static or flow environment, the cells were then fixed and stained for CD44, sdc1 and gpc1 via immunocytochemical staining (ICC). Confocal microscopy was ultimately used to image the fluorescently labeled HUVECs, and ImageJ, an Adobe-based image processor, was used to quantify fluorescence intensity. Fluorescence intensity was assumed to be indicative of GCX component expression. GCX expression levels were then displayed as mean ± standard error of mean (SEM) and statistical significance was determined using Mann-Whitney t-tests for α = 0.05 through GraphPad Prism software. All flow or static values (n=4) were normalized to the zero-hour static expression (Equation 1). This model, as well as its protocols, will then be translated to other relevant cell types, such as HCAECs.

RESULTS

Our analysis of HUVECs found that at 24 hours, the expression of sdc1 in flow and static conditions was not statistically different (p = 0.343) (Figure 1). Expression of CD44 was also observed at 24 hours and was shown to significantly increase in the flow condition (p = 0.0286) (Figure 1).

DISCUSSION

Our studies allowed us to confirm previous findings of sdc1 at the protein level³, and to establish CD44 protein expression in HUVECs. We also present the novel characterization of how these core proteins respond to the uniform flow environment found in the human vasculature. Studies on CD44 expression in a context relevant to CVDs are limited, and to our knowledge only one group has investigated CD44 expression in flow4. However, this study was in S12NL1 cancer cells and is not comparable for understanding the role of CD44 in EC GCX mechanobiology⁴. Conversely, our observation of unchanged sdc1 protein expression in flow follows the results seen by Liu et al., where sdc1 mRNA expression in HUVECs remained unchanged after 24 hours, compared to the control³. These trends may be explained by differences in how the core proteins themselves interact with the surrounding EC GCX^5,6. CD44 does not covalently bind to components of the GCX whereas sdc1 does interact via covalent binding⁵. Thus, it is possible that CD44 expression changes can be observed due to the simple fact that it is more accessible to antibody binding⁵. Sdc1 expression changes may not be detectable if the core protein is obstructed by covalently bound GCX components⁶. A potential study which may confirm this hypothesis would be to enzymatically degrade or silence surrounding EC GCX components to explore these core proteins free of surrounding obstruction. We also note that in our current in vitro model, the use of HUVECs are inherently a limitation because they are found in regions of the vasculature that are not often associated with atherosclerotic CVD. Rather, atheroprone regions are found in the coronary arteries and aortic arch, due to their branched and curved nature, respectively, and due to the associated disturbed flow. Thus, a more beneficial model would be the use of Human Aortic Endothelial Cells (HAECs) or HCAECs along with a disturbed flow environment to simulate GCX degradation. Mirroring the same methods performed with HUVECs, we plan to use HCAECs to study the effects of disturbed flow in addition to uniform flow on the GCX. In doing so, we hope to gather a more relevant understanding of core protein behavior in the diseased GCX condition.

ACKNOWLEDGEMENTS

We thank the Undergraduate Research and Fellowships office at Northeastern University for their funding of this work and support of Selina Banerjee, John Mwangi, and Theodora Stanley. We also acknowledge the NSF for providing REU funding to supplement CMMI-1846962 (awarded to E. Ebong) and support John Mwangi and Theodora Stanley. We also thank our mentors in the laboratory, and we thank the Institute for Chemical Imaging of Living Systems at Northeastern University for consultation and imaging support.

REFERENCES

1. Harding, I.C., et al., Pro-atherosclerotic disturbed flow disrupts caveolin-1 expression, localization, and function via glycocalyx degradation. Journal of Translational Medicine, 2018.
2. Benjamin Emelia, J., et al., Heart Disease and Stroke Statistics—2019 Update: A Report From the American Heart Association. Circulation, 2019. 139
3. Liu JX, et al. Hemodynamic shear stress regulates the transcriptional expression of heparan sulfate proteoglycans in human umbilical vein endothelial cell. Cell Mol Biol (Noisy-le-grand). 2016 Jul 31;62(8):28-34.
4. Qazi, H., et al.Cancer cell glycocalyx mediates mechanotransduction and flow-regulated invasion. Integrative biology : quantitative biosciences from nano to macro, 5(11), 1334–1343. (2013).
5. Nandi A, et al. Hyaluronan anchoring and regulation on the surface of vascular endothelial cells is mediated through the functionally active form of CD44. J Biol Chem. 2000 May 19;275(20):14939-48.
6. Zhang L, et al. Repetitive Ser-Gly sequences enhance heparan sulfate assembly in proteoglycans. The Journal of Biological Chemistry. 1995 Nov;270(45):27127-27135.