RESUMEN
BACKGROUND: Previous studies have demonstrated that the lack of lumican delayed corneal wound healing in lumican-null (Lum(-/-) ) mice. This defect is rescued by the addition of glycosylated lumican core protein to the injured corneas. OBJECTIVES: We examined the hypothesis that lumican is also required for the healing of cutaneous wounds using Lum(-/-) mice. METHODS: We demonstrated the basic thinner skin phenotypes in Lum(-/-) mice at different time points and the changes in arrangement of collagen fibres by transmission electron microscopy (TEM). A full skin thickness wound was generated by punch biopsy (6 mm diameter) in experimental Lum(-/-) and wild-type mice. The closure of injured skin was measured after various periods of time (3, 6, 12, 18 days). Specimens of injured and uninjured skin (serving as control) were then subjected to morphological examination with haematoxylin and eosin and Masson trichrome stains, and by TEM. Immunohistochemical staining with anti-CD68 antibody was used to assess the presence of macrophages in injured skin healing for various periods of time. Semiquantitative reverse transcription-polymerase chain reaction (RT-PCR) was used to elucidate the transforming growth factor (TGF)-ß1-induced myofibroblast phenotypic genes. RESULTS: Skin of adult Lum(-/-) mice (3 months and older) was much thinner (40% less) than that of age-matched wild-type mice. This phenomenon was aggravated in older mice. TEM revealed disoriented and irregular collagen fibrils in the dermis of Lum(-/-) mice. Delayed wound healing with an increase in inflammatory macrophages was compatible with the delayed response of the expression of TGF-ß1, type I collagen α1 and fibronectin at the mRNA level by semiquantitative RT-PCR in the Lum(-/-) mice. CONCLUSIONS: Our data demonstrate that lumican plays pivotal roles in skin collagen fibrillogenesis and wound healing.
Asunto(s)
Proteoglicanos Tipo Condroitín Sulfato/fisiología , Sulfato de Queratano/fisiología , Piel/fisiopatología , Cicatrización de Heridas/fisiología , Animales , Proteoglicanos Tipo Condroitín Sulfato/deficiencia , Proteoglicanos Tipo Condroitín Sulfato/genética , Colágeno/metabolismo , Colágeno/ultraestructura , Modelos Animales de Enfermedad , Fibronectinas/metabolismo , Inmunohistoquímica , Sulfato de Queratano/deficiencia , Sulfato de Queratano/genética , Lumican , Ratones , Ratones Noqueados , Microscopía Electrónica , Fenotipo , ARN Mensajero/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Piel/metabolismo , Piel/ultraestructura , Factor de Crecimiento Transformador beta1/metabolismo , Cicatrización de Heridas/genéticaRESUMEN
Tubedown-1 (tbdn-1) is a mammalian homologue of the N-terminal acetyltransferase subunit NAT1 of Saccharomyces cerevisiae and copurifies with an acetyltransferase activity. Tbdn-1 expression in endothelial cells becomes downregulated during the formation of capillary-like structures in vitro and is regulated in vivo in a manner which suggests a functional role in dampening blood vessel development. Here we show that tbdn-1 is expressed highly in the vitreal vascular network (tunica vasculosa lentis and vasa hyaloidea propria) during the pruning and remodeling phases of this transient structure. The vitreal blood vessels of mice harboring a targeted inactivation of TGF-beta2 fail to remodel and abnormally accumulate, a phenomenon reminiscent of the ocular pathology resembling persistent fetal vasculature (PFV) in humans. Since suppression of normal tbdn-1 expression has been previously observed in retinal vessel proliferation, we analyzed vitreal vascular changes and tbdn-1 expression in TGF-beta2(-/-) eyes. The nuclei of vitreal vessel endothelial cells in TGF-beta2(-/-) eyes express proliferating cell nuclear antigen (PCNA) and exhibit increased levels of active (P42/44)mitogen-activated protein kinase (phospho-(P42/44)MAPK), characteristics consistent with proliferative endothelial cells. In contrast to normal vitreal vessels, collagen IV expression exhibited a disorganized pattern in the TGF-beta2(-/-) vitreal vessels, suggesting vessel disorganization and possibly a breakdown of vessel basal laminae. Moreover, vitreal vessels of TGF-beta2(-/-) mice lack expression of pericyte markers (CD13, alpha smooth muscle actin) and show ultrastructural changes consistent with pericyte degeneration. The accumulating vitreal blood vessels of TGF-beta2(-/-) mice, while maintaining expression of the endothelial marker von Willebrand Factor, show a significant decrease in the expression of tbdn-1. We addressed the functional role of tbdn-1 in the regulation of vitreal blood vessels using an in vitro model of choroid-retina capillary outgrowth. Clones of the RF/6A fetal choroid-retina endothelial cell line showing suppression of tbdn-1 levels after overexpression of an antisense TBDN-1 cDNA display a significant increase in the formation of capillary-like structures in vitro compared with controls. These findings suggest that tbdn-1 inhibits capillary-like formation in vitro and may serve to dampen vitreal blood vessel formation preceding the regression of the vitreal vasculature during development. Our results also suggest that tbdn-1 may participate with TGF-beta2 in regulating normal development of the vitreal vasculature.