Erythropoietin ameliorates the reduced migration of human fibroblasts during in vitro hypoxia

Abstract

Erythropoietin promotes the formation of granulation tissue when administered to soft tissue wounds and it was shown to be most effective under tissue hypoxia. However, the action of erythropoietin on the cellular level is not well understood. In order to get a better insight into these processes, an in vitro wound healing assay was applied. Two main players of soft tissue healing-fibroblasts and microvascular endothelial cells-were used as mono- and co-cultures, subsequently inflicting in vitro wounds. Cell migration, proliferation, the differentiation of fibroblasts to myofibroblasts, and the release of vascular endothelial cell growth factor A and angiogenin were quantified in response to hypoxia and erythropoietin (5 IU/ml). Erythropoietin supplementation did neither affect proliferation nor migration of endothelial cells and fibroblasts under normoxia. Under hypoxia, the reduced fibroblast migration was ameliorated by erythropoietin. This effect coincided with an attenuated release of vascular endothelial growth factor A, whereas angiogenin release was unaffected by erythropoietin. The in vitro model applied in this study may represent an adequate approximation to certain aspects of the in vivo status of soft tissue regeneration and the results might serve to interpret the in vivo efficiency of erythropoietin at the cellular level: Erythropoietin has different impacts on the cells in normoxia and hypoxia. Its positive influence on fibroblast migration during hypoxia seems to support the strategies of applying erythropoietin in those chronic wounds, which exhibit fibroblastic dysfunction although good vascularisation is present.

Response of endothelial cells and pericytes to hypoxia and erythropoietin in a co-culture assay dedicated to soft tissue repair

“…In the present assay, we were not able to detect a shift of the MFP population after experimental hypoxia. In a similar assay, human dermal fibroblasts showed a clear response to hypoxia in terms of increasing the a-SMA positive MF [20]. Thus, the regulation of the hypoxia response is clearly distinct with respect to fibroblast-derived MF and MFP.…”

“…It was used for the examination of physiological and pathological aspects of soft tissue repair in different studies [10,19,20]. In the present study, the most adequate marker, namely a-SMA, served to identify a subset of differentiated pericytes, the MFP [9].…”

“…EPO is a 34 kDa glycoprotein hormone which is involved in the production of erythroid progenitor cells in response to hypoxia. A beneficial role of EPO for angiogenesis in the context of wound healing in vivo was documented [21], and the aforementioned assay already showed a promising effect of EPO on fibroblast migration in vitro [20].…”

“…Endothelial cells were identified by the expression of vWF, a protein which is absent on pericytes, so that the different cell types and subpopulations could be monitored over time and in response to the different stimuli. The stimulus hypoxia as a general factor occurring subsequent to tissue damage is provided by a special incubator using a 5 % CO 2 /95 % N 2 mixture at constant flow [10,19,20]. As demonstrated for the aforementioned CCSWMA, the present assay is also suitable to measure the influence of agents and drugs, which are assumed to be beneficial for the processes of tissue repair.…”

Evidence of synergistic/additive effects of sildenafil and erythropoietin in enhancing survival and migration of hypoxic endothelial cells

“…Some groups have reported on this phenomenon, suggesting that the data depend on the exact type of endothelial cells. For instance, Epo could promote migration of human umbilical vein endothelial cells (HUVEC) and bovine adrenal capillary endothelial cells (2) as well as mouse embryoid body endothelial cells and human endothelial progenitor cells (30), whereas migration of human dermal microvascular endothelial cells was reduced by Epo under normoxia and unaffected under hypoxia (4).…”