Nitric oxide triggers enhanced induction of vascular endothelial growth factor expression in cultured keratinocytes (HaCaT) and during cutaneous wound repair.
Recently, we demonstrated a large induction of inducible nitric oxide synthase (iNOS) during cutaneous wound repair. In this study, we investigated the role of nitric oxide (NO) for the expression of vascular endothelial growth factor (VEGF), which represents the most important angiogenic factor during the proliferative phase of skin repair. Since keratinocytes are the major source of VEGF production during this process, we used cultured keratinocytes (HaCaT cell line) as an in vitro model to investigate NO action on growth factor- and cytokine-stimulated VEGF expression. Exogenously added NO enhanced transforming growth factor-beta1-, keratinocyte growth factor-, interleukin-1beta-, tumor necrosis factor-alpha-, and interferon-gamma-induced VEGF mRNA and protein synthesis in keratinocytes. We could demonstrate that high-level expression of cytokine-induced VEGF mRNA in keratinocytes is dependent on endogenously produced NO, as inhibition of the coinduced iNOS by N(G)-monomethyl-L-arginine (L-NMMA) markedly decreased cytokine-triggered VEGF mRNA levels in the cells. We also established an in vivo model in mice to investigate the role of NO during wound healing. During excisional wound repair, mice were treated with L-N(6)-(1-iminoethyl)lysine (L-NIL), a selective inhibitor of iNOS enzymatic activity. Compared to control mice, L-NIL-treated animals were characterized by markedly reduced VEGF mRNA levels during the inflammatory phase of repair. Immunohistochemistry demonstrated reduced VEGF protein expression and a completely disorganized pattern of VEGF-expressing keratinocytes within the hyperproliferative epithelium at the wound edge in L-NIL-treated mice. We demonstrate that triggering of VEGF expression is a crucial molecular mechanism underlying NO function during wound healing.
Frank
S
S
Zentrum der Pharmakologie, Klinikum der Johann Wolfgang Goethe-Universität, D-60590 Frankfurt am Main, Germany.
Stallmeyer
B
B
Kämpfer
H
H
Kolb
N
N
Pfeilschifter
J
J
D000818:Animals; D016207:Cytokines / Q000494:pharmacology; D016228:Endothelial Growth Factors / Q000096:biosynthesis* / Q000235:genetics; D005260:Female; D051526:Fibroblast Growth Factor 10; D051523:Fibroblast Growth Factor 7; D005346:Fibroblast Growth Factors; D005978:Glutathione / Q000031:analogs & derivatives / Q000494:pharmacology; D006133:Growth Substances / Q000494:pharmacology / Q000502:physiology; D006801:Humans; D015603:Keratinocytes / Q000378:metabolism*; D008222:Lymphokines / Q000096:biosynthesis* / Q000235:genetics; D008239:Lysine / Q000031:analogs & derivatives / Q000494:pharmacology; D051379:Mice; D008807:Mice, Inbred BALB C; D009569:Nitric Oxide / Q000502:physiology*; D009603:Nitroso Compounds / Q000494:pharmacology; D012333:RNA, Messenger / Q000032:analysis; D026422:S-Nitrosoglutathione; D012867:Skin / Q000378:metabolism*; D014407:Tumor Cells, Cultured; D042461:Vascular Endothelial Growth Factor A; D042442:Vascular Endothelial Growth Factors; D014945:Wound Healing
D016207:Cytokines; D016228:Endothelial Growth Factors; C496453:FGF7 protein, human; C496454:Fgf7 protein, mouse; D051526:Fibroblast Growth Factor 10; D006133:Growth Substances; D008222:Lymphokines; C090663:N(6)-(1-iminoethyl)lysine; D009603:Nitroso Compounds; D012333:RNA, Messenger; D042461:Vascular Endothelial Growth Factor A; D042442:Vascular Endothelial Growth Factors; D051523:Fibroblast Growth Factor 7; D009569:Nitric Oxide; D026422:S-Nitrosoglutathione; D005346:Fibroblast Growth Factors; D005978:Glutathione; D008239:Lysine