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 underg....

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., ....

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

List of materials used in this article

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