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Cell-cell adhesion is an important factor in the regulation of angiogenesis and in the establishment and maintenance of the vascular barrier function. Importantly, loss of vascular barrier function is the hallmark of pathologic conditions such as edema and inflammation. Vascular endothelial-cadherin (VE-cad), a member of the cadherin family found in endothelial cells, plays an active role in cell-cell adhesion. The cytoplasmic tail of VE-cad is thought to participate in the process of adhesion strengthening by interacting with p120-catenin (p120). p120, a member of the armadillo protein family, binds to the juxtamembrane domain of the VE-cad tail and in turn activates a downstream mechanism that enhances cell-cell adhesion. The exact mechanism by which p120 contributes to adhesion is not fully understood. By expressing constructs containing wild type and mutant forms of the cytoplasmic tail of VE-cad in endothelial cells, the role of the tail and p120 can be isolated and studied through cell spreading and hydrodynamic shear assays. VE-cad mutants lacking the p120 binding site demonstrated decreased amounts of cell spreading, suggesting a weakened interaction with the actin cytoskeleton. The use of micropatterned surfaces in a hydrodynamic shear assay subjected the cells expressing the various constructs to a uniform gradient of shear stress. Results using this assay verify that adhesion strength is weakened by the loss of the VE-cad tail; however, it is unknown as to whether or not this loss of adhesion strength is specific to the loss of p120 binding. Further experiments using VE-cad mutants will show whether or not this loss of adhesion strength is p120-specific. This reduction in adhesion strength signifies that p120 may play an active role in adhesion strengthening and provides further insight into p120’s role in cell-cell interactions. By gaining a better understanding of how the VE-cad / p120 complex regulates adhesion strength, treatments could be developed to help modulate and strengthen vascular barrier function and angiogenesis.
Cadherins bind to one another in the extracellular domain to form cell-cell junctions.
Loss of cadherin function in vascular endothelial cells leads to edema and inflammation.
VE-cadherin is a member of the cadherin family and is specifically found in endothelial cells.
VE-cad is composed of an extracellular domain that interacts with other VE-cad molecules on adjacent cells and an intracellular domain that binds to a family of proteins known as armadillo proteins. This family includes p120-catenin and β-catenin.
The juxtamembrane domain (JMD) of the VE-cad cytoplasmic tail binds to p120-catenin whereas the distal domain of the VE-cad tail binds to β-catenin. β-catenin in turn interacts with α-catenin and the actin cytoskeleton. Rac1, a small Rho family GTPase, also interacts with the actin cytoskeleton to mediate cell spreading.
Catenins initiate a downstream process that mediates cadherin function. This process is not yet fully understood
Cell Spreading Assay
Cultured human microvascular endothelial cells (MECs).
Infected cells with adenoviruses expressing the chimeric constructs shown in Figure 2.
Used self assembled monolayer (SAM) on Au coated coverslips to bind to IL2 receptor antibody (Ab).
Seeded cells on IL2R Ab coated coverslips and allowed to sit for one hour.
Fixed and mounted cells. Acquired DIC images on microscope.
Using Simple PCI, measured area of cell spreading with a pixel count.
Compared cell spreading with graphs and Kruskal-Wallis statistical analysis
Hydrodynamic Shear Assay
Cells expressing IL2R constructs are seeded on coverslips where adhesion is limited to regularly spaced 20-micron islands
Allowed cells to sit for 24 hours and used a spinning disc apparatus to subject the cells hydrodynamic shear force, removing cells with weak adhesion strength off of the coverslips
Performed statistical analysis to determine what shear stress is required to remove the cells from the coverslips
The cytoplasmic tail of VE-cadherin plays an active role in cell spreading and adhesion strengthening.
Cells with p120 binding to VE-cad show increased spreading over those with the AAA mutation and the p120 null cells. This indicates that p120 binding to VE-cad is an integral step in the cell spreading process.
In cells expressing the AAA construct, the spreading effect can be rescued by introducing DA Rac1. Rac1 is a GTPase that is known to be activated downstream of p120. By using a constitutively active Rac1, we can bypass p120 to activate the actin cytoskeleton and mediate cell spreading.
Hydrodynamic shear assays on cells expressing the full length VE-cad tail show an increase in adhesion strength in comparison to cells where the full length tail is absent.
We can conclude that p120 binding to VE-cadherin is involved in cell spreading. Cell spreading is closely related to adhesion strengthening, so it is possible that p120 also plays a role in cell adhesion strengthening. The removal of the cytoplasmic tail results in decreased cell adhesion strength, but it is unknown as to whether or not loss of p120 binding is the reason for this decrease.
Future Studies
We hope to determine what portion of the cytoplasmic tail of VE-cadherin is necessary for adhesion strengthening.
Using the hydrodynamic shear assay on VE-cadherin constructs containing either the AAA mutation or a deletion the catenin binding domain (CBD) could provide a measurement into how much the JMD and CBD respectively affect adhesion strengthening.
We also hope to gain a better understanding of the signaling pathways that are engaged or required for adhesion strengthening.
Preliminary results suggest Rac1 is one pathway through which cell adhesion is strengthened, but there may be others.
Obtaining more knowledge on how cell adhesion strengthening works will lead to further experimentation and possible treatments for pathogenic cell adhesion strength disorders.
We also hope to perform further rescue treatments that could be used to recover cell spreading and adhesion strength.
This material is based upon work supported by the Howard Hughes Medical Institute under Grant No. 52005873 and by a fellowship award by the Atlanta Clinical and Translational Science Institute. This work was also supported by the National Institute of Health Grant No. 5R01AR050501-05. The authors would like to thank the Summer Undergraduate Research at Emory program for their help throughout the work of this project. The authors would also like to thank all of the members of the Garcia lab and the Kowalczyk lab for all of their guidance and assistance.
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