RESUMEN
Vascular endothelial growth factor (VEGF-A) is known for its effects on endothelial cells and as a positive mediator of angiogenesis. VEGF is thought to promote repair of cutaneous wounds due to its proangiogenic properties, but its ability to regulate other aspects of wound repair, such as the generation of scar tissue, has not been studied well. We examined the role of VEGF in scar tissue production using models of scarless and fibrotic repair. Scarless fetal wounds had lower levels of VEGF and were less vascular than fibrotic fetal wounds, and the scarless phenotype could be converted to a scar-forming phenotype by adding exogenous VEGF. Similarly, neutralization of VEGF reduced vascularity and decreased scar formation in adult wounds. These results show that VEGF levels have a strong influence on scar tissue formation. Our data suggest that VEGF may not simply function as a mediator of wound angiogenesis, but instead may play a more diverse role in the wound repair process.
Asunto(s)
Cicatriz/metabolismo , Factor A de Crecimiento Endotelial Vascular/fisiología , Factor A de Crecimiento Endotelial Vascular/uso terapéutico , Cicatrización de Heridas/fisiología , Animales , Anticuerpos/administración & dosificación , Anticuerpos/inmunología , Anticuerpos/farmacología , Cicatriz/tratamiento farmacológico , Cicatriz/patología , Colágeno/ultraestructura , Femenino , Feto/fisiopatología , Feto/cirugía , Fibroblastos/citología , Fibroblastos/metabolismo , Inmunoglobulina G/administración & dosificación , Inmunohistoquímica , Ratones , Ratones Endogámicos , Neovascularización Fisiológica/efectos de los fármacos , Neovascularización Fisiológica/fisiología , Molécula-1 de Adhesión Celular Endotelial de Plaqueta/metabolismo , Piel/irrigación sanguínea , Piel/ultraestructura , Factores de Tiempo , Factor A de Crecimiento Endotelial Vascular/genética , Vimentina/metabolismo , Cicatrización de Heridas/efectos de los fármacosRESUMEN
Wound healing in the oral mucosa is clinically distinguished by rapid healing and lack of scar formation compared with dermal wounds. Mechanisms of favorable mucosal healing are yet to be elucidated. Utilizing a murine model of equivalent-size mucosal and skin wounds, we verified the rapid reepithelializaton and reduction in scarring of oral wounds reported in humans. Collagen fibrillar structure in oral wounds rapidly approached the size of normal collagen fibrils, while the collagen ultrastructure in skin remained immature through the later phases of healing. To determine whether the transforming growth factor-beta (TGF-beta) contributes to the lack of scar formation in oral mucosa, we compared the expression and production in oral and skin wounds. The RNase protection assay demonstrated significantly lower levels of TGF-beta1 expression in oral wounds compared with dermal wounds, and no changes were observed in the expression levels of TGF-beta2 or TGF-beta 3. ELISA analysis confirmed that oral wounds contained lower levels of TGF-beta1 levels compared with dermal wounds, along with a significant increase in the ratio of TGF-beta 3 to -beta1. These findings showed reduced scarring in oral wounds at the ultrastructural level, and provide evidence that site-specific differences in TGF-beta production contributes to the superior healing of oral wounds.
Asunto(s)
Mucosa Bucal/metabolismo , Piel/metabolismo , Factor de Crecimiento Transformador beta/biosíntesis , Cicatrización de Heridas/fisiología , Heridas y Lesiones/metabolismo , Animales , Femenino , Ratones , Ratones Endogámicos BALB C , Mucosa Bucal/patología , Piel/patología , Factor de Crecimiento Transformador beta1/biosíntesis , Factor de Crecimiento Transformador beta3/biosíntesis , Heridas y Lesiones/patologíaRESUMEN
Scar and fibrosis are often the end result of mechanical injury and inflammatory diseases. One chemokine that is repeatedly linked to fibrotic responses is monocyte chemoattractant protein-1 (MCP-1). We utilized a murine fibrosis model that produces dermal lesions similar to scleroderma to evaluate collagen fibrillogenesis in the absence of MCP-1. Dermal fibrosis was induced by subcutaneous injection of bleomycin into the dorsal skin of MCP-1-/- and wild-type C57BL/6 mice. After 4 weeks of daily injections, bleomycin treatment led to thickened collagen bundles with robust inflammation in the lesional dermis of wild-type mice. In contrast, the lesional skin of MCP-1-/- mice exhibited a dermal architecture similar to phosphate-buffered saline (PBS)-injected control and normal skin, with few inflammatory cells. Ultrastructural analysis of the lesional dermis from bleomycin-injected wild-type mice revealed markedly abnormal arrangement of collagen fibrils, with normal large diameter collagen fibrils replaced by small collagen fibrils of 41.5 nm. In comparison, the dermis of bleomycin-injected MCP-1-/- mice displayed a uniform pattern of fibril diameters that was similar to normal skin (average diameter 76.7 nm). The findings implicate MCP-1 as a key determinant in the development of skin fibrosis induced by bleomycin, and suggest that MCP-1 may influence collagen fiber formation in vivo.
Asunto(s)
Quimiocina CCL2/genética , Dermis/patología , Enfermedades de la Piel/patología , Enfermedades de la Piel/fisiopatología , Animales , Antimetabolitos Antineoplásicos , Bleomicina , Quimiocina CCL2/deficiencia , Quimiocina CCL2/metabolismo , Colágeno Tipo I/genética , Colágeno Tipo I/metabolismo , Colágeno Tipo I/ultraestructura , Colagenasas/metabolismo , Decorina , Dermis/fisiopatología , Modelos Animales de Enfermedad , Proteínas de la Matriz Extracelular/genética , Fibroblastos/patología , Fibroblastos/ultraestructura , Fibromodulina , Fibrosis , Expresión Génica , Proteínas del Choque Térmico HSP47/genética , Metaloproteinasa 13 de la Matriz , Ratones , Ratones Endogámicos C57BL , Ratones Mutantes , Proteoglicanos/genética , Enfermedades de la Piel/inducido químicamente , Inhibidores Tisulares de Metaloproteinasas/metabolismo , Factor de Crecimiento Transformador beta/metabolismo , Factor de Crecimiento Transformador beta1RESUMEN
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
Transforming growth factor-beta (TGF-beta) is a potent stimulus of connective tissue accumulation, and is implicated in the pathogenesis of scleroderma and other fibrotic disorders. Smad3 functions as a key intracellular signal transducer for profibrotic TGF-beta responses in normal skin fibroblasts. The potential role of Smad3 in the pathogenesis of scleroderma was investigated in Smad3-null (Smad3(-/-)) mice using a model of skin fibrosis induced by subcutaneous injections of bleomycin. At early time points, bleomycin-induced macrophage infiltration in the dermis and local TGF-beta production were similar in Smad3(-/-) and wild-type mice. In contrast, at day 28, lesional skin from Smad3(-/-) mice showed attenuated fibrosis, lower synthesis and accumulation of collagen, and reduced collagen gene transcription in situ, compared to wild-type mice. Connective tissue growth factor and alpha-smooth muscle actin expression in lesional skin were also significantly attenuated. Electron microscopy revealed an absence of small diameter collagen fibrils in the dermis from bleomycin-treated Smad3(-/-) mice. Compared to fibroblasts derived from wild-type mice, Smad3(-/-) fibroblasts showed reduced in vitro proliferative and profibrotic responses elicited by TGF-beta. Together, these results indicate that ablation of Smad3 is associated with markedly altered fibroblast regulation in vivo and in vitro, and confers partial protection from bleomycin-induced scleroderma in mice. Reduced fibrosis is due to deregulated fibroblast function, as the inflammatory response induced by bleomycin was similar in wild-type and Smad3(-/-) mice.
Asunto(s)
Fibrosis/metabolismo , Esclerodermia Sistémica/metabolismo , Transducción de Señal/efectos de los fármacos , Transactivadores/metabolismo , Factor de Crecimiento Transformador beta/metabolismo , Actinas/biosíntesis , Animales , Bleomicina/farmacología , Western Blotting , Células Cultivadas , Colágeno/biosíntesis , Colágeno/efectos de los fármacos , Factor de Crecimiento del Tejido Conjuntivo , Modelos Animales de Enfermedad , Fibroblastos/efectos de los fármacos , Fibroblastos/metabolismo , Fibroblastos/patología , Fibroblastos/ultraestructura , Fibronectinas/biosíntesis , Fibrosis/inducido químicamente , Fibrosis/patología , Citometría de Flujo , Regulación de la Expresión Génica/efectos de los fármacos , Marcación de Gen , Hidroxiprolina/análisis , Proteínas Inmediatas-Precoces/efectos de los fármacos , Proteínas Inmediatas-Precoces/metabolismo , Inmunohistoquímica , Hibridación in Situ , Péptidos y Proteínas de Señalización Intercelular/metabolismo , Ratones , Ratones Noqueados , Esclerodermia Sistémica/patología , Piel/química , Piel/patología , Piel/ultraestructura , Transactivadores/genética , Factor de Crecimiento Transformador beta/genética , Factor de Crecimiento Transformador beta/farmacologíaRESUMEN
In previous studies, mice given a full-thickness scald injury had an influx of neutrophils into the skin that followed a local increase in a neutrophil chemoattractant. Because macrophages are known to infiltrate the wound area after neutrophils and are essential for normal wound repair, studies were designed to characterize the time course of macrophage accumulation in the wound and to identify the factor(s) responsible for this influx. A macrophage infiltrate into the wound was observed at 4 days post-injury and persisted through at least 10 days. This influx was preceded by an initial fourfold increase in dermal monocyte chemoattractant protein-1 levels at 24 hours post-injury (p < 0.05). This elevation in monocyte chemoattractant protein-1 was enhanced at 4 and 10 days postburn resulting in a sixfold increase over baseline (p < 0.01). Levels of tumor necrosis factor-alpha, a proinflammatory cytokine known to induce chemokine production, were elevated at 90 minutes after injury in burn- versus sham-injured groups (p < 0.05). Furthermore, administration of tumor necrosis factor-alpha neutralizing antibody in vivo reduced the dermal levels of monocyte chemoattractant protein-1 seen at 10 days postburn by 57% (p < 0.01); however, macrophage accumulation was not altered. Thus, elevated systemic TNF-alpha levels may influence the local chemokine milieu following burn injury.
Asunto(s)
Quemaduras/inmunología , Quimiocina CCL2/biosíntesis , Quimiotaxis de Leucocito/inmunología , Monocitos/inmunología , Factor de Necrosis Tumoral alfa/inmunología , Cicatrización de Heridas/fisiología , Animales , Quimiocina CCL2/análisis , Quimiocina CCL2/inmunología , Femenino , Macrófagos/fisiología , Ratones , Ratones Endogámicos BALB C , Piel/metabolismo , Factor de Necrosis Tumoral alfa/análisis , Factor de Necrosis Tumoral alfa/biosíntesisRESUMEN
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.