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

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.     

Keratinocytes control the pheo/eumelanin ratio in cultured normal human melanocytes

The pheo/eumelanin ratio of cultured normal human melanocytes is distinct from the ratio observed for the same cells in vivo where they are in close contact with keratinocytes. To study the possible involvement of keratinocytes in the control of melanogenesis, we compared quantitatively and qualitatively the melanin production in melanocyte mono-cultures, in melanocyte-keratinocyte co-cultures and in pigmented reconstructed epidermis. Pheomelanin and eumelanin contents were assessed by high-performance liquid chromatography with electrochemical and fluorometric detection of their specific degradation products and revealed striking differences in the presence of keratinocytes. In the absence of keratinocytes (melanocyte mono-cultures), we observed a very limited eumelanin production and a very high pheomelanin synthesis. The pheo/eumelanin ratio in mono-cultures could be slightly influenced by changing the composition of the culture medium, however, the very strong imbalance in favor of pheomelanin remained unchanged. An induction of eumelanin synthesis accompanied by an important reduction of pheomelanin formation was only observed in the presence of keratinocytes. The pheo/eumelanin ratio in melanocyte mono-culture dropped from 1043 down to about 25 in the presence of keratinocytes (co-cultures). The same observations were made when the melanocytes were integrated into a reconstructed human epidermis. Interestingly, under co-culture conditions resulting in only a partial contact between melanocytes and keratinocytes, the reduction of the pheo/eumelanin ratio were less pronounced. From these results we conclude that keratinocytes play an important role in the melanin production, affecting the melanogenic pathways.     

Culture of skin cells in 3D rather than 2D improves their ability to survive exposure to cytotoxic agents

In this study, we asked the question of whether cells in 3D culture cope more effectively with cytotoxic agents than cells in 2D. The sensitivities of human skin cells (keratinocytes, dermal fibroblasts and endothelial cells) to oxidative stress (hydrogen peroxide) and to a potentially toxic heavy metal (silver) when cultured under 2D and 3D conditions were investigated. The results show a marked resistance of cells to a given dose of hydrogen peroxide or silver nitrate causing a 50% loss of viability in 3D cultures, when compared to the same cells grown in 2D. There was also an improvement in the ability of cells to withstand both stresses when cells were in co-culture rather than in mono-culture. Foetal calf serum was found to have a mild protective effect in 2D culture but this was not extended to findings in 3D culture. This study suggests that dermatotoxicity testing using 3D co-cultures might be more likely to reflect true physiological responses to xenobiotic materials than existing models that rely on 2D mono-cultures.     

Different cell cycle responses of wound healing protagonists to transient in vitro hypoxia

Polyploidization is a process present in cells of many different human tissues. Since it is also prominent in human wound healing in vivo and in vitro, we focused on the influence of hypoxia on the cells proliferation and polyploidization response. The proliferation response of two major cell types, involved in human wound healing, human dermal microvascular endothelial cells (HDMEC) and normal human dermal fibroblasts (NHDF) was quite similar in the in vitro setup: proliferation significantly decreased under the influence of 18 h of hypoxia and was reinitiated after 72 h of reoxygenation. The cells response concerning their tendency towards the development of polyploidy was different: NHDF did not generate any polyploid cells, which stands in contrast to former in vitro studies with human wound-derived fibroblasts, but HDMEC were characterized by the presence of both mononuclear and binuclear tetraploid cells. The number of tetraploids was downregulated during hypoxia and increased during reoxygenation, accompanied by proliferation onset. The immunomicroscopic survey of HDMEC opened up a cell cycle model, which might be useful in the future to evaluate cell cycle modulations leading to polyploidy without the need to apply any additional cell cycle inhibitors.     

In vitro wounding: effects of hypoxia and transforming growth factor β<Subscript>1</Subscript> on proliferation,migration and myofibroblastic differentiation in an endothelial cell-fibroblast co-culture model

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.     

Cocoa procyanidins inhibit proliferation and angiogenic signals in human dermal microvascular endothelial cells following stimulation by low-level H2O2

Procyanidins extracted from cocoa play a role in the defense against oxidative stress, as well as in vascular and immune functions. We previously reported that pentameric procyanidins isolated from cocoa inhibit the expression of the tyrosine kinase ErbB2 gene, thus slowing the growth of cultured human aortic endothelial cells. We herein investigate the further consequences of such inhibition by cocoa procyanidins, particularly regarding the protein level in phosphorylation patterns and the effects on the proliferation of human dermal microvascular endothelial cells (HDMECs) following angiogenic stimulation with low-level H2O2. We report herein that both the pentameric and octameric procyanidin fractions of cocoa inhibit the proliferation of HDMECs, whereas the pentameric fraction modulates the activity of several crucial proteins in angiogenic signaling by altering their tyrosine phosphorylation. Similar to aortic endothelial cells, the pentameric procyanidin fraction down-regulates the expression of ErbB2 tyrosine kinase in HDMECs. In conclusion, we report evidence suggesting that polyphenols may influence endothelial growth signaling, thus affecting angiogenesis in vitro. If these observations are applicable in vivo, they suggest a beneficial effect for cells overexpressing ErbB2, such as in specific neoplasias     

Low molecular weight hyaluronan mediated CD44 dependent induction of IL-6 and chemokines in human dermal fibroblasts potentiates innate immune response

Complex regulation of the wound healing process involves multiple interactions among stromal tissue cells, inflammatory cells, and the extracellular matrix. Low molecular weight hyaluronan (LMW HA) derived from the degradation of high molecular weight hyaluronan (HMW HA) is suggested to activate cells involved in wound healing through interaction with HA receptors. In particular, receptor CD44 is suggested to mediate cell response to HA of different MW, being the main cell surface HA receptor in stromal tissue and immune cells. However, the response of dermal fibroblasts, the key players in granulation tissue formation within the wound healing process, to LMW HA and their importance for the activation of immune cells is unclear. In this study we show that LMW HA (4.3 kDa) induced pro-inflammatory cytokine IL-6 and chemokines IL-8, CXCL1, CXCL2, CXCL6 and CCL8 gene expression in normal human dermal fibroblasts (NHDF) that was further confirmed by increased levels of IL-6 and IL-8 in cell culture supernatants. Conversely, NHDF treated by HMW HA revealed a tendency to decrease the gene expression of these cytokine and chemokines when compared to untreated control. The blockage of CD44 expression by siRNA resulted in the attenuation of IL-6 and chemokines expression in LMW HA treated NHDF suggesting the involvement of CD44 in LMW HA mediated NHDF activation. The importance of pro-inflammatory mediators produced by LMW HA triggered NHDF was evaluated by significant activation of blood leukocytes exhibited as increased production of IL-6 and TNF-α. Conclusively, we demonstrated a pro-inflammatory response of dermal fibroblasts to LMW HA that was transferred to leukocytes indicating the significance of LMW HA in the inflammatory process development during the wound healing process.     

Apoptotic primary normal human dermal fibroblasts for in vitro models of fibrosis

Recent studies show that apoptosis affects surrounding tissue, playing a role in diseases such as fibrosis, a significant global disease burden. Elucidating the mechanisms by which the different apoptotic cells present during fibrotic wound healing affect their environment would enable development of new therapies. We describe here a simple, rapid, and cost-effective method for inducing apoptosis of primary normal human dermal fibroblasts without affecting the overall cell viability of the population. Such population could be used for in vitro models of fibrotic wound healing in co-culture with other cells involved in this process to study events such as apoptosis-induced proliferation.     

Origin of periendothelial cells in microvessels derived from human microvascular endothelial cells

In microvessels, periendothelial cells expressing alpha smooth muscle actin (alphaSMA) interact with the endothelial cells and are essential for vessel maturation and stabilization. In adult tissues, the cellular origin of the periendothelial cells is still not clear, in particular in humans. To determine the origin of human periendothelial cells, we used a recently developed 3D co-culture system that mimics human skin connective tissue. This system is composed of normal human dermal fibroblasts (NHDF), human dermal microvascular endothelial cells (HMEC-1), and a collagen matrix. In this system, "microvessels" composed of an endothelial lumen associated with periendothelial cells develop. Using this co-culture system, we (i) labelled fibroblasts with the vital dye CFDA-SE, cultured them with unlabelled endothelial cells, and observed that only endothelium-associated CFDA-SE-labelled cells express alphaSMA; (ii) infected endothelial cells with a retrovirus stably expressing eGFP, cultured them with unlabelled fibroblasts, and observed that cells expressing alphaSMA did not co-express eGFP, but were associated with the eGFP-expressing endothelial cells of the microvessels. Together, these results indicate that periendothelial cells arise by differentiation from fibroblasts and that they require interaction with endothelial cells to do so.     

Soft tissue fibroblasts from well healing and chronic human wounds show different rates of myofibroblasts in vitro

Due to an increasing life expectancy in western countries, chronic wound treatment will be an emerging challenge in the next decades. Because therapies are improving slowly appropriate diagnostic tools enabling the early prediction of the healing success remain to be developed. We used a well-established in vitro assay in combination with the analysis of 27 cytokines to discriminate between fibroblasts from chronic (n = 6) and well healing (n = 8) human wounds. Proliferation and migration of the cells as well as their response to hypoxia and their behaviour in co-culture with microvascular endothelial cells were analyzed. Myofibroblast differentiation, a time-limited essential process of regular wound healing, was also quantified. Besides weaker proliferation and migration significantly higher rates of myofibroblasts were detected in chronic wounds. With respect to the cytokine release, there was a clear trend within the group of chronic wound fibroblasts, which were releasing interferon-γ, monocyte chemotactic protein-1, granulocyte–macrophage colony stimulating factor and basic fibroblast growth factor in higher amounts than fibroblasts from healing wounds. Although the overall response of both groups of fibroblasts to hypoxia and to the contact with endothelial cells was similar, especially chronic wound fibroblasts seemed to benefit from the endothelial interaction during hypoxia and displayed better migration characteristics. The study shows (1) that the assay can identify specific features of fibroblasts derived from different human wounds and (2) that wound fibroblasts are varying in their response to the chosen parameters. Thus, current therapeutic approaches and individual healing prediction might benefit from this assay.     

VEGF Stimulates RCAN1.4 Expression in Endothelial Cells via a Pathway Requiring Ca2+/Calcineurin and Protein Kinase C-δ

Background

Vascular endothelial growth factor (VEGF) has previously been shown to upregulate the expression of the endogenous calcineurin inhibitor, regulator of calcineurin 1, variant 4 (RCAN1.4). The aim of this study was to determine the role and regulation of VEGF-mediated RCAN1.4 expression, using human dermal microvascular endothelial cells (HDMECs) as a model system.

Methodology/Principal Findings

We show that VEGF is able to induce RCAN1.4 expression during cellular proliferation and differentiation, and that VEGF-mediated expression of RCAN1.4 was inhibited by the use of inhibitors to protein kinase C (PKC) and calcineurin. Further analysis revealed that siRNA silencing of PKC-delta expression partially inhibited VEGF-stimulated RCAN1.4 expression. Knockdown of RCAN1.4 with siRNA resulted in a decrease in cellular migration and disrupted tubular morphogenesis when HDMECs were either stimulated with VEGF in a collagen gel or in an endothelial/fibroblast co-culture model of angiogenesis. Analysis of intracellular signalling revealed that siRNA mediated silencing of RCAN1.4 resulted in increased expression of specific nuclear factor of activated T-cells (NFAT) regulated genes.

Conclusions/Significance

Our data suggests that RCAN1.4 expression is induced by VEGFR-2 activation in a Ca²⁺ and PKC-delta dependent manner and that RCAN1.4 acts to regulate calcineurin activity and gene expression facilitating endothelial cell migration and tubular morphogenesis.     

Salmon nasal cartilage proteoglycan enhances growth of normal human dermal fibroblast through Erk1/2 phosphorylation

Proteoglycan (PG) is a heavily glycosylated protein, localized to cell surface and extracellular matrix, and has various functions. Recently, it has been gradually revealed that PG interacts with various growth factors and morphogens and regulates cellular functions. Although salmon nasal cartilage PG (Salmon-PG) increases proliferation of immortalized cells, its mechanism remains unclear. In this study, we confirmed the effect of Salmon-PG on normal human dermal fibroblast (NHDF) and investigated the mechanism of PG action on NHDF. Salmon-PG dose- and time-dependently increased NHDF proliferation. Receptor tyrosine kinase array revealed that Salmon-PG increased only Erk1/2 signaling. Erk1/2 phosphorylation was significantly increased by Salmon-PG in a time-(10 min) and dose-(400 or 800 μg/mL) dependent manner. MEK inhibitor suppressed the enhancement of NHDF proliferation by Salmon-PG. The overall findings indicate that Salmon-PG plays a role as a growth factor in NHDF via Erk1/2 activation, suggesting that Salmon-PG contributes to the maintenance of skin homeostasis.     

Granulocyte macrophage colony stimulating factor (GM-CSF) biological actions on human dermal fibroblasts.

Fibroblasts are involved in all pathologies characterized by increased ExtraCellularMatrix synthesis, from wound healing to fibrosis. Granulocyte Macrophage-Colony Stimulating Factor (GM-CSF) is a cytokine isolated as an hemopoietic growth factor but recently indicated as a differentiative agent on endothelial cells. In this work we demonstrated the expression of the receptor for GM-CSF (GM-CSFR) on human normal skin fibroblasts from healthy subjects (NFPC) and on a human normal fibroblast cell line (NHDF) and we try to investigate the biological effects of this cytokine. Human normal fibroblasts were cultured with different doses of GM-CSF to study the effects of this factor on GM-CSFR expression, on cell proliferation and adhesion structures. In addition we studied the production of some Extra-Cellular Matrix (ECM) components such as Fibronectin, Tenascin and Collagen I. The growth rate of fibroblasts from healthy donors (NFPC) is not augmented by GM-CSF stimulation in spite of increased expression of the GM-CSFR. On the contrary, the proliferation of normal human dermal fibroblasts (NHDF) cell line seems more influenced by high concentration of GM-CSF in the culture medium. The adhesion structures and the ECM components appear variously influenced by GM-CSF treatment as compared to fibroblasts cultured in basal condition, but newly only NHDF cells are really induced to increase their synthesis activity. We suggest that the in vitro treatment with GM-CSF can shift human normal fibroblasts towards a more differentiated state, due or accompanied by an increased expression of GM-CSFR and that such "differentiation" is an important event induced by such cytokine.     

HIF1A/miR-20a-5p/TGFβ1 axis modulates adipose-derived stem cells in a paracrine manner to affect the angiogenesis of human dermal microvascular endothelial cells

Angiogenic cytokines secreted by the adipose-derived stem cells (ADSCs) might promote the angiogenesis of endothelial cells. In the present study, we hypothesize that miR-20a targets TGFB1 to modulate the transforming growth factor β1 (TGFβ1) secretion by ADSCs, therefore affecting the angiogenesis. We found that hypoxia-inducible factor 1A (HIF1A) and TGFβ1 expressions were increased by hypoxia, accompanied with promoted ADSC cell viability. Incubation with conditioned medium from ADSCs treated with hypoxia significantly enhanced the angiogenesis capacity of human dermal microvascular endothelial cells (HDMECs), while TGFB1-silenced ADSCs medium significantly reverses HDMECs angiogenesis. miR-20a suppresses the expression of TGFB1 and secretion of TGFβ1 by ADSCs via binding to its 3′untranslated region, therefore modulating the HDMEC angiogenesis via affecting the paracrine from ADSCs; the effects of miR-20a-overexpressed conditioned medium on HDMEC angiogenesis were significantly reversed by TGFB1-overexpressed conditioned medium. Finally, HIF1A suppressed the expression of miR-20a via targeting its promoter region, subsequently promoting the paracrine from ADSCs and HDMEC angiogenesis.     

Mesenchymal stem cell-conditioned medium accelerates skin wound healing: An in vitro study of fibroblast and keratinocyte scratch assays

We have used in vitro scratch assays to examine the relative contribution of dermal fibroblasts and keratinocytes in the wound repair process and to test the influence of mesenchymal stem cell (MSC) secreted factors on both skin cell types. Scratch assays were established using single cell and co-cultures of L929 fibroblasts and HaCaT keratinocytes, with wound closure monitored via time-lapse microscopy. Both in serum supplemented and serum free conditions, wound closure was faster in L929 fibroblast than HaCaT keratinocyte scratch assays, and in co-culture the L929 fibroblasts lead the way in closing the scratches. MSC-CM generated under serum free conditions significantly enhanced the wound closure rate of both skin cell types separately and in co-culture, whereas conditioned medium from L929 or HaCaT cultures had no significant effect. This enhancement of wound closure in the presence of MSC-CM was due to accelerated cell migration rather than increased cell proliferation. A number of wound healing mediators were identified in MSC-CM, including TGF-β1, the chemokines IL-6, IL-8, MCP-1 and RANTES, and collagen type I, fibronectin, SPARC and IGFBP-7. This study suggests that the trophic activity of MSC may play a role in skin wound closure by affecting both dermal fibroblast and keratinocyte migration, along with a contribution to the formation of extracellular matrix.     

Engineering the migration and attachment behaviour of primary dermal fibroblasts

The availability of primary cells present in pathological conditions is often very limited due to stringent ethical regulation and patient consent. One such condition is chronic wounds, where dermal fibroblasts show a deficient migration. In vitro models with cellular tools that mimic the in vivo scenario would be advantageous to test new therapies for these challenging wounds. Since the availability of primary dermal fibroblasts present in chronic wounds is restricted and their “shelf-life” limited due to the increased senescence, our aim was to engineer human dermal fibroblasts with impaired migration using synthetic Arg-Gly-Asp (RGD) peptides. We studied fibroblast behaviour on three different two dimensional (2D) surfaces, representative of the dermal extracellular matrix and the materials used in the development of dermal scaffolds, in addition to commercially available, collagen-based 3D dermal scaffolds, demonstrating that the concentration of synthetic RGD peptides necessary to impair migration of dermal fibroblasts should be tailored to the particular surface/material and cell population used. The described technology could be translated to other cell types including established cell lines. A wide range of synthetic peptides exists, which differ in the amino acid sequence, thus increasing the possibilities of this technology.     

Mesenchymal stem cells induce dermal fibroblast responses to injury

Although bone marrow-derived mesenchymal stem cells have been shown to promote repair when applied to cutaneous wounds, the mechanism for this response remains to be determined. The aim of this study was to determine the effects of paracrine signaling from mesenchymal stem cells on dermal fibroblast responses to injury including proliferation, migration and expression of genes important in wound repair. Dermal fibroblasts were co-cultured with bone marrow-derived mesenchymal stem cells grown in inserts, which allowed for paracrine interactions without direct cell contact. In this co-culture model, bone marrow-derived mesenchymal stem cells regulate dermal fibroblast proliferation, migration and gene expression. When co-cultured with mesenchymal stem cells, dermal fibroblasts show increased proliferation and accelerated migration in a scratch assay. A chemotaxis assay also demonstrated that dermal fibroblasts migrate towards bone marrow-derived mesenchymal stem cells. A PCR array was used to analyze the effect of mesenchymal stem cells on dermal fibroblast gene expression. In response to mesenchymal stem cells, dermal fibroblasts up-regulate integrin alpha 7 expression and down-regulate expression of ICAM1, VCAM1 and MMP11. These observations suggest that mesenchymal stem cells may provide an important early signal for dermal fibroblast responses to cutaneous injury.     

Fibroblast state switching orchestrates dermal maturation and wound healing

Murine dermis contains functionally and spatially distinct fibroblast lineages that cease to proliferate in early postnatal life. Here, we propose a model in which a negative feedback loop between extracellular matrix (ECM) deposition and fibroblast proliferation determines dermal architecture. Virtual‐tissue simulations of our model faithfully recapitulate dermal maturation, predicting a loss of spatial segregation of fibroblast lineages and dictating that fibroblast migration is only required for wound healing. To test this, we performed in vivo live imaging of dermal fibroblasts, which revealed that homeostatic tissue architecture is achieved without active cell migration. In contrast, both fibroblast proliferation and migration are key determinants of tissue repair following wounding. The results show that tissue‐scale coordination is driven by the interdependence of cell proliferation and ECM deposition, paving the way for identifying new therapeutic strategies to enhance skin regeneration.     

Ellagitannins from Phyllanthus muellerianus (Kuntze) Exell.: Geraniin and furosin stimulate cellular activity, differentiation and collagen synthesis of human skin keratinocytes and dermal fibroblasts

Leaves from Phyllanthus muellerianus (Kuntze) Exell. are traditionally used for wound healing in Western Africa. Aqueous extracts of dried leaves recently have been shown to stimulate proliferation of human keratinocytes and dermal fibroblasts. Within bioassay-guided fractionation the ellagitannins geraniin (1), corilagin (2), furosin (3), the flavonoids quercetin-3-O-β-d-glucoside (isoquercitrin), kaempferol-3-O-β-d-glucoside (astragalin), quercetin-3-O-d-rutinoside (rutin), gallic acid, methyl gallate, caffeic acid, chlorogenic acid, 3,5-dicaffeoylquinic acid and caffeoylmalic acid (phaselic acid) have been identified in P. muellerianus for the first time. Geraniin was shown to be the dominant component of an aqueous extract.Suitable analytical methods for quality control of geraniin in P. muellerianus extract (methanol/water, 70/30) have been developed and validated based on ICH guidelines (ICH-compliant protocol).Geraniin and furosin increased the cellular energy status of human skin cells (dermal fibroblasts NHDF, HaCaT keratinocytes), triggering the cells towards higher proliferation rates, with fibroblasts being more sensitive than keratinocytes. Highest stimulation of NHDF by geraniin was found at 5 μM, and of keratinocytes at 50-100 μM. Furosin stimulated NHDF at about 50 μM, keratinocytes at about 150-200 μM. Necrotic cytotoxicity of geraniin, as measured by LDH release, was observed at 20 μM for NHDF and 150 μM for keratinocytes. Toxicity of furosin - less than that of geraniin - was observed at >400 μM.Furosin and geraniin stimulated the biosynthesis of collagen from NHDF at 50 μM and 5-10 μM respectively. Geraniin at 105 μM significantly stimulated the differentiation in NHEK while furosin had a minor influence on the expression of involucrin and cytokeratins K1 and K10. The study proves clearly that hydrophilic extracts from P. muellerianus and especially the lead compound geraniin exhibit stimulating activity on dermal fibroblasts and keratinocytes, leading to increased cell proliferation, barrier formation and formation of extracellular matrix proteins. From these findings the traditional clinical use of such extracts for wound healing seems to be justified.