RESUMEN
Acute ethanol exposure represents an increased risk factor for morbidity and mortality associated with surgical or traumatic injury. Despite clinical observations suggesting that ethanol exposure before injury alters tissue repair processes, little direct evidence about the mechanism by which ethanol affects the wound healing process is available. In this study, excisional wounds from female BALB/c mice with or without circulating ethanol levels of 100 mg/dl were used to assess wound closure, angiogenesis, and collagen content. Ethanol exposure resulted in a significant but transient delay in wound closure at day 2 postwounding (28 +/- 4% vs. 17 +/- 1%). In addition, total collagen content was significantly reduced by up to 37% in wounds from ethanol-treated mice compared with controls. The most significant effect of ethanol exposure on wounds was on vascularity because angiogenesis was reduced by up to 61% in wounds from ethanol-treated mice. The reduction in vessel density occurred despite near-normal levels of proangiogenic factors VEGF and FGF-2, suggesting a direct effect of ethanol exposure on endothelial cell function. Further evidence for a direct effect was observed in an in vitro angiogenesis assay because the exposure of endothelial cells to ethanol reduced angiogenic responsiveness to just 8.33% of control in a cord-forming assay. These studies provide novel information regarding the effect of a single dose of ethanol on multiple parameters of the wound healing process in vivo and suggest a potential mechanism by which ethanol impairs healing after traumatic injury.
Asunto(s)
Depresores del Sistema Nervioso Central/farmacología , Etanol/farmacología , Neovascularización Fisiológica/efectos de los fármacos , Cicatrización de Heridas/efectos de los fármacos , Animales , Quemaduras/fisiopatología , División Celular/efectos de los fármacos , Colágeno/metabolismo , Células Epiteliales/efectos de los fármacos , Células Epiteliales/fisiología , Femenino , Factor 2 de Crecimiento de Fibroblastos/metabolismo , Ratones , Ratones Endogámicos BALB C , Piel/irrigación sanguínea , Piel/lesiones , Piel/metabolismo , Factor A de Crecimiento Endotelial Vascular/metabolismoRESUMEN
The expression of chemokines has been suggested to involve an interdependent network, with the absence of a single chemokine affecting the expression of multiple other chemokines. Monocyte chemoattractant protein (MCP-1), a member of C-C chemokine superfamily, plays a critical role in the recruitment and activation of leukocytes during acute inflammation. To examine the effect of the loss of MCP-1 on expression of the chemokine network, we compared the mRNA expression profiles of MCP-1(-/-) and wild type mice during the acute inflammatory phase of excisional wounds. Utilizing a mouse cDNA array containing 514 chemokine and chemokine related genes, the loss of MCP-1 was observed to cause a significant upregulation of nine genes (Decorin, Persephin, IL-1beta, MIP-2, MSP, IL1ra, CCR5, CCR3, IL-11) and significant downregulation of two genes (CCR4 and CD3Z) in acute wounds. The array data was confirmed by semi-quantitative RT-PCR. The effect of MCP-1 deletion on chemokine expression was further examined in isolated macrophages. Compared to wild type, LPS-stimulated peritoneal macrophages from MCP-1(-/-) mice showed a significant increase in the expression of RANTES, MIP-1beta, MIP-1alpha and MIP-2 mRNA. The data suggest that loss of a single chemokine perturbs the chemokine network not only in the setting of acute inflammation but even in an isolated inflammatory cell, the macrophage.
Asunto(s)
Quimiocina CCL2/metabolismo , Quimiocinas/genética , Regulación de la Expresión Génica , Inflamación/genética , Receptores de Quimiocina/genética , Animales , Quimiocina CCL2/genética , Quimiocina CCL2/farmacología , Modelos Animales de Enfermedad , Femenino , Regulación de la Expresión Génica/efectos de los fármacos , Macrófagos Peritoneales/efectos de los fármacos , Macrófagos Peritoneales/inmunología , Ratones , Ratones Endogámicos C57BL , Ratones Mutantes , Análisis de Secuencia por Matrices de Oligonucleótidos , Piel/lesiones , Heridas y Lesiones/inmunologíaRESUMEN
Vascular endothelial growth factor (VEGF-A), a potent stimulus for angiogenesis, is up-regulated in the skin after wounding. Although studies have shown that VEGF is important for wound repair, it is unclear whether this is based solely on its ability to promote angiogenesis or if VEGF can also promote healing by acting directly on non-endothelial cell types. By immunohistochemistry and reverse transcriptase-polymerase chain reaction, expression of VEGF receptor-1 (VEGFR-1), but not VEGFR-2, was detected in murine keratinocytes during wound repair and in normal human epidermal keratinocytes (NHEKs). The presence of VEGF receptors on NHEKs was verified by binding studies with 125I-VEGF. In vitro, VEGF stimulated the proliferation of NHEKs, an effect that could be blocked by treatment with neutralizing VEGFR-1 antibodies. A role for VEGFR-1 in keratinocytes was also shown in vivo because treatment of excisional wounds with neutralizing VEGFR-1 antibodies delayed re-epithelialization. Treatment with anti-VEGFR-1 antibodies also reduced the number of proliferating keratinocytes at the leading edge of the wound, suggesting that VEGF sends a proliferative signal to these cells. Together, these data describe a novel role for VEGFR-1 in keratinocytes and suggest that VEGF may play several roles in cutaneous wound repair.
Asunto(s)
Queratinocitos/fisiología , Receptor 1 de Factores de Crecimiento Endotelial Vascular/fisiología , Cicatrización de Heridas/fisiología , Animales , Línea Celular , Femenino , Expresión Génica , Humanos , Inmunohistoquímica , Isótopos de Yodo/metabolismo , Queratinocitos/química , Ratones , Ratones Endogámicos BALB C , Unión Proteica , ARN Mensajero/análisis , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Piel/patología , Factor A de Crecimiento Endotelial Vascular/metabolismo , Receptor 1 de Factores de Crecimiento Endotelial Vascular/genética , Receptor 1 de Factores de Crecimiento Endotelial Vascular/metabolismoRESUMEN
BACKGROUND: Normal wound healing is a regulated sequence of events that successfully restore tissue integrity. Previous studies have suggested that wound healing is impaired in a septic host. The current study examines the effect of sepsis on the inflammatory and proliferative phases of wound healing at a remote site of secondary injury. METHODS: Polyvinyl alcohol sponges, either inoculated with a standard dose of Pseudomonas aeruginosa (experimental) or soaked in normal saline (control), were placed subcutaneously in the anterior abdominal region of male B6D2F1 mice. Immediately following sponge placement, full thickness excisional dermal wounds were created on the dorsum. Wound healing was examined at days 3, 5, and 7 postinjury. The infiltration of neutrophils and macrophages into wounds was quantified, and the reepithelialization rate and collagen content were measured. RESULTS: Peripheral neutrophil counts were significantly elevated in infected mice, yet neutrophil content of the remote wound of infected animals was significantly reduced (5% of control, P < 0.05). Wounds of infected mice also showed a 30% reduction in the macrophage content. Wounds of infected animals exhibited delayed reepithelialization (76 +/- 3 vs 97 +/- 3% at day 5, P < 0.05) and collagen synthesis (55.3 +/- 9.5 vs 105 +/- 13.0 microg/wound, P < 0.05). CONCLUSION: Systemic infection alters both the inflammatory and the proliferative processes at remote sites of injury. Multiple factors seem likely to contribute to the increased incidence of wound complications in septic patients.
Asunto(s)
Sepsis/inmunología , Sepsis/fisiopatología , Cicatrización de Heridas/inmunología , Animales , División Celular/fisiología , Colágeno/análisis , Recuento de Leucocitos , Macrófagos/inmunología , Masculino , Ratones , Ratones Endogámicos , Neutrófilos/inmunología , Infecciones por Pseudomonas/inmunología , Infecciones por Pseudomonas/mortalidad , Infecciones por Pseudomonas/fisiopatología , Pseudomonas aeruginosa , Sepsis/mortalidad , Piel/lesiones , Piel/microbiología , Pérdida de PesoRESUMEN
Upon stimulation, mast cells release a heterogeneous group of factors that promote inflammation and influence cell proliferation. Mast cells accumulate at sites of injury, further suggesting a critical role in wound healing. To assess the importance of mast cells in tissue repair, we compared wound healing in mast cell-deficient WBB6F1/J-KitW/KitW-v (KitW/KitW-v) and wild type WBB6F1/++ (WT) mice. During the inflammatory phase, neutrophil infiltration into wounds of the KitW/KitW-v mice was significantly less than that of WT mice (84.6 +/- 10.3 vs. 218 +/- 26.0 cells/10 high-power fields at day 3, p < 0.001), while wound macrophage and T cell infiltration were similar in both strains. The decrease in neutrophils could not be explained by changes in tumor necrosis factor-alpha or macrophage inflammatory protein-2 levels, because the amounts of these two neutrophil chemoattractants were similar in both KitW/KitW-v and WT mice. Surprisingly, the absence of mast cells had no effect on the proliferative aspects of wound healing, including reepithelialization, collagen synthesis, and angiogenesis. Although mast cells are known to release proangiogenic mediators, vascular endothelial growth factor levels were similar in WT and KitW/KitW-v mice. Moreover, levels of fibroblast growth factor-2 were increased in KitW/KitW-v mice (4206 +/- 107 vs. 1865 +/- 249 pg/ml, p < 0.01). These results suggest that mast cells modulate the recruitment of neutrophils into sites of injury, yet indicate that mast cells are unlikely to exert a major influence on the proliferative response within healing wounds.