Analysis of VE-cadherin Expression in Primary Human Endothelial Cells (Z’ > 0.5)

VE-cadherin (vascular endothelium cadherin, also known as Cadherin-5), is a member of cadherin superfamily, expressed by vascular endothelial cells (reviewed in 1-3). In these cells it is the major adherens junction protein, which, by modulating cell-cell adhesion, regulates angiogenesis, and vascular permeability. It is also required for differentiation into vasculature-like structures by endothelial cells in vitro (4, 5). Like other cadherins, VE-cadherin provides Ca2+-dependent homotypic adhesion between neighboring cells and serves as a link between plasma membrane and actin cytoskeleton. Additionally, unlike cadherins in epithelial cells, VE-cadherin also forms specialized adhesion structures linking to intermediate filaments (6, 7). Similar to other cadherins, VE-cadherin participates in signal transduction through Cdc42 (8), RhoA (9) and Rac (10) pathways, as well as via its dynamic associations with Î²-catenin and p120ctn. Endothelial monolayer permeability and the stability of the endothelial cell-cell adhesions can be modulated via endocytosis and degradation of VE-cadherin in a p120ctn –dependent manner (11, 12; reviewed in 3, 13). VE-cadherin has been shown to be the key factor in preventing proliferation of contact-inhibited endothelial cells (14). Conversely, inhibition of VE-cadherin correlates with increased cellular motility (15).

Since VE-cadherin is crucial for controlling the state of adherens junctions, which in turn regulate endothelial cell-cell adhesion, cell motility, morphogenesis and intracellular signaling pathways, this molecule has many clinical implications. For example, VE-cadherin expression is markedly reduced in vasculature-derived cancers, suggesting that this protein is important for tumor progression (16). On the other hand, highly aggressive melanomas (which are not endothelial in origin), acquire an ability to “mimic” blood vessel formation, and this process is accompanied by induction of VE-cadherin [removed]17). Similarly, tumor-induced angiogenesis has been shown to involve upregulation of VE-cadherin in the neighboring vasculature (18, 19). Futhermore, VE-cadherin is actively involved during transendothelial migration of metastatic cancer cells (20, 21; reviewed in 22). Since blocking angiogenesis is a common strategy to inhibit tumor growth and extravasation, several approaches for inhibiting VE-cadherin function have been suggested as possible anti-tumor therapies (23-26). Finally, disruption of endothelial function during inflammation and pre-eclampsia has been shown to involve redistribution of VE-cadherin from the plasma membrane (27, 28).

Figure 1

Visualization of VE-cadherin in human microvascular endothelial cells. Upper left, Cells visualized for VE cadherin. Lower left, Mask generated by CyteSeer. Membrane associated VE-cadherin for cells cultured in EGTA or maintained in serum-free media. Data are the mean +/- SD for n=8 wells per condition. The wells were imaged (4 fields of view/well) with a Beckman IC100 high content microscopy workstation.

Vala Sciences Inc has developed a reagent plus software kit to visualize and quantify VE-cadherin in cultured cells. The kit features staining reagents, optimized for staining cells cultured in the 96-well dish, and Vala’s CyteSeerTM image analysis software platform. CyteSeer is an affordable image cytometry program designed to analyze images acquired with virtually any digital microscopy workstation, and is compatible with most computer operating systems. To demonstrate the capabilities of our kit, confluent primary human microvascular endothelial cells (HDMECs) were cultured under three condition: 1) in DMEM plus 10% FBS (control), 2) the same media containing 3 mM EGTA (which strongly reduces the calcium concentration of the culture media) for 3 hr, and 3) Overnight in serum-free media. The cells were then visualized for VE-cadherin using Vala’s reagent kit components, imaged using a Beckman IC100 robotic microscopy workstation (4 fields of view/well), and analyzed for VE-cadherin expression at the plasma membrane utilizing CyteSeer. Incubation with EGTA led to an approximate 25% reduction in VE-cadherin association with the plasma membrane (Figure 1). Interestingly, overnight serum-withdrawal led to an approximate 100% increase in VE-cadherin visualized at the plasma membrane. For this experiment, the Z’ value for the minimal and maximal responses (EGTA vs serum-starved cells) was 0.50, indicating that the assay is robust and suitable for quantitative image cytometry applications related to drug discovery or chemical genomics (29).

References

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