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In vitro wounding: effects of hypoxia and transforming growth factor β1 on proliferation, migration and myofibroblastic differentiation in an endothelial cell-fibroblast co-culture model

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Abstract

The adequate reconstitution of human soft tissue wounds requires the coordinated interaction of endothelial cells and fibroblasts during the proliferation phase of healing. Endothelial cells assure neoangiogenesis, fibroblasts fill the defect and provide extracellular matrix proteins, and myofibroblasts are believed to support the reconstitution of microvessels. In the present study, we combined in vitro-wound size measurement and multicolour immunocytochemical staining of co-cultured human dermal microvascular endothelial cells and normal human dermal fibroblasts, recently introduced as co-culture scratch-wound migration assay. Applying antibodies for α-smooth-muscle actin, von Willebrand factor, extra domain A fibronectin and endothelin-1, we were able to monitor proliferation, migration and the differentiation process from fibroblasts to myofibroblasts as a response to hypoxia. Furthermore, we verified, whether transforming growth factor β1 (TGFβ1) and endothelin-1 are able to mediate this response. We show, that proliferation and migration of endothelial cells and fibroblasts decreased under hypoxia. The additional administration of TGFβ1 did not significantly attenuate this decrease. Solely the myofibroblast population in co-culture adapted well to hypoxia, when cultures were supplemented with TGFβ1. Considerating the data concerning TGFβ1 and endothelin-1, we propose a model explaining the cellular interaction during early and late proliferation phase of human wound healing.

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Abbreviations

α-SMA:

α-Smooth-muscle actin

bFGF:

Basic fibroblast growth factor

CCSWMA:

Co-culture scratch-wound migration assay

DAPI:

4′,6-Diamidino-2-phenylindole

ECM:

Extracellular matrix

ED-A-fn:

Extra domain A fibronectin

ET-1:

Endothelin-1

HDMEC:

Human dermal microvascular endothelial cell/s

NHDF:

Normal human dermal fibroblast/s

MF:

Myofibroblast/s

sd:

Standard deviation

TGFβ1 :

Transforming growth factor β1

vWF:

von Willebrand factor

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Authors and Affiliations

  1. Clinic of Trauma-, Hand- and Reconstructive Surgery, Saarland University, Homburg, Germany

    Martin Oberringer, Claudia Meins, Monika Bubel & Tim Pohlemann

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  1. Martin Oberringer
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  2. Claudia Meins
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  3. Monika Bubel
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  4. Tim Pohlemann
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Correspondence to Martin Oberringer.

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Oberringer, M., Meins, C., Bubel, M. et al. In vitro wounding: effects of hypoxia and transforming growth factor β1 on proliferation, migration and myofibroblastic differentiation in an endothelial cell-fibroblast co-culture model. J Mol Hist 39, 37–47 (2008). https://doi.org/10.1007/s10735-007-9124-3

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  • DOI: https://doi.org/10.1007/s10735-007-9124-3

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