Assessment of Endothelial Cell Migration After Exposure to Toxic Chemicals

Summary

Investigation of early endothelial cell (EEC) migration is important to understand the pathophysiology of certain illnesses and to potentially identify novel strategies for therapeutic intervention. The following protocol describes techniques to assess cell migration that have been adapted for the investigation of EEC.

Abstract

Exposure to chemical substances (including alkylating chemical warfare agents like sulfur and nitrogen mustards) cause a plethora of clinical symptoms including wound healing disorder. The physiological process of wound healing is highly complex. The formation of granulation tissue is a key step in this process resulting in a preliminary wound closure and providing a network of new capillary blood vessels – either through vasculogenesis (novel formation) or angiogenesis (sprouting of existing vessels). Both vasculo- and angiogenesis require functional, directed migration of endothelial cells. Thus, investigation of early endothelial cell (EEC) migration is important to understand the pathophysiology of chemical induced wound healing disorders and to potentially identify novel strategies for therapeutic intervention.

We assessed impaired wound healing after alkylating agent exposure and tested potential candidate compounds for treatment. We used a set of techniques outlined in this protocol. A modified Boyden chamber to quantitatively investigate chemokinesis of EEC is described. Moreover, the use of the wound healing assay in combination with track analysis to qualitatively assess migration is illustrated. Finally, we demonstrate the use of the fluorescent dye TMRM for the investigation of mitochondrial membrane potential to identify underlying mechanisms of disturbed cell migration. The following protocol describes basic techniques that have been adapted for the investigation of EEC.

Introduction

Cell migration is important in many physiological and pathophysiological processes including development, various diseases, and wound healing after skin injury.

Following skin injury, inflammation removes damaged or necrotic tissue and granulation drives preliminary wound closure and allows formation of a network of new capillaries through vasculogenesis (novel formation) or angiogenesis (sprouting of existing vesicles)^1-3. Both vasculo- and angiogenesis require migration of endothelial cells. The growing network of blood vessels is essential to transport oxygen and nutrients to proliferating keratinocytes which ultimately undergo keratinization, form a new epithelium and provide wound closure.

Impaired migration of endothelial cells is an underlying cause of wound healing disorder^4,5. Thus, methods to assess migration of early endothelial cells are required to explore the pathophysiology of cell migration disorders and to identify novel strategies for therapeutic intervention.

Dermal exposure to alkylating agents (e.g., sulfur and nitrogen mustards) causes wound healing disorder⁶. Such compounds were used as chemical warfare agents in several conflicts in the 20th century and remain reason for strong concern due to existing stockpiles in politically unstable regions and the relatively simple synthesis. Although sulfur mustard was first synthesized in 1822, the molecular and clinical pathology of SM exposure is not understood in detail and no antidote for SM exposure has been identified.

Several studies have been conducted to understand and to model impaired wound healing after SM exposure and to test for potential candidate compounds capable of reserving that effect. Schmidt et al. (2009) tested the effect of chlorambucil, an alkylating compound with properties similar to SM in mouse embryoid body models and found a dramatic, sometimes more than 99% reduction in vessel formation⁷. This adverse effect was most pronounced at a stage of development which, under physiological conditions, is dominated by the proliferation and migration of vascular endothelial precursor cells. Thus, these cells were identified to be particularly sensitive to alkylating agents. Steinritz et al. (2010) tested scavengers of reactive oxygen species (ROS), in particular, N-acetylcysteine (NAC) and alpha linolenic acid (ALA) for their ability to reduce SM toxicity in mouse embryoid body models and in particular, to restore vessel formation⁸. Temporary protective effects were observed, indicating that excessive ROS formation was likely to contribute to the adverse effects of SM on wound healing. These effects were not permanent and the two candidate compounds may not be capable of restoring vessel formation and wound healing in the long term⁸. However, those experiments were conducted in a complex 3D model which did allow investigation of cell migration. Thus, we subsequently tested NAC and ALA for beneficial effects on cell migration of EEC that have a key role in the process of vessel formation⁹.

Moreover, there is evidence that cell polarity is required for cell migration. Mitochondrial dysfunction leading to ROS formation was shown to impair cell polarity and may thus adversely affect cell migration. Therefore, live cell imaging with regard to mitochondrial function was performed and the effects of ROS scavengers were examined. The following protocol describes general requirements for the cultivation of EEC, the Boyden chamber assay, the wound healing assay including cell tracking analysis and the use of TMRM for assessment of mitochondrial function in detail. Important aspects of experimental protocols for EEC cultivation and migration are highlighted.

Protocol

The following protocol describes techniques for the investigation of early endothelial cell migration. The proper cultivation of vascular endothelial cells requires pre-coating of cell culture flasks with gelatin to ensure proper proliferation and maintenance of an endothelial phenotype.

1. Pre-coating of Cell Culture Flasks

1. Dissolve gelatin in 0.1 M PBS to a final concentration of 0.1%.
2. Autoclave the solution with parameters for liquid autoclaving (for details see instructions of the specific autoclave).
3. Add sufficient volume of the autoclaved gelatin solution to a sterile cell culture flask (e.g., at least 5 ml for a T25 cell culture flask).
4. Transfer the flasks into an incubator (37° C, 5% CO₂ is not required but does not interfere) for at least 30 min.
5. Remove the remaining gelatin solution. Use the pre-coated flasks subsequently (see cell cultivation) or store under sterile conditions until use.

2. Cell Cultivation of Early Endothelial Cells

Note: Embryonic stem cell derived early endothelial cells (EEC) were obtained from differentiated murine embryoid bodies by magnetic-activated cell sorting of the PECAM-1 positive cell fraction as described earlier^10,11.

1. Culture EEC on gelatin-coated cell culture dishes in DMEM supplemented with 15% FCS, 50 U/ml penicillin, 50 U/ml streptomycin, 200 µM L-glutamine, 100 µM ß-mercaptoethanol and 1% non-essential amino acids. Handle cells under sterile conditions. Cultivate the cells with 5% CO₂ at 37 °C and 95% humidity until sub-confluence (max. 80%).
2. At sub-confluence, split the cells at a 1:5 ratio. Note: Detachment of endothelial cells is a critical step.
   1. Harvest the cells with RT accutase. Remove the media, rinse with PBS and add 1 ml of accutase per 25 cm².
   2. Incubate the flask at RT for 2-10 min until the cells have detached. Disperse the cells and transfer them to the desired application. Note: A chemical neutralization of the accutase is not required as it takes place when the seeded cells are stored in the incubator at 37 °C. However, accutase activity can also be decreased by adding DMEM containing FCS.

3. Boyden Chamber

Note: Boyden chamber assays are performed by using light-opaque polyethylene terephthalate insert systems with 8 µm pore size.

1. Pre-coat the filter inserts (that fit inside cell culture wells thus creating a Boyden chamber) by adding 500 µl of 0.1% gelatin dissolved in 0.1 M PBS for at least 30 min.
2. If cells are to be exposed to toxic chemicals, expose according to the specific experimental design before cell harvesting. Note: Cells were exposed to 12.5 µg chlorambucil/ml DMEM for 24 hr. With regard to the specific experimental design, instructions may vary.
3. Harvest EEC and determine the cell number by using a counting cell chamber. Note: Automatic counting devices can be used, but should be used with caution: manual cell counting is more accurate and is highly recommended.
4. Add 500 µl cell culture medium into the lower chamber compartment of the Boyden Chamber.
5. Add exactly 10⁴ EEC in 500 µl cell culture medium per filter insert in the upper chamber compartment. Eliminate bubbles.
6. Incubate the filter inserts in the incubator for exactly 8 hr.
7. Rinse with PBS once and replace the medium with 0.5 ml 4% paraformaldehyde in both compartments for 25 min for cell fixation. Wash the filter extensively but at least 3 times with 0.1 M PBS.
8. Excise the membranes with a scalpel.
9. Mount the membrane between two glass cover slips with mounting medium containing DAPI for nuclear staining. Pay attention to the orientation. Ensure that only cells that have migrated towards the lower compartment side of the membrane are counted.
10. Count cells that have migrated towards the lower compartment side of the membrane with a fluorescence microscope at 400X magnification. Do not confuse membrane pores with migrated cells (Figure 1A, 1B). Investigate a reasonable number of biological replicates (at least 3 biological replicates per condition).

4. Wound Healing Assay

1. Depending on the available equipment, carefully choose the cell culture dishes or plates: When using DIC microscopy, avoid plastic surface based culture dishes or well plates but use glass based devices instead.
   Note: If using phase-contrast microscopy, plastic based dishes can also be used.
2. Cultivate EEC in a suitable cell culture device (e.g., 4 cm glass bottom petri dish suitable for live cell imaging) until 80% confluence. Important: do not cultivate the cells to complete confluence.
3. Scratch the monolayers with sterile 10 µl pipette tips. Push the tip gently without too much pressure onto the dish surface and move it in a straight line smoothly from one side to the other.Wash the cells twice with 0.1 M PBS to remove detached cells.
4. Add a sufficient volume of medium to the culture dish. If applicable, add compounds that should be investigated.
   Note: 1.5 ml medium containing 15 ng/ml alpha linolenic acid was added.
5. Mount the culture dish under a microscope capable of live cell imaging. Ensure 5% CO₂, 37 °C and a humidified atmosphere.
   Note: Humidification is especially important to avoid medium evaporation.
6. Acquire time-lapse images over 24 hr at 10 min intervals. Plan for large file sizes. Note: A resolution of 512 x 512 pixels is usually sufficient; however, we recommend using images of at least 1,024 x 1,024.
7. Measure the gap width at t = 0 hr and at t = 24 hr using the length tool of the software provided with the microscope or use open-source software (e.g., ImageJ). Note: In general specific image acquisition and analysis software is provided by the manufacturer. Therefore, for technical details regarding the use of the software, refer to the manual.

5. Cell Tracking

1. Depending on the available equipment, carefully choose the cell culture dishes or plates: When using DIC microscopy, avoid plastic surface based culture dishes or well plates but use glass based devices instead.
   Note: If using phase-contrast microscopy, plastic based dishes can also be used.
2. Seed 5 x 10⁴ EEC in supplemented DMEM in a suitable cell culture device (e.g., 24-multiwell plate) and cultivate the cells with 5% CO₂ at 37 °C and 95% humidity for 1-2 days.
3. When cells have grown up to a 80% confluence, remove the media and culture the cells with new media in the presence of the respective test substances (e.g., 12.5 µg chlorambucil / ml DMEM). Always include control cells (treated with the solvent, e.g., ethanol) at 37 °C for a certain period of time (24 hr, depending on the individual assay system).
4. Mount the culture dish under a microscope capable of live cell imaging (37 °C, 5% CO₂ and 95% humidity). Acquire time-lapse images over 24 hr at predefined intervals. Acquire images at 10 min intervals.
5. Perform manually tracking of EEC by the use of the ImageJ plugin MTrackJ. Choose 10 cells randomly from the field of view and track their movements by adding a data point per point in time using the “Add” command of MTrackJ.

Note: MTrackJ is available for free at [Meijering, \Mtrackj." http://www.imagescience.org/meijering/software/mtrackj/ “] and ImageJ is available at [Rasband, \Imagej." http://rsbweb.nih.gov/ij/ “]. A detailed manual about the MTrackJ plugin is available at “http://www.imagescience.org”.

6. Live Cell Imaging/Assessment of Mitochondrial Membrane Potential

1. Cultivate EEC in a suitable cell culture device (e.g., 4 cm glass bottom petri dish suitable for live cell imaging) up to a 80% confluence.
   Important: Do not cultivate the cells to complete confluence.
2. If applicable, expose the cells to chemicals. Note: Cells were exposed to 12.5 µg/ml chlorambucil for 24 hr. With regard to the specific experimental design, instructions may vary.
3. Prepare a 10 mM stock solution of tetramethylrhodamine (TMRM) in DMSO. Protect from light. Note: The stock solution can be stored at -20 °C.
4. Dilute the stock solution in cell culture medium to a working solution with a concentration of 10 µM (1:1,000 dilution). Protect from light and use as soon as possible. Note: The working solution can be kept at RT for some time (>1 hr), however, preparation of a fresh working solution is highly recommended.
5. Add 2 µl of the working solution to 1 ml fresh cell culture medium (loading solution).
6. Load the cells by replacing the cell culture medium with the loading solution. Incubate for 15 min at 37 °C, 5% CO₂ and humidified atmosphere (incubator). Caution: Almost all fluorescence indicators are exported by living cells over time; therefore avoid prolonged loading or delayed analysis.
7. Without washing, place the dish under a microscope suitable for live cell imaging. Important: fluorescence indicators are highly sensitive to light, therefore, avoid unnecessary light exposure.
8. Acquire images without changing the acquisition parameters to ensure comparability between different images.

Results

Dermal exposure to alkylating agents provokes erythema, blister formation and dermal ulceration that is associated with a wound healing disorder. Wound healing requires angio- and vasculogenesis which are based on migration of endothelial cells. Quantitative migration can be assessed by use of the Boyden chamber assay. As shown in Figure 1C exposure of EEC to the alkylating agent chlorambucil resulted in a significant decrease in cell migration⁹. Addition of the ROS-scavenger alpha linolenic a...

Discussion

Dermal exposure to toxic chemicals often results in severe wound healing disorder. The underlying mechanisms are largely unknown. Wound healing is a complex process that consists of different phases (hemostasis, inflammation, proliferation and remodeling). Cell migration is involved in every phase, however, it is of utmost importance for the formation of the granulation tissue. Here, new blood vessels are formed either by angio- or vasculogenesis.

Both processes require unaffected migration of...

Materials

Name	Company	Catalog Number	Comments
Boyden Chamber
Corning FluoroBlok Tissue Culture (TC)-treated Inserts, 24 well - 3 µm	Corning Incorporated	#351151
Corning FluoroBlok Tissue Culture (TC)-treated Inserts, 24 well - 8 µm	Life Sciences	#351152
(for use with Falcon Insert 24 well Companion Plate (353504)
Wound  healing assay
Glass bottom dishes	Word Precision Instruments, Inc.	#FD35-100
Assessment of mitochondrial potential
TMRM (tetramethylrhodamine methyl ester)	Life Technologies	#T669
Cell culture
Accutase	PAA, Pasching, Austria	# L11-007
α-Linolenic acid	Fluka (Sigma), Steinheim, Germany	# L2376
Chlorambucil	Fluka (Sigma), Steinheim, Germany	# 23125
Gelatin	Sigma-Aldrich, Steinheim, Germany	# G2500-100G