Assessment of Endothelial Cell Migration After Exposure to Toxic Chemicals.
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.
Bundeswehr Institute of Pharmacology and Toxicology; Walther Straub Institute of Pharmacology and Toxicology, Ludwig-Maximilians-Universität München; DirkSteinritz@bundeswehr.org.
Dirk
D
Steinritz
Bundeswehr Institute of Pharmacology and Toxicology; Department of Molecular and Cellular Sports Medicine, German Sports University Cologne.
Annette
A
Schmidt
Bundeswehr Institute of Pharmacology and Toxicology.
Frank
F
Balszuweit
Bundeswehr Institute of Pharmacology and Toxicology.
Horst
H
Thiermann
Department of Molecular and Cellular Sports Medicine, German Sports University Cologne.
Marwa
M
Ibrahim
Department of Molecular and Cellular Sports Medicine, German Sports University Cologne.
Birgit
B
Bölck
Department of Molecular and Cellular Sports Medicine, German Sports University Cologne.
Wilhelm
W
Bloch
D000477:Alkylating Agents / Q000633:toxicity*; D000818:Animals; D054443:Cell Migration Assays; D002465:Cell Movement / Q000187:drug effects* / Q000502:physiology; D058948:Cell Tracking / Q000379:methods*; D002478:Cells, Cultured; D002699:Chlorambucil / Q000633:toxicity; D042783:Endothelial Cells / Q000187:drug effects* / Q000502:physiology; D053078:Membrane Potential, Mitochondrial; D051379:Mice; D018919:Neovascularization, Physiologic / Q000187:drug effects / Q000502:physiology; D012867:Skin / Q000098:blood supply / Q000187:drug effects / Q000293:injuries; D014945:Wound Healing / Q000187:drug effects* / Q000502:physiology
D000477:Alkylating Agents; D002699:Chlorambucil