RESUMEN
Hypertrophic scars are a pathological process characterized by an excessive deposition of extracellular matrix components. Using a tissue-engineered reconstructed human skin (RHS) method, we previously reported that pathological keratinocytes induce formation of a fibrotic dermal matrix. We further investigated keratinocyte action using conditioned media. Results showed that conditioned media induce a similar action on dermal thickness similar to when an epidermis is present. Using a two-dimensional electrophoresis technique, we then compared conditioned media from normal or hypertrophic scar keratinocytes and determined that TIMP-1 was increased in conditioned media from hypertrophic scar keratinocytes. This differential profile was confirmed using ELISA, assaying TIMP-1 presence on media from monolayer cultured keratinocytes and from RHS. The dermal matrix of these RHS was recreated using mesenchymal cells from three different origins (skin, wound and hypertrophic scar). The effect of increased TIMP-1 levels on dermal fibrosis was also validated independently from the mesenchymal cell origin. Immunodetection of TIMP-1 showed that this protein was increased in the epidermis of hypertrophic scar biopsies. The findings of this study represent an important advance in understanding the role of keratinocytes as a direct potent modulator for matrix degradation and scar tissue remodeling, possibly through inactivation of MMPs.
Asunto(s)
Cicatriz Hipertrófica/enzimología , Queratinocitos/enzimología , Inhibidor Tisular de Metaloproteinasa-1/metabolismo , Células Cultivadas , Cicatriz Hipertrófica/patología , Medios de Cultivo Condicionados , Electroforesis en Gel Bidimensional , Ensayo de Inmunoadsorción Enzimática , Fibrosis , Humanos , Inmunohistoquímica , Queratinocitos/patología , Cicatrización de Heridas/fisiologíaRESUMEN
Interactions between cells are a crucial mechanism to correctly heal a wounded tissue. Myofibroblasts have a central role during healing but their means to communicate with other cells is unknown. Microparticles (MP) have demonstrated a potential role as mediators of cellular interactions during various diseases. We have analyzed the production of MP by normal (Wmyo) and pathological (hypertrophic scar, Hmyo) myofibroblasts and human dermal fibroblasts (Fb) when treated with serum or plasma as examples of body fluids. We have shown that the presence of these body fluids induced a very significant increase in MP production by Wmyo while no MP production was denoted for Hmyo and Fb. These effects were at least due to thermally sensitive protein(s) with a molecular mass >30 kDa. Furthermore, the increase in MP production was not linked to an increase in apoptotic Wmyo. MP characterization showed that VEGF and FGF2 were present in MP and that endothelial and (myo)fibroblast cell growth can be stimulated by MP treatment. We postulated that MP production by myofibroblasts could modulate mesenchymal cell growth and angiogenesis during normal healing.
Asunto(s)
Comunicación Celular , Micropartículas Derivadas de Células/metabolismo , Cicatriz Hipertrófica/metabolismo , Fibroblastos/metabolismo , Tejido de Granulación/metabolismo , Piel/metabolismo , Cicatrización de Heridas , Adulto , Apoptosis , Proliferación Celular , Micropartículas Derivadas de Células/patología , Células Cultivadas , Cicatriz Hipertrófica/patología , Factor 2 de Crecimiento de Fibroblastos/sangre , Fibroblastos/patología , Tejido de Granulación/patología , Humanos , Peso Molecular , Fenotipo , Desnaturalización Proteica , Piel/patología , Factor A de Crecimiento Endotelial Vascular/sangre , Adulto JovenRESUMEN
Hypertrophic scarring is a pathological process characterized by fibroblastic hyperproliferation and by excessive deposition of extracellular matrix components. It has been hypothesized that abnormalities in epidermal-dermal crosstalk explain this pathology. To test this hypothesis, a tissue-engineered model of self-assembled reconstructed skin was used in this study to mimic interactions between dermal and epidermal cells in normal or pathological skin. These skin equivalents were constructed using three dermal cell types: normal wound (Wmyo) or hypertrophic wound (Hmyo) myofibroblasts and normal skin fibroblasts (Fb). Epidermis was reconstructed with normal skin keratinocytes (NK) or hypertrophic scar keratinocytes (HK). In the absence of keratinocytes, Hmyo formed a thicker dermis than Wmyo. When seeded with NK, the dermal thickness of Hmyo (121.2 +/- 31.4 microm vs 196.2 +/- 27.8 microm) and Fb (43.7 +/- 7.1 microm vs 83.6 +/- 16.3 microm) dermis was significantly (p < 0.05) reduced, while that of Wmyo (201.5 +/- 15.7 microm vs 160.7 +/- 21.1 microm) was increased. However, the presence of HK always induced significantly thicker dermis formation than observed with NK (Wmyo: 238.8 +/- 25.9 microm; Hmyo: 145.5 +/- 22.4 microm; Fb: 74.2 +/- 11.2 microm). These results correlated with collagen and MMP-1 secretion and with cell proliferation, which were increased when keratinocytes were added, except for the collagen secretion of Hmyo and Fb in the presence of NK. The level of dermal apoptosis was not different when epidermis was added to the dermis (<1% in each category). These observations strongly suggest that hypertrophic scar keratinocytes play a role in the development of pathological fibrosis by influencing the behaviour of dermal cells.
Asunto(s)
Cicatriz Hipertrófica/patología , Piel/patología , Cicatrización de Heridas , Apoptosis , Estudios de Casos y Controles , Comunicación Celular , Diferenciación Celular , Cicatriz Hipertrófica/enzimología , Dermis/enzimología , Dermis/patología , Epidermis/enzimología , Epidermis/patología , Fibroblastos/patología , Fibrosis , Humanos , Queratinocitos/patología , Metaloproteinasa 1 de la Matriz/metabolismo , Modelos Biológicos , Piel/enzimología , Ingeniería de Tejidos/métodosRESUMEN
During wound healing, the transition from granulation to scar tissue shows a decrease in myofibroblast cellularity. Previous results have correlated the disappearance of these cells with the induction of apoptotic cell death by some unknown stimuli. In contrast, hypertrophic scar appearance after wound healing is thought to be linked to a disorder of apoptotic function which induces myofibroblast persistence in granulation tissue. Oxidative stress being an important mediator of apoptosis, we have evaluated the apoptotic response of normal and pathological wound myofibroblasts (WMyo and HMyo respectively) in their interaction with two oxidative stress inducers: hydrogen peroxide, using a high concentration as a single dose, and sodium ascorbate which induced a continuous release of H2O2 at a low concentration. Our results showed that, according to the H2O2 treatment type, HMyo were more sensitive (after ascorbate treatment) or less sensitive (after H2O2 treatment) when compared to WMyo and Fb. We next assessed the presence of several molecules known to be involved in the antioxidant network protecting cells against H2O2 injury and found HMyo to have a higher level of activity of glutathione peroxidase and a lower level of activity of catalase than WMyo. These results can help explain the contradictory responses of myofibroblasts according to the oxidative stress treatment. This is the first study linking refractory oxidative stress mediated cell death to cellular phenotype in hypertrophic myofibroblasts, and indicates a pivotal role for the antioxidant enzyme system in this type of resistance.
Asunto(s)
Antioxidantes/metabolismo , Apoptosis/efectos de los fármacos , Fibroblastos/fisiología , Peróxido de Hidrógeno/farmacología , Oxidantes/metabolismo , Adulto , Anexina A5/metabolismo , Ácido Ascórbico/metabolismo , Ácido Ascórbico/farmacología , Caspasas/metabolismo , Catalasa/metabolismo , Células Cultivadas , Relación Dosis-Respuesta a Droga , Activación Enzimática/efectos de los fármacos , Fibroblastos/citología , Fibroblastos/patología , Citometría de Flujo , Fluoresceína-5-Isotiocianato , Colorantes Fluorescentes , Glutatión/análisis , Glutatión Peroxidasa/análisis , Glutatión Reductasa/análisis , Humanos , Peróxido de Hidrógeno/metabolismo , Concentración de Iones de Hidrógeno , Estrés Oxidativo/efectos de los fármacos , Propidio , Piel/citología , Coloración y Etiquetado , Cicatrización de HeridasRESUMEN
During wound healing, myofibroblasts play a central role in matrix formation and wound contraction. At the end of healing, there is evidence that myofibroblasts disappear via apoptotic pathways. Hypertrophic scars are a fibroproliferative disorder that leads to considerable morbidity. It has been postulated that a defect in myofibroblast apoptosis could be responsible for the pathological scar formation, but no evidence exists. We have isolated and cultured human normal wound (Wmyo) and hypertrophic scar (Hmyo) myofibroblasts and compared their basal apoptotic rates and their sensitivity to serum starvation and Fas antibody-induced apoptosis to that obtained for dermal fibroblasts (Fb). A higher rate of apoptosis as evidenced by morphological criteria and a propidium iodide assay was observed for Wmyo in comparison to Fb and Hmyo. These results came along with a low level of the anti-apoptotic proteins Bcl-2 and Bclx(L) in Wmyo, whereas there was an increase in the level of the pro-apoptotic molecule Bax when compared to the results obtained for Fb and Hmyo. Hmyo showed a higher level of Bcl-2 compared to Fb but no difference in the Bax or Bclx(L) level. After serum starvation, Wmyo revealed an increased apoptotic rate, whereas Hmyo and Fb did not show any difference. Anti-Fas treatment did not modify the levels of apoptosis but strongly increased the cell growth of Hmyo as compared to Wmyo. This is the first study presenting a broad vision of the apoptotic sensitivity of normal and pathological myofibroblasts. These results confirmed the hypothesis of defects in apoptosis and growth during pathological scar formation impeding myofibroblast disappearance at the end of healing.