Endothelial Cells

Endothelial cells are a specialized type of epithelial cell which forms the inner layer of blood vessels.

These cells play a key role in angiogenesis, the development of new blood vessels from pre-existing vessels. Angiogenesis is a multi-step process that is important for both physiological and pathological development. During angiogenesis, endothelial cells are activated and express matrix metalloproteinases (MMPs), which degrade the vascular basement membrane. In response to environmental cues, endothelial cells secrete MMPs and then invade through the basement membrane to form new capillary networks.

Endothelial cells are tested in a variety of assays for functions that contribute to the angiogenesis process. Collagen I coated surfaces are suitable for culturing endothelial cells such as fetal bovine heart endothelial cells (FBHECs) and human umbilical vein endothelial cells (HUVECs) (Figure 1). In vitro assays of endothelial cell function include cell migration1, invasion2, and tubule formation3-9. Both the BD BioCoat™ Angiogenesis System: Endothelial Cell Invasion and the BD BioCoat Angiogenesis System: Endothelial Cell Migration allow for rapid data collection without multiple handling steps. These quantitative assays utilize BD FluoroBlok™ microporous polyethylene terephthalate (PET) membranes (3 μm pore size) which effectively block the fluorescence signal from labeled cells that have not invaded or migrated through the membrane, respectively, thereby allowing the selective detection of cells that reside on the underside of the membrane (Figure 2). To perform fluorescence detection, cells may be pre-labeled or post-labeled with a fluorescent dye (Figure 3). The pre-labeling technique enables real-time kinetic measurements of cell migration or invasion. Endothelial cells must be able to migrate and enzymatically degrade the basement membrane in order for angiogenesis to occur. The wells of the BD BioCoat Angiogenesis System: Endothelial Cell Invasion are evenly coated with BD Matrigel™ Matrix, which allows researchers to examine the ability of endothelial cells to invade through reconstituted basement membrane in response to chemoattractants, such as VEGF, in the presence or absence of anti-angiogenic agents (Figure 4).

BD BioCoat Angiogenesis System: Endothelial Cell Migration consists of BD FluoroBlok inserts evenly coated with human fibronectin (Figure 5). Studies conducted using the post-labeling technique demonstrated that BD™ HUVEC-2 cells migrate towards VEGF in a concentration dependent manner (Figure 6).

During angiogenesis, endothelial cells form capillaries once they have invaded through the basement membrane. The correct culture surface is critical for successful endothelial cell tube formation in vitro. Both primary endothelial cells and endothelial cell lines have been demonstrated to form tubules on the BD BioCoat Angiogenesis System: Endothelial Cell Tube Formation (Figures 7-9) which is comprised of a 3D gel of BD Matrigel Matrix. The BD BioCoat Angiogenesis Systems are available in 24- and 96-Multiwell formats, which can be used for moderate to high throughput compound screening. BD Matrigel™ Matrix has also been extensively used to study in vivo angiogenesis4-5, 10-12 as a less technically challenging alternative to the corneal implantation model. A "plug" of material is placed subcutaneously, followed by histological quantification 7-10 days later. These in vitro and in vivo assays give researchers multiple options for exploring endothelial cell functions that are essential during angiogenesis.

* BD BioCoat Angiogenesis System: Endothelial Cell Tube Formation offers a standardized and robust assay for studying endothelial cell tubulogenesis. For customers interested in establishing an assay for tube formation using vialed BD Matrigel Matrix, we recommend pre-testing lots to ensure optimal performance.

Figure 1. Effects of BD Biocoat Endothelial Cell Growth Environment on HUVEC

Figure 1. Effects of BD Biocoat Endothelial Cell Growth Environment on HUVEC

BD BioCoat Endothelial Cell Growth Environment utilizes BD BioCoat Collagen I Cellware and BD™ Endothelial Cell Culture Medium to enhance endothelial attachment and proliferation. HUVECs grown for five days using the BD BioCoat Endothelial Cell Growth Environment form a confluent monolayer and show numerous mitotic cells.(A) HUVECs grown for five days in basal medium containing 10% FBS on tissue culture-treated plastic show sparse growth (B).

Figure 2. Labeling Cells Post-Invasion with Calcein AM

Figure 2. Labeling Cells Post-Invasion with Calcein AM

A fluorescence plate reader quantifies cells post-invasion by measuring fluorescence which correlates to cell number. Cells on top of the BD FluoroBlok™ membrane are not detected by a bottom-reading fluorometer.


 
Figure 3. Labeling Methods for Endpoint or Real-Time Kinetic Migration and Invasion Assays

Figure 3. Labeling Methods for Endpoint or Real-Time Kinetic Migration and Invasion Assays

BD FluoroBlok Inserts can be used for endpoint or real-time kinetic assays. For endpoint assays, the cell migration or invasion assay is performed with unlabeled cells. At the end of the assay the cells are labeled with a fluorescent dye, such as BD™ Calcein AM, and the data is collected using a bottom reading fluorescent plate reader. For real time kinetic assays, the cells are pre-labeled with a fluorescent dye, such as BD DiIC12(3). After labeling, the migration or invasion assay is run with data collected over a time course using a bottom reading fluorescent plate reader.

Figure 4. Effects of TIMP-2 and 1'10' Phenathanthroline in VEGF-Mediated HMVEC Invasion

Figure 4. Effects of TIMP-2 and 1'10' Phenathanthroline in VEGF-Mediated HMVEC Invasion

Human microvascular endothelial cells (HMVECs) were assayed in the BD BioCoat Angiogenesis System: Endothelial Cell Invasion in the presence of VEGF (4 μg/ml) with varying concentrations of (left) TIMP-2 or (right) 1’10’ phenanthroline in the bottom chamber. Cells were allowed to invade for 22 ± 1 hour. Cells were labeled post-invasion with BD Calcein AM (4 μg/ml) and then analyzed for invasion through BD Matrigel™ Matrix using an Applied Biosystems CytoFluor® 4000 plate reader [485/540 nm (Ex/Em) wavelengths]. Data represents the mean of n=3 inserts ± S.D.


 
Figure 5. HUVEC Migration on Uncoated and Human Fibronectin-coated Inserts

Figure 5. HUVEC Migration on Uncoated and Human Fibronectin-coated Inserts

Migration assays were conducted using HUVECs in the BD BioCoat Angiogenesis System: Endothelial Cell Migration and compared with uncoated BD FluoroBlok 24-Multiwell Inserts using both FBS (5%) and VEGF (10 μg/ml) as chemoattractants. The cells were allowed to migrate for 22 ± 1 hour. Cells were labeled post-migration with BD Calcein AM (4 μg/ml) and measured by detecting the fluorescence of the cells that migrated through the BD FluoroBlok membrane using an Applied Biosystems CytoFluor® 4000 plate reader [485/530 nm (Ex/Em) wavelengths]. The results indicate a marked increase in migration in response to VEGF when the assay was performed on the fibronectin coated inserts included in the system. Data represents the mean of n=3 inserts ± S.D.

Figure 6. BD HUVEC-2 Cells exhibit concentration-dependent migration towards VEGF

Figure 6. BD HUVEC-2 Cells exhibit concentration-dependent migration towards VEGF

BD HUVEC-2 cells assayed in the BD BioCoat Angiogenesis System: Endothelial Cell Migration (96-Multiwell format) in response to increasing concentrations of VEGF. Samples were incubated for 22 hours. Cells were labeled post-migration with BD Calcein AM and measured by detecting the fluorescence of cells that migrated through the fibronectin-coated BD FluoroBlok membrane with the Victor2™ plate reader (PerkinElmer) at 485 nm emission. Data represents the mean of n=4 inserts ± S.D.


 
Figure 7. Human Endothelial Cell Types exhibit Tube Formation

Figure 7. Human Endothelial Cell Types exhibit Tube Formation

HUVEC, HMVEC, and the human endothelial cell line HMEC-1 exhibit tube formation on BD BioCoat Angiogenesis System: Endothelial Cell Tube Formation. For this study, 20,000 cells of each cell type were added to wells containing pre-solidified BD Matrigel Matrix. The assay was incubated for 18 hours. Each bar represents the mean of n=32 wells ± S.D.

Figure 8. Confocal Image of BD HUVEC-2 Cell Tube Formation

Figure 8. Confocal Image of BD HUVEC-2 Cell Tube Formation

BD HUVEC-2 cells were assayed using the BD BioCoat Angiogenesis System: Endothelial Cell Tube Formation. Cells were stained using BD Calcein AM. Confocal images were captured using the BD Pathway™ Bioimager in confocal mode using the 4x objective (NA 0.13) for quantification of tubule formation.


 
Figure 9. Suramin inhibits HMEC-1 Tube Formation

Figure 9. Suramin inhibits HMEC-1 Tube Formation

HMEC-1 cells (40,000 cells/ml) were treated with Suramin at concentrations ranging from 0-40 μm and then analyzed for tube formation using BD BioCoat Angiogenesis System: Endothelial Cell Tube Formation. 50 μl of cells plus compound were added to wells containing pre-solidified BD Matrigel Matrix. Samples were incubated at 37°C, 5% CO2 for 18 hours before staining with BD Calcein AM. Images were acquired with a 2x objective lens and the total tube length was measured using MetaMorph® (Universal Imaging Corporation™). Each bar represents the mean of n=8 wells ± S.D.

References

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AlexaFluor is a trademark of Invitrogen Corporation.
CytoFlour is a trademark of Applied Biosystems.
MetaMorph is a registered trademark of Universal Imaging Corporation (UIC).
mTeSR1 and WiCell is the property of WiCell Research Institute.
PuraMatrix is a registered trademark of 3DM, Inc.
Victor2 is a trademark of PerkinElmer, Inc.


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