Endothelial glycocalyx-mediated intercellular interactions: mechanisms and implications for health and disease.

Permanent URL: http://hdl.handle.net/2047/D20328715
Title:
Endothelial glycocalyx-mediated intercellular interactions : mechanisms and implications for health and disease
Creator:
Mensah, Solomon Arko (Author)
Contributor:
Ebong, Eno (Advisor)
Coury, Arthur (Committee member)
Carrier, Rebecca (Committee member)
Monaghan, James (Committee member)
Language:
English
Publisher:
Boston, Massachusetts : Northeastern University, 2019
Date Accepted:
October 2019
Date Awarded:
December 2019
Type of resource:
Text
Genre:
Dissertations
Format:
electronic
Digital origin:
born digital
Abstract/Description:
The endothelial glycocalyx (GCX) plays a critical role in the health of the vascular system. Degradation of the GCX has been implicated in the onset of diseases like atherosclerosis and cancer because it disrupts endothelial cell (EC) function that is meant to protect from atherosclerosis and cancer. Intercellular interactions are physiologically relevant activities that ensure proper EC function. Various intercellular interactions including those mediated by gap junction proteins, like connexin, for maintaining cell to cell communication, and adhesion molecules, like those mediated by E-selectin and integrins for regulating cell to cell contact between ECs and leukocytes, cancer cells, or other circulating cells. To-date, limited progress has been made to best understand the role of the GCX in intercellular interactions. Previous work demonstrated that GCX degradation disrupts EC gap junction connexin (Cx) proteins, likely blocking interendothelial communication that maintains EC and vascular tissue homeostasis to resist disease. Other reports suggest that the ability of immune cells to interact with EC could be a model for the way cancer cells interact with EC, and these interactions are modulated by the GCX. We hypothesize that the GCX controls the opening and closing of Cx containing gap junction proteins for regulating communication and also controls accessibility to receptors on the surface of the endothelium for regulating intercellular interactions. To test our hypothesis, we performed multiple EC experiments to investigate the role of GCX in intercellular interactions. To understand GCX involvement in gap junction regulation we tested the effect of different GCX conditions on the expression of Cx isotype 43 (Cx43) containing gap junctions. Expression of Cx43 at EC borders was characterized immunocytochemically, and the function of Cx-containing gap junctions were assessed by measuring interendothelial spread of gap junction permeable Lucifer Yellow dye. We further examined the activities of the gap junctions and Cx43 after applying various regeneration techniques for GCX. GCX regeneration was achieved via treatment with exogenous heparan sulfate (HS), a major component of GCX. HS was applied with or without the GCX regenerator and protector sphingosine 1- phosphate (S1P). With intact HS, 60% of EC borders expressed Cx43 and dye spread to 2.88 ± 0.09 neighboring cells. HS degradation decreased Cx43 expression to 30% and reduced dye spread to 1.87± 0.06 cells. Artificial HS recovery with exogenous HS partially restored Cx43 expression to 46% and yielded dye spread to only 1.03 ± 0.07 cells. Treatment with both HS and S1P, recovered HS and restored Cx43 to 56% with significant dye transfer to 3.96 ± 0.23 cells. This is the first evidence of GCX regeneration in a manner that effectively restores vasculoprotective EC communication. This work validates the importance of GCX in Cx activities. We also investigated the importance of GCX in concealing or uncovering receptors that mediate cancer-endothelial cell interactions. While it is known that cancer cell interactions with vascular EC drive metastatic cancer cell extravasation from blood vessels into secondary tumor sites, the mechanisms of action are still poorly understood. It is known that GCX structure depends on vascular flow patterns, which are irregular in tumor environments. This dissertation presents evidence that disturbed flow (DF) induces GCX degradation, and leads to circulating tumor cells (CTC) homing to the endothelium, a first step in secondary tumor formation. To understand the role of GCX in cancer-EC interactions, a two-pronged experiment was performed. First, we tested the hypothesis that neuraminidase-induced degradation of EC GCX, particularly the sialic acid (SA) residue components of the GCX, will substantially increase metastatic cancer cell attachment to ECs. To our knowledge, our study is the first to isolate the role of GCX SA residues in cancer cell attachment to the endothelium, which were found to be differentially affected by the presence of neuraminidase and to indeed regulate metastatic cancer cell homing to ECs. Second, we investigated the effect of DF on cancer-EC interactions. A 2-fold greater attachment of CTCs to human ECs was found to occur in DF conditions, compared to uniform flow (UF) conditions. These results corresponded to an approximately 50% decrease in wheat germ agglutinin (WGA) labeled components of the GCX in DF conditions, versus UF conditions. E-selectin receptor expression was similar in DF and UF conditions. Neuraminidase induced degradation of WGA-labeled GCX in UF cell culture conditions or in Balb/C mice led to an over 2-fold increase in CTC attachment to ECs or to Balb/C mouse lungs, respectively, compared to non-enzymatic conditions. These experiments confirm that GCX degradation enables CTC attachment, providing new insight into a possible GCX-mediated pathway to secondary tumor formation. Collectively, we have shown that GCX indeed plays a significant role in gap junction mediated intercellular communication and adhesion molecules-mediated intercellular interactions. We reported that the expression and functionality of Cx43 protein and gap junctions depend on the structural stability of GCX, specifically the HS component. In addition, we also demonstrated that destabilizing subcomponents of the GCX specifically SA, results in increased receptor-mediated cancer-EC interactions. Finally, we showed that DF-induced GCX degradation, or, alternatively, enzymatic degradation of GCX, results in increased attachment, clustering and migration of cancer cells through the endothelium.
Subjects and keywords:
Bioengineering
DOI:
https://doi.org/10.17760/D20328715
Permanent URL:
http://hdl.handle.net/2047/D20328715
Use and reproduction:
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